U.S. patent application number 09/749588 was filed with the patent office on 2002-08-15 for isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof.
Invention is credited to Beasley, Ellen M., Chandramouliswaran, Ishwar, Di Francesco, Valentina, Guegler, Karl, Webster, Marion, Yan, Chunhua.
Application Number | 20020110889 09/749588 |
Document ID | / |
Family ID | 25014370 |
Filed Date | 2002-08-15 |
United States Patent
Application |
20020110889 |
Kind Code |
A1 |
Chandramouliswaran, Ishwar ;
et al. |
August 15, 2002 |
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: |
Chandramouliswaran, Ishwar;
(Silver Spring, MD) ; Guegler, Karl; (Menlo Park,
CA) ; Webster, Marion; (San Francisco, CA) ;
Yan, Chunhua; (Boyds, MD) ; Di Francesco,
Valentina; (Rockville, MD) ; Beasley, Ellen M.;
(Darnestown, MD) |
Correspondence
Address: |
CELERA GENOMICS CORPORATION
45 West Gude Dr. C2-4#20
Rockville
MD
20850
US
|
Family ID: |
25014370 |
Appl. No.: |
09/749588 |
Filed: |
December 28, 2000 |
Current U.S.
Class: |
435/194 ;
435/325; 435/6.16; 435/69.1; 435/7.1; 514/1; 536/23.2; 800/8 |
Current CPC
Class: |
C12N 9/1205 20130101;
C12Y 207/11001 20130101; A61P 17/06 20180101; A61P 29/00 20180101;
A01K 2217/05 20130101; A61P 9/10 20180101; A61K 38/00 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
435/194 ; 435/6;
514/1; 800/8; 435/325; 435/7.1; 536/23.2; 435/69.1 |
International
Class: |
A61K 031/00; C12P
021/02; C12N 005/06; C12Q 001/68; G01N 033/53; A01K 067/00; C12N
009/12; C07H 021/04 |
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 homeodomain-interacting protein 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] The mitogen-activated protein kinases (MAP) are also members
of the STK family. MAP kinases also regulate intracellular
signaling pathways. They 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).
[0010] 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. Altered MAP kinase expression is
implicated in a variety of disease conditions including cancer,
inflammation, immune disorders, and disorders affecting growth and
development.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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
N K (1992) Annu. Rev. Cell. Biol. 8:463-93). Regulation of PTK
activity may therefore be an important strategy in controlling some
types of cancer.
[0015] Homeodomain-Interacting Protein Kinases
[0016] The novel human protein, and encoding gene, provided by the
present invention is related to the family of
homeodomain-interacting protein kinases (HIPKs). HIPKs are nuclear
kinases that act as transcriptional co-repressors for homeodomain
transcription factors. HIPKs enhance the repressor functions of NK
homeoproteins. The HIPK family comprises at least three previously
described members: HIPK1, HIPK2, and HIPK3. HIPKs comprise a
conserved protein kinase domain and a separate
homeoprotein/homeodomain interaction domain. HIPK2 has been found
to significantly increase the DNA binding activity of the NK-3
homeoprotein (Kim et al., J Biol Chem Oct. 2,
1998;273(40):25875-9).
[0017] HIPKs show a high degree of similarity to yeast YAK1
proteins, human PKY (protein kinase YAK1 homolog), and Myak (mouse
YAK homolog) proteins, which play important roles in cellular
regulation and signaling, multidrug resistance, and in restricting
cell growth. For a further review of HIPKs, YAK1, and PKY proteins,
see Begley et al., Gene 200: 35-43, 1997; Nupponen et al.,
Cytogenet. Cell Genet. 87: 102-103, 1999; and Sampson et al., J.
Cell. Biochem. 52: 384-395, 1993.
[0018] Kinase proteins, particularly members of the
homeodomain-interacting protein 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 homeodomain-interacting protein kinase
subfamily.
SUMMARY OF THE INVENTION
[0019] 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 homeodomain-interacting protein 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 testis, brain
medulloblastomas, infant brain, schizophrenic brain, retina,
germinal center B cells, colon, and liver.
DESCRIPTION OF THE FIGURE SHEETS
[0020] 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 testis, brain
medulloblastomas, infant brain, schizophrenic brain, retina,
germinal center B cells, colon, and liver.
[0021] 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.
[0022] 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, the following SNPs were identified: T4452A, A5330G, A9256C,
A11773G, G12886A, and a T insertion/deletion ("indel") at position
14131.
DETAILED DESCRIPTION OF THE INVENTION
[0023] General Description
[0024] 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 homeodomain-interacting protein 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 homeodomain-interacting protein 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.
[0025] 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 homeodomain-interacting protein kinase
subfamily and the expression pattern observed. Experimental data as
provided in FIG. 1 indicates expression in humans in testis, brain
medulloblastomas, infant brain, schizophrenic brain, retina,
germinal center B cells, colon, and liver. 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 homeodomain-interacting
protein kinase family or subfamily of kinase proteins.
[0026] Specific Embodiments
[0027] Peptide Molecules
[0028] 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
homeodomain-interacting protein 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.
[0029] 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.
[0030] 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).
[0031] 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.
[0032] 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.
[0033] 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 testis, brain medulloblastomas,
infant brain, schizophrenic brain, retina, germinal center B cells,
colon, and liver. 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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 encoding the novel kinase protein
of the present invention is located on a genome component that has
been mapped to human chromosome 1 (as indicated in FIG. 3), which
is supported by multiple lines of evidence, such as STS and BAC map
data.
[0046] 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 encoding the novel kinase protein of the present invention
is located on a genome component that has been mapped to human
chromosome 1 (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.
[0047] 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 seen: T4452A, A5330G, A9256C, A11773G,
G12886A, and a T insertion/deletion ("indel") at position 14131.
SNPs in introns, such as these, may affect control/regulatory
elements.
[0048] 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.
[0049] 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.
[0050] 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).
[0051] 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.
[0052] 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.
[0053] 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)).
[0054] 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.
[0055] 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.
[0056] 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).
[0057] 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.
[0058] 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)).
[0059] 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.
[0060] Protein/Peptide Uses
[0061] 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.
[0062] 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.
[0063] 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
testis, brain medulloblastomas, infant brain, schizophrenic brain,
retina, germinal center B cells, and colon, as indicated by virtual
northern blot analysis. In addition, PCR-based tissue screening
panels indicate expression in human liver. A large percentage of
pharmaceutical agents are being developed that modulate the
activity of kinase proteins, particularly members of the
homeodomain-interacting protein 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 testis, brain medulloblastomas, infant brain,
schizophrenic brain, retina, germinal center B cells, colon, and
liver. Such uses can readily be determined using the information
provided herein, that which is known in the art, and routine
experimentation.
[0064] 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 homeodomain-interacting protein
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
testis, brain medulloblastomas, infant brain, schizophrenic brain,
retina, germinal center B cells, and colon, as indicated by virtual
northern blot analysis. In addition, PCR-based tissue screening
panels indicate expression in human liver.
[0065] 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
testis, brain medulloblastomas, infant brain, schizophrenic brain,
retina, germinal center B cells, colon, and liver. In an alternate
embodiment, cell-based assays involve recombinant host cells
expressing the kinase protein.
[0066] 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.
[0067] 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.
[0068] 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).
[0069] 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.
[0070] 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.
[0071] 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 testis, brain medulloblastomas, infant
brain, schizophrenic brain, retina, germinal center B cells, and
colon, as indicated by virtual northern blot analysis. In addition,
PCR-based tissue screening panels indicate expression in human
liver.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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 testis, brain
medulloblastomas, infant brain, schizophrenic brain, retina,
germinal center B cells, colon, and liver. 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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 testis, brain medulloblastomas, infant brain,
schizophrenic brain, retina, germinal center B cells, colon, and
liver. 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.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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 or therapeutic 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.
[0086] 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 testis, brain medulloblastomas,
infant brain, schizophrenic brain, retina, germinal center B cells,
colon, and liver. Accordingly, methods for treatment include the
use of the kinase protein or fragments.
[0087] Antibodies
[0088] 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.
[0089] 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.
[0090] 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).
[0091] 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.
[0092] 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.
[0093] 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).
[0094] 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.
[0095] Antibody Uses
[0096] 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
testis, brain medulloblastomas, infant brain, schizophrenic brain,
retina, germinal center B cells, and colon, as indicated by virtual
northern blot analysis. In addition, PCR-based tissue screening
panels indicate expression in human liver. 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.
[0097] 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 testis, brain
medulloblastomas, infant brain, schizophrenic brain, retina,
germinal center B cells, colon, and liver. 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.
[0098] 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 testis, brain medulloblastomas, infant
brain, schizophrenic brain, retina, germinal center B cells, colon,
and liver. 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.
[0099] 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.
[0100] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression in
humans in testis, brain medulloblastomas, infant brain,
schizophrenic brain, retina, germinal center B cells, colon, and
liver. 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.
[0101] 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.
[0102] 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 nuleic acid arrays and
similar methods have been developed for antibody arrays.
[0103] Nucleic Acid Molecules
[0104] 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.
[0105] 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 5KB, 4KB, 3KB, 2KB, or 1KB 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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).
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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 encoding
the novel kinase protein of the present invention is located on a
genome component that has been mapped to human chromosome 1 (as
indicated in FIG. 3), which is supported by multiple lines of
evidence, such as STS and BAC map data.
[0120] 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 seen: T4452A, A5330G, A9256C, A11773G,
G12886A, and a T insertion/deletion ("indel") at position 14131.
SNPs in introns, such as these, may affect control/regulatory
elements.
[0121] 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 6X 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.
[0122] Nucleic Acid Molecule Uses
[0123] 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, the following SNPs were identified: T4452A, A5330G, A9256C,
A11773G, G12886A, and a T insertion/deletion ("indel") at position
14131.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0128] 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 encoding the
novel kinase protein of the present invention is located on a
genome component that has been mapped to human chromosome 1 (as
indicated in FIG. 3), which is supported by multiple lines of
evidence, such as STS and BAC map data.
[0129] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0130] 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.
[0131] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0132] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0133] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0134] 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 testis, brain medulloblastomas, infant
brain, schizophrenic brain, retina, germinal center B cells, and
colon, as indicated by virtual northern blot analysis. In addition,
PCR-based tissue screening panels indicate expression in human
liver. 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.
[0135] 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.
[0136] 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 testis, brain medulloblastomas, infant
brain, schizophrenic brain, retina, germinal center B cells, and
colon, as indicated by virtual northern blot analysis. In addition,
PCR-based tissue screening panels indicate expression in human
liver.
[0137] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate kinase nucleic acid
expression.
[0138] 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 testis, brain medulloblastomas,
infant brain, schizophrenic brain, retina, germinal center B cells,
colon, and liver. 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.
[0139] 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.
[0140] 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.
[0141] 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
testis, brain medulloblastomas, infant brain, schizophrenic brain,
retina, germinal center B cells, and colon, as indicated by virtual
northern blot analysis. In addition, PCR-based tissue screening
panels indicate expression in human liver. Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or nucleic acid expression.
[0142] 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 testis, brain
medulloblastomas, infant brain, schizophrenic brain, retina,
germinal center B cells, colon, and liver.
[0143] 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.
[0144] 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.
[0145] 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 seen: T4452A, A5330G, A9256C, A11773G, G12886A, and
a T insertion/deletion ("indel") at position 14131. SNPs in
introns, such as these, may affect control/regulatory elements. The
gene encoding the novel kinase protein of the present invention is
located on a genome component that has been mapped to human
chromosome 1 (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.
[0146] Alternatively, mutations in a kinase gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0147] 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.
[0148] 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)).
[0149] 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.
[0150] 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 seen: T4452A,
A5330G, A9256C, A11773G, G12886A, and a T insertion/deletion
("indel") at position 14131. SNPs in introns, such as these, may
affect control/regulatory elements.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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
testis, brain medulloblastomas, infant brain, schizophrenic brain,
retina, germinal center B cells, and colon, as indicated by virtual
northern blot analysis. In addition, PCR-based tissue screening
panels indicate expression in human liver. 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.
[0156] Nucleic Acid Arrays
[0157] 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).
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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 seen: T4452A, A5330G, A9256C,
A11773G, G12886A, and a T insertion/deletion ("indel") at position
14131. SNPs in introns, such as these, may affect
control/regulatory elements.
[0164] 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 (1 982), 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).
[0165] 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, Ma.) 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 (Kurjan 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 1 70:31-39 (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,
hydroxylapatitc 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 G.sub.0 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 3565 DNA Human 1 ttcttccttt ctctcaatat aggtatggca tcacagctgc
aagtgttttc gcccccatca 60 gtgtcgtcga gtgccttctg cagtgcgaag
aaactgaaaa tagagccctc tggctgggat 120 gtttcaggac agagtagcaa
cgacaaatat tatacccaca gcaaaaccct cccagccaca 180 caagggcaag
ccaactcctc tcaccaggta gcaaatttca acatccctgc ttacgaccag 240
ggcctcctcc tcccagctcc tgcagtggaa catattgttg taacagccgc tgatagctcg
300 ggcagtgctg ctacatcaac cttccaaagc agccagaccc tgactcacag
aagcaacgtt 360 tctttgcttg agccatatca aaaatgtgga ttgaaacgaa
aaagtgagga agttgacagc 420 aacggtagtg tgcagatcat agaagaacat
ccccctctca tgctgcaaaa caggactgtg 480 gtgggtgctg ctgccacaac
caccactgtg accacaaaga gtagcagttc cagcggagaa 540 ggggattacc
agctggtcca gcatgagatc ctttgctcta tgaccaatag ctatgaagtc 600
ttggagttcc taggccgggg gacatttgga caggtggcta agtgctggaa gaggagcacc
660 aaggaaattg tggctattaa aatcttgaag aaccacccct cctatgccag
acaaggacag 720 attgaagtga gcatcctttc ccgcctaagc agtgaaaatg
ctgatgagta taattttgtc 780 cgttcatacg agtgctttca gcataagaat
cacacctgcc ttgtttttga aatgttggag 840 cagaacttat atgattttct
aaagcaaaac aaatttagcc cactgccact caagtacatc 900 agaccaatct
tgcagcaggt ggccacagcc ttgatgaagc tcaagagtct tggtctgatc 960
cacgctgacc ttaagcctga aaacatcatg ctggttgatc cagttcgcca gccctaccga
1020 gtgaaggtca ttgactttgg ttctgctagt cacgtttcca aagctgtgtg
ctcaacctac 1080 ttacagtcac gttactacag agctcctgaa attattcttg
ggttaccatt ttgtgaagct 1140 attgatatgt ggtcactggg ctgtgtgata
gctgagctgt tcctgggatg gcctctttat 1200 cctggtgctt cagaatatga
tcagacacct gaagaacacg aactggagac tggaataaaa 1260 tcaaaagaag
ctcggaagta catttttaat tgcttagatg acatggctca ggtgaatatg 1320
tctacagacc tggagggaac agacatgttg gcagagaagg cagaccgaag agaatacatt
1380 gatctgttaa agaaaatgct cacaattgat gcagataaga gaattacccc
tctaaaaact 1440 cttaaccatc agtttgtgac aatgactcac cttttggatt
ttccacatag caatcatgtt 1500 aagtcttgtt ttcagaacat ggagatctgc
aagcggaggg ttcacatgta tgatacagtg 1560 aatcagatca agagtccctt
cactacacat gttgccccaa atacaagcac aaatctaacc 1620 atgagcttca
gcaatcagct caatacagtg cacaatcagg ccagtgttct agcttccagt 1680
tctactgcag cagctgctac tctttctctg gctaattcag atgtctcact actaaactac
1740 cagtcagctt tgtacccatc atctgctgca ccagttcctg gagttgccca
gcagggtgtt 1800 tccttgcagc ctggaaccac ccagatttgc actcagacag
atccattcca acagacattt 1860 atagtatgtc cacctgcgtt tcaaactgga
ctacaagcaa caacaaagca ttctggattc 1920 cctgtgagga tggataatgc
tgtaccgatt gtaccccagg caccagctgc tcagccacta 1980 cagattcagt
caggagttct cacgcaggga agctgtacac cactaatggt agcaactctc 2040
caccctcaag tagccaccat cacgccgcag tatgcggtgc cctttactct gagctgcgca
2100 gccggccggc cggcgctggt tgaacagact gccgctgtac tgcaggcgtg
gcctggaggg 2160 actcagcaaa ttctcctgcc ttcaacttgg caacagttgc
ctggggtagc tctacacaac 2220 tctgtccagc ccacagcaat gattccagag
gccatgggga gtggacagca gctagctgac 2280 tggaggaatg cccactctca
tggcaaccag tacagcacta tcatgcagca gccatccttg 2340 ctgactaacc
atgtgacatt ggccactgct cagcctcaat gttggtgttg cccatgttgt 2400
ctgacaacaa caatccagtt ccctcccttc gaagaagaat aagcagtcag ctccagtctc
2460 ttccaagtcc tctctagatg ttctgccttc ccaagtctat tctctggttg
ggagcagtcc 2520 cctccgcacc acatcttctt ataattcctt ggtccctgtc
caagatcagc atcagcccat 2580 catcattcca gatactccca gccctcctgt
gagtgtcatc actatccgaa gtgacactga 2640 tgaggaagag gacaacaaat
acaagcccag tagctctgga ctgaagccaa ggtctaatgt 2700 catcagttat
gtcactgtca atgattctcc agactctgac tcttctttga gcagccctta 2760
ttccactgat accctgagtg ctctccgagg caatagtgga tccgttttgg aggggcctgg
2820 cagagttgtg gcagatggca ctggcacccg cactatcatt gtgcctccac
tgaaaactca 2880 gcttggtgac tgcactgtag caacccaggc ctcaggtctc
ctgagcaata agactaagcc 2940 agtcgcttca gtgagtgggc agtcatctgg
atgctgtatc acccccacag ggtatcgagc 3000 tcaacgcggg gggaccagtg
cagcacaacc actcaatctt agccagaacc agcagtcatc 3060 ggcggctcca
acctcacagg agagaagcag caacccagcc ccccgcaggc agcaggcatt 3120
tgtggcccct ctctcccaag ccccctacac cttccagcat ggcagcccgc tacactcgac
3180 agggcaccca caccttgccc cggcccctgc tcacctgcca agccaggctc
atctgtatac 3240 gtatgctgcc ccgacttctg ctgctgcact gggctcaacc
agctccattg ctcatctttt 3300 ctccccacag ggttcctcaa ggcatgctgc
agcctatacc actcacccta gcactttggt 3360 gcaccaggtc cctgtcagtg
ttgggcccag cctcctcact tctgccagcg tggcccctgc 3420 tcagtaccaa
caccagtttg ccacccaatc ctacattggg tcttcccgag gctcaacaat 3480
ttacactgga tacccgctga gtcctaccaa gatcagccag tattcctact tatagttggt
3540 gagcatgagg aagggcgaat tctgt 3565 2 1170 PRT Human 2 Met Ala
Ser Gln Leu Gln Val Phe Ser Pro Pro Ser Val Ser Ser Ser 1 5 10 15
Ala Phe Cys Ser Ala Lys Lys Leu Lys Ile Glu Pro Ser Gly Trp Asp 20
25 30 Val Ser Gly Gln Ser Ser Asn Asp Lys Tyr Tyr Thr His Ser Lys
Thr 35 40 45 Leu Pro Ala Thr Gln Gly Gln Ala Asn Ser Ser His Gln
Val Ala Asn 50 55 60 Phe Asn Ile Pro Ala Tyr Asp Gln Gly Leu Leu
Leu Pro Ala Pro Ala 65 70 75 80 Val Glu His Ile Val Val Thr Ala Ala
Asp Ser Ser Gly Ser Ala Ala 85 90 95 Thr Ser Thr Phe Gln Ser Ser
Gln Thr Leu Thr His Arg Ser Asn Val 100 105 110 Ser Leu Leu Glu Pro
Tyr Gln Lys Cys Gly Leu Lys Arg Lys Ser Glu 115 120 125 Glu Val Asp
Ser Asn Gly Ser Val Gln Ile Ile Glu Glu His Pro Pro 130 135 140 Leu
Met Leu Gln Asn Arg Thr Val Val Gly Ala Ala Ala Thr Thr Thr 145 150
155 160 Thr Val Thr Thr Lys Ser Ser Ser Ser Ser Gly Glu Gly Asp Tyr
Gln 165 170 175 Leu Val Gln His Glu Ile Leu Cys Ser Met Thr Asn Ser
Tyr Glu Val 180 185 190 Leu Glu Phe Leu Gly Arg Gly Thr Phe Gly Gln
Val Ala Lys Cys Trp 195 200 205 Lys Arg Ser Thr Lys Glu Ile Val Ala
Ile Lys Ile Leu Lys Asn His 210 215 220 Pro Ser Tyr Ala Arg Gln Gly
Gln Ile Glu Val Ser Ile Leu Ser Arg 225 230 235 240 Leu Ser Ser Glu
Asn Ala Asp Glu Tyr Asn Phe Val Arg Ser Tyr Glu 245 250 255 Cys Phe
Gln His Lys Asn His Thr Cys Leu Val Phe Glu Met Leu Glu 260 265 270
Gln Asn Leu Tyr Asp Phe Leu Lys Gln Asn Lys Phe Ser Pro Leu Pro 275
280 285 Leu Lys Tyr Ile Arg Pro Ile Leu Gln Gln Val Ala Thr Ala Leu
Met 290 295 300 Lys Leu Lys Ser Leu Gly Leu Ile His Ala Asp Leu Lys
Pro Glu Asn 305 310 315 320 Ile Met Leu Val Asp Pro Val Arg Gln Pro
Tyr Arg Val Lys Val Ile 325 330 335 Asp Phe Gly Ser Ala Ser His Val
Ser Lys Ala Val Cys Ser Thr Tyr 340 345 350 Leu Gln Ser Arg Tyr Tyr
Arg Ala Pro Glu Ile Ile Leu Gly Leu Pro 355 360 365 Phe Cys Glu Ala
Ile Asp Met Trp Ser Leu Gly Cys Val Ile Ala Glu 370 375 380 Leu Phe
Leu Gly Trp Pro Leu Tyr Pro Gly Ala Ser Glu Tyr Asp Gln 385 390 395
400 Thr Pro Glu Glu His Glu Leu Glu Thr Gly Ile Lys Ser Lys Glu Ala
405 410 415 Arg Lys Tyr Ile Phe Asn Cys Leu Asp Asp Met Ala Gln Val
Asn Met 420 425 430 Ser Thr Asp Leu Glu Gly Thr Asp Met Leu Ala Glu
Lys Ala Asp Arg 435 440 445 Arg Glu Tyr Ile Asp Leu Leu Lys Lys Met
Leu Thr Ile Asp Ala Asp 450 455 460 Lys Arg Ile Thr Pro Leu Lys Thr
Leu Asn His Gln Phe Val Thr Met 465 470 475 480 Thr His Leu Leu Asp
Phe Pro His Ser Asn His Val Lys Ser Cys Phe 485 490 495 Gln Asn Met
Glu Ile Cys Lys Arg Arg Val His Met Tyr Asp Thr Val 500 505 510 Asn
Gln Ile Lys Ser Pro Phe Thr Thr His Val Ala Pro Asn Thr Ser 515 520
525 Thr Asn Leu Thr Met Ser Phe Ser Asn Gln Leu Asn Thr Val His Asn
530 535 540 Gln Ala Ser Val Leu Ala Ser Ser Ser Thr Ala Ala Ala Ala
Thr Leu 545 550 555 560 Ser Leu Ala Asn Ser Asp Val Ser Leu Leu Asn
Tyr Gln Ser Ala Leu 565 570 575 Tyr Pro Ser Ser Ala Ala Pro Val Pro
Gly Val Ala Gln Gln Gly Val 580 585 590 Ser Leu Gln Pro Gly Thr Thr
Gln Ile Cys Thr Gln Thr Asp Pro Phe 595 600 605 Gln Gln Thr Phe Ile
Val Cys Pro Pro Ala Phe Gln Thr Gly Leu Gln 610 615 620 Ala Thr Thr
Lys His Ser Gly Phe Pro Val Arg Met Asp Asn Ala Val 625 630 635 640
Pro Ile Val Pro Gln Ala Pro Ala Ala Gln Pro Leu Gln Ile Gln Ser 645
650 655 Gly Val Leu Thr Gln Gly Ser Cys Thr Pro Leu Met Val Ala Thr
Leu 660 665 670 His Pro Gln Val Ala Thr Ile Thr Pro Gln Tyr Ala Val
Pro Phe Thr 675 680 685 Leu Ser Cys Ala Ala Gly Arg Pro Ala Leu Val
Glu Gln Thr Ala Ala 690 695 700 Val Leu Gln Ala Trp Pro Gly Gly Thr
Gln Gln Ile Leu Leu Pro Ser 705 710 715 720 Thr Trp Gln Gln Leu Pro
Gly Val Ala Leu His Asn Ser Val Gln Pro 725 730 735 Thr Ala Met Ile
Pro Glu Ala Met Gly Ser Gly Gln Gln Leu Ala Asp 740 745 750 Trp Arg
Asn Ala His Ser His Gly Asn Gln Tyr Ser Thr Ile Met Gln 755 760 765
Gln Pro Ser Leu Leu Thr Asn His Val Thr Leu Ala Thr Ala Gln Pro 770
775 780 Leu Asn Val Gly Val Ala His Val Val Arg Gln Gln Gln Ser Ser
Ser 785 790 795 800 Leu Pro Ser Lys Lys Asn Lys Gln Ser Ala Pro Val
Ser Ser Lys Ser 805 810 815 Ser Leu Asp Val Leu Pro Ser Gln Val Tyr
Ser Leu Val Gly Ser Ser 820 825 830 Pro Leu Arg Thr Thr Ser Ser Tyr
Asn Ser Leu Val Pro Val Gln Asp 835 840 845 Gln His Gln Pro Ile Ile
Ile Pro Asp Thr Pro Ser Pro Pro Val Ser 850 855 860 Val Ile Thr Ile
Arg Ser Asp Thr Asp Glu Glu Glu Asp Asn Lys Tyr 865 870 875 880 Lys
Pro Ser Ser Ser Gly Leu Lys Pro Arg Ser Asn Val Ile Ser Tyr 885 890
895 Val Thr Val Asn Asp Ser Pro Asp Ser Asp Ser Ser Leu Ser Ser Pro
900 905 910 Tyr Ser Thr Asp Thr Leu Ser Ala Leu Arg Gly Asn Ser Gly
Ser Val 915 920 925 Leu Glu Gly Pro Gly Arg Val Val Ala Asp Gly Thr
Gly Thr Arg Thr 930 935 940 Ile Ile Val Pro Pro Leu Lys Thr Gln Leu
Gly Asp Cys Thr Val Ala 945 950 955 960 Thr Gln Ala Ser Gly Leu Leu
Ser Asn Lys Thr Lys Pro Val Ala Ser 965 970 975 Val Ser Gly Gln Ser
Ser Gly Cys Cys Ile Thr Pro Thr Gly Tyr Arg 980 985 990 Ala Gln Arg
Gly Gly Thr Ser Ala Ala Gln Pro Leu Asn Leu Ser Gln 995 1000 1005
Asn Gln Gln Ser Ser Ala Ala Pro Thr Ser Gln Glu Arg Ser Ser Asn
1010 1015 1020 Pro Ala Pro Arg Arg Gln Gln Ala Phe Val Ala Pro Leu
Ser Gln Ala 1025 1030 1035 1040 Pro Tyr Thr Phe Gln His Gly Ser Pro
Leu His Ser Thr Gly His Pro 1045 1050 1055 His Leu Ala Pro Ala Pro
Ala His Leu Pro Ser Gln Ala His Leu Tyr 1060 1065 1070 Thr Tyr Ala
Ala Pro Thr Ser Ala Ala Ala Leu Gly Ser Thr Ser Ser 1075 1080 1085
Ile Ala His Leu Phe Ser Pro Gln Gly Ser Ser Arg His Ala Ala Ala
1090 1095 1100 Tyr Thr Thr His Pro Ser Thr Leu Val His Gln Val Pro
Val Ser Val 1105 1110 1115 1120 Gly Pro Ser Leu Leu Thr Ser Ala Ser
Val Ala Pro Ala Gln Tyr Gln 1125 1130 1135 His Gln Phe Ala Thr Gln
Ser Tyr Ile Gly Ser Ser Arg Gly Ser Thr 1140 1145 1150 Ile Tyr Thr
Gly Tyr Pro Leu Ser Pro Thr Lys Ile Ser Gln Tyr Ser 1155 1160 1165
Tyr Leu 1170 3 36159 DNA Human misc_feature (1)...(36159) n = A,T,C
or G 3 aaagtgggga gatgttggaa ggcagcaagc agattttgga gtgcatttta
aggcaggttg 60 agacaggttt tttttttgag ataggatcta gctctgttgc
ccaggctaga gtgcaatgga 120 gtaatcacaa ctcactgtag cctcaatgtc
ccagactcag atgattttcc tgcttcagcc 180 tcctgagtag ctgggaccac
aggcatgtgc cacttacact tggctttttt tttttttttt 240 tcccggtaga
gatggagtct ccccatgttg ctctggctgg tcttgaactc gtggactcaa 300
gtgatccttc caccttgggt tcctaaagtg ccaggattac aggcgtgagc caccacatct
360 ggcctaattt ttcttttctt ttcttttttt tttttgttaa tgcttcccag
gctgatcttg 420 aactcctgag ctcaagtgat cctcctgcct gggcctccca
aagtgcggga attacaggct 480 tgagcgacca tggccagcca agttgagaat
cttggacatt atctccaaag caatgagaaa 540 ccccgggaga aggggaagca
agatggttaa gaagagagag cagttatctg atttgcattg 600 tttaaaagca
aagatctact gagtggattt aaggagatta gtttggaagc tactggcagt 660
ttgaactaga atggtgccaa taagggtagg gaaaaagggc tgatttgaaa tatacttagg
720 aggccagggg cagtggctca cgcctgtaat cccagcactt tgggagggtg
aggggggtgg 780 atcacttgag ctcaggagtt tgagaccacc caggcaacat
ggtgaaaacc catctctact 840 aaaaatacaa agaaattaac tgggtgtggt
cgtgcacgcc tctactctca gctacttggg 900 aggctgaggc aggagaattg
cttgagcccc agaggtgaag gttgcagcga gccaagattg 960 caccattgca
ctccagcttg ggctacagag tgagactctg tctcaaaaaa aaaaaaaaaa 1020
aatatacaca cacacacaca cacacacaca cacacacaca cacacacact taggagatgg
1080 aatggataag atagagatta gatgtggagg aataagggag aggaatgagc
caggataata 1140 gtattaaata tgtggtagac actatcattt tacatgtatt
aaattattta attctcagaa 1200 caaccccatg aggtaggtat tgctatcacc
attatgtagc tgaggaaaca gacatcccta 1260 attttttttc ttttttttga
gacagggtgt cattctttca ccctggctgg agtgcagtgg 1320 cacgatcaca
gctcactgca gcctctacct ctgggctcag gtgatcactt ctgccttctg 1380
agtagctagg actacaggca tgtgccacca tgcctggcta actttttttt ctgttttttt
1440 ttttttttgt tgttgttgtt gtttgtttgt tttagagacg gtttcaccat
gttgcccagg 1500 ctggagctcc ctgatttctg gcttgaggcg ttggatgtgt
gatggcatct cataattaag 1560 atagaaaact gaaggtgggg tggaggctcg
taagtttaat tctgaatgta ttgaatatga 1620 ggtgtttgtg aaatgtccaa
gttgcagggt taagtagtca tttggatata gggtttggag 1680 ttcaaagcga
gagaatcctg ggttagagat agagatttta gtcttttgaa gatgactaat 1740
tttgaggagt aattattaaa aaggggcaaa gggtagagca aggaaagagg gattactata
1800 gagccccaag gcatatgaga tcggatggtt gaaaatggag aagataaata
tgaaatggct 1860 tgtgtgctat gtcagatgtt tggactttat tctgaaaaaa
tagctttcag tttaatctgt 1920 gggcttattg agctggaagt gtctgtggga
tattcaggga cagaaagctg gactgttctt 1980 acatcctttc ctcatatttt
tctgctaact ttcctcggtt cttcagaata tactctagct 2040 ttctcatcat
cctggttact tttttttttt tttttttcaa ttttagtatt tttagagaca 2100
gggtctcact acattgccta ggctggtctc gaactcctca gctcaggaga tcttcctgcc
2160 ttggcctccc aaagtgctgg aattaaaggc ttgagccact gtgcctggcc
catactggtt 2220 acttttttat cttaaaatgt ggtagacaat tgaatgcatt
ttatgtatga cctgagcaga 2280 gtggataatc ttcactttgt ccagcacgtt
ctgtacactg tttctatgaa tataggtcaa 2340 gattgaatta gtttttgaga
agaggagaac attattacat catgtttctt ttatcaagta 2400 aaagtgtgtg
tgtgtgtttg tgtgttttaa atctaagcct tgtatctttt atccttgtgg 2460
tctaattctt cctttctctc aatataggta tggcatcaca gctgcaagtg ttttcgcccc
2520 catcagtgtc gtcgagtgcc ttctgcagtg cgaagaaact gaaaatagag
ccctctggct 2580 gggatgtttc aggacagagt agcaacgaca aatattatac
ccacagcaaa accctcccag 2640 ccacacaagg gcaagccaac tcctctcacc
aggtagcaaa tttcaacatc cctgcttacg 2700 accagggcct cctcctccca
gctcctgcag tggagcatat tgttgtaaca gccgctgata 2760 gctcgggcag
tgctgctaca tcaaccttcc aaagcagcca gaccctgact cacagaagca 2820
acgtttcttt gcttgagcca tatcaaaaat gtggattgaa acgaaaaagt gaggaagttg
2880 acagcaacgg tagtgtgcag atcatagaag aacatccccc tctcatgctg
caaaacagga 2940 ctgtggtggg tgctgctgcc acaaccacca ctgtgaccac
aaagagtagc agttccagcg 3000 gagaagggga ttaccagctg gtccagcatg
agatcctttg ctctatgacc aatagctatg 3060 aagtcttgga gttcctaggc
cgggggacat ttggacaggt ggctaagtgc tggaagagga 3120 gcaccaagga
aattgtggct attaaaatct tgaagaacca cccctcctat gccagacaag 3180
gacagattga agtgagcatc ctttcccgcc taagcagtga aaatgctgat gagtataatt
3240 ttgtccgttc atacgagtgc tttcagcata agaatcacac ctgccttgtt
tttgaaatgt 3300 tggagcagaa cttatatgat tttctaaagc aaaacaaatt
tagcccactg ccactcaagt 3360 acatcagacc aatcttgcag caggtggcca
cagccttgat gaagctcaag agtcttggtc 3420 tgatccacgc tgaccttaag
cctgaaaaca tcatgctggt tgatccagtt cgccagccct 3480 accgagtgaa
ggtcattgac tttggttctg ctagtcacgt ttccaaagct gtgtgctcaa 3540
cctacttaca gtcacgttac tacaggcaag tggcaaatgc tgaaaatcgt atcttaggct
3600 agagttctgt ccttatattt aacatatacc ccgtaggcta catatagcaa
tgaatttgtt 3660 tatagattct gagatagaaa taggatatgt tttagctcat
tctatgtgtg tggcattcct 3720 atatatgaca tttatttctg aaattttatc
tagcactgga aaaattaact cagtctgatt 3780 ctgaaagttg ttactagttg
aattatacta gcacctggtt ctttagtatt attttacctc 3840 attttcccat
tttatttatt ttatttattt atttatttat ttagagacag aatctcgctc 3900
tgtcgcccag gctggagtgc agtggcgtga tatcagctca ctgcaagccc cacctcctgg
3960 gttcacgcca ttctcctgcc tcagcctcct gagtagctgg gaccacaggc
acccgccatc 4020 acgcccggct aatttttttt gtatttttag tagagacggg
gtttcaccgt gttagccagg 4080 atggtctcga tatcctgacc tcgtgatcca
cccgtctcgg cctcccaaag cgctgggatt 4140 acaggcgtga
gccaccgtgc ccagcctatt tatttatttt tttaagatgg agtttcactt 4200
gccacccagg ctagagtgca gtggtgtgac attgactcac ggcagcctcc acctcctggg
4260 gtcaagtgat ttctcctgca actcctgtcc tgagtagctg ggactacagg
cacctgctac 4320 cacgcccggc taattttttt gtttttaata gagatggggt
ttcaccatgt tgaccgggct 4380 ggttttgaac tcctgacctc aggtgatcca
cccgcctcag cctcccaaag tgattagagg 4440 cgtgagccac catgcccagc
cattttccca ttttgaagag tcttgaaata cacaaagata 4500 tttacttatt
tgtaatgaat ctgagcatat gttgctgttt tttcgaacct cttatcttgg 4560
caggtaaaat aacgtgggaa taactctagg tttaactcta gtaacatttt attcttttac
4620 attttcttct gtagtagcat gaattgaatt acatggttgc tacaatctct
tcctgtttta 4680 actttctcta aatactttga acttaatggg ttatcctaga
atggccttga cccaagtacc 4740 ttatacttta atgatatata tttctagatt
gatactttta atgtagctac cattttaata 4800 tataataatt attgggacag
tatgtaaatg ctgatatata caattttgtc tgtaccataa 4860 ccaaggcttt
taaaatgtgc tttttatcag cacccattta cttacttgcc tagttattaa 4920
ttttaaggaa tctaatattt agttttaatg gccatacatt aaatacaaat catgtaagca
4980 tccaatcaag aagtgaaata ataaaaacat agataaccta taaaatatgt
ttataaggag 5040 ctatacatgc cagatgcctg taataaaatt tgaggaaata
gaaattctga attaagaatt 5100 ttatattatg tgtgaaaata atgtgaggat
attttaaccc atacaaggac tcagaaaaaa 5160 tgtcatctgc atgtttcctt
ttttaaaaac attgtggtaa gatgtttata ataggaaatt 5220 tacaatttta
accatttggt accactcatt gtgttaagta cattcatagt gttgtgtaac 5280
catcactgct gtctgttaag tatattcaca atgttgtgta accatcacca ctatttccaa
5340 atgttttcat cacccaaaac agaaattcta accattaagc aataactccc
tattctctct 5400 tcttcctacc actggtaatc ttgatttgac tttctgtctc
tatgaatttg cctattctag 5460 atactgcatg taagtggaat catacaatat
ttgtcttttt gtgtctagtt tatttcactt 5520 agtgtaatgc ttttgaggct
aatccatgct gtaacatgta tcagaacttc attcctttta 5580 tggctgtata
atattccatt gtttgtatat accacatttt gtttatgcat tcatctgttg 5640
gtagatattt gggttgttgc tacctttagg ctgttgtgaa taatgctgct atgaacattg
5700 gtgtacaagt atcctagtcc ctattttcag ttactttggg gatatagcta
ggagggaatt 5760 gctgggtcac atgataattc tatgtttaac tttttgcaga
attaccaaat tattttccac 5820 agaggctgca ctattttaca ttcctaccag
cagtggatgt gcattccaaa tttctccaca 5880 ttttctctaa catttgttat
tttttttatt taaaaatatt gtttgtttat ttttacagag 5940 acaggggctg
cctctattgc tcatgctgga gtacagtggc acgatcatag ttcactgtag 6000
cctccaactc ctggacttga gcagtcctcc cacttcagcc tcccaagtag ctaggactgc
6060 agtcacactc caccatacct ggctaattac tattatttta ttttttgtgg
cgacagtgtt 6120 ttgagggtct cattttgttg cccaatctgg tctcaaacta
ctggcctcaa gccatcctcc 6180 tgcctcagtc tcccaaagtt ctgggattac
aggtgtgaac taccactcct ggccttgttt 6240 tgttttttaa ataatagcca
tgggtttttt tttttttttt tttttttttt ttttttttgg 6300 aaagggagtt
tcacttttgt tgcctaggct ggagggcagg ggggcaatct cggttaactg 6360
gaacctttgc ctcccaggat tttcctgcct aaacctccca agtagctggg attacagggg
6420 cctgccacca cacccagtta atttttgttt ttttaaaaaa aatggggttt
taccatgttg 6480 gccagggggg gctccaactc ctgacctcag gggatctgcc
caccttggcc tcccaaagtg 6540 ctgggattac aggcatgagc cactatgcct
ggccaataat agtttttttt tgtttttttt 6600 ttgttttttt tttgagatgg
agtcttgctc tgttgccagg ctggagtgca gtggcacaat 6660 ctcggttcac
tgcaacctcc acctcacagg ttcaagcagt tctcctagct tggcctcctg 6720
agtagctggg aatacaggtg ccaccatgcc cagctaattt ttgtattttt agtagagaca
6780 gggtttcacc atgttggccg ggatggtctc gatctcttga cctcgtgatg
aagtgctggg 6840 attacaggca tgagccaccg ggcccggtca ataatagcca
ttcttatggg tgtgaagtgg 6900 tatctcattg tggttttgat ttgtatttcc
ctaatgatta atgatgttga gcatttgttt 6960 tattttgttt gtttgagaca
gagtcccact ttgtcaccca ggctggggtg cagttgtgca 7020 atcatggctt
actgcagcca tgacctctca ggctcaagca gtcctcccac cttagccttt 7080
cgggtacctg agactacggg catgcacccc cacacctgac tagtgttttg tatttttagt
7140 agagacgggg tttcactgtg ttgcccaggc tggtctcaaa ctcataggct
caagtgatat 7200 gcccgcctcg gcaacccaaa gtgctgggat tacagacatg
agccaccatg cccagcctgg 7260 cattttttta tgtgcccgac atctgtatat
cttctttgga gaaatgtcta tttaagtcct 7320 ttcctcattt cttgaattgg
gctttttgtt gttgagttgt atactctata tacttaattt 7380 tcatctattc
cttgggttgc ctttttacct gttgatagtg tttgacacag aaaagttttt 7440
aactttggtg aagtgcagtt tgtctacttt ttctttggtt gcttgtgctt ttggtgacat
7500 atccaagaag tcactgttaa gtcgaaatca tacagatttt cccctatgtt
ttctgctaag 7560 agttttatag ttttagctct tatattttgg tctttgattc
tttgttgatt tttgtctatg 7620 gtccaaggta caaatccagt gtaattcttt
ggcatgtgac tattcagttc ttcaaacacc 7680 atttgctaag aagattgtcc
tttctgcatt gggtggtttg ggcacccttg ttggaatcat 7740 ttgaacatat
atacaaataa ttcttatctt ctattgcttt cccattttca atgttgggct 7800
ctctattcca ttaatctata tatgtcttta tgccagtacc acattgtttt gattattgta
7860 gctttgtagt aagtttgaaa tcaggaagtg tgagacctcc aactttgttc
tttttcaaga 7920 ttgttttggc tatttggggt ctttgagggt ccatataaat
tttaggatgg gtttttctat 7980 tttatacaaa aaccataatt gctttttatt
aaggatagcg atgaatctgt agatgacttt 8040 gggtagtatt gacagcttaa
tagtaagtca gtccatcctt atttctttat atcttttcac 8100 agttttataa
aactggtatt ttttacttga ggtaaggtaa ataaactctt agagcctttg 8160
ttttctggtt ttatgctgcc ctaggcaacc ttggctaact ttaagaatgt catctccatt
8220 tatttattta tgccttggga gatttaggtt ccaactacat ttgcttttta
atgctctcct 8280 ttgagcagtc tcaccaccag ccacaccaac ataacatata
tataacatac tctacaggtg 8340 cactgaagaa ttcagcgcag catcccattt
tgagtcctca ggagggacta ggcagaccac 8400 agctgaagga agagggctga
tcagctcttc ctttctgtct tgactctgtg cctgcaggtg 8460 ttctttaact
atttgtttgc cctgtcaaag acaaagtgca ttctcttgtg ataccagagt 8520
agtttttaaa ttgaaaaagg gagagcaata ggagataaaa attatttggc tttgttataa
8580 ttggggcatg ttaatacaaa ataaaatgaa ttatttggct gcttagcttt
ctgtaatgtg 8640 taattcattt gaataatttt cagtgttagg ttgctgatct
ttgtattttt tatcctttta 8700 atttaagctg tcaattgatt cattttggtt
ttgtttttta tagaaactaa ggttttcaaa 8760 tcttcaaagt tacccttcga
caaagctttc tttaaattca ctgcaatata gttgttgact 8820 ataattttaa
gtggagctaa gtttgcctct taaaaacagg agttcattct gtgtattact 8880
gagtaattac tctgtatact gaagttcagt gcccagggct tgacagtgtt taggatttaa
8940 catgagtgtt ctgttgtgtc acagtaatac ttgataatga gcctaaggca
gatggaacag 9000 cagctcaggc attccttctt tatcactagt tttacccgca
gtggtcctct taagcttctt 9060 tgatgttgct ttgttgccat attagagtcc
attaagtcct cacccttctg tctttcaaaa 9120 aacctctgtg aaatctgttt
ggctggtgaa gcattttgac tccaaagcta gtccttctct 9180 tacccacctt
tttagtttac cttgctttgt ctttctgata atatgccagt attatcagct 9240
cacataaatt tgccaccttg ctgtgtccat tggccctggg catggctaaa tgattcaggc
9300 agtggaaata atatacttta ctctctggct cactgtaaat gtgcacagac
tccaagcaaa 9360 gctcctgtct ttcgggcttg agttttagag acaaaggttt
gccatgctta caggctgaat 9420 gtttttccct actgaacaaa ctagccagcc
tttatttcaa gctgaatcac tttgttactt 9480 acggaaggaa aaggtctaga
gaaggaaaac gtatcttcca tttatcctag acaaacaaat 9540 aatctaattt
cctctggcag tcaaaatata gtttcaccta agccactgat gcaggaagtt 9600
aggttttatg taacctctct aattggtaag taagtaggtt tgatgtctct gagataggaa
9660 gaaagaaacg aaaatgttca tgaaaataat cagagtgatt tgtgttaagt
gatcctaacc 9720 ttagcttgct ctgggtgcca gtgaaattaa cctcaacaat
gttggttgga agaatttttc 9780 aacttaaaga agtttgaagt tggggaatca
aaaggcaggg attgttgttt ctatcactta 9840 gctgtaataa ccagagcctg
tttagtattt gttttttaag gatgggatgt gtcttcaaag 9900 aggagacttg
ccatgttcaa agcacaatta atgccatttt cctactgaag tgaacactgc 9960
cagtttttaa cagtttcttt cactttcctg tgcttctgta gataaccttt tttactgccc
10020 agttgttgga atgttacagc tggaaaggga cctagaagag taattatcta
actctgtttc 10080 cttattacac aaatgaggta gaaccagagt tttacgtgtc
tatagagtnn nnnnnnnnnn 10140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnca gtggcgcaat catggctcac 10200 tgcagccttg acctcctggg
ctcaagtgat ccttccacct cagcttcctg agtagctaga 10260 actgcaggca
tgcaccacca tacctggcta attttttaaa tttttttggt agagattggg 10320
tcttgctgtg ttgcctaggc tggtctcaaa ctcctgctca agtgatcctc ctgccttggc
10380 ttcccaaagt gctgggatta taggtgtgaa ccgccgtttc tggccgtatt
tttttttttt 10440 ttaaactgct ttcctttttt tgtttttcct tttgctattt
gcctttttat aaggtagata 10500 cggtagagtt gctgttttga ctgagctttt
gctggaagtc cttagctgct tttgtcactc 10560 caaaagcagg gtgagaccaa
atgaaaaaac tttcagctaa tcttcagttt ttttttttaa 10620 ttaaatggga
cttgggggct gtggaagtga tattttcctt aatttcccag aaaactttaa 10680
gctcgagaca gttatcatat attattgcta ccttttttat ttttttctga gacggagttt
10740 cgctcttacg cccagtctgg agtgaattga cgtaatcttg gctcactgca
acctctgccc 10800 cccgggttca agcgattctc ctgcctcagc ctctggagta
gttgggatta caggcgcctg 10860 ccaccatgcc cagctaattt ttgtattttt
agtagagact gggtttcatc atgttggcca 10920 ggctggactt gaactcctga
cctcaggcca tccacgtgcc ttggccaccc acagtgctag 10980 gattataggc
gtgagccacc gcgcctggcc tgttatcgct accttttaaa gaagaaagtt 11040
atagagtggc ctggccattc tctgtgacct accttttgga ctttaaaatg tctttctagt
11100 atggtaggaa tagcaaaatc aatatgctgc cctccaattg atgctttgga
atgttctaaa 11160 cccagttttt attttggctc attgccagtt gtgctccccc
tgccagctat ttttggcatt 11220 gtcatgaact tggaaattaa tgattctgct
cactaggagt agaaaatttt gttccttttc 11280 aattttagaa aagcttttat
gttgtttctg ggtagtctta ctagcttatt aatgcgcctg 11340 tcaaacttgt
gcagtgttga aaacatgcca ttatggttga gatttcactg aactctgaaa 11400
ttccatcagt aatatgtgcc tctagccacc tgcaacagga atatcacttt tgagggttac
11460 ttcttttctt ttcttttttt tttttttttg agacagagtc ttactctgtc
acccaggctg 11520 gagtgcagtg gcactgtgtc agctcactgc aacctccgtc
tcctgggttc aagcaattct 11580 cctgcctcag cctcctgagt agctgggatt
acaggtgccc gccaccacac ctggctgatt 11640 ttttatattt ttagtagaga
tggggtttca ccatgttggc caggctggtc tcaaactcct 11700 gacttcaggt
gatccagcct cctcggcctc ccaaagtgct tgtattacag gcatgagcca 11760
ccgcacccgg ccagttattt ctttggtaaa caaaaccaca gttcaaatta aatactacaa
11820 aaactgatga tattggtggt tccacccata gctgttaaaa agtgtaagcc
cgaagaggcc 11880 tgggtcgact gctatttgta tttaaggatc agaaaagctt
tcaggccctg tggagtcccc 11940 agatttactg ttttctaagg gccactattt
atgtataaat actaggggag gatttttttt 12000 ttcttctatg cctagtttgc
ttagatgggg agggatattc ttatttaggg aaattatatt 12060 ctccttgctt
aatccttgcc ttccctcaac cccctgcaca tatacacaaa acaacaataa 12120
gcacatatca cttgaaagta gtttgagaaa cctgggtatt ctgtgaggag acacggccag
12180 ttagatggtt cttcacggaa agatctgttt tccatttaac tcctgtaaca
tgaggaatct 12240 gaatctggat ctggatctgg atctggagtg tccttataga
ttataattcc atgacttgta 12300 agaaagaaag gaaattattt ggagaagatg
atccagtgcc taagagctga aatcttggat 12360 ccaaatagct tgggtaccat
cctttttctt ttgttgagat ggagtctcgc tctgtcgccc 12420 aggctggagt
gcagtggcag atctcaactc attgcaactt atgcctccca agtttaagta 12480
attctcttgc ctcagcctct gagtagttgg gattacagct gcccgccacc atgcccagct
12540 aatttttgta tttttagtag agatggagtt tcaccgtgtt gaccaggctg
ttctcgaact 12600 cgtgacctca agcaatccac tcaccttggc ttcccaaagt
gctgggatta caggtgtgag 12660 ccactgcgct ggccaagggc accatctttt
agtagttatg tagccttggg caagttactt 12720 taacctctta gtgccagttt
cttcatctgt aaaatggagg taatactttt attgcataga 12780 atctaacatg
ccattgatta taagatacat tatttttgta ccacttaaaa aaggaaaagc 12840
gctgccatta aactaaaata taccatcatt tgtaagaatc ttcttaattt cagaggtgct
12900 aacatgtaaa aagatgtgta tcttagaatt gatgacatca taataatact
ttgataggat 12960 tgttgtaagg attaagtgac tcaacattta taaagaactt
agaacagtgc atggcacata 13020 gtaggcaata tttatgtgtc atttattatt
attgctatta gtgttaccta ttattttctt 13080 tttgaaccca cttattgcct
aattagtcat agtttgacaa ttgcccttgt attccaccat 13140 gtcaaatata
aatttacata gatgagtatg tacttttact tattgagaaa cagtgtaata 13200
tataatatac tcaattctgg agccagattg cagggattca aatcctagct ctgccactta
13260 tttgactgtg actctaggcc aataacttaa tctttctttt tctcagtttc
ttcttctgta 13320 atggggataa taattctatt ttagatgtgt tcgtatatat
aaacgtctga gttatggatt 13380 accttagtca tctctttaaa gttccctagc
attttatttc tcacttggac tgttaatgaa 13440 tatttctaaa aagcacacta
agagttcaaa gttttaaaat aatggtaaca taatactgtt 13500 attatatcta
acacctacta gtatttacca tgtgccatgc actggtctaa aagctttcat 13560
atatttattt aagcttcaca acaactctat gtggtgggaa ctcttactgt ctccatttta
13620 tagatgagga acctgaggca cagagagatc aagtaatata cctgcagcta
ttaaatgatg 13680 gaactaggat tcagaccctg acaggctggc tctagagagt
gtgctgtcaa caccatgtct 13740 cttcagaagg catttctttt tctttttttt
ttttccagaa ggcatttctg tcatgaaggg 13800 gttatttatt gaccagggct
tttttttttt tttttggact gtcttgggtc tcctaggctg 13860 gagtgtagtg
gtgtgatctt ggcttactgc aacctctacc tcctgggttc aagcgattct 13920
tttgtctcaa cctcctgagt agctgggatt acaggcgccc accaccacac ctgactcatt
13980 tttgtatttt tagtagattt ggggtttcac catattggcc aagctggtct
tgaacttctg 14040 acctcaggtg atccacccgc ttcggcttcc caaagtgctg
ggattacagg catgagccac 14100 tgtgcccagt tgaccaggcc ttttaattga
tttttttttt ttatgattga aatggtgcta 14160 ggaataataa taaaaaaaat
ctattatccc ttatctgtga ttgcaaaatt cagaaagctc 14220 tcataaatga
aaatttttct ttaagtttgg tataaattca tttaattggc aagacctgac 14280
ttgaaccatt gttaagctat ttgaagtctt tatttagcca acttagtatg actgttccca
14340 tgttttgttg cagaaatatt aatgtgtttg attatagtgc actgccctga
actccactgg 14400 gattattata taatgtgtac tttgtgttct gcattacctt
tctgaaattc aaaatattct 14460 gaattccaaa acacatctgg ccctgagctt
cagatgatgg attgtatacc aaagattttt 14520 tttccatttc attgaataaa
tgttccttta gctaatacta aaacaggact tagtcatgta 14580 gttaattttc
ccaaataatg tttttttttt tttaatctga tattttttgt ttttgctaga 14640
gctcctgaaa ttattcttgg gttaccattt tgtgaagcta ttgatatgtg gtcactgggc
14700 tgtgtgatag ctgagctgtt cctgggatgg cctctttatc ctggtgcttc
agaatatgat 14760 caggtaaaag tgtttatttg aatggaaata gaatgcaaat
agttacttgt gaattagatt 14820 ctggagaaag agaagtacta agtactactg
aagtatttag ataataggga aagagtagtc 14880 caattgctac taaagaactt
tttaaagaat agtataattt tctcttcctg cttcttaggc 14940 taaagtatgt
ttgcaattct ataataaaaa aaagaatttt attttatatt aaggttgata 15000
ctttgctaga tctgtaatga tttattagag acttaacttt cattaactta tgtgctttgt
15060 gagttaggaa agtagagtaa agatgaggaa ctggaatttt aaaagagaac
ttctacttac 15120 tcggagctat tataatttac ttttacgcat gacacactga
agtactttct tcagactgaa 15180 acacttcagg tcccagaagc tgtgacatac
tgggtcgcta agataggatt tagaaaggaa 15240 atcatcctga gttgtagtaa
tatatgatct gtatctaaaa atagacaaac ttaggagata 15300 tggtataatt
tcctaaggaa ttggtccgtt aagggaaaat gttttactat ggaagtaaat 15360
tgtgaattct catttcttgt tattttttct tttttctttt tatttgtttg agatggagtc
15420 ttgctctgtc acccaggctg gagtgcagtg gcgcgatctc ggctcactgc
aacctctgcc 15480 tccctggttc aagcgattct cctgcctcag cctcctgagt
agctgggatt acaggtgcct 15540 gccaccatga ccaactaatt tttgtatttt
ttagtagaga cggggtttca ccatgttggc 15600 caggctggtt tcgaactcct
gacttcaggt gatccacctg cctcagcctc ccaaagtgtt 15660 gggattacag
gtgtgagtca ccgtgcctgg ccttcttctt attttttaaa aatgttcctg 15720
ccctttatga tgtaagctcc ttgagggtag agattgtttc acatcaccag tgtatcctca
15780 gctcctaaca ctgtgtctgg tacacagtaa gtacaccagt ttttttgttg
tggtttttaa 15840 gtttttattt ttttagagac agagtcttgc tcagtcaccc
aggctggcat gtaggcctgt 15900 cacagcttac tgtaacctct agctcctggg
cttaagtgat cctcccacct cagcctccca 15960 ggtagatggg actataggtg
catgccacct tgcctagcta attcttttat tttttgtaga 16020 gtcggggatc
ttgctatatc agcctagggt ggtctcaaac tcccaggctc aagctatcct 16080
cctaccttgg cctcccaaag tactgggatt acaggtgtga gccaccatgc ctggcctata
16140 ttgtcaaata tctttacttg tccgtaaata cacttctacc ttgtcattta
caatgtctgc 16200 atggtatttt ggtttccagc tacaggattt agaaaggaag
ttatctgagt tgtagtagat 16260 tccacagatt tgaagtatta gaagtcaaca
ggaaaagcaa aaaagattat agccaaaatt 16320 ttcaaaactg gatttccttg
taaataatag atacagtagc tgtggatgga ttagtatata 16380 taggtattta
cagataaatt tcagttgtat tgattaaaga tttgatttct tcctttgcct 16440
aaagttaatg atgttttagt gtaaaagcct ttaataattt cccttttcac tccaaatagt
16500 tgtttgatgg ttttgatgtt tcagattcgt tatatttcac aaacacaagg
cttgccagct 16560 gaatatctta tcagtgccgg aacaaaaaca accaggtttt
tcaacagaga tcctaatttg 16620 gggtacccac tgtggaggct taaggtctgt
cttccctact atgcttccga ctcctgtact 16680 ccacccctca ctccccaatt
ttgaattcaa agtttagtta ttaaattctt caggtagaga 16740 agggaaagga
gagggggaag cattttgaaa aattatttct ttgtacctgt ttggccttat 16800
cctcagttga aaaacaaaac attaattgct agttcagttg gctgaggtta ttttgtatat
16860 gttcaatcca cagctgatag aaagtttgga gggtagtgct caccattaag
cgatagaact 16920 agagacatat agtaatgact gatttttaga gaattctcaa
tgaacatgat aaaatcacaa 16980 attttctaac tgcccacatt caggacttct
atattttttc ttgaaacaaa tacctgcttt 17040 ttacttctga gcctactctg
tcaggttcag aaatatctga gtaatttgac taaccctgtg 17100 actgtgtgtc
tgagtctgtt gaacagttag catttgagat atcgatttat ttgaaagtag 17160
ctttaagaga acaatggtag tgtccccttt tacctgacat tctttaggaa ctgtgctgta
17220 tcattacttg catgtttatc actgttgaaa gggtagctag atatcaaggt
cacatctctc 17280 cactggaaga ttttctggtt gtgaattact ttcatgtttg
ccatctatgg ttggcaaggt 17340 gaccacactt gtctcttgta ttctggcttt
ggttttgaat aaaatgtgaa aataacatac 17400 agatggaatt taaggaggaa
aatctttatt ttatagacac ctgaagaaca tgaactggag 17460 actggaataa
aatcaaaaga agctcggaag tacattttta attgcttaga tgacatggct 17520
caggtgagta cggaaagttt cagaaagtca gacatttatt tttaatcaga gacacttctg
17580 ttgattatac taaagacaaa tttaatgtta tctttctagt atttgttttc
agtttttata 17640 aaaaatgcat taatattcca ccatgtagta aaggaacatt
taaatcctaa ccaagtatat 17700 ttttagaatt acatatttct ctcttgcttt
acttgtcttg ttacatagca gtgttttaaa 17760 atattactta tgaaagtttc
ttgtcccatt ttctctatta aatacttaag aattatattt 17820 attgagcgcc
tattatgtta cgaactctga acacttcaca cttatgtcat ttaatttttt 17880
caacagtaga agcttttata tttaggtagt aacaatcact attgcttagc tacttgttcc
17940 attttttttt tttttttttt tttttttgag acagagtctc actctattgc
ccaggctgga 18000 gtgcagtggc gtaatctcag ctcactgcaa cctctgcctc
ccgggttcaa gcgattctcc 18060 tgcctcagcc tcccaagtag ctgggattac
aggcacgtgc caccatgccc ggctaatttt 18120 tgtatttttt ttagtagaga
cggggttttg ccatgttggc caggctgggt ctcaaactcc 18180 tgacctcagg
tgatccacct gccttggctt cccaaaatgc tgggattaca ggcatgagcc 18240
accgcgccca gcccccaaat ttttaatgac aagaaattgt ttagctttct tctaccactc
18300 aatttagatg aagattttaa ttaaacagca taaaaagagc ttcctcctct
gaaaatgatt 18360 agattttcat aaaaagaatt tccccaggtt tctcttttga
ttacatatat acacacacac 18420 atagtttgga gggaaagcag ctatgtagtg
tcagtgccaa aggttaagtg aagaagtata 18480 attctgaatt ttctttggaa
ggtgaatatg tctacagacc tggagggaac agacatgttg 18540 gcagagaagg
cagaccgaag agaatacatt gatctgttaa agaaaatgct cacaattgat 18600
gcagataaga gaattacccc tctaaaaact cttaaccatc agtttgtgac aatgactcac
18660 cttttggatt ttccacatag caatcagtga gtatggaata ttctggggct
tttgccatgt 18720 ggttctttgt tgagttaccg ccttatcaat ggcactatca
aatgagcccg ccactttggt 18780 gcttataaat ctggctcagc agtgcttttc
tttctcattg aaacatcata agataaaaat 18840 tagatgtgta tttttcttcc
ctatgattat acaaattctt gatttatttt atctgaaagt 18900 gattgggaaa
aaaagctttg atccatgttc atcttgagtt atttgctgtc tgtttaaatc 18960
tcagcattca tttaatgaat ctttaatctc cttttcagtg ttaagtcttg ttttcagaac
19020 atggagatct gcaagcggag ggttcacatg tatgatacag tgagtcagat
caagagtccc 19080 ttcactacac atgttgcccc aaatacaagc acaaatctaa
ccatgagctt cagcaatcag 19140 ctcaatacag tgcacaatca ggtattcaat
aaataatttt ggaaactcaa gcttaagtgg 19200 gatagaaact
agtaagaata cagggcaagg taaagaacca atttttgttt tggtggtctt 19260
gttgcttctt agaaattctc cacttgacaa aagttgatgg aaaacagggt agactgataa
19320 tacttaccag gcacaggcta actaaagtta aatataaagg cctaatccat
gccctcatat 19380 gttcagcatc gtcaaataaa tggggtctga ctattatgct
acttactgct gttagtttta 19440 ctgactttag ccaaatgact ttctccctgt
taggagaagg attttatatc tcttgttact 19500 gtattgaaag gtttccagtc
attaacttta agggtggttt tgcatttgtt tgctagccag 19560 tgatatttgc
atttaggttt atttctgaag atgtaagctt cccagtttct tggctgggtc 19620
tactttttta atggaagagc ctatgagatt tggtgggatc ttccatccag taattttttg
19680 tgcagaagta gttggggttt gtgtagccac agccaacata ggaccattcg
tttttttttt 19740 tttatttgct tatttgacca tataatatgc cttcaattta
gggactaggg aagtttctta 19800 agcagagagt tatttcagag gcagttaaca
ttacatttta aaacattatt ctacgttttt 19860 ctggataaat tctgtatata
taaaattatt gtgtgtctct acttaataca agtgtacaaa 19920 tataatcctt
ttgtttttag gccagtgttc tagcttccag ttctactgca gcagctgcta 19980
ctctttctct ggctaattca gatgtctcac tactaaacta ccagtcagct ttgtacccat
20040 catctgctgc accagttcct ggagttgccc agcagggtgt ttccttgcag
cctggaacca 20100 cccagatttg cactcagaca gatccattcc aacagacatt
tatagtatgt ccacctgcgt 20160 ttcaaagtaa gtggggaaac tcctgtatca
tatggtattg tatcagacct acctgcttta 20220 ggcagctcta gttgtttagt
tctgatcttt acaagtttaa actctgtctc tgatgaagaa 20280 ggtaactaaa
attgggtaat atcgcaaaat ggattttctc tttttacata ggctatttat 20340
ctaattatga tgctatctga tgcattgtaa gagctcactt tatgtttcct taattgaatt
20400 gcctgatacc agttttcttg cccattgagt ccttgtgtca atgtcgtacg
tcttgtataa 20460 gcatgtatct gtcaatatgc aaaatctata caacttgaaa
aaatttgttg taagcagaat 20520 tgctaaatan nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20580 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20640 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20700
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
20760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 20820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 20880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20940 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21000 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21060
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
21120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 21180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 21240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21300 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21360 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21420
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
21480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 21540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 21600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21660 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21720 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21780
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
21840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 21900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 21960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22020 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22080 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22140
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
22200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 22260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 22320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22380 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22440 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22500
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
22560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 22620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 22680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22740 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22800 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22860
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
22920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 22980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 23040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23100 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23160 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23220
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
23280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 23340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 23400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23460 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23520 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23580
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
23640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 23700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 23760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23820 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23880 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23940
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
24000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 24060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 24120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24180 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24240 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24300
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
24360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 24420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 24480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24540 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24600 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24660
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
24720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 24780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn ncaagttttt 24840 tttccaccta gtggattaaa aagtgaataa
tgctgggcac agtggctcac acctgtaatc 24900 ccggcacttt gggaggccaa
gatgggcaga tcatgaggtc aggagttcga gaccagcctg 24960 gccaacatgg
tgaaaccccg tctctactga aaatataaaa attagacggg cgtggtggcg 25020
cactcctgta gtcgcagcta cttgggaggc tgtggcagaa gaatcgcttg aacttgggag
25080 cagagtttgc agtgagccca gatagcatca ctgcactcca gcctgggtga
cagagcgaga 25140 ctccatctca naaaaaaaaa aaaaaaaaaa aaaaaaaatg
aaagtgaatt taatttacta 25200 agtgaagatt tttttgtttt tgcctgtact
cttaatggag atgggatgaa tattgtgttt 25260 taaattttgt agcataaaaa
aaatttagaa ttcttttaaa ccatccctca ctatatcaaa 25320 catctttcca
ttaacctaac ctgaggggaa gtctcccttt tcttaacttc caagacttct 25380
atgaatgaaa cttctttgtc ttaagtgtct aggattaaaa cctgaacagt ggattatttg
25440 aacagagaag ctgaaaacaa aataatctta aaacagtact cccagacctt
gcaaaactat 25500 ttaactgtga tgctgttttc aatagctgga ctacaagcaa
caacaaagca ttctggattc 25560 cctgtgagga tggataatgc tgtaccgatt
gtaccgcagg caccagctgc tcagccacta 25620 cagattcagt caggagttct
cacgcaggta aaagctagag caatgtggat actcagtatt 25680 gctaaacact
attgagattc agatattttg tcctagaaaa tggtatttcc tttgactata 25740
agatctttct tggtcatgat tcagtggact taaaatgaaa catctctatg gaacaatata
25800 ctaattccta acactattgc aactctgcca ttgtcttcct tagacttgca
gggaaaaaat 25860 atccagacat tcttgagaaa tggtcttctg agtaagttta
ctctaatttt gcggggtgaa 25920 gcgttttttt ttgttgttgt ttgtttttta
aatgttggag ctcatataaa gataagtata 25980 tatgtagcat tttgattcta
aaatataagc ttccactttt gcaccctttg tgtccctttt 26040 gctcactctt
ttagaatttc atcacagttg agaggctgga tcacatcagg atgcctcttc 26100
acatattaca ttccttcatt ccgtgtgttt aaccatattg tagaaagctt taaaacttta
26160 tacttgctat ggaacattcg actaagatga tagagaaaat tgtaaagtat
ttaaatagca 26220 acaaacagta tattttatat tttatatata aatgtttgtg
gcctatgaca cataggaaat 26280 tctcaaatcc aaaaactcta ttttgtgaac
aggaggaaga attcttaata aatgtctctg 26340 tttcatagaa ctgatcccct
gaatctagcc caaggaggat tcctatactt ctttgtttta 26400 ggccattggc
atttgacttt gtgggccaat gccatacaaa gttgaagggg gaaagttgtg 26460
tttggtatat gcattttaga tgctatcaaa ttactgttca ttgtcagtaa taacttgggg
26520 ggaatgagaa cccttctgaa tccagatatc ctgcctccgt gtttgtttca
ctcacctgcc 26580 taatctacgt ttcagggaag ctgtacacca ctaatggtag
caactctcca ccctcaagta 26640 gccaccatca caccgcagta tgcggtgccc
tttactctga gctgcgcagc cggccggccg 26700 gcgctggttg aacagactgc
cgctgtactg gtaattcccc tcacttgatt gtgttactaa 26760 cggagtttct
ttggtttctt tcttttttgt tttttcctgt ttgtttttgt tctgtttttt 26820
tctggtttat ttttcaaaaa aattttttag cttagtcttt gcagagggca tggaagcatg
26880 tgccatcttg tggctgtgtt cttgctacat cttttaatgt ctcattgttt
tcctccccca 26940 acatttgctg tagcactgtc tgactgatgg ttatgtggct
ctgtaaggag ccagatccct 27000 tgattcttct ttgccagcct agtgtgataa
aagctctcga tgtagcctca gaagagcttc 27060 aacactgtta cttgttttct
gccttttacc ctctgatcct tgagaatgaa ggaaatggcc 27120 tttaatttgg
ctcagctaca ggtacccgga tggccaaaat tgagctcgtt ggcaagatag 27180
ctttctcctt tttttgctgt tttaaggttc taaaactttt ctgcatgtgg aaatgcttaa
27240 gatgctctta atttattgct ttctcagttg atactaacca tccttctatg
gacacatgaa 27300 gatagctctt tttgccactc atgatcagtg gcagcttata
tcttacattt aaagatattc 27360 cctttagaat gtagatcata ttgtaggtca
tattgttggt tgaaattaaa agtggacaca 27420 tttttaggag ttttgggagg
aattaattat tttacaaata ggtctttata caaaggttga 27480 aagtatggcc
aacaaactat gcaaatacaa tatctttgtt attaaaagtc atggggaata 27540
gttacttaat gtagctgcaa ttgtagtact gggcttagag atagaaaaaa caaccaagac
27600 tttgaggtgt gtttcaggat ttgtaacctt aagagtgatt ttttggatgt
gttttggttt 27660 atatggggag ttagaattgt ttattggata tccagcattt
tctcatttga aatttaaaat 27720 taggtaagca tccaaaaacc tgaaaggcac
tattgaatcc tgttatttct ctgttttcaa 27780 agttgcttta aatttctttt
gttgtttctt aagtattcat gtgagtgaaa acacttccag 27840 aaacatactt
tggttggaat ttggttttca ttttaaaagt ctttgtcact tccagattaa 27900
ttttcttgta agcagaaaac ctgagtgtga gggagtcaga tatatttcca acttgattga
27960 tccaagccaa tggttttaac tcactcatct gtagtttcga ggttggaatg
ttagttgaca 28020 cttctccttt gctgcttctc ttaagtttgc actagccatt
tgctaagctt tgcatatttt 28080 tgttcttcct ctgtcaaaac agttcgtcgc
ttggaacgcc agatttctgc ctcacattgg 28140 aatgttattt cttgctttgt
attaaactac atgctttgtc ttgtgcaggt gttggcttat 28200 ggaaagacgc
atggtgtgac ttaacatact tcttttattt tttgttttgt tttattttgt 28260
tttaaattct gcttatggta gcagacgtgt ttgcagtttc tctgctttga aggcttcttg
28320 tttggaacca gtatttgtaa caagtagatc tgttacttgc agaaatattt
ttaaaacagt 28380 gtgttgatgg ccttgcaatt tgaaattcaa gaaacagaac
catatgtacc caagcattgt 28440 aggtaattgt actgcagaat tcaagtttaa
aaaggaaact tccaaatccg ttcccatttt 28500 ttttttcaaa aaatgccaga
atttctgtga ggaagaagta cagaaaacat ttgttgctca 28560 gtttattgca
agtgacatgg cttttttaaa actagaaatc atgtattttc ttgtagtgat 28620
tagtttttat gtggaaatat tcctgcaaat gatacataaa aacatatttt aggtaactat
28680 tagaaacaaa gtatgatgct ttctgcttct aaaacatctt actttttgcc
ttattttgaa 28740 attccattat gtggctataa atgatgaatt agctttttct
tgctggcata agattttttc 28800 cccaaaagga ttaggccttt ggtcatccac
atctggctcc attttccaaa tactaccctt 28860 ttaaaaaagg gaacagttct
gccttttgtt ttatgggttg aattgatctg ataccttatc 28920 tgattggcag
tcagattaaa aaattttaat ctccagggag tcctcttaac tctttctagg 28980
gatttatttt agaattgttg caaaaatgaa actccagcat ttaaccagct cttatctctg
29040 aactctcaga ctccttttct ctacagtata atagaagctc ttaaccgcag
ggatcttctt 29100 ttctttaata cccgggtggt caggttatgg gggtgagcaa
gagaaagcag tatgttttct 29160 ttccccattc ataagacttg tagcttgagc
ccctctgacc tttctttttt aatctctgca 29220 agttaacaat ctacaagcaa
cttcttttaa gatatgaatt tttctttctt taaaaaaaca 29280 gaagaaaaag
ccaagaatga atcaagtctg gatgtttttt atgtgtgttt ccttacagca 29340
ggcgtggcct ggagggactc agcaaattct cctgccttca acttggcaac agttgcctgg
29400 ggtagctcta cacaactctg tccagcccac agcaatgatt ccagaggcca
tggggagtgg 29460 acagcagcta gctgactgga ggcaagtgtc ctgtgttact
ctgggagatt tgtaagggcc 29520 gatcccatag ggtgggagca cttggtaata
aggagagaga ctagtaagaa aataaaggaa 29580 aatttgacac tgttggaatc
ctttaagaac ccatatcagg ctaggagatg gtgttataag 29640 aaaactttga
atataggaaa gcagtaggtt ctgaaggtca ggaatcattt cttctagatt 29700
ttttaaagag agtcttaagt gattagaaac catacagtga gatcctaaag ccttgtaatc
29760 taggtcccca actttatctt ttataatgaa aattcttttt ttctaatgtt
taatttttgt 29820 gattacatac taggtatata tatttatggg gtacatgaga
tgttttgaca caggcatgta 29880 atgtgaaata agcacatcat ggaaaagggg
gtgtccatcc cctcaagcat ttatcctttg 29940 agttacaaat aatccaatta
cactctttaa gtcatttaaa aatgtacaat taagttatta 30000 ctgactataa
tcacctattg cgctatcaaa tagtagttct tattcttttt tttttttttt 30060
tgtacccatt aaccatccct acctccccac tagccctcca ctactcttac cagcctctgg
30120 taaccatcct actctctatg tccatgaatt aaattgtttt gatttttaga
tcccataaat 30180 aagtgagaac atgtggtttg tctttctgtg tctggcttat
ttcacttaac atgatgatct 30240 tgagttccat ccatgttgtt gcaaatgaca
acgtgtactt tttgtggctg agtagtactc 30300 cattgtgtat atgtaccata
ttttctttat ccattcatct gttgatggac acttaggctg 30360 cttccaaatc
ttactgtgaa cagtgctgca acataggagt gcaggtatct ctttgatata 30420
ctgatttcct ttcttttggg tatataccca gcagtgggat tgctggatca tatggtagct
30480 caatttttag tttttacagg aacctccaaa ctgttctcca taatagttgt
actaacttac 30540 attcctacca acagtgtaca attgttccct tttttccata
tccttggcag tgtttattat 30600 tgcttgtctt ttggatataa gccattttaa
ctggggtgag ataatatctc attgcagttt 30660 tgatctgcat ttctctaatg
atcagagatg ttgagcacct tttcatatgc ctgtttgtca 30720 tttgtaagtc
atcttttgga aaatgtctat tcaattcttt tgcccatttt ttgatcgtat 30780
tattagattt ttttccatag agttgtttga gctgcttacg tattctggtt attaatccct
30840 tatcagatgg taggtgctca actttaaaaa aataaaatgc agctgcattt
tggctaattg 30900 cttttgatgt ctgtttggtc ctgattcttc agtggttttg
gaattcactc ttctctttct 30960 ttctgttggt acaggaatgc ccactctcat
ggcaaccagt acagcactat catgcagcag 31020 ccatccttgc tgactaacca
tgtgacattg gccactgctc agcctctgaa tgttggtgtt 31080 gcccatgttg
tcagacaaca acaatccagt tccctccctt cgaagaagaa taagcagtca 31140
gctccagtct cttccaagtg agtctgtgtt acagctgata gttaaaactg tgccagtttg
31200 agagatatgt tgccttgcat ttggaatatt gtatagacat ataatataga
tatgaagcag 31260 caagtagctg ccaaattgag gaagagcaaa tcatttcatc
tgggcatgta caccaggtgt 31320 gtcctggttt tatgatggtc ctttgtctct
gctgccactt tgaatctagg gcattttatg 31380 atgtttttat tttactttac
agagtgaaat ttaatcctgg gataaagggc ttataaaagt 31440 aaaatgtctt
ttgtattttg gtgttcttgt ccctggaaac tcttgccagc atggtgctta 31500
ttttcactgg aacttatata gttaaatgta tttgcttaat gattatgtaa aaaggaatca
31560 atgagtaaat tggaaagcag tctggggaaa agatacacaa tttggaaggc
caaggactga 31620 atcatctttc catgtgaact tttcctacag gtcctctcta
gatgttctgc cttcccaagt 31680 ctattctctg gttgggagca gtcccctccg
caccacatct tcttataatt ccttggtccc 31740 tgtccaagat cagcatcagc
ccatcatcat tccagatact cccagccctc ctgtgagtgt 31800 catcactatc
cgaagtgaca ctgatgagga agaggacaac aaatacaagc ccagtaggta 31860
agataagtga atggttcctg gctctattgg ttttagactg ttggcctcag gcaagtgggc
31920 ccagtttggc ctgtgaaaga aaggaccggt ggggcatggt ggctcacgcc
tgtaatccca 31980 gcactttggg aggttgaggc cagcagatca cctgaggtga
ggagttcaag accagcctgg 32040 ccaacatggc acaaccctgt ctctactaaa
aatacaaaaa ttagcagggc cgtggtggca 32100 catgtttgta tcccagctac
tcgggaggct gaggcaggag aatcacttga acccaggagg 32160 cggaggttac
agtgagctga gatcgtgcca ctgtactcca gcctgagtga cagagcaaga 32220
ctgcatcccc tcccgccccc acccaaaaaa aagggccgaa gaaaaannnn nnnnnnnnnn
32280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnncaat ggtaagaaat
aaaggctagg 32340 aaaaattcaa atttactgac gccagaatag ggtgagttga
gtcaccagtt attaacttgc 32400 aggagtgggg aaccctatga tctcttgatt
ctccctcttc ctgtcatact tacaagcaaa 32460 atgctgttac ctgagccaga
ttaggatcta tcttgctaag aggcagaagg aggggtggct 32520 gatttctgac
agcattcaat gccagtgtgg gagattatgt gctataccca ttcgaaaaca 32580
gtacaagtca gaacctgagc tcttaacttg gcctttggtg ccctgagctg gagtgacctc
32640 aggattcctc acttcttcct tctttcttcc agaaccagca gtcatcggcg
gctccaacct 32700 cacaggagag aagcagcaac ccagcccccc gcaggcagca
ggcgtttgtg gcccctctct 32760 cccaagcccc ctacaccttc cagcatggca
gcccgctaca ctcgacaggg cacccacacc 32820 ttgccccggc ccctgctcac
ctgccaagcc aggctcatct gtatacgtat gctgccccga 32880 cttctgctgc
tgcactgggc tcaaccagct ccattgctca tcttttctcc ccacagggtt 32940
cctcaaggca tgctgcagcc tataccactc accctagcac tttggtgcac caggtccctg
33000 tcagtgttgg gcccagcctc ctcacttctg ccagcgtggc ccctgctcag
taccaacacc 33060 agtttgccac ccaatcctac attgggtctt cccgaggctc
aacaatttac actggatacc 33120 cgctgagtcc taccaagatc agccagtatt
cctacttata gttggtgagc atgagggagg 33180 aggaatcatg gctaccttct
cctggccctg cgttcttaat attgggctat ggagagatcc 33240 tcctttaccc
tcttgaaatt tcttagccag caacttgttc tgcaggggcc cactgaagca 33300
gaaggttttt ctctggggga acctgtctca gtgttgactg cattgttgta gtcttcccaa
33360 agtttgccct atttttaaat tcattatttt tgtgacagta attttggtac
ttggaagagt 33420 tcagatgccc atcttctgca gttaccaagg aagagagatt
gttctgaagt taccctctga 33480 aaaatatttt gtctctctga cttgatttct
ataaatgctt ttaaaaacaa gtgaagcccc 33540 tctttatttc attttgtgtt
attgtgattg ctggtcagga aaaatgctga tagaaggagt 33600 tgaaatctga
tgacaaaaaa agaaaaatta ctttttgttt gtttataaac tcagacttgc 33660
ctattttatt ttaaaagcgg cttacacaat ctcccttttg tttattggac atttaaactt
33720 acagagtttc agttttgttt taatgtcata ttatacttaa tgggcaattg
ttatttttgc 33780 aaaactggtt acgtattact ctgtgttact attggagatt
ctctcaattg ctcctgtgtt 33840 tgttataaag tagtgtttaa aaggcagctc
accatttgct ggtaacttaa tgtgagagaa 33900 tccatatctg cgtgaaaaca
ccaagtattc tttttaaatg aagcaccatg aattcttttt 33960 taaattattt
tttaaaagtc tttctctctc tgattcagct taaatttttt tatcgaaaaa 34020
gccattaagg tggttattat tacatggtgg tggtggtttt attatatgca aaatctctgt
34080 ctattatgag atactggcat tgatgagctt tgcctaaaga ttagtatgaa
ttttcagtaa 34140 tacacctctg ttttgctcat ctctcccttc tgttttatgt
gatttgtttg gggagaaagc 34200 taaaaaaacc tgaaaccaga taagaacatt
tcttgtgtat agcttttata cttcaaagta 34260 gcttcctttg
tatgccagca gcaaattgaa tgctctctta ttaagactta tataataagt 34320
gcatgtagga attgcaaaaa atattttaaa aatttattac tgaatttaaa aatattttag
34380 aagttttgta atggtggtgt tttaatattt tacataatta aatatgtaca
tattgattag 34440 aaaaatataa caagcaattt ttcctgctaa cccaaaatgt
tatttgtaat caaatgtgta 34500 gtgattacac ttgaattgtg tacttagtgt
gtatgtgatc ctccagtgtt atcccggaga 34560 tggattgatg tctccattgt
atttaaacca aaatgaactg atacttgttg gaatgtatgt 34620 gaactaattg
caattatatt agagcatatt actgtagtgc tgaatgagca ggggcattgc 34680
ctgcaaggag aggagaccct tggaattgtt ttgcacaggt gtgtctggtg aggagttttt
34740 cagtgtgtgt ctcttccttc cctttcttcc tccttccctt attgtagtgc
cttatatgat 34800 aatgtagtgg ttaatagagt ttacagtgag cttgccttag
gatggaccag caagcccccg 34860 tggaccctaa gttgttcacc gggatttatc
agaacaggat tagtagctgt attgtgtaat 34920 gcattgttct cagtttccct
gccaacattg aaaaataaaa acagcagctt ttctccttta 34980 ccaccacctc
tacccctttc cattttggat tctcggctga gttctcacag aagcattttc 35040
cccatgtggc tctctcactg tgcgttgcta ccttgcttct gtgagaattc aggaagcagg
35100 tgagaggagt caagccaata ttaaatatgc attcttttaa agtatgtgca
atcactttta 35160 gaatgaattt ttttttcctt ttcccatgtg gcagtccttc
ctgcacatag ttgacattcc 35220 tagtaaaata tttgcttgtt gaaaaaaaca
tgttaacaga tgtgtttata ccaaagagcc 35280 tgttgtattg cttaccatgt
ccccatacta tgaggagaag ttttgtggtg ccgctggtga 35340 caaggaactc
acagaaaggt ttcttagctg gtgaagaata tagagaagga accaaagcct 35400
gttgagtcat tgaggctttt gaggtttctt ttttaacagc ttgtatagtc ttggggccct
35460 tcaagctgtg aaattgtcct tgtactctca gctcctgcat ggatctgggt
caagtagaag 35520 gtactgggga tggggacatt cctgcccata aaggatttgg
ggaaagaaga ttaatcctaa 35580 aatacaggtg tgttccatct gaattgaaaa
tgatatattt gagatataat tttaggactg 35640 gttctgtgta gatagagatg
gtgtcaagga ggtgcaggat ggagatggga gatttcatgg 35700 agcctggtca
gccagctctg taccaggttg aacaccgagg agctgtcaaa gtatttggag 35760
tttcttcatt gtaaggagta agggcttcca agatggggca ggtagtccgt acagcctacc
35820 aggaacatgt tgtgttttcn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 35880 nnnnnnnnna agatgggagg atagcttgag cccagaggtt
gaggtcgcag cgagctgtga 35940 tcactccact gcactccagc ctgggtgaca
gaacaagacg ctgtcacaca cacaaaaaag 36000 aacaattcaa ttttcatgta
tttttctttt cctcagctct ggactgaagc caaggtctaa 36060 tgtcatcagt
tatgtcactg tcaatgattc tccagactct gactcttctt tgagcagccc 36120
ttattccact gataccctga gtgctctccg aggcaatag 36159 4 1209 PRT Mus
musculus 4 Met Ala Ser Gln Leu Gln Val Phe Ser Pro Pro Ser Val Ser
Ser Ser 1 5 10 15 Ala Phe Cys Ser Ala Lys Lys Leu Lys Ile Glu Pro
Ser Gly Trp Asp 20 25 30 Val Ser Gly Gln Ser Ser Asn Asp Lys Tyr
Tyr Thr His Ser Lys Thr 35 40 45 Leu Pro Ala Thr Gln Gly Gln Ala
Ser Ser Ser His Gln Val Ala Asn 50 55 60 Phe Asn Leu Pro Ala Tyr
Asp Gln Gly Leu Leu Leu Pro Ala Pro Ala 65 70 75 80 Val Glu His Ile
Val Val Thr Ala Ala Asp Ser Ser Gly Ser Ala Ala 85 90 95 Thr Ala
Thr Phe Gln Ser Ser Gln Thr Leu Thr His Arg Ser Asn Val 100 105 110
Ser Leu Leu Glu Pro Tyr Gln Lys Cys Gly Leu Lys Arg Lys Ser Glu 115
120 125 Glu Val Glu Ser Asn Gly Ser Val Gln Ile Ile Glu Glu His Pro
Pro 130 135 140 Leu Met Leu Gln Asn Arg Thr Val Val Gly Ala Ala Ala
Thr Thr Thr 145 150 155 160 Thr Val Thr Thr Lys Ser Ser Ser Ser Ser
Gly Glu Gly Asp Tyr Gln 165 170 175 Leu Val Gln His Glu Ile Leu Cys
Ser Met Thr Asn Ser Tyr Glu Val 180 185 190 Leu Glu Phe Leu Gly Arg
Gly Thr Phe Gly Gln Val Ala Lys Cys Trp 195 200 205 Lys Arg Ser Thr
Lys Glu Ile Val Ala Ile Lys Ile Leu Lys Asn His 210 215 220 Pro Ser
Tyr Ala Arg Gln Gly Gln Ile Glu Val Ser Ile Leu Ser Arg 225 230 235
240 Leu Ser Ser Glu Asn Ala Asp Glu Tyr Asn Phe Val Arg Ser Tyr Glu
245 250 255 Cys Phe Gln His Lys Asn His Thr Cys Leu Val Phe Glu Met
Leu Glu 260 265 270 Gln Asn Leu Tyr Asp Phe Leu Lys Gln Asn Lys Phe
Ser Pro Leu Pro 275 280 285 Leu Lys Tyr Ile Arg Pro Ile Leu Gln Gln
Val Ala Thr Ala Leu Met 290 295 300 Lys Leu Lys Ser Leu Gly Leu Ile
His Ala Asp Leu Lys Pro Glu Asn 305 310 315 320 Ile Met Leu Val Asp
Pro Val Arg Gln Pro Tyr Arg Val Lys Val Ile 325 330 335 Asp Phe Gly
Ser Ala Ser His Val Ser Lys Ala Val Cys Ser Thr Tyr 340 345 350 Leu
Gln Ser Arg Tyr Tyr Arg Ala Pro Glu Ile Ile Leu Gly Leu Pro 355 360
365 Phe Cys Glu Ala Ile Asp Met Trp Ser Leu Gly Cys Val Ile Ala Glu
370 375 380 Leu Phe Leu Gly Trp Pro Leu Tyr Pro Gly Ala Ser Glu Tyr
Asp Gln 385 390 395 400 Ile Arg Tyr Ile Ser Gln Thr Gln Gly Leu Pro
Ala Glu Tyr Leu Leu 405 410 415 Ser Ala Gly Thr Lys Thr Thr Arg Phe
Phe Asn Arg Asp Pro Asn Leu 420 425 430 Gly Tyr Pro Leu Trp Arg Leu
Lys Thr Pro Glu Glu His Glu Leu Glu 435 440 445 Thr Gly Ile Lys Ser
Lys Glu Ala Arg Lys Tyr Ile Phe Asn Cys Leu 450 455 460 Asp Asp Met
Ala Gln Val Asn Met Ser Thr Asp Leu Glu Gly Thr Asp 465 470 475 480
Met Leu Ala Glu Lys Ala Asp Arg Arg Glu Tyr Ile Asp Leu Leu Lys 485
490 495 Lys Met Leu Thr Ile Asp Ala Asp Lys Arg Ile Thr Pro Leu Lys
Thr 500 505 510 Leu Asn His Gln Phe Val Thr Met Ser His Leu Leu Asp
Phe Pro His 515 520 525 Ser Ser His Val Lys Ser Cys Phe Gln Asn Met
Glu Ile Cys Lys Arg 530 535 540 Arg Val His Met Tyr Asp Thr Val Ser
Gln Ile Lys Ser Pro Phe Thr 545 550 555 560 Thr His Val Ala Pro Asn
Thr Ser Thr Asn Leu Thr Met Ser Phe Ser 565 570 575 Asn Gln Leu Asn
Thr Val His Asn Gln Ala Ser Val Leu Ala Ser Ser 580 585 590 Ser Thr
Ala Ala Ala Ala Thr Leu Ser Leu Ala Asn Ser Asp Val Ser 595 600 605
Leu Leu Asn Tyr Gln Ser Ala Leu Tyr Pro Ser Ser Ala Ala Pro Val 610
615 620 Pro Gly Val Ala Gln Gln Gly Val Ser Leu Gln Pro Gly Thr Thr
Gln 625 630 635 640 Ile Cys Thr Gln Thr Asp Pro Phe Gln Gln Thr Phe
Ile Val Cys Pro 645 650 655 Pro Ala Phe Gln Thr Gly Leu Gln Ala Thr
Thr Lys His Ser Gly Phe 660 665 670 Pro Val Arg Met Asp Asn Ala Val
Pro Ile Val Pro Gln Ala Pro Ala 675 680 685 Ala Gln Pro Leu Gln Ile
Gln Ser Gly Val Leu Thr Gln Gly Ser Cys 690 695 700 Thr Pro Leu Met
Val Ala Thr Leu His Pro Gln Val Ala Thr Ile Thr 705 710 715 720 Pro
Gln Tyr Ala Val Pro Phe Thr Leu Ser Cys Ala Gly Arg Pro Ala 725 730
735 Leu Val Glu Gln Thr Ala Ala Val Leu Gln Ala Trp Pro Gly Gly Thr
740 745 750 Gln Gln Ile Leu Leu Pro Ser Ala Trp Gln Gln Leu Pro Gly
Val Ala 755 760 765 Leu His Asn Ser Val Gln Pro Ala Ala Val Ile Pro
Glu Ala Met Gly 770 775 780 Ser Ser Gln Gln Leu Ala Asp Trp Arg Asn
Ala His Ser His Gly Asn 785 790 795 800 Gln Tyr Ser Thr Ile Met Gln
Gln Pro Ser Leu Leu Thr Asn His Val 805 810 815 Thr Leu Ala Thr Ala
Gln Pro Leu Asn Val Gly Val Ala His Val Val 820 825 830 Arg Gln Gln
Gln Ser Ser Ser Leu Pro Ser Lys Lys Asn Lys Gln Ser 835 840 845 Ala
Pro Val Ser Ser Lys Ser Ser Leu Glu Val Leu Pro Ser Gln Val 850 855
860 Tyr Ser Leu Val Gly Ser Ser Pro Leu Arg Thr Thr Ser Ser Tyr Asn
865 870 875 880 Ser Leu Val Pro Val Gln Asp Gln His Gln Pro Ile Ile
Ile Pro Asp 885 890 895 Thr Pro Ser Pro Pro Val Ser Val Ile Thr Ile
Arg Ser Asp Thr Asp 900 905 910 Glu Glu Glu Asp Asn Lys Tyr Glu Pro
Asn Ser Ser Ser Leu Lys Ala 915 920 925 Arg Ser Asn Val Ile Ser Tyr
Val Thr Val Asn Asp Ser Pro Asp Ser 930 935 940 Asp Ser Ser Leu Ser
Ser Pro His Ser Thr Asp Thr Leu Ser Ala Leu 945 950 955 960 Arg Gly
Asn Ser Gly Thr Leu Leu Glu Gly Pro Gly Arg Pro Ala Ala 965 970 975
Asp Gly Ile Gly Thr Arg Thr Ile Ile Val Pro Pro Leu Lys Thr Gln 980
985 990 Leu Gly Asp Cys Thr Val Ala Thr Gln Ala Ser Gly Leu Leu Ser
Ser 995 1000 1005 Lys Thr Lys Pro Val Ala Ser Val Ser Gly Gln Ser
Ser Gly Cys Cys 1010 1015 1020 Ile Thr Pro Thr Gly Tyr Arg Ala Gln
Arg Gly Gly Ala Ser Ala Val 1025 1030 1035 1040 Gln Pro Leu Asn Leu
Ser Gln Asn Gln Gln Ser Ser Ser Ala Ser Thr 1045 1050 1055 Ser Gln
Glu Arg Ser Ser Asn Pro Ala Pro Arg Arg Gln Gln Ala Phe 1060 1065
1070 Val Ala Pro Leu Ser Gln Ala Pro Tyr Ala Phe Gln His Gly Ser
Pro 1075 1080 1085 Leu His Ser Thr Gly His Pro His Leu Ala Pro Ala
Pro Ala His Leu 1090 1095 1100 Pro Ser Gln Pro His Leu Tyr Thr Tyr
Ala Ala Pro Thr Ser Ala Ala 1105 1110 1115 1120 Ala Leu Gly Ser Thr
Ser Ser Ile Ala His Leu Phe Phe Pro Gln Gly 1125 1130 1135 Ser Ser
Arg His Ala Ala Ala Tyr Thr Thr His Pro Ser Thr Leu Val 1140 1145
1150 His Gln Val Pro Val Ser Val Gly Pro Ser Leu Leu Thr Ser Ala
Ser 1155 1160 1165 Val Ala Pro Ala Gln Tyr Gln His Gln Phe Ala Thr
Gln Ser Tyr Ile 1170 1175 1180 Gly Ser Ser Arg Gly Ser Thr Ile Tyr
Thr Gly Tyr Pro Leu Ser Pro 1185 1190 1195 1200 Thr Lys Ile Ser Gln
Tyr Ser Tyr Leu 1205
* * * * *
References