U.S. patent application number 09/734673 was filed with the patent office on 2002-07-18 for isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof.
Invention is credited to Beasley, Ellen M., Di Francesco, Valentina, Guegler, Karl, Ketchum, Karen.
Application Number | 20020094946 09/734673 |
Document ID | / |
Family ID | 24952636 |
Filed Date | 2002-07-18 |
United States Patent
Application |
20020094946 |
Kind Code |
A1 |
Guegler, Karl ; et
al. |
July 18, 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: |
Guegler, Karl; (Menlo Park,
CA) ; Ketchum, Karen; (Germantown, MD) ; Di
Francesco, Valentina; (Rockville, MD) ; Beasley,
Ellen M.; (Darnestown, MD) |
Correspondence
Address: |
CELERA GENOMICS CORP.
ATTN: WAYNE MONTGOMERY, VICE PRES, INTEL PROPERTY
45 WEST GUDE DRIVE
C2-4#20
ROCKVILLE
MD
20850
US
|
Family ID: |
24952636 |
Appl. No.: |
09/734673 |
Filed: |
December 13, 2000 |
Current U.S.
Class: |
514/1 ; 435/194;
435/325; 435/6.16; 435/69.1; 435/7.1; 536/23.2; 800/8 |
Current CPC
Class: |
C12N 9/1205
20130101 |
Class at
Publication: |
514/1 ; 435/194;
435/6; 435/7.1; 435/325; 800/8; 536/23.2; 435/69.1 |
International
Class: |
A61K 031/00; A01K
067/00; C12Q 001/68; G01N 033/53; C12N 009/12; C07H 021/04; C12P
021/02; C12N 005/06 |
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 or3; (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 SNF-like 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. 2 71:19402-8). 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] SNF Kinases
[0016] The kinase proteins of this invention are homologous to the
SNF kinases, involved in signal transduction and development. SNF
kinases derepress glucose-repressible genes in yeast and in higher
plants. SNF-like kinases are also found in mammals. For example,
the hunkI kinase is expressed in murine mammary gland and may play
a role in tissue development and carcenogenesis. Also, msk
(myocardial SNF1-like kinase) is expressed in developing heart.
Experimental evidence indicates that msk regulates HEK kinase, an
ephrin receptor type kinase. For more information on SNF-like
kinases, see Bielke W, et al., Gene Feb. 25, 1994;139(2):235-9; Le
Guen L, et al., Gene Oct. 21, 1992;120(2):249-54; Gardner H P, et
al., Genomics Jan. 1, 2000;63(1):46-59; Ruiz J C, et al., Mech Dev
December 1994;48(3):153-64; and Yoshida E N, et al., Genome
December 1999;42(6): 1077-87.
[0017] Kinase proteins, particularly members of the SNF-like 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 SNF-like
kinase subfamily.
SUMMARY OF THE INVENTION
[0018] 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 SNF-like 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 lung, liver, kidney, thyroid, brain,
infant brain, fetal brain, placenta, bone marrow, germ cell tissue,
germ cell tumor tissue, and primary cancers.
DESCRIPTION OF THE FIGURE SHEETS
[0019] FIG. 1 provides the nucleotide sequence of a cDNA molecule
or transcript sequence 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 lung, liver, kidney, thyroid, brain, infant brain,
fetal brain, placenta, bone marrow, germ cell tissue, germ cell
tumor tissue, and primary cancers.
[0020] 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.
[0021] 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, known SNP variations include C1681A, T6888-, A7939G, G9790A,
G11689A, A12145G, Al2437-, A12437G, G12437-, C22982T, C23239T,
A23859G, G24862A, G25097A, C25751T, T25934-, G26179A, A26277G,
-26354T, T26356-, T26359G, G29484A, A29885T, C30601T, C30920T,
C31057A, C31057G, G31097A, T31204C, T32660A, T32875-, C33477G,
C34059T, - 36681A, C36821T, A38079G, and A38432T.
DETAILED DESCRIPTION OF THE INVENTION
[0022] General Description
[0023] 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 SNF-like 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 SNF-like 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.
[0024] 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 SNF-like kinase subfamily and the expression
pattern observed. Experimental data as provided in FIG. 1 indicates
expression in lung, liver, kidney, thyroid, brain, infant brain,
fetal brain, placenta, bone marrow, germ cell tissue, germ cell
tumor tissue, and primary cancers. 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 SNF-like kinase family or subfamily of kinase
proteins.
[0025] Specific Embodiments
[0026] Peptide Molecules
[0027] 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 SNF-like
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.
[0028] 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.
[0029] 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).
[0030] 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.
[0031] 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.
[0032] 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 lung, liver, kidney, thyroid, brain, infant
brain, fetal brain, placenta, bone marrow, germ cell tissue, germ
cell tumor tissue, and primary cancers. 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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 ofSequence 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, 4:1 1-17 (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.
[0043] 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.
[0044] Full-length pre-processed forms, as well as mature processed
forms, of proteins that comprise one of the peptides of the present
invention can readily be identified as having complete sequence
identity to one of the kinase peptides of the present invention as
well as being encoded by the same genetic locus as the kinase
peptide provided herein. The gene provided by the present invention
is located on a genome component that has been mapped to human
chromosome 5 (as indicated in FIG. 3), which is supported by
multiple lines of evidence, such as STS and BAC map data.
[0045] Allelic variants of a kinase peptide can readily be
identified as being a human protein having a high degree
(significant) of sequence homology/identity to at least a portion
of the kinase peptide as well as being encoded by the same genetic
locus as the kinase peptide provided herein. Genetic locus can
readily be determined based on the genomic information provided in
FIG. 3, such as the genomic sequence mapped to the reference human.
The gene provided by the present invention is located on a genome
component that has been mapped to human chromosome 5 (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.
[0046] FIG. 3 provides information on SNPs that have been found in
a gene encoding the kinase proteins of the present invention. The
following variations were seen: C1681A, T6888-, A7939G, G9790A,
G11689A, A12145G, A12437-, A12437G, G12437-, C22982T, C23239T,
A23859G, G24862A, G25097A, C25751T, T25934-, G26179A, A26277G,
-26354T, T26356-, T26359G, G29484A, A29885T, C30601T, C30920T,
C31057A, C31057G, G31097A, T31204C, T32660A, T32875-, C33477G,
C34059T, -36681A, C36821T, A38079G, and A38432T. The changes in the
amino acid sequence that these SNPs cause can readily be determined
using the universal genetic code and the protein sequence provided
in FIG. 2 as a base.
[0047] 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.
[0048] 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 manunals, 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.
[0049] 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).
[0050] 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.
[0051] 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.
[0052] 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)).
[0053] 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.
[0054] 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.
[0055] 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).
[0056] 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.
[0057] 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)).
[0058] 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.
[0059] Protein/Peptide Uses
[0060] 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.
[0061] 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.
[0062] 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
expression in lung, liver, kidney, thyroid, brain, infant brain,
fetal brain, placenta, bone marrow, germ cell tissue, germ cell
tumor tissue, and primary cancers. Specifically, a virtual northern
blot shows expression in lung, infant brain, germ cell tissue, germ
cell tumor tissue, and primary cancers. In addition, PCR-based
tissue screening panel indicates expression in liver, kidney, bone
marrow, thyroid, brain, fetal brain, and placenta. A large
percentage of pharmaceutical agents are being developed that
modulate the activity of kinase proteins, particularly members of
the SNF-like 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 lung, liver,
kidney, thyroid, brain, infant brain, fetal brain, placenta, bone
marrow, germ cell tissue, germ cell tumor tissue, and primary
cancers. Such uses can readily be determined using the information
provided herein, that which is known in the art, and routine
experimentation.
[0063] 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 SNF-like 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 expression in lung, liver, kidney, thyroid, brain,
infant brain, fetal brain, placenta, bone marrow, germ cell tissue,
germ cell tumor tissue, and primary cancers. Specifically , a
virtual northern blot shows expression in lung, infant brain, germ
cell tissue, germ cell tumor tissue, and primary cancers. In
addition, PCR-based tissue screening panel indicates expression in
liver, kidney, bone marrow, thyroid, brain, fetal brain, and
placenta.
[0064] 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 lung, liver,
kidney, thyroid, brain, infant brain, fetal brain, placenta, bone
marrow, germ cell tissue, germ cell tumor tissue, and primary
cancers. In an alternate embodiment, cell-based assays involve
recombinant host cells expressing the kinase protein.
[0065] 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.
[0066] 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.
[0067] Candidate compounds include, for example, I) 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).
[0068] 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.
[0069] 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.
[0070] 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 expression in lung, liver, kidney, thyroid,
brain, infant brain, fetal brain, placenta, bone marrow, germ cell
tissue, germ cell tumor tissue, and primary cancers. Specifically ,
a virtual northern blot shows expression in lung, infant brain,
germ cell tissue, germ cell tumor tissue, and primary cancers. In
addition, PCR-based tissue screening panel indicates expression in
liver, kidney, bone marrow, thyroid, brain, fetal brain, and
placenta.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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 lung, liver, kidney, thyroid, brain,
infant brain, fetal brain, placenta, bone marrow, germ cell tissue,
germ cell tumor tissue, and primary cancers. 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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
lung, liver, kidney, thyroid, brain, infant brain, fetal brain,
placenta, bone marrow, germ cell tissue, germ cell tumor tissue,
and primary cancers. 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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 lung, liver, kidney, thyroid, brain, infant
brain, fetal brain, placenta, bone marrow, germ cell tissue, germ
cell tumor tissue, and primary cancers. Accordingly, methods for
treatment include the use of the kinase protein or fragments.
[0086] Antibodies
[0087] 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.
[0088] 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.
[0089] 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).
[0090] 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.
[0091] 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.
[0092] 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).
[0093] 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.
[0094] Antibody Uses
[0095] 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 expression in
lung, liver, kidney, thyroid, brain, infant brain, fetal brain,
placenta, bone marrow, germ cell tissue, germ cell tumor tissue,
and primary cancers. Specifically, a virtual northern blot shows
expression in lung, infant brain, germ cell tissue, germ cell tumor
tissue, and primary cancers. In addition, PCR-based tissue
screening panel indicates expression in liver, kidney, bone marrow,
thyroid, brain, fetal brain, and placenta. 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.
[0096] 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 lung, liver, kidney, thyroid,
brain, infant brain, fetal brain, placenta, bone marrow, germ cell
tissue, germ cell tumor tissue, and primary cancers. 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.
[0097] 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 lung, liver, kidney, thyroid, brain, infant brain,
fetal brain, placenta, bone marrow, germ cell tissue, germ cell
tumor tissue, and primary cancers. 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.
[0098] 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.
[0099] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression in
lung, liver, kidney, thyroid, brain, infant brain, fetal brain,
placenta, bone marrow, germ cell tissue, germ cell tumor tissue,
and primary cancers. 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.
[0100] 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.
[0101] 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.
[0102] Nucleic Acid Molecules
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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).
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. As described in the Peptide
Section, these variants comprise a nucleotide sequence encoding a
peptide that is typically 60-70%, 70-80%, 80-90%, and more
typically at least about 90-95% or more homologous to the
nucleotide sequence shown in the Figure sheets or a fragment of
this sequence. Such nucleic acid molecules can readily be
identified as being able to hybridize under moderate to stringent
conditions, to the nucleotide sequence shown in the Figure sheets
or a fragment of the sequence. Allelic variants can readily be
determined by genetic locus of the encoding gene. The gene provided
by the present invention is located on a genome component that has
been mapped to human chromosome 5 (as indicated in FIG. 3), which
is supported by multiple lines of evidence, such as STS and BAC map
data.
[0119] FIG. 3 provides information on SNPs that have been found in
a gene encoding the kinase proteins of the present invention. The
following variations were seen: C1681A, T6888-, A7939G, G9790A,
G11689A, A12145G, A12437-, A12437G, G12437-, C22982T, C23239T,
A23859G, G24862A, G25097A, C25751T, T25934-, G26179A, A26277G,
-26354T, T26356-, T26359G, G29484A, A29885T, C30601T, C30920T,
C31057A, C31057G, G31097A, T31204C, T32660A, T32875-, C33477G,
C34059T, -36681A, C36821T, A38079G, and A38432T. The changes in the
amino acid sequence that these SNPs cause can readily be determined
using the universal genetic code and the protein sequence provided
in FIG. 2 as a base.
[0120] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a peptide at
least 60-70% homologous to each other typically remain hybridized
to each other. The conditions can be such that sequences at least
about 60%, at least about 70%, or at least about 80% or more
homologous to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent
hybridization conditions are hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45C, followed by one or more
washes in 0.2.times.SSC, 0.1% SDS at 50-65C. Examples of moderate
to low stringency hybridization conditions are well known in the
art.
[0121] Nucleic Acid Molecule Uses
[0122] 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, known SNP variations include C1681A, T6888-, A7939G,
G9790A, G11689A, A12145G, A12437-, A12437G, G12437-, C22982T,
C23239T, A23859G, G24862A, G25097A, C25751T, T25934-, G26179A,
A26277G, -26354T, T26356-, T26359G, G29484A, A29885T, C30601T,
C30920T, C31057A, C31057G, G31097A, T31204C, T32660A, T32875-,
C33477G, C34059T, -36681A, C36821T, A38079G, and A38432T.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0127] The nucleic acid molecules are also useful as probes for
determining the chromosomal positions of the nucleic acid molecules
by means of in situ hybridization methods. The gene provided by the
present invention is located on a genome component that has been
mapped to human chromosome 5 (as indicated in FIG. 3), which is
supported by multiple lines of evidence, such as STS and BAC map
data.
[0128] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0129] 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.
[0130] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0131] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0132] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0133] 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 expression in lung, liver, kidney, thyroid, brain, infant
brain, fetal brain, placenta, bone marrow, germ cell tissue, germ
cell tumor tissue, and primary cancers. Specifically, a virtual
northern blot shows expression in lung, infant brain, germ cell
tissue, germ cell tumor tissue, and primary cancers. In addition,
PCR-based tissue screening panel indicates expression in liver,
kidney, bone marrow, thyroid, brain, fetal brain, and placenta.
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.
[0134] 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.
[0135] 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 expression in lung, liver, kidney, thyroid, brain,
infant brain, fetal brain, placenta, bone marrow, germ cell tissue,
germ cell tumor tissue, and primary cancers. Specifically , a
virtual northern blot shows expression in lung, infant brain, germ
cell tissue, germ cell tumor tissue, and primary cancers. In
addition, PCR-based tissue screening panel indicates expression in
liver, kidney, bone marrow, thyroid, brain, fetal brain, and
placenta.
[0136] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate kinase nucleic acid
expression.
[0137] 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 lung, liver, kidney, thyroid, brain,
infant brain, fetal brain, placenta, bone marrow, germ cell tissue,
germ cell tumor tissue, and primary cancers. 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.
[0138] 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.
[0139] 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.
[0140] 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 expression in
lung, liver, kidney, thyroid, brain, infant brain, fetal brain,
placenta, bone marrow, germ cell tissue, germ cell tumor tissue,
and primary cancers. Specifically, a virtual northern blot shows
expression in lung, infant brain, germ cell tissue, germ cell tumor
tissue, and primary cancers. In addition, PCR-based tissue
screening panel indicates expression in liver, kidney, bone marrow,
thyroid, brain, fetal brain, and placenta. Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or nucleic acid expression.
[0141] 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 lung, liver, kidney, thyroid,
brain, infant brain, fetal brain, placenta, bone marrow, germ cell
tissue, germ cell tumor tissue, and primary cancers.
[0142] 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.
[0143] 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.
[0144] 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 a gene
encoding the kinase proteins of the present invention. The
following variations were seen: C1681A, T6888-, A7939G, G9790A,
G11689A, A12145G, A12437-, A12437G, G12437-, C22982T, C23239T,
A23859G, G24862A, G25097A, C25751T, T25934-, G26179A, A26277G,
-26354T, T26356-, T26359G, G29484A, A29885T, C30601T, C30920T,
C31057A, C31057G, G31097A, T31204C, T32660A, T32875-, C33477G,
C34059T, -36681A, C36821T, A38079G, and A38432T. The changes in the
amino acid sequence that these SNPs cause can readily be determined
using the universal genetic code and the protein sequence provided
in FIG. 2 as a base. The gene provided by the present invention is
located on a genome component that has been mapped to human
chromosome 5 (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.
[0145] Alternatively, mutations in a kinase gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0146] 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.
[0147] 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)).
[0148] 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.
[0149] 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 a gene encoding the kinase proteins of the
present invention. The following variations were seen: C1681A,
T6888-, A7939G, G9790A, G11689A, A12145G, A12437-, A12437G,
G12437-, C22982T, C23239T, A23859G, G24862A, G25097A, C25751T,
T25934-, G26179A, A26277G, -26354T, T26356-, T26359G, G29484A,
A29885T, C30601T, C30920T, C31057A, C31057G, G31097A, T31204C,
T32660A, T32875-, C33477G, C34059T, -36681A, C36821T, A38079G, and
A38432T. The changes in the amino acid sequence that these SNPs
cause can readily be determined using the universal genetic code
and the protein sequence provided in FIG. 2 as a base.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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 expression in
lung, liver, kidney, thyroid, brain, infant brain, fetal brain,
placenta, bone marrow, germ cell tissue, germ cell tumor tissue,
and primary cancers. Specifically , a virtual northern blot shows
expression in lung, infant brain, germ cell tissue, germ cell tumor
tissue, and primary cancers. In addition, PCR-based tissue
screening panel indicates expression in liver, kidney, bone marrow,
thyroid, brain, fetal brain, and placenta. 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.
[0155] Nucleic Acid Arrays
[0156] 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).
[0157] 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.
[0158] 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.
[0159] 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.
[0160] 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 WO95/25 1116 (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.
[0161] 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.
[0162] 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 a gene encoding the kinase proteins of the present invention.
The following variations were seen: C1681A, T6888-, A7939G, G9790A,
G11689A, A12145G, A12437-, A12437G, G12437-, C22982T, C23239T,
A23859G, G24862A, G25097A, C25751T, T25934-, G26179A, A26277G,
-26354T, T26356-, T26359G, G29484A, A29885T, C30601T, C30920T,
C31057A, C31057G, G31097A, T31204C, T32660A, T32875-, C33477G,
C34059T, -36681A, C36821T, A38079G, and A38432T. The changes in the
amino acid sequence that these SNPs cause can readily be determined
using the universal genetic code and the protein sequence provided
in FIG. 2 as a base.
[0163] 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, FL Vol. 1 (1 982), Vol. 2 (1983), Vol. 3
(1985); Tijssen, P., Practice and Theory ofEnzyme Immunoassays:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
[0164] 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.
[0165] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0166] 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.
[0167] 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.
[0168] Vectors/host Cells
[0169] 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.
[0170] 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.
[0171] 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).
[0172] 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.
[0173] 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.
[0174] 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.
[0175] 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).
[0176] 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).
[0177] 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.
[0178] 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.
[0179] 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.
[0180] As described herein, it may be desirable to express the
peptide as a fusion protein. Accordingly, the invention provides
fusion vectors that allow for the production of the peptides.
Fusion vectors can increase the expression of a recombinant
protein, increase the solubility of the recombinant protein, and
aid in the purification of the protein by acting for example as a
ligand for affinity purification. A proteolytic cleavage site may
be introduced at the junction of the fusion moiety so that the
desired peptide can ultimately be separated from the fusion moiety.
Proteolytic enzymes include, but are not limited to, factor Xa,
thrombin, and enterokinase. Typical fusion expression vectors
include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New
England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway,
N.J.) which fuse glutathione S-transferase (GST), maltose E binding
protein, or protein A, respectively, to the target recombinant
protein. Examples of suitable inducible non-fusion E. coli
expression vectors include pTrc (Amann et al., Gene 69:301-315
(1988)) and pET 11d (Studier et al., Gene Expression Technology:
Methods in Enzymology 185:60-89 (1990)).
[0181] 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)).
[0182] 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.).
[0183] The nucleic acid molecules can also be expressed in insect
cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL
series (Lucklow et al., Virology 170:31-39 (1989)).
[0184] 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
(Kaufinan et al., EMBO J. 6:187-195 (1987)).
[0185] 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.
[0186] 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).
[0187] 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.
[0188] 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).
[0189] 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.
[0190] 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.
[0191] 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.
[0192] 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.
[0193] 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.
[0194] Where the peptide is not secreted into the medium, which is
typically the case with kinases, the protein can be isolated from
the host cell by standard disruption procedures, including freeze
thaw, sonication, mechanical disruption, use of lysing agents and
the like. The peptide can then be recovered and purified by
well-known purification methods including ammonium sulfate
precipitation, acid extraction, anion or cationic exchange
chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0195] 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.
[0196] Uses of Vectors and Host Cells
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.o 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.
[0206] 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.
[0207] 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
6 1 1544 DNA Human 1 ctaccacgtt cactgccttc ctctcactaa agccgagagg
gaggctgctc agctctcagg 60 aaaactcttt tgaaccctgg gcacctgctg
tcctcagttg gcatctccca ccctctgagc 120 ctcttctgtt cctgcacaac
ctgcctcttc gctgagatgg agacgtgagc ccccgtggac 180 gatgactgca
gtgtatatga atggaggtgg cctggtgaac ccccactatg cccggtggga 240
tcggcgcgac agtgtagaaa gtggctgtca gaccgagagt agcaaggagg gtgaggaggg
300 acagccccgc cagctgacgc ccttcgagaa actgacacag gacatgtccc
aggatgagaa 360 ggtggtgagg gagatcacgc tggggaaacg gataggcttc
taccgaattc gaggggaaat 420 cggaagtgga aacttctccc aagtgaagct
tgggattcac tccctaacca aagaaaaggt 480 ggccattaag atcctggaca
agaccaagtt agaccagaaa acccagaggc tactatcccg 540 agaaatctcc
agcatggaaa agctgcacca tcccaacatc atccgccttt acgaagtggt 600
ggagacccta tccaagctgc acttggtgat ggagtatgca gggggtgggg agctcttcgg
660 aaaaattagc actgagggga agctctctga accagaaagc aagctcatct
tctcccagat 720 tgtgtctgcc gtgaagcaca tgcatgaaaa ccaaattatt
catagagatc tgaaagcaga 780 aaatgtattc tataccagta atacttgtgt
gaaggtgggc gattttggat tcagcacagt 840 aagcaaaaaa ggtgaaatgc
tgaacacttt ctgtgggtct cctccctacg ctgcgcctga 900 actcttccgg
gacgagcact acatcggcat ttacgtggat atctgggcct tgggggtgct 960
tttgtacttc atggtgactg gcaccatgcc atttcgggca gaaaccgtgg ccaaactaaa
1020 aaagagcatc ctcgagggca catacagtgt accgccgcac gtgtcagagc
cctgccaccg 1080 actcatccga ggagtccttc agcagatccc cacggagagg
tacggaatcg actgcatcat 1140 gaatgatgaa tggatgcaag gggtgccata
ccctacacct ttggaacctt tccaactgga 1200 tcccaaacat ttgtcggaaa
ccagcactct caaggaagaa gaaaatgagg tcaaaagcac 1260 tttagaacat
ttgggcatta cagaagagca tattcgaaat aaccaaggga gagatgctcg 1320
cagctcaatc acaggggtct atagaattat tttacataga gtccaaagga agaaggcttt
1380 ggaaagtgtc ccagtcatga tgctaccaga ccctaaagaa agagacctca
aaaaagggtc 1440 ccgtgtctac agagggataa gacacacatc caaattttgc
tcgattttat aaattgcact 1500 agactgcttg taactaacca agatgattgt
tgctgcttct aaat 1544 2 436 PRT Human 2 Met Thr Ala Val Tyr Met Asn
Gly Gly Gly Leu Val Asn Pro His Tyr 1 5 10 15 Ala Arg Trp Asp Arg
Arg Asp Ser Val Glu Ser Gly Cys Gln Thr Glu 20 25 30 Ser Ser Lys
Glu Gly Glu Glu Gly Gln Pro Arg Gln Leu Thr Pro Phe 35 40 45 Glu
Lys Leu Thr Gln Asp Met Ser Gln Asp Glu Lys Val Val Arg Glu 50 55
60 Ile Thr Leu Gly Lys Arg Ile Gly Phe Tyr Arg Ile Arg Gly Glu Ile
65 70 75 80 Gly Ser Gly Asn Phe Ser Gln Val Lys Leu Gly Ile His Ser
Leu Thr 85 90 95 Lys Glu Lys Val Ala Ile Lys Ile Leu Asp Lys Thr
Lys Leu Asp Gln 100 105 110 Lys Thr Gln Arg Leu Leu Ser Arg Glu Ile
Ser Ser Met Glu Lys Leu 115 120 125 His His Pro Asn Ile Ile Arg Leu
Tyr Glu Val Val Glu Thr Leu Ser 130 135 140 Lys Leu His Leu Val Met
Glu Tyr Ala Gly Gly Gly Glu Leu Phe Gly 145 150 155 160 Lys Ile Ser
Thr Glu Gly Lys Leu Ser Glu Pro Glu Ser Lys Leu Ile 165 170 175 Phe
Ser Gln Ile Val Ser Ala Val Lys His Met His Glu Asn Gln Ile 180 185
190 Ile His Arg Asp Leu Lys Ala Glu Asn Val Phe Tyr Thr Ser Asn Thr
195 200 205 Cys Val Lys Val Gly Asp Phe Gly Phe Ser Thr Val Ser Lys
Lys Gly 210 215 220 Glu Met Leu Asn Thr Phe Cys Gly Ser Pro Pro Tyr
Ala Ala Pro Glu 225 230 235 240 Leu Phe Arg Asp Glu His Tyr Ile Gly
Ile Tyr Val Asp Ile Trp Ala 245 250 255 Leu Gly Val Leu Leu Tyr Phe
Met Val Thr Gly Thr Met Pro Phe Arg 260 265 270 Ala Glu Thr Val Ala
Lys Leu Lys Lys Ser Ile Leu Glu Gly Thr Tyr 275 280 285 Ser Val Pro
Pro His Val Ser Glu Pro Cys His Arg Leu Ile Arg Gly 290 295 300 Val
Leu Gln Gln Ile Pro Thr Glu Arg Tyr Gly Ile Asp Cys Ile Met 305 310
315 320 Asn Asp Glu Trp Met Gln Gly Val Pro Tyr Pro Thr Pro Leu Glu
Pro 325 330 335 Phe Gln Leu Asp Pro Lys His Leu Ser Glu Thr Ser Thr
Leu Lys Glu 340 345 350 Glu Glu Asn Glu Val Lys Ser Thr Leu Glu His
Leu Gly Ile Thr Glu 355 360 365 Glu His Ile Arg Asn Asn Gln Gly Arg
Asp Ala Arg Ser Ser Ile Thr 370 375 380 Gly Val Tyr Arg Ile Ile Leu
His Arg Val Gln Arg Lys Lys Ala Leu 385 390 395 400 Glu Ser Val Pro
Val Met Met Leu Pro Asp Pro Lys Glu Arg Asp Leu 405 410 415 Lys Lys
Gly Ser Arg Val Tyr Arg Gly Ile Arg His Thr Ser Lys Phe 420 425 430
Cys Ser Ile Leu 435 3 38564 DNA Human misc_feature (1)...(38564) n
= A,T,C or G 3 aaagcacaag ttcctgccca cctggtgctt tttttttttt
tcagatggcg tttcactctt 60 gtcgcccacg ctggagtgca atggcatgat
ctcagctcac tgcaccctct gcctcctggg 120 ttcaaacaat tctcctgcct
cagcctccca agtagctggg attacaggtg tgcgccacca 180 tgcttggcta
atttttgtat ttttagtaga gatggagttt caccatgttg gccaggctgg 240
tctcaaactc ctaacctcag gtgatccacc cacctcagcc tcccaaagtg ttgggattac
300 aggcttgagc cactgtgccc ggcgccctcc tagtgctgat gtagttgtgg
cccaagtaca 360 ttagtgacaa ggacagcaca gtaatgagga cacagagcca
gtaagttctt cagagagtct 420 gttagaacac aaagtgatca aagtgatcaa
aagtactgca ttctatggaa aatctatatt 480 tttctttttc cttaggttaa
gctaaagttt cttgcagaat tactacttca ttctagtctg 540 tcataagctg
catggtgttt ttctgccagg aatctgggga gatctagtgc cacttgacct 600
tcacagagat tcttaacctg atgggtttca gaatgtcatc ttaaaattgg atgcaaattg
660 tatgggtgag ggtttttttt tttttcctcc tggggataaa gtcataggtt
ttgttagcat 720 ctcaaaggag tctgtgagcc caggatgatt aagaatcagt
gattttggct gagtacagtg 780 gttcacacct gtaattccag cactttggga
ggtagaggtg ggtaaagaaa aggaagaaga 840 atcagtgact tagattttct
tcactctaat taaggacctt tacctttttt gtttgtttgc 900 tttgctttgt
ttttttgaga cagggtccgg cattgtcacc caggctggag tacagtggct 960
tgatctcagc tcactgcaac ctccacttcc tgggctcaag ccatcctccc acctcagtct
1020 gtaggtgtag ctgagactac aggtgcacac cctgctaagg ggtacaccac
cacaccctgc 1080 taggaggtac aacaccacac cctgctaagg ggaactttat
acttaaaaat gtttttaaaa 1140 aacatcctaa ttggtctcta acctagagat
gtgcttagtt actgatacct ggtccctttt 1200 aagcctatgt tcgtcatcct
tcttccagat tcctttctct ctatccagaa ataggagcag 1260 tactgggcat
caacctcctc tttacttccc ttatttatgt ttatttggca accccatact 1320
taaccttaag tataagctta ggaagcctta tttatactta attgtaataa tgtgatatta
1380 ttgagcagag ttggaaatcc atacttccaa ggcagtctct catgttttca
gtcaaatttt 1440 tggtttggaa tgctcaagtc caaaaatcaa gtttaacaca
tatttactta gtaaacactt 1500 tctatatgta agaacttata ctccattttg
aaggagagag gaggtgagca ggacatacct 1560 acaagtcaaa accagttccc
accttcactg agatgggaca ttttggaaaa tgcacgaagt 1620 gctgtgggaa
ttcagagagg aagagagtat ttcctgctgg agagtgcaga aaaaaaacat 1680
cccgatgaca tcgtaggtgg gcagcatttc ttgtcactgg caatgctgta aaccctcctt
1740 ttccacatct tcaaaataag gtgctgggcc tagaaactga cacctgctca
gaaaaattca 1800 aaaagtgaat gtaggaggaa aaatcagcct ttgtcataaa
atgacatagg aagaaagagt 1860 ggaagcaact agtataggac aagttgtgca
agaacatgaa gtagtaaagg agataaaata 1920 ttattatttg aataaatatg
agaaattttg aaaaccatta gtaatccgga atctttcatc 1980 tttgctatca
tattattttt aaaatttggt tttaaagcaa cacaaggcaa tgtgcatatt 2040
ttcttaaact ttaagaaata gaaaaattgt tttgctagaa atatgtacgg accttagggg
2100 cttttctaaa atgtaggttg agaggtgtgt gtatatgctc ttttcttctt
actagatcac 2160 atgtaatcaa gcatctacta ctgatatgag tcataaattt
gtggggtact tttagatcaa 2220 tgcttacaaa gcagttgcac attcattaca
aatcctgtac agtacattag cataagctaa 2280 taaatttctt actctctatt
cttaggttga accagccaaa ttttcgagac agctcacggc 2340 ttagaggaag
gttcatctaa ataaaggccg gctaaagtga cattgcaggg attaaatcct 2400
tctttggctg cctgtgtgac cagaaggctt atttgcaagt ttcttctttc ctggggtcca
2460 gattattagg tctccagcgc cctgcagctt gacagaaaga gaagcatgaa
atgaaggtca 2520 gagatgagat cccgcagcag ggacgtgggg gcctcccagg
ggcatttacg caccagagtg 2580 caagattctc tggccatcaa gggaaatagc
aaacagaagc ctttgtcctg gggcacagcc 2640 acctaccaca aagcatcaga
ctccacgtct ggccagaaag ttcctggagt cccatcaggc 2700 cagtgggtat
gtaacatgtg cctaattgta cagctagagc ctgcaagttc aacgtgaggg 2760
aaggtgggaa atgtcttgag tgaggcgagc agctcctggc tgggctgggc agactcagct
2820 accacgttca ctgccttcct ctcactaaag ccgagaggga ggctgctcag
ctctcaggaa 2880 aactcttttg aaccctgggc acctgctgtc ctcagttggc
atctcccacc ctctgagcct 2940 cttctgctcc tgcacaacct gcctcttcgc
tgagatggag acgtgagccc ccgtggacga 3000 tgactgcagt gtatatgaat
ggaggtggcc tggtgaaccc ccattatgcc cggtgggatc 3060 ggcgcgacag
tgtagaaagt ggctgtcaga ccgagagtag caaggagggt gaggagggac 3120
agccccgcca gctgacgccc ttcgagaaac tgacacagga catgtcccag gatgagaagg
3180 tggtgaggga gatcacgctg gggaaacgga taggcttcta ccgaattcga
ggggaaatcg 3240 gaagtggaaa cttctcccaa gtgaagcttg ggattcactc
cataaccaaa ggtaggatcc 3300 gacttcccaa gggtcatccc tggcagtatt
gggacctagt gtaggaaagg ggttaggtgg 3360 ccagggccaa ggaagcaagt
aaagtgacct cagcagagcc cctgcaaggc ccacatcctg 3420 tgccagccgc
cttctgtggt cttctcagtt aattttcaca gtaaccatgt gaggtcaata 3480
tttttttcca ttttgcagat aaagaaactg agatccaaag aaggcaaatg tgtcttcagt
3540 tcnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 3600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 3660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3720 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3780 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3840
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
3900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 3960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 4020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4080 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4140 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4200
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
4260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 4320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 4380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4440 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4500 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4560
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
4620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 4680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 4740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4800 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4860 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4920
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
4980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 5040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 5100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5160 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5220 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5280
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
5340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 5400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 5460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5520 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5580 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5640
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
5700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 5760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 5820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5880 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5940 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6000
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
6060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 6120 nnnnnttgac acataaaata aaccatcaga gttggtaact
gattggatgt agaactgaag 6180 atggaaggat ggacgaaggc aatgaccttg
ctttagttga ctatgtgaac caaaacaggt 6240 gatgccttta ataggaatag
gagaaagagc aaatttggga gggaaaaagt tgagtgaaaa 6300 cactgacatc
cagaaggagc tgtctactgg gcatgctcca gtcatggaca agggcatgag 6360
gtcagaccag gtatttgcat tgagaagatt gtagaaatgg atgcagtcac tgaaggagac
6420 tgtggagaag ggtgatgagc aaccttttta cacttagtaa ctagttctac
atcctagttg 6480 acattttatg tatggattag tttttttttt tttttttttt
tgagatggag tctcgctctg 6540 ttgcccacgc tggagtgtag tggtgtgatc
ttggctcact gcaagctctg cctcctgggt 6600 tcatgccgtt ctcctgcctc
aacctcccga ggagctggga ctacagacac ccaccaccac 6660 gcctggctaa
ttttttgtat ttttagtaga gacggggttt caccatgtta gccaggatgg 6720
tctcgatctc ctgacctcgt gatctgcccg cctcggcctc ccaaagtgct gggattacag
6780 gcatgagcca ccgcgcctgg ccggattagt tttaaatatg ctaatggaat
cattcactgt 6840 tgctccatga acactatgat ttttcaatgt cattaaatat
agatttttaa aatatattta 6900 tgtgtatata ttatctatat atgtgtattt
attatctata ctctcatctt ttaaagagga 6960 catattgttt cacatatagc
tgtggcataa attctttaac ttagccacta ttggttaaca 7020 tttggatcat
ttctaaagtt ttgctcttaa ttataatgct acagagatta ttcttttcca 7080
tacttctttg catatacgta tgatttctct cagcatacat tcctagaagt tagctgtctt
7140 ggtcagaggg catgcattga aaaattttaa cacacagcac caaaagagca
gcttttgaat 7200 ccttactcat agagtacatc tatctgtaac tcaaatctca
cagggactaa tgttgctttt 7260 caatagtcta gagccagcat gaaagacaaa
agatgaaaca catagagcga tgaaaggcca 7320 aaaatcgaga cagaagatgt
gatttggaag gttacaagag aagttggata aagccaaaca 7380 aggaaaggaa
tttagaaaaa agtgtccaat gctgcagaag ggtcacagag ctacagtctg 7440
agcaaaggct actgcataag gccaactttg agaagtttca tacaagtgtg ggttcagatg
7500 ttagattcca atagaaagga agcaggatgg gacagtagga aaagcgtgga
cttggggacc 7560 agacaaattt atctgaaata ctcattcagt aacttactag
gcttctgact ttaggcatat 7620 taatctcttt ggtcctcagt tgcctcagct
gtaaaataga gataatgatt tttagaattg 7680 ttggggaaat tggaaataat
atatgtgaag tacatgacat atagtaacca ctcaaaatgg 7740 tagctgccat
tgctataact ttacttttta attaatctta tttcaaaaag gtatttaatg 7800
ttattacaac aataaaaacc agtatcactt actatgaata gaaggtgatg gtaaaaataa
7860 atacaatgca aacaaattaa aaagcattaa aggaaaatta caagtaacaa
agtaattttt 7920 aaaacatgct aacaatcaat ctcagacttt ctctttgcct
taattaggag aattaaagga 7980 gagctgaaat gggaatcatg tttttacgct
gtgattcagt gctatttaag gctgtttcca 8040 cgccctaaaa tcatttcaac
ctcatgagtc ttaaaggttc cactttggga agcactgaca 8100 tcatctagtc
cttccatgtt ttacaaacga ggagatggag gctcagaaga ggctgtccca 8160
aggtttagaa cctgtttaca ggaattcaca tggaattgag tactcacatg tattgagtac
8220 aggggaattc tatggaccca cagcagggaa gtagggccgg gtcttaagat
gatattcttt 8280 tgatcttcac tttctctggg gctgtgtgct ttctcatttc
tacttttctt gatgaatttg 8340 ttccatcttt atacctcttt ctgcaaaaag
gttttccaca cttaattatc agaaggtata 8400 tcatggccaa aaatggttgt
tgccagtccc acatctacat aaactctcaa tgttattgac 8460 cgcctccatc
ccatgtgttt cattctgttt cagttcaaat tttaaaaaga aagaatatac 8520
ttaattacta gccagctgaa aatgaactac ctttgagtca agggcatgcc cgtgtcagtg
8580 agctgtaacc ctttgagtca cgtgggccac tactgtctca gcagagccaa
aggtccaaag 8640 aagtctctgg gaagggagaa aattgacaga catatccacc
atagagacat tagctagcta 8700 agccgagaag tttctacaag ccgggagcaa
gacagtgccc aattctgtag acaggatatt 8760 atgtgaaagg acaaaaaaaa
aaggtggaat aataattagt gtttatatac tgacttactt 8820 ctcgggttcc
aaagacttct tgtctgttgt attacaaaat taacttcaaa cttgtcccca 8880
tgagtttcag agagcatggc attttcccac tgtgtgggtg tgggtgggtg ccttgtgtgg
8940 cctcactgac atgccctcct tatacctaac ctgctacaaa gtagctctat
tgtcaaagaa 9000 acagtctcac ccgctggttc tgggacagta tgtgcaaaaa
gggacaggaa aataaatagc 9060 agtttgtaca aatatggccc ccacaccaaa
ccaacttgcc cagaattgtc atccggtttt 9120 ctaggaatat aaggacagtt
tgaataagat aatagaggag attcccagga aagcagagag 9180 tttcagtgcc
aggagcagct cccaggacag tagtctaaaa aattgaaaaa aaaaaaaata 9240
aagaaataca aagcaccaag tgctctggat ctctacaaag gaggctccaa tatgggtccc
9300 tgctggctgg ggtttatttt tatagctcac agtttggaat tcccttgggc
tctccaagct 9360 gatagtgaag gttgacagtc atcatgagcc cccttttagg
ctgattagaa atttctcata 9420 agagatgata cacttagctt ctgaatattt
tatgtcccat ctgttgtcac aaacaaaaga 9480 agtggcagcg aatgtgggtg
tttttttctg ttgctgccct tctgacttct tccttctgct 9540 actctagtct
catgtttcta ggatcctttc ttcctcccac tttatttatc tatttatcta 9600
ttaattcatt ctttccttcc ttcctttttt ttttttttat cttttgagga agagttttgc
9660 ttcattgccc aggctggagt gcagtgatgc gatcaaagct cactgctgcc
ttggactcct 9720 gggctcaagt gattcaccca cctcagcctc ccaaagcact
gggcattaca ggtgtgagac 9780 accatgactg gccttcattc ctttatatat
atatataata taatatataa tgtaatatat 9840 aatataatat ataatataat
atatgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtaa 9900 gcacatgcct
gataaggaac ttacagcagt ttaacagagg cagacaggaa aaaattgatg 9960
agtgctgtcc agagtaaggc agcaagaatg tgcttacctg tgccccgtgg atccaggaag
10020 ggtctattga gcaggaaaag cttgcaatgg gtcttgaggt ctgaggagca
atttcacagg 10080 agaaggagga aagagaactc cagaaagaga gataggatat
aaagtacatg gaagcatgaa 10140 ccagtgtggg gagtatttgg agatttaaaa
gtaattcagc aaagctagag catgaggtgc 10200 acccacagag tcatgggaaa
gagataaggt ggatggggct tgatcatgga aaatcttctt 10260 gcatatctgg
attttgtcat ttaggtattg gaaagctact gaagagtttt tttttttttt 10320
gagacggagt cttgccctgt cacccagcct ggagtacaat ggtgtgatct ctgttcattg
10380 aaacctctgc ctcctgggtt caagtgattc tcctgcctca gtctcctgag
tagctgggat 10440 tacaggcaca cgccaccatg ctcggcaatt tttttttgta
tctttagtag agatggggtt 10500 tcaccatctt ggccaggctg gtctcgaatt
cctgacctcg tgatccgccc gcctcagcct 10560 cccaaagtgc tgggattaca
ggcatgagcc actgcgctca gtcgtttagg agctttaagc 10620 aagccattaa
tgcaagtgat taatatgatc ttatttcagt gcggaatgtg aattgaagtt 10680
ggaaagtaag gagagtcaag gaagtcagaa aaaaatgtta gtcaaatggt
ccagatggat 10740 gatgttgttc atctcaacta aggagaagtg gcagtgagaa
tggagagaag agcatgtatt 10800 tgaaaaatgt cttaggatat tgaaaaacct
tgtgatcaat ggtatgggtg agagcaggac 10860 gggacatggg agaggaagaa
ttgacgatga ctcccagttt ccagttttaa tgccttacca 10920 gtaaataatt
acaggagaag tggatttgag agtaaatata tggaagcact gacttggagg 10980
tatatggaat tcatgaaatg aatgggtcta gcagctttct tggagcccaa gagaaagtat
11040 gatacataaa gatttgaggc ttaacaaact ctgggatgta gtgacatctt
gggagtccta 11100 gttacaagct ccttggtgtc agatactata tcacattgat
ccttaaagtt cctccatgtg 11160 tgttataaat aataaacact cgacaaatat
ttggtgagtt aagttagtat aacttctcat 11220 gaaaatccag gagtggtgga
ggaagaaata ttcgagggag caatgcatag cagcaccttc 11280 tggtaggtgc
tgtatgctgt gatctccctg gaggacaggg ctagagcacg aactcagtct 11340
gtagcagagc ttcataaaga tagcccaatg gttttgtcgt tttgcttttt ctagagatga
11400 agtctcaact atggttatcc aggccgttca agaacttctg ggctcaagca
atcctcctgc 11460 ctcagtttcc taagtagcat gggggacagc tatgcaccac
tgcacctgac tgctcactgg 11520 ttttaaaaat tgattccccc ctcttgaata
cattcattaa aatagcaatt caaaattgta 11580 ttttcttact ttaaaaatgg
ttagttagtg atttagggaa ttcaccctaa aaataaatca 11640 tatattggaa
atactacaac caaaaaaatg ttcattgtcg tgttatttat agtgtcgaga 11700
actagtaata acttatggga tccaatgata taggaatggt caggtaaagc atatacactg
11760 ggcagaagat tccatagcca tgagaaatca tgattatgaa gactctgtgg
taacatggaa 11820 gatgggcagg acatgagtgt aagtgaaaag ccaggtgcaa
atggagcgca cactattatg 11880 ataatactta ctccaggttc ttcagtgaga
ttattcactc agcaaatact tgttgagcac 11940 ctactatgca atagccacta
ttctagagac tggtgataca gcagtgaaca aaatagacag 12000 caatctctgt
ccttatggaa cttacactct agtgagaggg aagccagaca aaaataaatg 12060
tgtaaaatat tcattgtatt atgtagcgat aagtgataag aaagaaaaga gggccgggcg
12120 cggtcggtca cgcctgtaat cccagcactt tgggaggcca aggtgggtgg
attatgagat 12180 caagagatgg aggccatcct ggccgacatg gtgaaaccac
aattctacta aaaatacaaa 12240 aattagctgg gcatggtggt gtgcaccttt
agtctcagct actcaggaga ctgaggcagg 12300 aaaatcgctt gaacccagga
gggggaggtg gcagtgagcc aagatcgtgc cactgtactc 12360 cagcctggtg
acagagccag actctgtctc aaaaaaaaaa aagaaagaaa gaaagaaaga 12420
aagaaagaaa gaaagagcag gaggggagat agaaagcatt gaggatgaca gtttttagat
12480 aagttggcca ggacagcctt actgaggagg taataattaa actgaaagaa
atgagggaaa 12540 tggctataca tgggggaact ccatcaagca gaaggcgtag
caggcgtaag agtgtccaga 12600 tgggagatgc ctgcagtact cttgaaacag
ctgaggggaa agggcagaga gtaacaggga 12660 acagattgtg gaggaccaag
tttgtcaaag taagaagttt ggcttttcct gggattttac 12720 aggagccatc
agaaggaact ggggatttca caaagggaac cagggatttc ctgggatttc 12780
acaggagcca tcagagggaa ctgggggagt gacatgatct ggcttannnn nnnnnnnnnn
12840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnncttg ggcaacatag
caagaccctg 12900 tctttaaaac caaaaccaaa accaaaaaaa ccacacacat
ataacctttt tatgtttaac 12960 gcatatcaat accaaaaaag tttaaaaatc
atgagtattc attttgatga attttccaaa 13020 ctgaaaagac ccatggaagc
accaccaaca tcaagacaca gaacacttct ggtactcaga 13080 gcgctgccct
ccacctgctc gtcatcctcc attctattct ctacatcctg aagtgtaacc 13140
actattctga cttctaatac catagattgc ttttgactgt tttaaacttt acataaatgg
13200 catcatccaa tgtgtattct tttgtcttac ttcaattgtc caattttgag
tttgtgacta 13260 ccatccatac tgttttatgt agttcagatg tttcatgctc
attgccatat atacagtaat 13320 tcattgtatg aatgtagtac cacaatttat
ttatccattc aactgttgct gatgtgacta 13380 gtttctagtt tccagtttgg
ggctattatg aataacattg atgtgaacat tctagtgcaa 13440 gcgtttttgg
taaacttttt tttttttttt gaggcagggt tttgctttgt cacccaggct 13500
ggaatgtggt ggcacaatca cagctcactg caaccttgac tttctgtatt tgagtgatcc
13560 tccaacctca gcctctggag tagctggaac cacaggggca tgccatcatg
cctggctaat 13620 tttttaaatg ttttgtagat atggggtcct gctatgttgc
ccagactggt ctcaaactcc 13680 agggctcgag taatcctctt gccttggcct
cccagagtgt tggtattaca gatgtgagcc 13740 actgagtctg gctcgtatgc
atttttattg gggacatttt tacttataaa ttttttttca 13800 atgacttttg
gggtacaagt ggtttttggt tacaatgatg aactgtatac tggtgaaatc 13860
ttagatttta gtgcacctat cacctgagta gtgtacattg tacccaatac gtagttttta
13920 atccctcacc ccctcccacc ctaccccttc taagtctcca aagcccatgc
cttttcatac 13980 ccatagctaa actctcattt ctaagtaaga acatacagta
tttgattttc cattcctgag 14040 ttacttcact tggaacaatg gcctccagct
ccatccaagt tgctgcaaaa tacattattt 14100 tattcttttt catagctgag
tagtattcta tggtatatac accacgtttt ctttacccac 14160 tcattggttg
gttgacactt aggttggttc cagatctttg caattgtgaa ttgtgctgcc 14220
ataaacatgc atgcaggtgt tttttgatat aaggacttct tttcctttgg ggagataccc
14280 agtaggggat tgttggatca aatggtagat ctactctcag atttagattt
taatttactt 14340 ttagatttta gatttagaaa tctccattct gttttccata
gaggctgtac taatttacat 14400 tcccaccagc agtgtataaa tgttaccttt
tcaccacatt catgccaaca tctactgttt 14460 tttgactttt taataatgac
cattctggct ggggtaaagc ggtatctcac tgtggtttta 14520 atttggattt
ttgtgatgat tagtgatgta gagtgttttt ccatatgttt gttggccatt 14580
tgtatgtctt cttttgagaa atatttattc atatcatttg cccacttttt aatgagatct
14640 ttttttgttt tttgtttgct aatttgtttg agttccctgt aaattctgga
tattagttct 14700 ttgtcagata catagtttgc aaatattttc tcccattctg
tggattgcct gtttgctttg 14760 atgattattt tttctgttgt gcagaagctt
tcagtttaat gaggtcccat ttatttattt 14820 ttgtttttgt tgcatttact
tttggggtct tagtcacaaa ttcttatcta agtcaatgtc 14880 cagtagagtt
tttcctgttt tcttctataa tttttgtggt ttcaggtctt agatttaagt 14940
ctttaatcat attgagttga tttttgtata aggtgagtgg gatccagttt tattctccta
15000 catggggcta tccagttttc ccagcaacat ttactaaata gggtgtcctt
tccctaattt 15060 atgtttttgt atgctttgtt gaagatcagt tggttgttan
nnnnnnnnnn nnnnnnnnnn 15120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15180 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15240 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15300
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
15360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 15420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 15480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15540 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15600 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15660
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
15720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 15780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 15840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15900 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15960 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16020
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
16080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 16140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 16200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16260 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16320 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16380
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
16440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 16500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 16560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16620 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16680 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16740
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
16800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 16860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 16920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16980 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17040 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17100
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
17160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 17220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 17280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17340 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17400 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17460
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
17520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 17580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 17640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17700 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17760 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17820
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
17880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 17940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 18000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18060 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18120 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18180
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
18240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 18300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 18360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18420 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18480 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18540
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
18600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 18660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 18720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18780 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18840 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18900
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
18960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 19020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 19080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19140 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19200 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19260
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
19320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 19380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 19440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19500 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19560 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19620
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
19680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 19740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 19800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19860 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19920 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19980
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
20040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 20100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 20160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20220 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20280 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20340
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
20400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 20460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 20520 nnnnnnnnnn 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 nctgtggggg gctccaccca gtttgagctt cccagctgct
21120 ttgtttacct actcaagcct cagcaatggc ggatgtccct ccaccagcct
cattgccacc 21180 ttgcagttca atctcagact gctgtgctag cagtgagtga
ggctccatgg gcgtgggacc 21240 ctctgagcca ggtgcgggat atgatctcct
ggtgtgccat ttgctaggac tgttggaaga 21300 gcacagtatt agggtgggag
tgtcccgatt ttccaggtac cttctgtcat cacttccctt 21360 ggctaagaaa
ggggattccc tgaccccttg cacttcctgg gtgaggcgat gccctgccct 21420
agttcagctc acacttcgtg ggctgcaccc actgtccaac aagccccagt gcgatgaacg
21480 cggtacctca gttggaaatg cagaaatcac ctgtcttctg cgttgctcat
gctgggagct 21540 gtagactgga gctgttccta tttggccatc tttgaacctc
ccccctgact tgcgtatgtt 21600 aaatcttccc tctatctctg ggacaaagcc
catttgatca tgatgtatta tgtttttgat 21660 ttgctattgg attctgtttg
ctggtatttt gttgaggact tttgcatcta tgttcatcag 21720 gaatattgat
ctgtagtttt cttttttgtt atgtcatttc ctgggtttga cacagggtga 21780
tactggcttc atagaatgag tttgggagga ttttctcttt ctcaatcttt ttgaataatc
21840 tcaatagtat tgcttccaat tcttctttga ctgtctggta gaattcagct
atgaatccat 21900 ctggccctag gcttttttgt tgctggcaat ttcaatcagt
aaattttact tagtcagtct 21960 cactgcttgt tatctggtct gctcaggatt
tctatttctt cctgatttaa tctgggatgg 22020 ttgtaaattt ccaggaattt
atccatttct tctatgtttt ttagtttgtg tgaatagaga 22080 tgttcatagt
aatctcaact gatcttttgt atttctgttt cattggttgt agtgtctcta 22140
ttttcatttc taattgaact tattcaaaac ttctctcctc ttagttaatc tagttaatgg
22200 tctatcaatt ttgtttatct tttcaaagaa ccaacttttt gttttactaa
ccttgtgtaa 22260 tatttttgtt tcaatttcat ttatttctgc tctggtcttt
gttgtttctt ttcttctcta 22320 gctttgggtt cctttgttcc tgtttcttta
gctccttgag gtgcgatgtt agggtgtcaa 22380 gttgtgatct ttcagacttt
ttgatgtagg catttagtgc tataaacttt cctcttagca 22440 ccacttttgc
tgtatctcag aagttttgac aactgtgtca tattatcatt catttcaagt 22500
aatttttaaa tgttcatctt tatttcattg ttaacccaaa atttatacag gagcagattt
22560 tttaatttcc atgtatttgt atagttttga gaattccttt tggagttggt
ttctagtttt 22620 ttcctactgt ggtctgagaa gatgcttgat atgattttga
tttttaaaaa ttattgatac 22680 ttgttttgta gccaatcgta tggcctatgt
tggaatatat tccatgtgct gatgagaaga 22740 aagtatattc tatagttttt
gggtaggatg ttctgtaaat atctgttagg tctacttgtt 22800 ttagagtatt
gtttaagtca ttgtttcttt gttgactttc tgcctcgatg atctgtctag 22860
tgctgtcagt ggggtgttga agtatcccac tattattggg ttgctcatta tctctttcct
22920 taggtctagc agtaattgtt ttatgaattt tggagctcca tagttaggta
cacatatatt 22980 ttggattata atattctctt gtcagattaa tccttttatc
attatataat tgtaaccacc 23040 taacaggtta tttctgccca ctgcacaagc
aaaatcaatt catggcattg tggtgaagaa 23100 agtgtttaat tgatgcaagg
ccagtcatgc catgtggtca aactaggatg gagatattac 23160 tcaaatcaat
ctcatcaaag gcttgttggt taggggtttt tcaaaggcag ttttggggaa 23220
gggattgggc tgaccagacc aggtgcttgc tgctgattgt ttgggttgga gatgaaatca
23280 tatggagttg aagctgtcct cttgtgctga gtcacttctg tggggccaca
ggagtggttg 23340 gtgggtccat atgggtccaa gtgaagccat cggtgtcaga
catgcaaaaa acctgaaaag 23400 atatctcaaa aggctaatct taggttctac
aatagtgatg ttatctgcat gagtaattgg 23460 gggagttgca tatctgtgac
ctctggaata atggctgtca atcatttata tccacacctt 23520 agcagaattc
aggctcttct cctcctccta agtctggtgg tttctcatta gctttacaaa 23580
ggtgactgag ttttgggaaa gattattatc atttaaacta caaactaaat gtctcccaaa
23640 gctagccaag actaagccca ggaataatta aggcagcttg aaggctaaag
gcaagaggga 23700 ggttggctag atcagatgtc ccccactgcc ataattgtct
cagtgatata atttttgcaa 23760 aggtggtttt ataatgacct tctttgtctt
tttctttttt ttaactgttg ttgctttaaa 23820 gtctgtttta tttaatataa
gaatagccac tcctacccag gcacggtggc tcacacctgt 23880 aaatcccagc
actttgggag gctgaggcag gtatatcacc tgaggtcagg agttcaagac 23940
cagccagaca gagatggtga aactccatct ctactaaatt tacaaaaaca ttagccaggc
24000 atggtggggg gcacctgtaa tcccagctac tcaggaggct gaggcaggag
aattgcttgt 24060 acccgggagg cagaggttgc agtgagccaa gattgcgcct
tgctctccag cctgggcaac 24120 aaagtgagac ttcatctcaa aaaaaaaaaa
aaaaaaaaaa aagaatagct actcctgcta 24180 gcctttggtt tccatataca
cagaacatct tttcccaccc ctttaccttg tgcttgtgtg 24240 aatccttatg
cattagatga gtctcttgaa gacagcagat atttggtttg tgatatttta 24300
tccattctgc caatctgtat gttttcagtg gagcatttat gccatttatg tttaatatta
24360 acattgagat gtaaggtacg attctcctca tcatgttgat tattacctgg
atccttttgt 24420 gtgtgtgtgt gtgtgttatt gtttttattt ttttatttta
atttttttga gacagagtct 24480 tgctctgtca cccaggctgg agtgcagtgg
cgagatctct gctcactgca agctccgcct 24540 cctgggttca tgccattctc
ctgcctcagc ctcccgagta gctgggacta cagacacccg 24600 ccactacgcc
tggctaattt tttgtatttt tagtagagac ggggtttcac catgttagcc 24660
aggatggtct cgatctcctg acctcgtgat ccacccccct tggcctccca aagtgctggg
24720 attacaggtg tgagccaccg tgcccggcca tgtgtgtgtg ttattgtttt
ataggccctg 24780 tgagttttat gctttcaaga ggttttattc tgatgcatgt
caacttttgg tttcaagatt 24840 tagaagtcct tttagaattt tgtgtatggc
tggtctggta gttacaaatt ccttcatcat 24900 ttgcttgtct ggaaaagact
ttatttctct ttcatttatg aaacttagtt ttgctggaca 24960 caaaattctt
gactgacact tattctgttt aaggaggcta aagattggac cccaatccct 25020
tctggcttgt aaggtttctg ctgaggggaa gatttttatc aatgacataa atgtgtgatg
25080 atggaaacaa tccagtggga agagaaaatt gatgcaggag agaagaaaga
aagctttttt 25140 ggagggctgc tctttagtat atgagagtag atggaatcta
ttcaacatgt aaaggagtct 25200 tagctaggcc ttagatagga gtaggaattg
tttatccaaa gtaaaatgaa ggaagagaga 25260 ttccagtagg ttggaagaca
tggtgggaag gacttgagga aattctcctc tattgcttat 25320 tttactcaat
aaagtaggaa gcaaggttac cagctgtgaa tgaggagtgg aagaaggcat 25380
tagagacttg atgagagaaa agaagccatg aaagagttat acaaaagtgt atgaggttta
25440 ttggactagg gaaagtatgt ctgccaggcg ctttaaaggg ccaactgaag
ttagtgacca 25500 gaaattcaaa gtgagacaag ccattatgtc caacagcatg
ggtgcaggca gagagtagtg 25560 ggaagctgga tttaaccaga gtcatggttt
agcaaagtga actcaacaac ctgggagaga 25620 gacaggggaa cttagggagt
gcccgaaggc atgataacat taatagagat cctcctccag 25680 tgaatgtcac
cagcctccca gttgctcaac tgagaaacct tgaccttttc ctcaacccct 25740
aacacccaaa caccaagtgg tattgatttc acttcctaaa tatctcctcc
tttcctcatt 25800 ggattaagct actgtaattt gtcttcatgt ttatatcatt
tcccccatag cagccagagg 25860 tgatgtttta gaaattacaa gcctgatcat
gtaactacct acttacaagc ttccagcagc 25920 ttcttttttt ttttcaagtg
gcagttttaa gtttgtgggt ttttaattta tatatgtgaa 25980 ataccttttt
ataggcaaaa ataataatag catgcttctt actgccctta atgtgaaatg 26040
actcacaaga ccttggtgtg gccaactctc cagcctcttt tttggcttat atttttcctt
26100 cttccccata tgccagccct actttcttct ttcaattctt caaagtcagt
catctctgag 26160 ggtgcatata cccccagaga atgcccaaga tgattcactg
ggatgaggaa aaaaaatact 26220 agaacttcct tttatattta tttgtgatta
tatattttta atttctgtga ttatatgtat 26280 tgaaaatgtg taaatcagta
gtacatggat acactttata aatacatata catatattag 26340 agtagtgctc
aaatatattt tttattatgc acatgcagtt gtaagaatat gtacagagac 26400
cacatgtacc atttacccag tttcacacaa acttgcatct tgcaaaacta tagtcataac
26460 cttgatatta atattgatct aattcactga tcttattaag attttcccag
ttcttgtatt 26520 catgcatgtg tatgtatttt aactctatgg aatgttatca
cataaatagg ttgttatatt 26580 ccccaccaca gtaaagatac aggacaagtc
cattatcaca agggcccttc aagtggctct 26640 tctgtagcca cctcctaacc
ccctataccc tgtccctgac tcctgccagc cactaatccg 26700 ttctccattt
ctataatttt gtcattccaa gaaaattata tacaagaaat catacagtag 26760
taacctttta ggattggcat ttttcactca ccatacttct cgggcaattc atctaaactg
26820 ttgtatcaat agttggttca tttttattgc tgagtggtat tccacgtatg
taggtaccac 26880 aatttgttta accattcacc agctgaggac atgtagattc
tttgcagtta ggagctatta 26940 tcaataaagc aactaatgat gttgaatatt
aaaaaaatgc tttattgaca aaggaagcca 27000 atcaaaaaag cttgtagacc
ataaccctaa tgtttcaggt ctttcctacc tcaagatctt 27060 ctgtcctacc
catccctact tttagaacct ctccctactt ttctctagct aactcttgga 27120
ctccagagaa agtgttaaaa gtcttcccta acccccagac caggagtaca tccccctgtt
27180 ttatgctcct aatgctcctg gcacttttcc ccctaagccc actgtacctg
atattttcta 27240 tctgcccctc cagatctact ctccaccctt cttcattctg
ctctcagccc aggactacat 27300 caaatctcct tagtcctctg ggttccagtt
gactttgatc tctagggagc ccttgcaggg 27360 gaaggaagga gaaggagggc
ctgattttat tcccccagcc cctcacggta aggctgactc 27420 aggcaacttt
tgtgtctaga aggaaagcct cagcttctct ctacacaaaa ctatcttcct 27480
aggttttaat atctgctccc ttccctttgt ccccttggtc ctgtagacgt attttgcttg
27540 gtctaatcct aggttttaaa atttgaatta gttgttgata cttaataatt
ggaagatttt 27600 gcataaagtt tcagatgtct ggcttctctt gaaaaaaaat
cagaaggttt gacaacactg 27660 agcctccatt ccaaatagta gccattgtgt
ggccttgagt agagccaccc cttccactgg 27720 ggcagctact cttaaggatt
caccatcccc actaactccc tgttccatac aataaccttg 27780 ctttgctatt
accaccctgg cactaggagt cctggtttaa agaatgtgtt gcccgctagg 27840
tgggacctgt gtcagacttc tcatcagatt tggagaaagg gcatgctggc ctctcccatc
27900 tgtctacttc tctctgtctc cccttgcacc acatagagca ccctttctct
ggggtttcta 27960 tcatcccctc ctagactccc acttgctccc aggcttgctg
ctcctctctg cacttctcac 28020 atcttgccag agtgcttttt tttttttctt
ttttttgagg tggagtctca ctctgtcacc 28080 taggctggag tgcagtggtg
tgatcttggc tcactgcaac ctctgccccc tgggttcaag 28140 caattctcct
gtctcagcct cccaagtagc cagaattaca ggcggctgcc accatgcctg 28200
gctaattttt tgtatttata gtagagacgg ggtttcactt tcaccatctt gaccaggctg
28260 gtcttgaact cttgaccttg tgatccaccc gcctcggcct cccaaagtgc
tgggattaca 28320 ggtgtgaggc atcacacccg gcccagagtg ctctttttaa
atgggactca gattacaacc 28380 ctttttctta aaaccttgct atgttttctc
attgccctat agttaaagcc tgcatttcta 28440 aagcagtccc ctggcttatc
tgaccttttc ctactaccag gctgatctgg ggccacccct 28500 cctcattctg
cttttccaga acagtgggtt cctcaagcac acaccttatg tcttgcctgg 28560
gcagcacact tgccaagctc ctaggccaag tatgaggcct ctctcctcac ttcctggtca
28620 caatttaagt gcccttaccc agatagtgcc tttccattct ctgtctgtat
ctcttcttaa 28680 taggtgctat gcttgtaaga agggattttt tttttagtct
ttctctcatt ggactgtgag 28740 aatagtgttt atctttttca ctgcctggtg
tatagtaggt gctcattgaa catttattga 28800 aagagcagat ttagagatct
agctcttcat ggaatagcag tttctaccac ctagcgacaa 28860 tgtggaatac
cagggcttct aaagtgttta gtatgatcat acatttatat tatgaggcct 28920
gcctgtgggg tgattgcaca tgaggtgagg ccattgaggc acctagggca gaggatttaa
28980 ggaagctttc acccttacaa tgcctctcct gccacactct agacctggct
ctgggaggga 29040 agcctttgct aagaaccatg aaaggaaagg agaaaaagaa
aactagggtg gtgctaaagg 29100 gacatttgtg atggcagcct gacacctacc
acctgattct tgaacactca ctgtggatag 29160 aatggtgtat gatggccaca
gggcaggagg gggcagcaga gtgatgatac cttaactgac 29220 acctgagtca
gtaatgggag aactgaggag aagcagtggg ttcttctaca ggaaatcgaa 29280
gggtggaatt cattaatcta gcccttaaaa caacagctta cccaacatgc tacacaaaac
29340 caggtttctg tttcagataa ataggtgaag ggactcttaa tcctaaagac
tgaaaagtag 29400 aaaagaaagg ggaagaagag tgcctcaagg attgccattg
gaggttcttt tgctggggct 29460 gattgccagc tgagattatt caagccccag
agcaaacatt ctgctcctgc tcccttagag 29520 ctgccctccc accgctcagt
attgcctcct gcgaggggcg ggctggctgc cgcagacacc 29580 agtgaaccct
ttttccattc cagaagtccc agtggaccta ctttaatata ccaataacac 29640
tcctatttta aactagctgt atccattttc gttttaatag tcccagtgct aaagtttttc
29700 aaagcagtta ttttgtaagt aggtcaaaca ggtactttgg gatcctgttc
tgtctgtttg 29760 cttgccaggt aacctctttg ttatctaatt caaagtctgg
tacagtttga accaaaacaa 29820 aaaaggaatg atgtttcact ttggagtcaa
gattcattca ttttctaaca ttaatcattt 29880 tcgtaataca gtaagtctat
attcatgata aaaaatagaa aatatgaata agcaaaacta 29940 aattgaaagg
aaaaccatct gtgatctgcc aattagaaaa tctctattct aaacattttg 30000
gtaaatatgc taccagattt ttatctatgc aaatgtgtat ctgtattttt cctcacttgt
30060 atagtggaca tcttttcata ttaataaata agttagcatc attacttttg
atagcttcat 30120 ggtgtgaaat tataaaatta gacctaccac actctatttt
aactatctct cttgctgggt 30180 acttaagcta ttcccaatat cacagaagcc
tttttacaca tgcatccttg tacatgcatc 30240 agattcttta agattgccaa
aggtgaaatg gttgggtccc agggtgcaac tattttttag 30300 catttcaata
cataatacca aagtgcaatc cagaaccatt gtaccaatta atgctcccgc 30360
cagcagcaca ttgcagtgtt gatttcctcc tggcctcacc agcacgctgt caacactggg
30420 tactggtgtg tcgtgtacat gacattacat gccaggcact gagagtgaaa
taaaacagtt 30480 catgttggga atggggcaag agagggagat aaacaaggga
ggtgacatct acagaccggt 30540 tagctgagat tacagtacat tggggctcca
agtgcacaga atctgaacat catgttcatc 30600 ctaggacaag tgttgcagag
gcttcctgaa gaggttcatt ctgaggctta ccttgaagga 30660 tcatcaggaa
ttaatgatga gtgtattaag ccattctcgc atttctataa agaaacgcct 30720
gaaattgagt aatttttttt tttttttttt tttctgagac ggagtcttgc tttcttacct
30780 aggctggaga gcagtggtgc gatctcggct cactgcaacc tctgcctccc
tgggttcaag 30840 tgattctcct gcctcagcca cccgagtagc acaggtgcct
gccaccgggt ccatctaatt 30900 tttgtatttt tagtagagac agggtttcac
catgttggcc aggctggtct caaactccca 30960 acctcgtgat cctcccgcct
tggcctcccg aagtgctggg attacaggcg tgagctgccg 31020 cgcccagcca
aaattgagta atttataaga aaaaaaggtt ttattagctc atgattctgc 31080
aggctgtaca ggaagcgtgg tggcatctgc ttctgtagag gcctcaggaa gctccaatca
31140 tggtggaagg tggaaggcac atcacactgt gaaagcagga gcaagagttg
ggggaacatg 31200 ccataagatc ccatcacttt taaagaacca gatctcatgt
gaactcagag cgagagctca 31260 cttatcacta aggagatggc ccaagccatt
catgagacat cagcctccat gacccaaaca 31320 cgtcccacca gactccacct
ccaacattgg ggattacatt tcaatgagat ttgggtgggg 31380 acaaatgtcc
aaactatatt gatgagtgaa agcaagagta aagaacaggg aaggaatgat 31440
ggagagaatg aaccaggtag agaaaatctt ctaggcatga atgttcagcc ccttttcagg
31500 gagcaacaaa aagttctgta ttggttgcaa tgaagggtgc atggggcatt
ggggatggtg 31560 gacaggaatg atggacagtg aggctgatga gatgagctgg
gtctcatcaa gaagttctct 31620 ccgggcttag caaaggagct ctagctttat
ctggaaactt tccttgggag gcaggacttg 31680 gcatcaaaac ctgtgtgtgg
aagactcaca ccgatccaag ggaagggcac tgatgctggg 31740 cttaagcatt
gctttgtcat tccctgatga gctctctcag cttgggaaca gccactctct 31800
gtctagtaaa ggaaaggagc tgatccaggt cctctccagt tctaactatg ctcctgtgaa
31860 ttctggcaca atttttactt tttttccttg cagaaaaggt ggccattaag
atcctggaca 31920 agaccaagtt agaccagaaa acccagaggc tactatcccg
agaaatctcc agcatggaaa 31980 agctgcacca tcccaacatc atccgccttt
acgaagtggt ggagacccta tccaagctgc 32040 acttggtgat ggagtatgca
gggggtgggg agctcttcgg aaaaattagc actgagggga 32100 agctctctga
accagaaagc aagctcatct tctcccagat tgtgtctgcc gtgaagcaca 32160
tggtgagcag gggtgacgag tgagaacctt gctcccattg cactgacact gggagcacag
32220 ggctttaggt tactaaccct caagtgtccc agagggcttt tgtcctacaa
agcagacagt 32280 agtcccttct gagagtcaga agtctggctg ggatcatgct
ccttgcctgt gaagcaacat 32340 ccagggatgt ccagttacac gtcaacctcc
tagcttctgc agacattggc aggaatcact 32400 gggagcagct gatgaatatc
acccactccc tggccatacc catttctcca acctcatctt 32460 ccactatctg
gatagctggc tcctcactgc ttcttggttt tgctttccta cttctgtcta 32520
ggatgccctc atatgacatc tcacattcta aatcctatcc atacttcatg cccagcttgg
32580 ttctctctct ctctctctcc cccacccccc ctctccgtgt gtgtgtgtgt
gtgtgtgtgt 32640 gtgtgtgtgt gtgtgagaga gagagagaga gagagagaga
tgttcttcaa tctcacatca 32700 gttatactga tattttgcct tcagtaagat
cccctagctc ttattgcctc tgtgcccagt 32760 agattaacac ttggttgtgt
ctttcttttt tcccttcctt cctgcctgcc ttccatcctt 32820 ccttccttcc
ttttcttcct cccttccttt cttttccttc cttccttcct tcctttcttt 32880
ctgtttcttt tttttttttt tagacagagt cttcctctgt cacccaggat ggagtgcagt
32940 ggcatgatct tggctcactg caacctccgc ctcccgggtt caagcgattc
tcctgcctca 33000 gcctcccaag tagctgggat tacaggcatg tgccaccatg
cctagctgat tttgtgtttt 33060 taatagagat gaggtttcac catgtttgtc
aggcttgtct tgaactcctg acctcaagcg 33120 atccacctgc ctcggtctcc
caaagtagtg ggattacagg cgtgagccac cgtgcctggc 33180 cgcttgtgtc
attcttattt tagttctttg gttacttctt tagataaatc tggactccta 33240
atgcacactc tgtagttctc ccaagaactg gctgttttgc tgcctcctta attttggccg
33300 tattgcgggc catacaggag gaacctgact tgcaggcctg agtatcactt
ttactaccaa 33360 aagttttagt cagtcaccat ctttactcag atcccagttg
aacggatctg tcagaaaaga 33420 gacacttggc tagggtctat attagttgag
aggctatttc caattcactt ctcagcctgc 33480 atggttgtag gagcaagaac
tgggatcagt gagccacagt gaacctcatt acatgccagg 33540 cactgaaaaa
gtcagataaa acagtccatg ctagcaatgg ggcaaaagag ggagaatttg 33600
caagggaatc tgtggcctag agggcccaaa atagcagtag ggaaggagag gaggaagaaa
33660 aagaagtgag ccagggagaa gagcttgact gtagtggtgg tggatgtttc
agacctgtga 33720 tatatgacca atcatcattt aagtggcttt gggctgtggt
gagctttccc agggccagag 33780 aataagccag acccatacca agtaatagaa
cagatgggca aagagtcttc cacattctac 33840 ttggaaaagt aacttggtgg
gagaacagaa gagaatccag caatagaagg tacagatgtt 33900 agtcttcagc
agctctactc cctgccattt ctccaacgct ctgcaaacag gaagtgtgtt 33960
tcatagccat agatccacat cttattgagt ttgtttatat gccaagacca tgctgagcac
34020 tgttctcatc atacgtagtt agcgtcatat atagttctca ccacagccta
tggttatcag 34080 tatcccactt tacagagaag gaaactgaca ttcacagtac
ccagctggta tagttgaacc 34140 tagaattgac cagactgtcc cctccaaagc
ctggattctt attccaggag ggccatcagg 34200 aaagctgaca aacacaaagc
cattttccaa gagcccttgg gactgaacag gtcaagggtc 34260 ctctgaagat
tgtctggatt caggatgcaa gggtgggagt ggagcatgtg cccacaatcc 34320
acagtgtgtt ctgtggctag atccttgcca aatgcaacca cctcccttcg gctgaattct
34380 gtaaggataa aagagtccac cccaaaaagc atggccggaa agtcagggga
gggctccaag 34440 ccttcctggt caaacgatcc atcagccaga aaaacccatg
tatgacctca acaactaagg 34500 attactgttt cattgtattt cagaatgtgt
agtttcataa gatctgggtc tgatttctgg 34560 tgttagtttc tgagtccaca
tgtgtggaac agactccaac ccttaccaca tagaatagga 34620 actttggtct
atttggggag gtgtaggcat tacattgggc taaaggttat gacagggttt 34680
gctatcatga cttaaggtga tcctcactga atttgttatt ccaccatcat tatctcactg
34740 tttcagagca actgatacat tttttatttt gacattttac tgtaaaaatg
acttttctct 34800 atgtcttctt ccacagcatg aaaaccaaat tattcataga
gatctgaaag cagaaaatgt 34860 attctatacc agtaatactt gtgtgaaggt
gggcgatttt ggattcagca cagtaagcaa 34920 aaaaggtgaa atgctgaaca
ctttctgtgg gtctcctccc tacgctgcgc ctgaactctt 34980 ccgggacgag
cactacatcg gcatttacgt ggatatctgg gccttggggg tgcttttgta 35040
cttcatggtg actggcacca tgccatttcg ggcagaaacc gtggccaaac taaaaaagag
35100 catcctcgag ggcacataca gtgtaccgcc gcacgtgtca gagccctgcc
accgactcat 35160 ccgaggagtc cttcagcaga tccccacgga gaggtacgga
atcgactgca tcatgaatga 35220 tgaatggatg caaggggtgc cataccctac
acctttggaa cctttccaac tggatcccaa 35280 acatttgtcg gaaaccagca
ctctcaagga agaagaaaat gaggtcaaaa gcactttaga 35340 acatttgggc
attacagaag agcatattcg aaataaccaa gggagagatg ctcgcagctc 35400
aatcacaggg gtctatagaa ttattttaca tagagtccaa aggaagaagg ctttggaaag
35460 tgtcccagtc atgatgctac cagaccctaa agaaagagac ctcaaaaaag
ggtcccgtgt 35520 ctacagaggg ataagacaca catccaaatt ttgctcgatt
ttataaattg cactagactg 35580 cttgtaacta accaagatga ttgttgctgc
ttctaaattt ttttcaagga caacttgagt 35640 ggagacattt ttgtaatttt
taaataaact taaatttgag atatgcattt tttttctcca 35700 aaaagtctat
tagctcagat tctggcttga tttgggatct tgttttatta tcaaatttca 35760
gcattcattc atttaatcaa gaaatattta tccagtgcct cctgtgtgtc aggcactatt
35820 taaggtgcta gggaaacagc aagaaataaa taggcaaggt ccctcctctc
atgtgcagga 35880 cgttctagcc caggacaaag gtactaacaa ctacattttt
taaaaaagag aaagatcagt 35940 gatagatgct atgcagagga ttcaaaaagg
gtgatctgat atcagaggct tgctactttg 36000 gattagtcat taaggaaggc
cactttgagt acatataagt ttaagatctg aatgagaagc 36060 aggagtgact
tttacaaaat gccagagcat tccaggcagc taggggtttc agactcagtt 36120
ccccccaaaa cagagcctaa gacaaaggct tccatataaa gtagtttatt tgggaactga
36180 tcccagagca caggagtgaa ggacagagga aacagggaaa gctaatacac
agacacatta 36240 gcaagttagc tagtgctaca gtttctggtg cttggttttg
caggatcctc cagaggagcc 36300 tgatgaaaca catctcaggt gtctgccctg
ggtatgaaag gggaaagcat ttaaccataa 36360 gctcttgctc accgctgatc
aagggtggcc ctttgtgtca tctgccctgc actcccagat 36420 tgggctgtgt
gaatgccaag tgggttccct aagcttccct tgccttcatg tcagagaagt 36480
cttggagcag gagggaagag gtacaccacc agtacgatac ctcctttctt tgcctgcttg
36540 tccactggca tggggagccc aaagtgacca tgtgacactt gtagattcca
attcactgga 36600 gcccttatca agtcccctga tgaaagcatc cctgatcccc
tgggaaccaa accttttaat 36660 ctagcctaga ttgtagaaaa aaaaagaata
gaaattaatc aagtgagtca ccaattgcgt 36720 gatggtgaca ggagtcaatc
tacttccacc tagtggtttc cagacctgtg ttttaatgat 36780 agggacatgt
atcagtcatt attaaagcat gttactgaat cctggaggac agcaccccaa 36840
ccctgcaggc tgctgtctca tctctggtgt cttagctgag tcattccatt gttctactca
36900 tgttggctgc ttctgatgtt gaggtatatg gtaagatgag gggatcccgt
tgtcatgtgc 36960 ccattgcacc acctccttta tcaaaaaagt ggtccttgag
gcaatgtttg agatatcatg 37020 acagtgagtc agttattctc tatgtcttag
tgtgcttgta tggaaagaca cattgcaggc 37080 agaaaaggca aacccatacc
ctgagatatc aattccaata aggatgaatc actcctgggc 37140 ccatctgaat
tcatgactcc atggatctga taatgcccag ctcatacgtt aacagctctg 37200
tttgcctagt cctcagtatc tcataatcag gttctcagcc tccactgggg cccagtagca
37260 catcgggagt ctgagtagtt ctgtcgcagg aagcatggcg ttactacagg
atcccacaga 37320 gctgtgctgt tatctcttat tggggctttc cagaggcccc
acactacaca gcctgagtag 37380 cagtttgctc agaagccaaa attgctgcaa
tctttctttt atgggatcca cttgaaactg 37440 gcagccgtgt taaatgggtg
ggaggaatac accagataca atatatgtgc ttccaaagtc 37500 cagagggacc
tactaaatcc tatgcctcta tcttagtgtt aagaggtggg aggtgcaaga 37560
aattgttctt tactatagaa gaaatatcta ggcatgcctc aactcattgg tcttctaaac
37620 tcttcatcaa tgtggcatgc cccagattct tcatagagtc tataatccac
accctggcac 37680 atgtaagcct tactagggca ttggtaatac ttgccacttc
ctgatagcca atccccctag 37740 catgctctca caatatagtg ggccagcatg
atgtcctgta gaatgtcaag atgatcaagg 37800 tccttttgga ttatattgtg
atatagagaa ggagatttaa gattgttttg gggcaataga 37860 gtaaatgaga
atcgctctcc atcccagaga aatgtgaact gcttttgatc ctcctttctg 37920
atggaaattt agaagaatga atttgccaaa tcaatagctg catatcaaat gccagaggct
37980 gccagtaaag ataccacatc caatatggca gctacaactg agactagcag
tagtccacca 38040 tcattctcca ttatccatct gctctttttc agaagtgtaa
tggtaactta aatgagatta 38100 tgatggagcc cattacactt gcatcccaaa
attttttgat ggtgacactg atgtctgcca 38160 ttcctcatgt ggattgctca
tgatttatta acatggccag aaagaggttt cctctttctc 38220 ccataatggt
tgttacttca caggtctaag acctccacag aggtctaagt atattgatcc 38280
caaatatgta cttagaggtt gggagaataa ttccaaggtg ggaaccatta tgtgatagac
38340 tttggccagg actcccttta tcatatggtt tccataaccc ctcatctacg
tacaaagcca 38400 taatggtatc cggttctgaa aaattctggg tatgtacatc
atatatactt gtgaaagcaa 38460 ggcctttcct caagaccagt cctccttttc
attgtgcact ggatctgtaa attgacttag 38520 ctctggaagc tgatgaagag
accgtgattt tcccattgtg acag 38564 4 290 PRT Caenorhabditis elegans 4
Gln Val Ala Leu Gly Arg Arg Ile Gly Phe Tyr Arg Leu Gly Lys Glu 1 5
10 15 Leu Gly Ala Gly Asn Phe Ser Lys Val Lys Leu Gly Val His Gln
Leu 20 25 30 Thr Lys Glu Lys Val Ala Val Lys Ile Met Asp Lys Ala
Lys Met Asp 35 40 45 Ala Lys Ala Gln Lys Leu Leu Ser Arg Glu Ile
Gln Ala Met Glu Glu 50 55 60 Met Asn His Pro Asn Ile Val Lys Leu
Phe Glu Val Val Glu Thr Leu 65 70 75 80 Thr Arg Val His Leu Val Ile
Glu Tyr Ala Ser Gly Gly Glu Leu Tyr 85 90 95 Thr Tyr Val His Glu
Arg Gly Lys Leu Thr Glu Gly Asp Ala Lys Pro 100 105 110 Leu Phe Ala
Gln Ile Val Ser Ala Val Ser His Met His Ser Arg Asn 115 120 125 Ile
Val His Arg Asp Ile Lys Ala Glu Asn Val Met Phe Ser Ser Pro 130 135
140 Asn Thr Val Lys Leu Val Asp Phe Gly Phe Ser Cys Leu Val Asp Arg
145 150 155 160 Glu Gln Met Leu Arg Thr Phe Cys Gly Ser Pro Pro Tyr
Ala Ala Pro 165 170 175 Glu Leu Phe Gln Asp Thr Ser Tyr Ala Gly Glu
Leu Val Asp Val Trp 180 185 190 Ala Leu Gly Val Leu Leu Phe Phe Met
Leu Ile Gly Val Thr Pro Phe 195 200 205 Lys Ala Glu Thr Val Pro Asp
Met Lys Val Leu Ile Thr Ala Gly Lys 210 215 220 Tyr Gln Ile Pro Asp
Tyr Val Ser Leu Leu Ala Thr Glu Leu Ile Lys 225 230 235 240 Ser Met
Leu Lys Thr Asp Thr Gly Gln Arg Ala Asp Ile Asp Ser Val 245 250 255
Lys Lys His Phe Trp Met Arg Asp Cys Arg Phe Thr Lys Ser Tyr Leu 260
265 270 Ser Ile Lys Ala Thr Ala Lys Ile Asp Asn Glu Glu Glu Lys Lys
Ala 275 280 285 Ile Asp 290 5 268 PRT Drosophila melanogaster 5 Gln
Val Thr Ile Gly Arg Arg Ile Gly Leu Tyr Arg Phe Cys Gly Asp 1 5 10
15 Ile Gly Arg Gly Asn Phe Ser Lys Val Lys Leu Ala Val His Gln Leu
20 25 30 Thr Arg Asp Lys Val Ala Ile Lys Val Val Asp Leu Asp Arg
Ala Gly 35 40 45 Leu Asp Ala Lys Ala Leu Arg Met Leu Ser Ser Glu
Ile Ala Thr Leu 50 55 60 Glu Cys Val His His Pro Asn Ile Leu Arg
Leu Phe
Glu Val Val Glu 65 70 75 80 Thr Leu Gly Arg Val Tyr Leu Val Thr Glu
Trp Ile Arg Gly Gly Glu 85 90 95 Leu Tyr Asn His Ile Thr Gln Gly
Gly Pro Leu Arg Glu Ile His Ala 100 105 110 Ala Pro Leu Leu Lys Gln
Leu Leu Leu Ala Val Lys His Met His Ser 115 120 125 Leu Gly Tyr Val
His Arg Asp Ile Lys Ala Glu Asn Val Leu Leu Leu 130 135 140 Ser Glu
Asp Arg Leu Lys Leu Ala Asp Phe Gly Phe Ser Thr Gln Leu 145 150 155
160 Ile Asn Gly Thr Gly Ala Asn Gln Lys Leu Asp Thr Phe Cys Gly Ser
165 170 175 Pro Pro Tyr Ala Ala Pro Glu Leu Phe Ser Asp Asp His Tyr
Ile Gly 180 185 190 Ala Pro Val Asp Val Trp Ala Leu Gly Ile Leu Leu
Tyr Phe Met Val 195 200 205 Val Gly Asn Met Pro Phe Arg Ala Pro Thr
Ile Pro Gly Leu Lys Ala 210 215 220 Ala Ile Leu Lys Gly Asp Tyr Leu
Leu Pro Gly Gln Leu Ser Leu Pro 225 230 235 240 Cys Ile Arg Leu Ile
Gln Arg Ile Leu Ile His Ile Pro Ala Gln Arg 245 250 255 Pro Thr Ile
Asp Asp Met Leu Asn Ser Gln Phe Val 260 265 6 74 PRT Human 6 Pro
Thr Asn Thr Glu Asp Leu Ser Gln Leu Gly Ala Leu Glu Phe Glu 1 5 10
15 Ala Arg Gln Ile Leu Ala Glu Leu Gly Leu Thr Ser Glu Met Leu Ile
20 25 30 Asn Ala Arg Gln Ser Gly Pro Arg Ser Asp Ile Ile Gly Ala
Tyr Arg 35 40 45 Ile Val Val Asn Arg Leu Gln Lys Gln Ser Trp Leu
Ala Arg Lys His 50 55 60 Val Glu Met Ala Leu His Ser Glu Pro Lys 65
70
* * * * *
References