Note: Descriptions are shown in the official language in which they were submitted.
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ISOLATED HUMAN KINASE PROTEINS, NUCLEIC ACID MOLECULES
ENCODING HUMAN KINASE PROTEINS, AND USES THEREOF
RELATED APPLICATIONS
The present application claims priority to provisional applications: U.S.
Serial No.
60/233,493, filed September 19, 2000(Atty. Docket CL0008S7-PROV), 60/247,031,
filed
November 13, 2000(Atty. Docket CL000904-PROV) and 09/729,995, filed December
6,
2000(Atty. Docket CL000904).
FIELD OF THE INVENTION
The present invention is in the field of kinase proteins that are related to
the
calcium/calmodulin-dependent protein kinase 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
Protein Kinases
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
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metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion
channel or pump, or
transcription factor.
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.
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 Kihase Facts
Books, Vol I:7-20 Academic Press, San Diego, Calif.).
The second messenger dependent protein lcinases 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.
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(1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York,
N.Y., pp. 416-431,
l 887).
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
S 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 (GaM kinase I and CaM lcinase 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
1 S another kinase as part of a "kinase cascade".
Another ligand-activated protein kinase is S'-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 cellulax
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.
2S 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).
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PRK (proliferation-related kinase) is a serumlcytokine inducible STK that is
involved in
regulation of the cell cycle and cell proliferation in human megakaroytic
cells (Li, B. et al.
(1996) ,I. Biol. Cheyra. 271:19402-8). PRK is related to the polo (derived
from humans polo gene)
family of STKs implicated in cell division. PRK is downregulated in lung tumor
tissue and may
be a proto-oncogene whose deregulated expression in normal tissue leads to
oncogenic
transformation. Altered MAP kinase expression is implicated in a variety of
disease conditions
including cancer, inflammation, immune disorders, and disorders affecting
growth and
development.
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.
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.
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.
Many of these PTKs were first identified as the products of mutant oncogenes
in cancer
cells where their activation was no longer subject to normal cellular
controls. In fact, about one
third of the known oncogenes encode PTKs, and it is well known that cellular
transformation
(oncogenesis) is often accompanied by increased tyrosine phosphorylation
activity (Carbonneau
H and Tonks NK (1992) A~hu. Rev. Cell. Biol. x:463-93). Regulation of PTK
activity may
therefore be an important strategy in controlling some types of cancer.
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Serine/Threonine Protein Kinases, including Calcium/Calmodulin-Dependent
Protein
Kinases
Calcium/calmodulin (CaM) dependent protein kinases are members of the
serine/threonine protein kinases (STK) family. Serine/threonine protein
kinases add phosphate
moiety to a serine or threonine residue of the substrate. Protein kinase
substrates include
elements of signal transduction pathways such as transcription factors or ion
channels, as well as
structural proteins such as filaments and cellular motors. The protein kinase
gene family is one
of the largest gene families in the genome. Classification of kinases are
based on their sequence,
tissue localization, and domain topology. Primary structures of kinases are
rather conserved. A
number of soluble and transmembrane proteins contain kinase domains along with
other
structural components; these mufti-domain proteins also are often referred to
as kinases. Tissue
specific expression of kinases is often defined by transcription regulatory
elements.
Calmodulin is a calcium receptor that mediates many calcium regulated
processes by
binding to target proteins in response to the binding of calcium. The
principal target protein in
these processes is CaM-dependent protein kinases (also referred to as CaM
kinases). CaM-
kinases are involved in regulating 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) EMBD 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 lcinase
as part of a "kinase
cascade" (Tokumitsu et al., J. Biol. Chem. 1995 270: 19320-19324).
The kinase provided by the present invention shows a high degree of similarity
to
calmodulin-dependent kinase kinase, an enzyme that activates CaM-kinase IV.
CaM-kinase IV is
markedly activated upon phosphorylation by CaM-kinase IV kinase. CaM kinase
IV, as well as its
activating kinase, CaM kinase IV kinase, are the key elements of the calcium-
dependent signal
transduction cascade in neurons and lymphocytes.
Northern and Western blot analyses of CaM-kinase IV kinase showed relatively
weak
reactions in the rat cerebellum, where the activity of CaM-kinase IV kinase
has been demonstrated
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to exist, indicating that CaM-kinase IV kinase isoforms distinct from the
enzyme cloned from the
cerebral cortex may exist in the cerebellum. Immunoprecipitation techniques
have indicated that at
least two distinct isoforms of CaM-lcinase IV kinase exist in the brain (Okuno
et al., J. Biochem
(Tokyo) 1996 Jun;119(6):1176-81 ).
Furthermore, the CaM kinase cascade in myeloid cells may play a critical role
in
mediating the effects of calcium on neutrophil function and maturation. It has
been found by
Western analysis that CaM protein kinase kinase alpha (CaMKKalpha) is
upregulated during
retinoic acid induced neutrophil maturation. In addition, neutrophil
progenitor cells express both
CaMKI and CaMKIV transcripts; CaMKIV is downregulated during neutrophil
maturation and
CaMKI is expressed in uninduced cells and is induced by all-trans retinoic
acid. (Lawson et al.,
Exp Hematol 1999 Nov;27(11):1682-90).
The gene provided by the present invention can be expressed in yeast to
identify possible
ligands or substrates of the kinase protein; this can be done by means of a
complementation
assay or a two-hybrid experiment. Artificially synthesized enzymes as well as
derived peptides
can be used to activate or inhibit cellular processes modulated by this
kinase. Immunoassay or
PCR may be used to measure the concentration of this protein and detect
abnormally developing
tissue or cancerous growth.
For a further review of calcium/calmodulin-dependent protein kinase kinases,
see Park et
al., JBiol Chem 1995 Dec 22;270(51):30464-9; Sakagami et al., By~ain Res Mol
Brain Res 1998
Mar 1;54(2):311-S; and Enslen ~t al., Biochem Biophys Res Commute 1995 Feb
27;207(3):1038-
43.
Kinase proteins, particularly members of the calcium/calmodulin-dependent
protein kinase
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
calcium/calmodulin-dependent protein kinase kinase subfamily.
SUMMARY OF THE INVENTION
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 calcium/calmodulin-
dependent protein
kinase kinase subfamily, as well as allelic variants and other mammalian
orthologs thereof.
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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 Figure 1 indicates expression in humans in the eye
(retinoblastomas) and brain.
DESCRIPTION OF THE FIGURE SHEETS
FIGURE 1 provides the nucleotide sequence of a cDNA molecule that encodes the
kinase
protein of the present invention. (SEQ ID NO:l) 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 Figure 1 indicates expression in humans in
the eye
(retinoblastomas) and brain.
FIGURE 2 provides the predicted amino acid sequence of the kinase of the
present
invention. (SEQ ID N0:2) 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.
FIGURE 3 provides genomic sequences that span the gene encoding the kinase
protein of
the present invention. (SEQ ID N0:3) 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
Figure 3, SNPs were identified at 34 different nucleotide positions.
DETAILED DESCRIPTION OF THE INVENTION
General Description
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/peptideldomains identified and
characterized
within the art as being a kinase protein or part of a kinase protein and axe
related to the
calcium/calmodulin-dependent protein kinase kinase subfamily. Utilizing these
sequences,
additional genomic sequences were assembled and transcript and/or cDNA
sequences were
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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
calcium/calmodulin-
dependent protein kinase 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.
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
calcium/calmodulin-
dependent protein kinase kinase subfamily and the expression pattern observed.
Experimental
data as provided in Figure 1 indicates expression in humans in the eye
(retinoblastomas) and
brain. 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
calcium/calmodulin-
dependent protein kinase kinase family or subfamily of kinase proteins.
Specific Embodiments
Peptide Molecules
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
calcium/calmodulin-dependent protein kinase kinase subfamily (protein
sequences are provided
in Figure 2, transcript/cDNA sequences are provided in Figure 1 and genomic
sequences are
provided in Figure 3). The peptide sequences provided in Figure 2, as well as
the obvious
variants described herein, particularly allelic variants as identified herein
and using the
information in Figure 3, will be referred herein as the kinase peptides of the
present invention,
kinase peptides, or peptides/proteins of the present invention.
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
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the Figure 2, (encoded by the nucleic acid molecule shown in Figure l,
transcript/cDNA or
Figure 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.
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).
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.
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.
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 Figure 1 indicates
expression in humans in the
eye (retinoblastomas) and brain. 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.
Accordingly, the present invention provides proteins that consist of the amino
acid
sequences provided in Figure 2 (SEQ ID N0:2), for example, proteins encoded by
the
transcript/cDNA nucleic acid sequences shown in Figure 1 (SEQ ID NO:1 ) and
the genomic
sequences provided in Figure 3 (SEQ ID N0:3). The amino acid sequence of such
a protein is
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provided in Figure 2. A protein consists of an amino acid sequence when the
amino acid sequence
is the final amino acid sequence of the protein.
The present invention fiu-ther provides proteins that consist essentially of
the amino acid
sequences provided in Figure 2 (SEQ ID N0:2), for example, proteins encoded by
the
transcript/cDNA nucleic acid sequences shown in Figure 1 (SEQ ID NO: l ) and
the genomic
sequences provided in Figure 3 (SEQ ID N0:3). 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.
The present invention further provides proteins that comprise the amino acid
sequences
provided in Figure 2 (SEQ ID NO:2), for example, proteins encoded by the
transcripdcDNA nucleic
acid sequences shown in Figure 1 (SEQ ID NO:1) and the genomic sequences
provided in Figure 3
(SEQ ID N0:3). 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.
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.
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.
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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., Cm°rent
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.
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.
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.
To determine the percent identity of two amino acid sequences or two nucleic
acid
sequences, the sequences axe 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
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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.
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;
Biocorrzputing:
Informatics and Geuome Projects, Smith, D.W., ed., Academic Press, New York,
1993; Compute
l 0 Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds.,
Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic
Press, 1987; and
Sequence Analysis Prime, Gribskov, M. and Devereux, J., eds., M Stockton
Press, New York,
I99I). 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
t 5 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 l, 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
?0 (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, S, or 6. In another
embodiment, the
percent identity between two amino acid or nucleotide sequences is determined
using the
algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated
into the ALIGN program (version 2.0), using a PAM120 weight residue table, a
gap length
?5 penalty of 12 and a gap penalty of 4.
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) ofAltschul, et al. (J. Mol. Biol. 215:403-10
(1990)). BLAST
30 nucleotide searches can be performed with the NBLAST program, score = 100,
wordlength = I2
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
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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.
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. As indicated by
the data presented in
Figure 3, the gene encoding the novel human kinase protein of the present
invention is positioned
on public BAC AC005940, which is known to be located on human chromosome 17.
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
Figure 3, such as the
genomic sequence mapped to the reference human. As indicated by the data
presented in Figure 3,
the gene encoding the novel human kinase protein of the present invention is
positioned on public
BAC AC005940, which is known to be located on human chromosome 17. 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.
Figure 3 provides information on SNPs that have been found in the gene
encoding the
novel human kinase protein of the present invention. SNPs were identified at
34 different
nucleotide positions, including a non-synonymous cSNP at position 16135 and
SNPs at two
positions (2082 and 2748) 5' of the ORF that may affect control/regulatory
elements. The change
in the amino acid sequence caused by the G16135A SNP is indicated in Figure 3
and can readily
be determined using the universal genetic code and the protein sequence
provided in Figure 2 as
a reference.
Paralogs of a kinase peptide can readily be identified as having some degree
of significant
sequence homologylidentity 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
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WO 02/24920 PCT/USO1/29161
typically at least about 70% or greater homology through a given region or
domain. Such
paxalogs 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.
Orthologs of a kinase peptide can readily be identified as having some degree
of significant
sequence homology/identity to at least a portion of the kinase peptide as well
as being encoded by a
gene from another organism. Preferred orthologs will be isolated from mammals,
preferably
primates, for the development of human therapeutic targets and agents. Such
orthologs will be
encoded by a nucleic acid sequence that will hybridize to a kinase peptide
encoding nucleic acid
molecule under moderate to stringent conditions, as more fully described
below, depending on
the degree of relatedness of the two organisms yielding the proteins.
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).
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. Figure 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.
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.
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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 Figure 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 i~
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)).
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 Figure 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.
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 Figure 2.
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 Figure 2).
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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
S 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.
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.,
Posttrahslatiohal Covalent
Modification ofProteins, B.C. Johnson, Ed., Academic Press, New York 1-12
(1983); Seifter et al.
(Meth. Enzyrnol. 182: 626-646 (1990)) and Rattan et al. (Ann. N. Y. Acad. Sci.
663:48-62 (1992)).
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.
Protein/Peptide Uses
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,
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CA 02422549 2003-03-14
WO 02/24920 PCT/USO1/29161
for example, in a kinase-effector protein interaction or lcinase-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.
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,
Bergen S. L. and A. R. Kimmel eds., 1987.
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
1 ~ as provided in Figure 1 indicates that kinase proteins of the present
invention are expressed in
humans in the eye (retinoblastomas) and brain. Specifically, a virtual
northern blot shows
expression in retinoblastomas and PCR-based tissue screening panels indicate
expression in the
brain. A large percentage of pharmaceutical agents are being developed that
modulate the
activity of kinase proteins, particularly members of the calcium/calmodulin-
dependent protein
kinase 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 Figure 1. Experimental data as provided in Figure 1 indicates
expression in humans
in the eye (retinoblastomas) and brain. Such uses can readily be determined
using the
information provided herein, that which is known in the art, and routine
experimentation.
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 calcium/calmodulin-dependent protein kinase 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 Figure 1 indicates that kinase proteins of
the present invention are
expressed in humans in the eye (retinoblastomas) and brain. Specifically, a
virtual northern blot
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WO 02/24920 PCT/USO1/29161
shows expression in retinoblastomas and PCR-based tissue screening panels
indicate expression in
the brain.
The proteins of the present invention are also useful in drug screening
assays, in cell-based
or cell-free systenns. 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
Figure 1 indicates
expression in humans in the eye (retinoblastomas) and brain. In an alternate
embodiment, cell-
based assays involve recombinant host cells expressing the kinase protein.
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.
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.
Candidate compounds include, for example, 1) peptides such as soluble
peptides, including
Ig-tailed fusion peptides and members of random peptide libraries (see, e.g.,
Lam et al., Nature
354:82-84 (1991); Houghten et al., Natuy~e 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')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).
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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 aff nity, or a fragment that
binds substrate but
does not allow release, is encompassed by the invention.
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.
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 Figure 2. Specifically, a
biological function of a
cell or tissues that expresses the kinase can be assayed. Experimental data as
provided in Figure 1
indicates that kinase proteins of the present invention are expressed in
humans in the eye
(retinoblastomas) and brain. Specifically, a virtual northern blot shows
expression in
retinoblastomas and PCR-based tissue screening panels indicate expression in
the brain.
Binding and/or activating compounds can also be screened by using chimeric
kinase
proteins in which the amino terminal extracellular doriiain, 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.
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
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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 pohypeptide that competes with the target kinase
region is designed to
contain peptide sequences corresponding to the region of interest.
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.
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. Fox example, ghutathione-S-transferase fusion proteins
can be adsorbed onto
ghutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione
derivatized microtitre
plates, which are then combined with the cell lysates (e.g., 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.
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
CA 02422549 2003-03-14
WO 02/24920 PCT/USO1/29161
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.
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 Figure 1 indicates
expression in humans in
the eye (retinoblastomas) and brain. 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.
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. Patent 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) Biotechhiques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;
and Brent
W094110300), 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 pxoteins or kinase targets as, for
example, downstream
elements of a kinase-mediated signaling pathway. Alternatively, such kinase-
binding proteins
axe likely to be kinase inhibitors.
The two-hybrid system is based on the modulax 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, ivy 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.
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
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as described herein (e.g., a kinase-modulating agent, an antisense lcinase
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.
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 Figure
1 indicates
expression in humans in the eye (retinoblastomas) and brain. 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.
One agent for detecting a protein in a sample is an antibody capable of
selectively binding to
protein. A biological sample includes tissues, cells and biological fluids
isolated from a subject, as
well as tissues, cells and fluids present within a subject.
The peptides of the present invention also provide targets for diagnosing
active protein
activity, disease, or predisposition to disease, in a patient having a variant
peptide, particularly
activities and conditions that are known for other members of the family of
proteins to which the
present one belongs. Thus, the peptide can be isolated from a biological
sample and assayed fox 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 electrophoxetic
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 technques useful for detecting
mutations in a protein.
Such an assay can be provided in a single detection format or a mufti-
detection format such as an
antibody chip array.
I~ 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
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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.
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. (Clip. Exp.
Pha~macol. 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, specif c
polymorphic peptides could be identified.
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 Figure 1
indicates expression in humans in the eye (retinoblastomas) and brain.
Accordingly, methods for
treatment include the use of the kinase protein or fragments.
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Antibodies
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.
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')Z, and
Fv fragments.
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).
In general, to generate antibodies, an isolated peptide is used as an
immunogen and is
adminstered 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 Figure 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.
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 functionlactivity
andlor kinase/binding
partner interaction. Figure 2 can be used to identify particularly important
regions while
sequence alignment can be used to identify conserved and unique sequence
fragments.
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 Figure 2).
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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, (3-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
lasly3ih sss or 3H.
Antibody Uses
The antibodies can be used to isolate one of the proteins of the present
invention by standard
techniques, such as affinity chromatography or iznmunoprecipitation. 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 Figure 1 indicates that kinase proteins of the present invention
are expressed in humans
in the eye (retinoblastomas) and brain. Specifically, a virtual northern blot
shows expression in
retinoblastomas and PCR-based tissue screening panels indicate expression in
the brain. 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.
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 Figure 1
indicates expression
in humans in the eye (retinoblastomas) and brain. If a disorder is
characterized by a specific
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mutation in the protein, antibodies specific for this mutant protein can be
used to assay for the
presence of the specific mutant protein.
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
Figure 1 indicates
expression in humans in the eye (retinoblastomas) and brain. 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.
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
irnmunological marker for
aberrant protein analyzed by electrophoretic mobility, isoelectric point,
tryptic peptide digest, and
other physical assays known to those in the art.
The antibodies are also useful for tissue typing. Experimental data as
provided in Figure 1
indicates expression in humans in the eye (retinoblastomas) and brain. 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.
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
Figure 2 for structural information relating to the proteins of the present
invention.
The invention also encompasses lcits for using antibodies to detect the
presence of a protein
in a biological sample. The kit can comprise antibodies such as a labeled or
Iabelable 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.
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Nucleic Acid Molecules
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.
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 SKB,
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.
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.
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 ih 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.
Accordingly, the present invention provides nucleic acid molecules that
consist of the
nucleotide sequence shown in Figure 1 or 3 (SEQ ID NO:1, transcript sequence
and SEQ ID N0:3,
genomic sequence), or any nucleic acid molecule that encodes the protein
provided in Figure 2,
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SEQ ID N0:2. A nucleic acid molecule consists of a nucleotide sequence when
the nucleotide
sequence is the complete nucleotide sequence of the nucleic acid molecule.
The present invention further provides nucleic acid molecules that consist
essentially of the
nucleotide sequence shown in Figure 1 or 3 (SEQ ID NO:1, transcript sequence
and SEQ ID N0:3,
genomic sequence), or any nucleic acid molecule that encodes the protein
provided in Figure 2,
SEQ ID N0:2. 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.
The present invention further provides nucleic acid molecules that comprise
the nucleotide
sequences shown in Figure 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID
N0:3, genomic
sequence), or any nucleic acid molecule that encodes the protein provided in
Figure 2, SEQ ID
N0:2. 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.
In Figures 1 and 3, both coding and non-coding sequences are provided. Because
of the
source of the present invention, humans genomic sequence (Figure 3) and
cDNA/transcript
sequences (Figure 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 Figures 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.
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
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generally is the case i~c situ, the additional amino acids may be processed
away from the mature
protein by cellular enzymes.
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.
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).
The invention fiuu thher 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.
The present invention fiu~ther provides non-coding fragments of the nucleic
acid molecules
provided in Figures 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 Figure 3.
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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.
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.
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. As indicated by the data presented in Figure 3, the gene
encoding the novel human
kinase protein of the present invention is positioned on public BAC AC005940,
which is known to
be located on human chromosome 17.
Figure 3 provides information on SNPs that have been found in the gene
encoding the novel
human kinase protein of the present invention. SNPs were identified at 34
different nucleotide
positions, including a non-synonymous cSNP at position 16135 and SNPs at two
positions (2082
and 2748) 5' of the ORF that may affect control/regulatory elements. The
change in the amino acid
sequence caused by the G16135A SNP is indicated in Figure 3 and can readily be
determined using
the universal genetic code and the protein sequence provided in Figure 2 as a
reference.
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
CA 02422549 2003-03-14
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known to those skilled in the art and can be found in Curs°ent
Protocols in Molecular Biology, John
Wiley & Sons, N.Y. (199), 6.3.1-6.3.6. One example of stringent hybridization
conditions are
hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45C,
followed by one or more
washes in 0.2 X SSC, 0.1 % SDS at 50-65C. Examples of moderate to low
stringency hybridization
conditions are well known in the art.
Nucleic Acid Molecule Uses
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 Figure 2 and to isolate cDNA
and genomic
clones that correspond to variants (alleles, orthologs, etc.) producing the
same or related peptides
shown in Figure 2. As illustrated in Figure 3, SNPs were identified at 34
different nucleotide
positions.
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.
The nucleic acid molecules axe 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.
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.
The nucleic acid molecules are also useful for expressing antigenic portions
of the proteins.
The nucleic acid molecules are also useful as probes for determining the
chromosomal
positions of the nucleic acid molecules by means of ih situ hybridization
methods. As indicated by
the data presented in Figure 3, the gene encoding the novel human kinase
protein of the present
invention is positioned on public BAC AC005940, which is known to be located
on human
chromosome 17.
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The nucleic acid molecules are also useful in making vectors containing the
gene regulatory
regions of the nucleic acid molecules of the present invention.
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.
The nucleic acid molecules are also useful for making vectors that express
part, or all, of the
peptides.
The nucleic acid molecules are also useful for constructing host cells
expressing a part, or
all, of the nucleic acid molecules and peptides.
The nucleic acid molecules are also useful for constructing transgenic animals
expressing
all, or a part, of the nucleic acid molecules and peptides.
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
Figure 1 indicates that kinase proteins of the present invention are expressed
in humans in the eye
(retinoblastomas) and brain. Specifically, a virtual northern blot shows
expression in
retinoblastomas and PCR-based tissue screening panels indicate expression in
the brain.
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.
Ivy vitro techniques for detection of mRNA include Northern hybridizations and
in situ
hybridizations. In vitro techniques for detecting DNA includes Southern
hybridizations and ih situ
hybridization.
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 Figure 1 indicates that kinase
proteins of the present
invention are expressed in humans in the eye (retinoblastomas) and brain.
Specifically, a virtual
northern blot shows expression in retinoblastomas and PCR-based tissue
screening panels indicate
expression in the brain.
Nucleic acid expression assays are useful for drug screening to identify
compounds that
modulate kinase nucleic acid expression.
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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 Figure 1 indicates expression in humans in
the eye
(retinoblastomas) and brain. 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.
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.
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.
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 Figure 1
indicates that kinase proteins of the present invention are expressed in
humans in the eye
(retinoblastomas) and brain. Specifically, a virtual northern blot shows
expression in
retinoblastomas and PCR-based tissue screening panels indicate expression in
the brain.
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Modulation includes both up-regulation (i.e. activation or agonization) or
down-regulation
(suppression or antagonization) or nucleic acid expression.
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 Figure 1 indicates expression in humans in
the eye
(retinoblastomas) and brain.
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.
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.
Individuals carrying mutations in the lcinase gene can be detected at the
nucleic acid level by
a variety of techniques. Figure 3 provides information on SNPs that have been
found in the gene
encoding the novel human kinase protein of the present invention. SNPs were
identified at 34
different nucleotide positions, including a non-synonymous cSNP at position
16135 and SNPs at
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two positions (2082 and 2748) 5' of the ORF that may affect control/regulatory
elements. The
change in the amino acid sequence caused by the G16135A SNP is indicated in
Figure 3 and can
readily be determined using the universal genetic code and the protein
sequence provided in Figure
2 as a reference. As indicated by the data presented in Figure 3, the gene
encoding the novel human
kinase protein of the present invention is positioned on public BAC AC005940,
which is known to
be located on human chromosome 17. 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 probelprimer in a polymerase
chain reaction (PCR)
(see, e.g. U.S. Patent 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., Sciev~ce 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.
Alternatively, mutations in a kinase gene can be directly identified, for
example, by
alterations in restriction enzyme digestion patterns determined by gel
electrophoresis.
Further, sequence-specific ribozymes (U.S. Patent 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.
Sequence changes at specific locations can also be assessed by nuclease
protection assays
such as RNase and S I 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.
Chromatog3°. 36:127-162 (1996); and Griffin et al., Appl. Biochem.
Biotechnol. 38:147-159 (I993)).
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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.
Euzy~raol. 217:286-295 (1992)), electrophoretic mobility ofmutant 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., Gehet. Ahal. 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.
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. Figure 3 provides information on SNPs that have been found in
the gene encoding
the novel human kinase protein of the present invention. SNPs were identified
at 34 different
nucleotide positions, including a non-synonymous cSNP at position 16135 and
SNPs at two
positions (2082 and 2748) 5' of the ORF that may affect control/regulatory
elements. The change in
the amino acid sequence caused by the G16135A SNP is indicated in Figure 3 and
can readily be
determined using the universal genetic code and the protein sequence provided
in Figure 2 as a
reference.
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.
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.
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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.
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.
The invention also encompasses kits for detecting the presence of a kinase
nucleic acid in a
biological sample. Experimental data as provided in Figure 1 indicates that
kinase proteins of the
present invention are expressed in humans in the eye (retinoblastomas) and
brain. Specifically, a
virtual northern blot shows expression in retinoblastomas and PCR-based tissue
screening panels
indicate expression in the brain. 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 fiuther comprise instructions
for using the kit to detect
kinase protein mRNA or DNA.
Nucleic Acid Arrays
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
Figures 1 and 3 (SEQ ID NOS:1 and 3).
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 US Patent 5,837,832,
Chee et al., PCT
application W095/11995 (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
37
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incorporated herein in their entirety by reference. In other embodiments, such
arrays are
produced by the methods described by Brown et al., US Patent No. 5,807,522.
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.
In order to produce oligonucleotides to a known sequence for a microarray or
detection
kit, the genes) 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.
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 W095/251116 (Baldeschweiler et al.) which is incorporated herein
in its entirety by
reference. In another aspect, a "gridded" array analogous to a dot (or slot)
blot may be used to
arrange and link cDNA fragments or oligonucleotides to the surface of a
substrate using a
vacuum system, thermal, UV, mechanical or chemical bonding procedures. An
array, such as
those described above, may be produced by hand or by using available devices
(slot blot or dot
blot apparatus), materials (any suitable solid support), and machines
(including robotic
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WO 02/24920 PCT/USO1/29161
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.
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.
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. Figure 3 provides
information on SNPs
that have been found in the gene encoding the novel human kinase protein of
the present
invention. SNPs were identified at 34 different nucleotide positions,
including a non-
synonymous eSNP at position 16135 and SNPs at two positions (2082 and 2748) 5'
of the ORF
that may affect controllregulatory elements. The change in the amino acid
sequence caused by
the G16135A SNP is indicated in Figure 3 and can readily be determined using
the universal
genetic code and the protein sequence provided in Figure 2 as a reference.
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
erriployed, and the
type and nature of the nucleic acid molecule used in the assay. One skilled in
the art will
39
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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
Radioirnmunoassay and Related Techniques, Elsevier Science Publishers,
Amsterdam, The
Netherlands (1986); Bullock, G: R. et al., Techniques in Inamuhocytochemistry,
Academic
Press, Orlando, FL Vol. 1 (1 982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P.,
Practice and
Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and
Molecular
Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
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 fox 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.
In another embodiment of the present invention, kits are provided which
contain the
necessary reagents to carry out the assays of the present invention.
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.
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.
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Vectors/host cells
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.
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.
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).
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.
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 ~., 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.
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.
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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 fox 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
Labo~atof y Manual. 2nd, ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY,
(1989).
A variety of expression vectors can be used to express a nucleic acid
molecule. Such
l0 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
baculoviz~.~ses,
papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses,
pseudorabies viruses, and
retxoviruses. Vectors may also be derived from combinations of these sources
such as those derived
L 5 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., Moleculaf° Cloning: A Labo~ato~y Manual. 2nd. ed., Cold Spring
Harbor Laboratory Press, Cold
Spring Harbor, NY, (I989).
The regulatory sequence may provide constitutive expression in one or more
host cells (i.e.
?0 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.
The nucleic acid molecules can be inserted into the vector nucleic acid by
well-known
?5 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 tile fragments together. Procedures for
restriction enzyme
digestion and ligation are well known to those of ordinary skill in the art.
The vector containing the appropriate nucleic acid molecule can be introduced
into an
30 appropriate host cell for propagation or expression using well-known
techniques. Bacterial cells
include, but are not limited to, E. coli, Sty°eptojuyces, and
Salmonella typhin2m°izana. Eukaryotic cells
include, but are not limited to, yeast, insect cells such as D~osophila,
animal cells such as COS and
CHO cells, and plant cells.
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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
S 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., Gehe 67:31-40 (I988)), pMAL
(New England
Biolabs, Beverly, MA) and pRITS (Pharmacia, Piscataway, NJ) 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 I 1d (Studier et al., Gene Expression
Technology.' Methods
in Enzymology 185:60-89 (1990)).
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., Gehe Expression Technology: Methods in Enzymology
185, Academic
Press, San Diego, California (1990) I 19-128). 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. 20:2111-2I 18 (1992)).
The nucleic acid molecules can also be expressed by expression vectors that
are operative in
yeast. Examples of vectors fox expression in yeast e.g., S. ce~evisiae include
pYepSecl (Baldari, et
al., EMBO J. 6:229-234 (1987)), pMFa (I~urjan et al., Cell 30:933-943(1982)),
pJRY88 (Schultz et
al., Gene 54:1 I3-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego,
CA).
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)).
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
(I~aufinan et al.,
EMBO J. 6:187-195 (1987)).
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
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CA 02422549 2003-03-14
WO 02/24920 PCT/USO1/29161
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.
MoleculaT° Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Labof°atory, Cold
Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, 1989.
The invention also encompasses vectors in which the nucleic acid sequences
described
herein are cloned into the vector in reverse orientation, but operabhy 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
inducibhe expression, tissue-specific expression).
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.
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, DEAF-dextran-
mediated
transfection, cationic lipid-mediated transfection, electroporation,
transduction, infection,
lipofection, and other techniques such as those found in Sambrook, et al.
(Molecular Cloni~g.~ A
Laboratoy y Manual. 2nd, ed , Cold Spring Harbor Laboratory, Cold Spring
Harbor Laboratory
Press, Cold Spring Harbor, NY, 1989).
Host cells can contain more than one vector. Thus, different nucleotide
sequences can be
introduced on different vectors of the same cell. Similarhy, 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 txans-acting factors for expression vectors.
When more than one
vector is introduced into a cehh, the vectors can be introduced independently,
co-introduced or joined
to the nucleic acid molecule vector.
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
rephication is defective,
replication will occur in host cells providing functions that complement the
defects.
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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.
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.
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.
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, amity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high performance
liquid
chromatography.
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.
Uses of vectors and host cells
The recombinant host cells expressing the peptides described herein have a
variety of uses.
First, the cells are useful for producing a lcinase protein or peptide that
can be further purif ed to
produce desired amounts of kinase protein or fragments. Thus, host cells
containing expression
vectors are useful for peptide production.
CA 02422549 2003-03-14
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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.
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.
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 axe 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.
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
~0 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.
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 sequences) can be operably linked to
the transgene to direct
expression of the kinase protein to particular cells.
Methods for generating transgenic animals via embryo manipulation and
micxoinjection,
particularly animals such as mice, have become conventional in the art and are
described, for
example, in U.S. Patent Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Patent 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 axe
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
46
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WO 02/24920 PCT/USO1/29161
carrying the transgene. Moreover, transgenic animals carrying a transgene can
further be bred to
other txansgenic 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.
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 crelloxP recombinase system of bacteriophage P 1. For a
description of the crelloxP
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. ce~evisiae (O'Gorman et
al. Science
251:1351-1355 (1991). If a c~elloxP 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.
Clones of the non-human transgenic animals described herein can also be
produced
according to the methods described in Wihnut, 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 Ga 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 morale 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.
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 ih 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.
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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.
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SEQUENCE LISTING
<110> PE CORPORATION (NY)
<120> ISOLATED HUMAN KINASE PROTETNS, NUCLEIC
ACTD MOLECULES ENCODING HUMAN KINASE PROTEINS, AND USES
THEREOF
<130> CL000904PCT
<140> TO BE ASSIGNED
<141> 2000-19-09
<150> 60/233,493
<151> 2000-19-09
<150> 60/247,031
<151> 2000-13-11
<150> 09/729, 995
<151> 2000-12-06
<160> 4
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 2190
<212> DNA
<213> Human
<400> 1
cgcccgcggg ctgagctcgg cgatctgggc cccagcgagg cggtggggcg gggcggggcg 60
gggcggggcg cgcagcagga gcgagtgggg ccgcccgccg ggccacggac actgtcgccc 120
ggcgcccagg ttcccaacaa ggctacgcag aagaaccccc ttgactgaag caatggaggg 180
gggtccagct gtctgctgcc aggatcctcg ggcagagctg gtagaacggg tggcagccat 240
cgatgtgact cacttggagg aggcagatgg tggcccagag cctactagaa acggtgtgga 300
ccccccacca cgggccagag ctgcctctgt gatccctggc agtacttcaa gactgctccc 360
agcccggcct agcctctcag ccaggaagct ttccctacag gagcggccag caggaagcta 420
tctggaggcg caggctgggc cttatgccac ggggcctgcc agccacatct ccccccgggc 480
ctggcggagg cccaccatcg agtcccacca cgtggccatc tcagatgcag aggactgcgt 540
gcagctgaac cagtacaagc tgcagagtga gattggcaag ggtgcctacg gtgtggtgag 600
gctggcctac aacgaaagtg aagacagaca ctatgcaatg aaagtccttt ccaaaaagaa 660
gttactgaag cagtatggct ttccacgtcg ccctcccccg agagggtccc aggctgccca 720
gggaggacca gccaagcagc tgctgcccct ggagcgggtg taccaggaga ttgccatcct 780
gaagaagctg gaccacgtga atgtggtcaa actgatcgag gtcctggatg acccagctga 840
ggacaacctc tatttggtgt ttgacctcct gagaaagggg cccgtcatgg aagtgccctg 900
tgacaagccc ttctcggagg agcaagctcg cctctacctg cgggacgtCa tcctgggcct 960
cgagtacttg cactgccaga agatcgtcca cagggacatc aagccatcca acctgctcct 1020
gggggatgat gggcacgtga agatcgccga ctttggcgtc agcaaccagt ttgaggggaa 1080
cgacgctcag ctgtccagca cggcgggaac cccagcattc atggcccccg aggccatttc 1140
tgattccggc cagagcttca gtgggaaggc cttggatgta tgggccactg gcgtcacgtt 1200
gtactgcttt gtctatggga agtgcccatt catcgacgat ttcatcctgg ccctccacag 1260
gaagatcaag aatgagcccg tggtgtttcc tgaggagcca gaaatcagcg aggagctcaa 2320
ggacctgatc ctgaagatgt tagacaagaa tcccgagacg agaattgggg tgccagacat 1380
caagttgcac ccttgggtga ccaagaacgg ggaggagccc cttccttcgg aggaggagca 1440
ctgcagcgtg gtggaggtga cagaggggga ggttaagaac tcagtcaggc tcatccccag 1500
ctggaccacg gtgatcctgg tgaagtccat gctgaggaag cgttcctttg ggaacccgtt 1560
tgagccccag gcacggaggg aagagcgatc catgtctgct ccaggaaacc tactggtgaa 1620
agaagggttt ggtgaagggg gcaagagccc agagctcccc ggcgtccagg aagacgaggc 1680
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tgcatcctga gcccctgcat gcacccaggg ccacccggca gcacactcat cccgcgcctc 1740
cagaggccca cccctcatgc aacagccgcc cccgcaggca gggggctggg gactgcagcc 1800
ccactcccgc ccctccccca tcgtgctgca tgacctccac gcacgcacgt ccagggacag 1860
actggaatgt atgtcatttg gggtcttggg ggcagggctc ccacgaggcc atcctcctct 1920
tcttggccct ccttggcctg acccattctg tggggaaacc gggtgcccat ggagcctcag 1980
aaatgccacc cggctggttg gcatggcctg gggcaggagg cagaggcagg agaccaagat 2040
ggcaggtgga ggccaggctt accacaacgg aagagacctc ccgctggggc cgggcaggcc 2100
tggctcagct gccacaggca tatggtggag aggggggtac cctgcccacc ttggggtggt 2160
ggcaccagag ctcttgtcta ttcagacgct 2190
<210> 2
<211> 505
<212> PRT
<213> Human
<400> 2
Met Glu Gly Gly Pro A1a Val Cys Cys Gln Asp Pro Arg Ala Glu Leu
1 5 10 15
Val Glu Arg Val Ala Ala Ile Asp Val Thr His Leu Glu Glu Ala Asp
20 25 30
Gly Gly Pro Glu Pro Thr Arg Asn Gly Val Asp Pro Pro Pro Arg Ala
35 40 45
Arg Ala Ala Ser Val Ile Pro Gly Ser Thr Ser Arg Leu Leu Pro Ala
50 55 60
Arg Pro Sex Leu Ser Ala Arg Lys Leu Ser Leu Gln Glu Arg Pro Ala
65 70 75 80
Gly Ser Tyr Leu Glu Ala Gln Ala Gly Pro Tyr Ala Thr Gly Pro Ala
85 90 95
Ser His Ile Ser Pro Arg Ala Trp Arg Arg Pro Thr Ile Glu Ser His
100 105 110
His Val Ala Ile Ser Asp Ala Glu Asp Cys Val Gln Leu Asn Gln Tyr
115 120 125
Lys Leu Gln Ser Glu Ile Gly Lys Gly Ala Tyr G1y Val Val Arg Leu
130 135 140
Ala Tyr Asn Glu Ser Glu Asp Arg His Tyr Ala Met Lys Val Leu Ser
145 150 155 160
Lys Lys Lys Leu Leu Lys Gln Tyr Gly Phe Pro Arg Arg Pro Pro Pro
165 170 175
Arg Gly Ser Gln Ala Ala Gln Gly Gly Pro A1a Lys G1n Leu Leu Pro
180 185 190
Leu Glu Arg Val Tyr Gln Glu I1e Ala Ile Leu Lys Lys Leu Asp His
195 200 205
Val Asn Val Val Lys Leu Ile G1u Val Leu Asp Asp Pro Ala Glu Asp
210 215 220
Asn Leu Tyr Leu Va1 Phe Asp Leu Leu Arg Lys Gly Pro Val Met Glu
225 230 235 240
Val Pro Cys Asp Lys Pro Phe Ser Glu Glu Gln Ala Arg Leu Tyr Leu
245 250 255
Arg Asp Val Ile Leu Gly Leu Glu Tyr Leu His Cys Gln Lys Ile Val
260 265 270
His Arg Asp Ile Lys Pro Ser Asn Leu Leu Leu Gly Asp Asp Gly His
275 280 285
Val Lys Ile Ala Asp Phe Gly Val Ser Asn Gln Phe Glu Gly Asn Asp
290 295 300
Ala Gln Leu Sex Ser Thr Ala Gly Thr Pro Ala Phe Met Ala Pro Glu
305 310 315 320
Ala Ile Ser Asp Ser Gly Gln Ser Phe Ser Gly Lys Ala Leu Asp Val
325 330 335
Trp Ala Thr Gly Val Thr Leu Tyr Cys Phe Val Tyr Gly Lys Cys Pro
340 345 350
Phe Ile Asp Asp Phe Ile Leu Ala Leu His Arg Lys Ile Lys Asn Glu
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355 360 365
Pro Val Val Phe Pro Glu Glu Pro Glu Ile Ser G1u Glu Leu Lys Asp
370 375 380
Leu Ile Leu Lys Met Leu Asp Lys Asn Pro Glu Thr Arg Ile Gly Val
385 390 395 400
Pro Asp Ile Lys Leu His Pro Trp Val Thr Lys Asn Gly Glu Glu Pro
405 410 415
Leu Pro Ser Glu Glu Glu His Cys Ser Val Val Glu Val Thr Glu Gly
420 425 . 430
Glu Val Lys Asn Ser Vah Arg Leu Ile Pro Ser Trp Thr Thr Val Ile
435 440 445
Leu Val Lys Ser Met Leu Arg Lys Arg Ser Phe Gly Asn Pro Phe Glu
450 455 460
Pro Gln Ala Arg Arg Glu Glu Arg Ser Met Ser Ala Pro Gly Asn Leu
465 470 475 480
Leu Val Lys Glu Gly Phe Gly Glu Gly Gly Lys Ser Pro Glu Leu Pro
485 490 495
Gly Val Gln Glu Asp Glu Ala Ala Ser
500 505
<210> 3
<211> 29629
<212> DNA
<213> Human
<400> 3
ccgcccgcgc atccatctgg gcctcagcgt gtcccgagca atcacaacag cagccgcaca 60
acaacaactc acttttacgg cctccttagt ggcaggcact gttctgagcg ccttacgggc 120
gttccctcct cagcatctca ccacgtgcgg tgaggtgagg cccgctagaa ccccatcttg 180
cgggcgagga aaacccaagg cacagaggcg aagccacctg ctcacgggct cccagccagg 240
aaagggtgca gcctggctgc ctggcttcag agcctgggcg ccaaaccggg taacagggct 300
caggctggaa caggaaacct tctgccccga cttgctgggt gaccccgggc ccatccccac 360
ccgctgggcc tccctctacc tatctaagaa aagcagggaa aggtgttcaa gggtaaagga 420
ggatggcctc ttgctggaat ggcaacctca aggaaatacg caaattttat gggcccgggc 480
agcctgtggc ttctgcctgt ggcggctctg agtcccgtag tccctgccta gggccaaaaa 540
gcaggagctc ctgactctgg agttcattct gttatatgtg ctggggcctg aggcttgctg 600
gggttgcctc tctgaggctg ctttctcatc tgtctaatgg ggacagggct gtaacgatca 660
ctatggcaac cactcattta ttcaacaaat atttatcgag ttcctatcac atgccaggca 720
ctgatgatct tttggagaca aggcagatga gcgtcctaat ctcatgaaac ttacattcgg 780
gagggaaaaa caaggcatgc ggagtgaggg gaaggggcgg aggggtgggc cacctgctgg 840
gaggagcctg gcgggtcctg gagggtgttc ccagctttgg cttcctcctt cctatgctgt 900
ctggtttcca agctctcccc gaagctccag ccccactcac tgtccctctc acctcctcca 960
gggaggcctc cctatgccac agcctctcac ctcctctggg gaggcctcct tatgccacag 2020
ccccactctc tgtcctctct cacctcctcc agggaggcct ccctgtgcca cagccccact 1080
ccctgtcccc tctcacctcc tccagggagg cctccttgtg ccacagccgc actcactgtc 1140
tcctgccctc tcttccaggg aggcctccct gatactctag cctcactcag cctcctcacc 1200
tccttcacct cctccaggga ggcctccttg atgttccagc ctcattaact ccctctcact 1260
cctctgggtc cagcttccat gacttttcct gttcctagtg tggagcctcc tctcttcctt 1320
tctccatgtc agcaccagcc ccaccgcctc caggcttcta ctcattcaac acactgcgta 1380
ccgggcacag ggggtctgga cctcaccctt accctcagtc tacctccaaa ccctgctgtg 1440
agcctggaaa atatgggaag gcagggaatc cacaggacaa gtcgggagac tggggctcag 2500
agtcgggaag gagctggtct agggcccctg gtgggtcagc aggcaggact ggaacccagt 2560
cctggctcct cagtggccgg tggactccag ccagccctgc ctcgctgaca tctgtcaaag 1620
caaggggatg gggaacgagc ggtagagcag gcgcttcacc atgcgtactc tgggtctccc 1680
tgagacccat gttctcagtt gctgtgtggg ttcggaggaa gttaccagca gacaggaagg 1740
atggagggtc aggagttcac tcacttcctt ctcctgagaa catgcagagt ccagcgcaag 1800
cagggggaag ggcatcaggt tgggcatggc cagcgctcta caagcctggg acagagatgg 1860
gggtctcagg ctgagtgtca gggttcagtc cggggtcagg atgtagccca gggtcatggc 1920
tgaaggtgag ggctgggggt cacctccctg atgtttcagc cgccacacag tgagtttgag 1980
aacatgagtc tcaggggatg tcatgcccct gtttcacccc tcattcccct cattcccatc 2040
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cccttgcttt tttttgaaac cgagtcttgc tccatcaccc aggctggagt gtagtggcgt 2100
gatcttggct cactgcaacc tccacctccc aagttcacac gattctcctg cctcagcctc 2160
ccgagtagat gggatttcag gtgcacgcca ccatgcctgg ctaatttttg tatttttaat 2220
agagacagag ttttgccatg ttagccaggc tagtctcgaa cttctgacct caggtgatcc 2280
acctgcctcg gcctcccaaa gtgctgggat tacaagtgtg agccaccatg tggggcccat 2340
ccccttgttt tgacagacgt caatgaggca gggctggctg gagtcgggag ccccagggaa 2400
gtcttcctgg aagcagtgag agggatgggg gtaggaggct gaaacatcaa ggagggctcc 2460
ctggaggagg cgggtgggtc tgaagcatca gcaaggcttc tgagttacta gtgtctagct 2520
cagcttccag gaggcagtgt cggagtgctc tgctgtcaag ggttgggact catgactcac 2580
agggctgcat gctgtgctgg ggctgagctg accctgggct ctgccccttc cagtgctgct 2640
gggcctccag gcttctgccc tgtctgtcct gattccagaa tatcagattc tctctgcttc 2700
cctgtgaagc cagcaggcag aagtgactgc ctctgttacc ggcagggata ctgaggccta 2760
gagggctggc atgcggcaga accgatgtga attcattcag gtcataggga cagacttgag 2820
tttgggtgtt ggcaatcccg gtagagggaa cagccagggc aaaggcatgg aggtgggacc 2880
cacagcgctg tggctacctt acctggtagc cagcctgaca cccaggagtg aagccttctc 2940
tgccttcttt tctcaggttc ccaacaaggc tacgcagaag aacccccttg actgaagcaa 3000
tggagggggg tccagctgtc tgctgccagg atcctcgggc agagctggta gaacgggtgg 3060
cagccatcga tgtgactcac ttggaggagg cagatggtgg cccagagcct actagaaacg 3120
gtgtggaccc cccaccacgg gccagagctg cctctgtgat ccctggcagt acttcaagac 3180
tgctcccagc ccggcctagc ctctcagcca ggaagctttc cctacaggag cggccagcag 3240
gaagctatct ggaggcgcag gctgggcctt atgccacggg gcctgccagc cacatctccc 3300
cccgggcctg gcggaggccc accatcgagt cccaccacgt ggccatctca gatgcagagg 3360
ttggtggggc agaacgaggg gttgttcatg agcccctcag tagtctgcaa tgaagactct 3420
ttcctgcccc tgtctgtgcc acacggctat ctagctttgg tttgcatacc ctcagagctg 3480
gggagatcac tacctaacaa tatagcttct tcccaaccag gggagctcca gctgagccaa 3540
aggctgcctt ccctaagtcc tgctattccc actcccagcc caggcctagg aaataggtct 3600
ctccctggtc ccctatgtag tcttcttaga gatgtgaaga tagatgctat gtcccccttc 3660
ccccctaact cttctccagc ttgcacccct cgcctctaat tctgcctctt agagtctgct 3720
gtgactcaga agcggccggc ctgcctccag cctctgggct tctgctggag ttcttgccat 3780
ttaggtctga aagtgaactc aggttccaag cagtctacag atgtcagggg ctgagctttc 3840
tgtgcctgaa cccaggctct cagcctctgt gcccagggct cctcatcttg tccttggagt 3900
ctagaccttc tcattcagct gcttctggaa atagttgctc atgggtttct catggattag 3960
ggtcttccag actccagaat ccagacagga attagcgttt tcccttcacc actgcttctg 4020
gggaacaagg cacagccatg gcgtcaccat ccatgttttc aaacatgagc cacgtcttct 4080
cgtcacatac gggggcgatg gcaccaccaa cttccccatc caaactcaaa agcttggtga 4140
gacctggggg tccgggaatg aggagcttat ggccagaatt ggaccctgaa cgggctctga 4200
ggtaggagca gtgctgcctc cggacccagc tccacctggt gctcgctctt cccccacagg 4260
actgcgtgca gctgaaccag tacaagctgc agagtgagat tggcaaggta ggagtgggca 4320
ggccgagagc agtgggggct tcgggattct ctgtttggcg ctgctccttc tctcgtgtgg 4380
gagggaacgg gaggcagagc caggcaagtc ctagcctgga ggtgaggaca gtttcgtgcc 4440
ctgtgggaag tacccaggta cccaggggga gggtggaaga tggctcctga ttcccgactc 4500
tctgagttct tgacagtgga caaggaggga ctgagggagg catggagcca tgtggagcca 4560
agcaggggca gttaccaggg cgcaggagtc ccctccccat ctgctacaat atttgcccgt 4620
gagccagctg gtggtgggta gtgcagatgg ggtgcaggag agaccagagc tgctcggctc 4680
cccacctcct gagctggtcc tgggaggggt tgccctgtcc aggtggggct gactgatgcc 4740
tatctgcagg gtgcctacgg tgtggtgagg ctggcctaca acgaaagtga agacagacac 4800
tatgtgagtc tggggatacg agggaggtgt tgcccaagcc aggccctgga agcctgaggg 4860
gtggggcagg agttgtgctt aggagataga ggacagggct gcctgagagt gagctccctg 4920
tccctagggg tatgcaaagg aatgagcttc ctaaccctgg ggatatgcaa gcagagactg 4980
gattcctctg aggggaaagc tccagaaagg cttgctgggg gaataagggg aagggctagg 5040
ctcagatatg gccaccccca accccgctta acacttacct gggccacacc ctcagggcca 5100
gtagcagatg tccagtgtgc ctctccggac ctcagtccac atgtaccagc ctgttctagc 5160
ccctggtggc tgcacagtag tgacatttct gtccctcctt ccttaggcaa tgaaagtcct 5220
ttccaaaaag aagttactga agcagtatgg ctttccacgt atgtatcttc tgatcctgtc 5280
cctgggagct cctagcctgg aggcagagga ggagacctcg atcctgagct agttttggct 5340
aggaatgggg tagagaggga gacagcgtga gcagaggcct ggggacagaa tgtgccctgt 5400
gggttgggac aagaccacgg gcatgcaaga ctcttgcttg agactggttt gggggccacg 5460
gtgaggccca gccacctgga acaggtgttt gagttctctt cctggtcaca ggtcgccctc 5520
ccccgagagg gtcccaggct gcccagggag gaccagccaa gcagctgctg cccctggagc 5580
gggtgtacca ggagattgcc atcctgaaga agctggacca cgtgaatgtg gtcaaactga 5640
tcgaggtagg gggtggtggt gagcaggtgg gaaccagcac ctgagtctca tgggagccgc 5700
4
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ttctggtgct ggggagcccc tagcacagac ccagggatct tgcccaggtg gcagatgtgg 5760
ctgaggcctc tgaggacagg gccagacttg gggtggggct gcaggaaggc tttgggggcc 5820
cagcctggtc agggatgttc ccaagttccc atggagggtg aggggctgcc ccagaggcaa 5880
gaagtgagcc cctcattgca gctggagggg aggaaggctg gatgtcgtgt ggcgggccag 5940
gttgggggtc ggtgacttct gaggccccat cagtctggca ccacctgtac acttcctgct 6000
tccttgtctg gggtggttgc atgcatacta agggttctgg ggctggcaag gaccaggagg 6060
cctgggacct CCaaCCCCdC gCCtCCtCaa gCCCCdCCCC CdtgtCtgCt CCCtCtgaCC 6120
aggtcctgga tgacccagct gaggacaacc tctatttggg tgagtgacct ggctcattcc 6180
cacagcagct cactcagggc tggcccaagg gctcccttgg gacatgtatg accttcaggt 6240
gggcggtgta aatgcactga cctcctgggg acagaagaaa aacacacgtt ctgaagccct 6300
ggattccctt gcccagccct gcagaaccag gcccagaata tccagttaga ttcaacaaat 6360
atcgccaagc cccactccct gcttccctct gagcagcaag acagtggatc cacgtgggct 6420
gcgcgctcag gtagatgcag gaagcaggct gcatgggttc ccagacactg tagctctgtg 6480
cctcagtttt cccacctata aaacagggat actagtggtg tctacctcat agggttcctg 6540
tgaagagtaa atgagtaatt atatgtaaag cacattcgtt attatccttg ttaatagtaa 6600
tgttattatt ttagttcctt gtgtctggtt cagggctggg cttagaggag gcctcagaaa 6660
atggggcaga agaagaactg gcttaggaat tagaggctga ggctttagtc tccactccct 6720
accctacctg cctgtctgct atgaccttta ggaaaatttc tgccccttct ctgtgcctca 6780
gtttccccct ctgtaaaagg gccccatgct gatgctgatg gttctcacct ggcacctgag 6840
gatcagatga gacaggtcca tagcagaccc cactctcatg catttatttg ctctcatatc 6900
ccagggtccc ctgtcctgtc cctgcctcga gtatgcctgc atgcctgccc cctctcctac 6960
cctccagaac agggagggac cttggcatcg gctgctttgc cagccagcta caccttacct 7020
tcttgtcttt tctttcagtg tttgacctcc tgagaaaggg gtgagttccc cgtcctgatc 7080
aggcaggtca attctcatcc aggccttcct tcctttccct ccctgtgtcc ccagcccagg 7140
ggtcagctac tctaggagaa gtcagagacg gaggccctgc ccttaggggt aaataagaga 7200
ccaagaggac cattctttga aggctgatgg gggtcagtga ggctgaaata gtcagggaga 7260
cctctggaaa aggggacgga ttttgaccca ggccttgaag aactaggaag atagggatgg 7320
aggagagggg gaagaaagga gtgtttttta ggtaaaagta tatagaggtg ggactcaact 7380
cttaccggta ttcaaatcac aaagggtttt tcagctttcc aacaagtctg tgaatggagt 7440
gggtgggatt ccagttgctc ccatttgtga gagggaaagc taaggaccag agaaggtacg 7500
tggcttgctc aaggtcacac~agcaagtcac tgatggagcc caggcttcca catgtctgcc 7560
ctatgcggct tttcagggta tttacagagc agatgacatg gagtaatgag cacggggctg 7620
ggtggtccgg gaccctcact gccaaggctt gaatgcagcc tgcggcttgt ccctttgcct 7680
gggcggctcc ctacagacca atctggggag aggggcaggg agtggtgtcc ctttaagact 7740
tggaggcttt caaatgtttt gacctctatc caaaacaaga aatatatatt tctattgcta 7800
tccatatctg taactgaaac caaaatttta caaagcagca tatatcttta ctacatgcaa 7860
tatattctga tatattctac ttatttagga aaaaaaaaaa aaagcagttg ccacccacta 7920
aattgatttc atgatcctct cttgggtctg gatccacggt ttgaaacagt gctctaaatg 7980
gcatctttgc aattgattat ggacaattaa gtacttagaa gaaggaatat caagccaatc 8040
agaaattaag agaaagctga tttgaaatta tgattgaaat gggatatgta tgagtatgtg 8100
tgctttaagt tttttattat gtagcagaaa aagctaatat cttgagttgt agggactcat 8160
gtgggcacag gtttcccggg acgtcccgac cacctgaatg gccgggtgcc ctgatttcag 8220
ctgaatgccc ctccccgcat ccttctccat aggcccgtCa tggaagtgcc ctgtgacaag 8280
cccttctcgg aggagcaagc tcgcctctac ctgcgggacg tcatcctggg cctcgagtac 8340
tgtgagtgcg gggcagcttg cccactgggg ctggggctag gggatctggc aggcggcaga 8400
gcccaggctg agcagactct gagcagctcc cgtcagtcag agctgacctg ccaatcagct 8460
tcagtgggag tggggcatgc acgtgtggcg gggccaaagg cctttttgtg gggtggggcg 8520
ggcggtggac tccactgggc atgtgccaga tccttcgtcg tgtctggtcc tgtgggtctg 8580
agtcctggct gttctgtatc tttcttctgc tgagttctta gcctagctta gcgttgccac 8640
ggggcttcaa gagatgtggg aaggaaggga tttatgtcca gctgctgggg agagtctgtc 8700
ctggcatggg gccggggcat ggtggcaggg tggatttacc tgtgaggggc cctagtctga 8760
taagagctca ggagggtgat gtgagcttgg cctctgtctc atttcattca ttagctacat 8820
tcacttgcct gggggcatag gggtgaaaga cccagacccg agttcacggc ctagtgggag 8880
ggacaggaat ctaggcaggc agataataca gcgtggtgcc tgccaaggct ggggagccta 8940
gaggctgtag gagtgccggg gggctgggga agtctccctg aagaggctac ttatgattcg 9000
ggtcctgagg gatgagtaga cttccctgct caggttttga gggatgggcg tggaagacga 9060
tgtgcctggc ataggcgtgt actctgagtc tggggagaag tggagtctgg ctgaagcctc 9120
cagtgggcag aggagggccg tggttagtga aagatgatgc tggaaacact gtccgggcca 9180
cagcatgagg gctgggaatc cctcccctga ggtctttgct gactgcatcc tgccagctct 9240
gtgaggccct gagagcttta agcatgggga ggggcgtgat gggatttgtg cctgagaaag 9300
ctctgtctgg cagctgtgtg gtggctggat tggagtgtgt catcggaggg tgagaggcag 9360
CA 02422549 2003-03-14
WO 02/24920 PCT/USO1/29161
ccagctggcc agggaggagg ctgtttctgc agcccaagtg acagatggtg aggcctggat 9420
taaggcagtg gcagcaggat ggggatagga aggaggtggg gtggtcagca tggagtgact 9480
tgccggtctg gggagaggag agcccctaga cacctagggt cctggcgtgg gttggggacc 9540
aggggagatg cccatctcta aaatcttagc ttgggccagg cgcaggggct catgcctgta 9600
atcccagcac tttgggaggc cgaggtgggt agatcacctg aggtcagggg tttgagacca 9660
gcctggccaa cgtggcaaaa gcctgtctct actacaaata caaaaattag ccttgtgtgg 9720
tggtgggcac ctgtaatccc agctactcgg gaggctgagg caggagaatc gcttgaacct 9780
gggaggtgga ggttgcagtg agccgagatc acgccattgc actccagcct gggtgacaag 9840
agtgaaactc catctcaaaa taaataaata aataaatgca tacatacata tatacataca 9900
tacataaaaa taaaaaataa aatcttagct tggtttcttg ggagcatatt ctttccctgg 9960
gggaacaggg tggggatctg gctgaggttt gacctgcagt gacagaaaca ggactgtctt 10020
tatcctgctc gagcctctcc tttgccttca gattaagact ctctttgcac atatggggaa 10080
actgaggcac acagagggga gggctttgca gaaaatccct accaagggcc tagaggcatg 10140
ggatgggaag gggacatttt accccggtac ggtcagtggc aggcacagtc ctgtaccagc 10200
ttggctccac ctcctttctg ttgtagtccc ttctttcccc tgaagtcctg ttgtctgcta 10260
tcccctagcc tccacaaaga aacgagttta tcttacctgg ttcttgggta aagcctcatc 10320
aggacccagc taatcacagt gaagggcttc cctggggcag aacggttagc gccaggggct 10380
ggacaggtgg atgaacagag gcacgagggc gctgaagacc tgccttgtga ttctggcccc 10440
aagaagagag agttgaggct gccatgagag ggctcggtgg tcagggcggc ccaggcctgg 10500
ttctcagttg atgggggcag gtgcaacgat gcagatgatg agaagcagtt ggatctggaa 10560
tagatgtgag aagctgagct cacagacctt gctgatgagc aggatgtggg gtctcagagg 10620
aggaattgag gatgatcctg aagtttttgg cctttcacag aatggaaaag aatggggagc 10680
agcaggggcg ttttgttttg ctttgttttg attttgttgg tggtaggcat tgcaggcaga 10740
gaaatcaagt tctgaattag acatgttatt gcactgtgtt cagatataca gagacatata 10800
tcgatgccta gctgcctagt tatctaccaa gatgtctatt ggaaatctat gtgggtaaag 10860
agctggagtt caagggagag gctagggttt gagataagaa catgagacca ctttccatgg 10920
tcaaatgtcc acccccctga gcttctgtgc cctgaagggt gtgtcagatt ccttgtgtgt 10980
gcctggcaca tagtaggcaa tcaagaaagt gccactggtt ttatggttat tgttatacgg 11040
CaCCCCJCCtt CtCtgCCCgC agcctccctc tCCtCttCtC CCttCCtCtt tCttCtCt Cg 11100
ccttctctcc tccctcctct ccagcatcct ggggtccgtt ggtccagatg aaggtacttg 11260
ccaaggaggg agcccacagg tcgatggtcg cgggatgggg tcagtggggt cattgtctct 11220
cttggctggg accttaccag tcatgtcagc ttgagccacc tgtcacttcg tggtggtgct 11280
gggcccagaa agcagggcag acctccagcc tattaggtca tttctgattt gggattcgtc 11340
ctactatatg tggctgacct tacaccccag ctgtgtcatc ctgcttgtcc caaggcctgg 11400
ggtgccatcc atctctctga aaccccatca gcccagatcc cgagggctga gatggtacct 11460
ctgtaggata gcagagtccc tacaatctta ctctcagtcc cagcagcagg gacatctttg 11520
cctagcctgg gtgggggatg gaactggaga aaggttttga ttggctttgg gcctgcagac 11580
ggcactcaca gggaaggggc agagctagcc taggaagaac tctgctccca gctgggggcg 11640
gtggctcacg cctgtaatcc cagcactttg ggaggccgag gtgggtggat cacctgaggt 11700
caggagttca agaccagcct gaccaacatg gcgaaaccct gtctctacta aaaatacaaa 11760
aagtagccgg gcgtggtggc agacacctgt aatcccaact actcgggagg ctgaggcagg 11820
agaatctctt gaacctggga ggtggaggct gcagtgagcc gagatcacgc cattgcactc 11880
cagcctgggg gacagagtga gactctgtct caaaaaaaaa aaaaaaaaac caaaaaaaaa 11940
aacagcaaca actctcctgc cctagtttcc tctgacctcc ccactcagca gcagatccct 12000
tgtttgtcat ggagagggtg ctggacttgg agtccaaaga ctcctaagat tccagtcctg 12060
gctctgctgc tcacagcctg ggctcagtgt ctgcacctgc gtggagcaga tggccctgac 12120
gtcctcctcc caggtcgtca ccagacgaaa gtgtgcatgg gctgggatgt cccggccggc 12180
gtccctggct gtgcaaggac gggtgtgggg tcctggccag cggtgcccag gccagcgctc 12240
agctcaagct ccccttctct gcagtgcact gccagaagat cgtccacagg gacatcaagc 12300
catccaacct gctcctgggg gatgatgggc acgtgaagat cgccgacttt ggcgtcagca 12360
accagtttga ggggaacgac gctcagctgt ccagcacggc gggaacccca gcattcatgg 12420
cccccgaggc catttctgat tccggccaga gcttcagtgg gaaggtgact cgcaggccct 12480
gggccaggct ggggttcaag tggggggcgt aatagcttgc cgcagtggcc cagtttctaa 12540
cctgagggtg ccagggtctt tgtgtctagg gagtgacata tttgcctctt ccttggagcc 12600
tgacaaactc cacaactttg gccttctcct gttttccagc aaagtggtcc caaatctccc 12660
ttgcagatat ttactgttgg ttgctctgtg ctgggttctg gaccggactg tggaagaggc 12720
agaaacaaag agaaccctgt ttcctgccct ctggatggtt tcgggggaag ttgggggtcc 12780
ccgcagatct tgggacatgg caggatttga actggccctt gaagaatggg gaggatctga 12840
gcaggacctg gagcctagag aataaaccag agaacagaag ggctcagggt ggggggcaga 12900
gggtataaag ggcctggaag tttgggcttt ctcctaagtg acaggagcgt aggcaaagtt 12960
gtctgaacaa gaggttacac ggtctggcgc agttccctgg gcacatggct gtttcaccta 13020
6
CA 02422549 2003-03-14
WO 02/24920 PCT/USO1/29161
tggagtgcca gccaccccac tgccagggag gctgtgggtg agaggcattt ggacacgtgt 13080
gagtatccag gaaagaggtc aggaggccgg gcacagtggc tcatgcctgt aatcccagtg 13140
ctttgggagg ccaaggtgga tctcttaagg ctaggaattt gagatgagcc tgggcaacat 13200
agcaagaccc catttctaca aaaaaaaaaa taaaaacatt agacaggtgt ggtagtgcac 13260
acctgtagtc ccagctactt gggaggccga ggtgggagga tcgcttgagt ccaggagttg 13320
ggggctgtag tgagctgtga tggtgtctag cctgagtgac tgagcgacac cttgtctcga 13380
agaaagaaag aaagacgttg gggatgttga taaagatttt ttgaaatgtt ttattttgat 13440
ataattctaa atttacagaa aagttggaag aatagtacaa agaaatcccc tatatctttt 13500
tacccagatt caccaattat tgacattttg tcccactggc tttttcatca tctttctttt 13560
tttttgagcc ggagtctcgc tcctgtcgcc caggctggag tgcagtggcg cgatctcagc 13620
tcactgcaag ctccacctcc tgggttcacg ccattctcct gcctcaacct cccgagtagc 13680
tgggactaca ggcgcccacc accacgcccg gctaattttt tgtatttttt agtagagacg 13740
gggtttcacc gtgttagcca ggatggtctg gatctcctga cctcgtgatc cgcccgcctc 13800
ggcctcccaa agtgctggga ttacaggtgt gagccaccac gcccagccag aaatttatca 13860
ttgataagac ttatatatcg gtcaggcatg gtggctcatg cctgtaattc cagccctttg 13920
ggaggccaag gtaggtggat cacctgaggt caggagtttg agaccagcct ggccaacgtg 13980
gtgaaacccc gtctctacta aaaaatacaa aaattagccg ggcatggtgg cgggcacctg 14040
taattccagc tacttgggag gccgaggcag gtggatcacc tgaggtcagg agtttgagac 14100
cagcctggcc aacgtggtga aaccccgtct ctactaaaaa atacaaaaat tagccgggca 14160
tggtggcagg cacctgtaat tccagctact tgggaggctg aggcagaaga atcgttcgaa 14220
cccaggaggc agaggttgca gtgagctaag atcgtgctat tgcactctag cctgggcgac 14280
agagtgagac tctgtctgaa aaaaaaaaga catacataat ccacagacct tatttaaatg 14340
ttatcagttg tcctgatact gtacttcata acttcttctt tttctggtcc cggaatccaa 14400
tcgaggacca cttgctgcat tcaccttctt gtctgtggta tcctttcatc tggaagaggg 14460
ccttggcctg ccgttgtctt tcctgatctt gacattttgg aagacaacca gcctgttatt 14520
ttgtagaatg ttgtcagttt gcatttgtct ggtgttccct ggttgggatt cagatgatgc 14580
atctggggca ggaatatgta ggtagagatc gagaatcact catataagcg agaaagtgga 14640
taccagaaga ggtggcgttc cggagcagaa ggtagagaga gcacacgctg gagtccaggg 14700
cgcggggagg cccaggggtg tttgggagcc cagaggagtt gttgcagtgg cggtggatga 14760
gggcgtgaga ggacagggcc tctgtgtggg caggggctgt ttgcaatatc aggaagaagg 14820
tggattatga ggagaaggga tgactccttg aagcccgagc tggtttagtg agcagaagtt 14880
ccatatatac catcattcct ggggtgcgtc tgtggcacgg gagcggcccg tgtgaccctc 14940
tggatgaagg aggttttgta cctgttgagt tggaaacgta cctggttaga gtctttccca 15000
aggaaaccca gaacccctgg agggtggagg ccttgttctg gccgcccctg tgtcctcagc 15060
actcagcacg gggcccagca tcgggcaagt accgcggagt gtttgtcgag tgagtacatg 15120
acagaggaaa gaggttccct gcaggcctct cctgcagccc gctggagctg ggtgggcaga 15180
ggtggctgtg cctgttgggg actgatgtga gcatgtttct ttccaggcct tggatgtatg 15240
ggccactggc gtcacgttgt actgctttgt ctatgggaag gtgagtgcca gggatgccag 15300
cagagctggg gcgggtccag tgaggcgggc acgggcgacg gatgcaggct cttccttttt 15360
gtccttaagt ggcttttgaa agagcccacc tggctcagag aaggctgaga gagaagaggc 15420
tttttctatc tttctctggt cccctgcgga gcgattctcg cgaaggagtc gcaggacagc 25480
agacacctaa ggggaggtgc cgacgatggt gttgccaccg ccccagccag agtgctcccc 15540
gtccctctgt cccttgacgc cattcactta ttgagccatg tgttcactcc cttgctcatt 15600
tattcgacaa attgtccttc acccctaccc tggctgaggc tggaccctgg ggacacccaa 15660
cgctgacgta tcggtgatcc ctgcccgcag gtgtgcctgc tctggtgacc acactaaggg 15720
gcagggggga atttcagtga acatgttccc aagccccagg ccctgggagt ggaggcctgg 15780
ccacaggtgg cggtaatggt ggtgggtgca cccagcctgg cctggcttgg ccgcgggtgg 15840
cagtaacggc ggtggatgca cccagcctca ttgttccctc agcaactcat tcattcagtc 15900
aacatttgtt gaacatttac agtgtgagtt gaggtccttc tcatgtaatg ggagcccaga 15960
cctgccccct acccctgccc ccaccaaggg aggggggttg atcccctggc acaggtcgag 16020
gccctggacc cacatccttt gtctgcctct ccaccccaca gtgcccgttc atcgacgatt 16080
tcatcctggc cctccacagg aagatcaaga atgagcccgt ggtgtttcct gaggggtgag 16140
ttgtccaccc aggggaacaa gggggctacc acccgctcct ggtgtctgag ttttagcaga 16200
gcttttgccc tctgaggacc ccaccccagc ctgcagatat gaaggtggcg gtgctgttcc 16260
ctgggaggga cccctgaata gatggacggg agggactctg gagccaaggg tctccgcaac 16320
gtcactgtgt ggatgggaac cctgagatcc agggttggcc agggatgacc acaggcatca 16380
ttcacaccac tccttcaccg caggcctgcc tggggtcagt ggcgccagcc ccacccagcc 16440
cctggactca aggggaactt ctccttcccc cactcagggt cagggaactt caagatgcca 16500
gtgcgtgctc cccatttcac agatggaaaa gaggatgctc tggaggagag cggtcagggg 16560
gctgggactc aagccactct tcctccccac tcttcccatt gtgaccgagg tctctgagcg 16620
tagcagggat gtcggggagg cctcttgctc atgcatggtt cgcctcatga cggccaccgt 16680
7
CA 02422549 2003-03-14
WO 02/24920 PCT/USO1/29161
ggcagccaca gcctgagctc ccaggctcct cttttcagca gtggatttca ggagtgaaat 16740
ggaggccggg tgcggtggct cacgcctgta atcccagcac tttgggaggc tgaggtgggc 16800
agatcacctg agttaggagt tagagaccag cctggccaac atggtgaaac cccatctcta 16860
ctaaaaatac aaaaattagc caggcgtggt ggcgcacatc tgtagtccca gctactcggg 16920
aggctgaagc acgagaattg cttgaaccca ggaggcagag gttgcagtga gcctgggcga 16980
cagagcaaga ctctgtctca aaaaaaaaaa acagaagaaa gaaactgaat aaggccgggt 17040
gcggtggctc acacctgtaa ttccagcact ttgggaggcc aaggagggcg gatcacgagg 17100
tcaggagatc gagaccatcc tggctaacac ggtgaaaccc catctctact aaaaatagaa 17160
aaaaaattag ccgggcgtgg tggcgggcgc ctgtagtccc agctactcga gaggctgagg 17220
caggagaacg gcgtgaaccc gggaggcaga ggtttcagtg agctgagacc gtgctactgc 17280
actccagcct gggcgacaga gcgagactct gtctcaaaaa aaaaaaaaaa aaaaaaaaac 17340
aaaaaaaaaa aacaaaaaac aacaaacaaa aaaagaaaat gaaacgggac ttgtactcag 17400
cgactcctgc tctcttCtgc ttatttcctg tgtggtcccc aagccctgct gagccctcct 17460
cttccctgtc tctgggcctt gttgccactt ataccccttg cctcattcag gcctcaggcc 17520
CCtCCCCaga CttatCtagC CaCCttCCCC CtggtCt CgC tgctgctggc ctccctccag 17580
tccagccaac acattcaggc ggggacagcc ctgataaagc acaacaaatc tgcctgcatc 17640
tcttgcctga agtttgtctg aagcttctca aagccacacc ctggcgctag cattcacacg 17700
tctccgggtt ctgccacccg ctcgtctggg gccgcctcac tccctttccc gagcaccagc 17760
cagctggctt ctgtccattt cctcctcatc ctgtggttgc cttccctccc tgcctccaca 17820
gttgtacccc tggtgcctct cttcctgcta taccccctgc tgaggggtgt ctttcccctc 17880
agcccaggaa ttttaaaagg gatgaagcat ctaagacaac agggggaacc gaagtcaaca 17940
gtcctgagag tggctttctg ctccctactc ttggaaggat gggctcccca agaccactgg 18000
tggcaaagaa acctggggtt tggccgggcg tggtggctca cgcctgtaat cccagcactt 18060
tgggaggcca aggcaggcgg atcatgagat caggagatcg agatcatcct ggctaacacg 18120
gtgaaacccc gtctctacta aaaatacaaa aaattagccg ggcacggtgg cgggcacctg 18180
tagtcccagc tactcgggag gctgaggcag gagaatggca tgaacctggg aggcggagct 18240
tgcagtgagc cgagattgcg ccactgcact ccagcctggg ccacagagcg agactccatc 18300
tcaaaaaaaa aaaagaaccc tggggtttgg gcagagagag ttggagctga tgtggcgctg 18360
agggggctgc tccctcccat ctgagtctcc catctctgcc tgcactcttc tggctggcac 18420
tgtgccagcc tgctaacctc cctgggcctc agtttcctcc tctgtcaaat gagagaggat 18480
cttctctggg tgtagaaaag gacgaggtgg tgagtgggtc tgaaggcctc tggtgtccca 18540
taaagcgact ctcctcacca tctttgccac ccattggggt gtccagcacc catggaactc 18600
tgtctgtgcc tctgtcctgg agggagactt gacctcctgc tcaggaaagg ctctccaagc 18660
ccttgttgtg aaattcctgc ctgctgtccg gaactcagtc ttcccatccg agggacgaag 18720
gtttcgggaa gagaggtgga eaggaagggg tcctcatcag cggtcccacc ctcctctcct 18780
tccttcgccc tctccaggcc agaaatcagc gaggagctca aggacctgat cctgaagatg 18840
ttagacaaga atcccgagac gagaattggg gtgccagaca tcaaggtcgg ggaactgggg 18900
gtcttgggct gggctgggac acagaaaaca ggagtcactt tccctttctg gagggatcaa 18960
caccaggatg catgtgtgtt gggtttgagt ctgtggactt tggacccctc caggtgattc 19020
tggtaatggc ctgacctctc CCCCtCtCCC tgCCCtCCCg gccccgacag ttgcaccctt 19080
gggtgaccaa gaacggggag gagccccttc cttcggagga ggagcactgc agcgtggtgg 19140
aggtgacaga ggaggaggtt aagaactcag tcaggctcat ccccagctgg accacggtgg 19200
taagagagcc ggggtagatg ctcccttgtc ctggagggcc tgggggacct gagccttgct 19260
ctgtgcctgg ctccttgggg ggacagaggc ctgcctggcc agccagctgt gatcctgggc 19320
cactggagcc gccattctgc tggaggccca tggagaggga ggtcttgtgg tcgggagacc 19380
aggaggcttg gtgaggagag tgactgattt aaagaaatag cgggcgtggg gccgggcgcg 19440
gtggctcacg cctgtaatcc cagcactttg ggaggccaag gcgggcagat cacgaggtca 19500
ggagatcgag accatccttg aaaccccgac tctactaaaa atatagaaaa ttagctgggc 19560
gtggtggcgg gcgcgtgtag tcccagctac tcgggaggct gaggcaggag aatggtgtga 19620
acccgggagg tggagtttgc cgtgagccga gatcgcgcca ctgcactcca gcctgggcca 19680
cagagcgaga ctgcgtctca aaaaaaaaaa aagaagaaaa gaaaagaaag aaataccggg 19740
cgcggtggct cacgcctgga atcccagcac tttgggaggc cgaggcgggt ggatcacgag 19800
gtcaggagat cgagaccatc ctggctaata cggcgaaacc ccacctctac taaaaataca 19860
aaaaaattag ccgggcgcag tggtgggcac ctgtagtccc agctactggg gaggccgagg 19920
caggagaatc gcttgaacct gggaggtgga ggttgtagtg agccaagatc acgccattgc 19980
actccagcct ggttgacaga acgagactcc atctcaaaaa aaaaaagaaa gaaatagatg 20040
gcccttgctc agcggcagca gtcaccgtga ctggaagaag catttcattc cgtccagaca 20100
gttactgagc ttccgttctc caggcactgc acaaggtgcc gaggacaagg caggggaacg 20160
gcctgggcag cctttggatt ggaggagtgg ccccaaagcc cacgtatcag ttaggcggcg 20220
cctgcgtctc ccccagagcc cacgtatcag ttaggcagca cctgcgtctc ccccagagcc 20280
cacatatcag ttagacggcg cctgcttctc ccccagcgcc cacgtatcag ttagacggcg 20340
CA 02422549 2003-03-14
WO 02/24920 PCT/USO1/29161
cctgcttctc ccccagagcc cacgtatcag ttagacggcg cctgcttctc ccccagatcc 20400
tgtgtatcag ttagactgcg cctgcttctc ccccagagcc cacgtatcag ttagacggcg 20460
cctgttactc ccccagagcc cacgtatcag ttagacggcg cttgcttctc ccccagatcc 20520
cgcgtatcag ttagacgggc ctgcgtctcc cccagatccc gcgtatcagt tagacgggcc 20580
tgcgtctccc ccagagccca cgtatcagtt agacgggcct gcgtctcccc cagagcccac 20640
gtatcagtta gacggcgcct gcttctcccc cagagcccac gtatcagtta gacgggcctg 20700
cgtctccccc agagcccacg tatcagttag acggcgcctg cttctccccc agagcccgcg 20760
tatcagttag acggtgcctg catctccccc gtgcccacgt atcagttaga cggcgcctgc 20820
ttctccccca gagcccacgt atcagttaga cgggcctgcg tctcccccag atcctgcgta 20880
tccattagac agtgcctgtg tctcccctag tgcccgctca catttcggtt ttgctcctct 20940
tcctctgctc agcttctgtg ttggcacttg gaaagtgatt cacatagtcc cccgtggcca 21000
cctggggcca ctgagagccc tgccctgccc ctgcctgaca gtcaagtgag tcagggcaag 21060
cacaaggcca ggaggagagc cagggccact gccgttggcg gggcctggcc ttgcacttta 21120
tccccctctg cagggtcccg gcccagctgg gaccagctgg ctcaatccct gccccctatg 21180
cttacttgac tctgtggggt cgctggaacc aggcaactcc cacggggtcc ccatgaccac 21240
ttgcctgatc ttagccacca tctcctctct ctcagaccac tggaacaacc tcccacgctg 21300
tcccttgctt ctactctcac tccctgtccc cctggtcaat gctcaactca gcacccagca 21360
tggtcccagt ggcatgagtg tgtcacctcc cagctcagag cctgcttctc actcgggctg 21420
ctgtgtccct cagaatcaga cctccagcct gtgccccacc acccgccctg tttttctgcg 21480
gggctcgtgc accgtcccgc catcatgcac tcgtctctgg ccacgtgcca tggaaggggc 21540
tgccccagag ccttcagact tcgcttccct ctgcccgggg agtcccaccc ccgatggcca 21600
cgggactcgc tccctcactt ccttcggctt tttacgccag ggtcccctcc tagagagaag 21660
cgagccttcc ctgaccctgt agcttcagcc tcccctgctt cacacctcat cgccattccc 21720
ttgttttatt ttttcctttc cacttactga catacataat ttactgattt ttcttcttta 21780
cttatcgcct gtctccccca actagaatat aagctgtatg atggctgggc gcagtggctc 21840
acgcctgtaa tcccagcact ttgggaggcc aaggcgggag gatcaCttga ggtcaggagt 21900
ttgagaccag cctggccaac atgctgaagc cccgtctctt ctaaaaatac aaaaaattag 21960
ccgggtgtgg tggtggacgc ctgtaatccc agctattcag gagactgagg cggaaggatc 22020
atttgaagcg gggaggcaga ggttgcagtg agccgagatt gtgccactgc actccagccc 22080
tgggcaacaa gagcaaaact. ccgtctgaaa aaaaaaaagg ctatatgagg gcaggaattc 22140
tggcctcagt gtggccccag ggcctagagt agtggccagc acccagtagg cagccagtgg. 22200
tgaccagtgt tgacgggatg gatggacaca agcgagggag tgaagggact ggcaagtgtg 22260
ccgctgcctc tctgcatgcg tgtgagtcgg cgtgtctgtg ggcacggcat ggaaccgtcc 22320
ttgtcacgga ggagggacaa aggcagagag ccaggctgcg gcagctgttc ccctcctggc 22380
agccccactg actgggccac cggctgcggc tcagccgctt cccgggccgc cctgcagtag 22440
catcttggca tcttctcggc ggccggaagg cgggaaggat ggcacagcat ccctccatgg 22500
cattgctgcc gtagcgagaa ggtatcttct aatggactcc cacttccagc cctggccctc 22560
cccactcttt cagcctggcc ttgcggaccc ttcatgggct ggtcccggcc ccctcctcat 22620
gtaccagtgg catccggctc ctcaccattc caggaatatg cccccagctg ccagcgcccc 22680
gtgttcttgc ctctgccatt tcatgctgtg ctgattgaga tgggacccgc actgcggccc 22740
ccttggcagc tgctctcggg gaatcggagc agaggctgcg tgtctgggag cctgggacct 22800
gtgctcctca cgctgccttg tcctcctcag atcctggtga agtccatgct gaggaagcgt 22860
tcctttggga acccgtttga gccccaagca cggagggaag agcgatccat gtctgctcca 22920
ggaaacctac tggtgtaagt actggtgggc cagggactgc cgggcactcc ctggagttgg 22980
gtggggaggt ctgaggccca tcctcccact ctcactgtcg ttgggccaag gccagagcct 23040
ggggacttgg ccaggtctcg gtgttggccc catttgcatc tctgtcccca aggttagtcg 23100
gggctagaag ggaccttttg ggcccagctc ttgcttcatt cctggggcca gcatccctca 23160
cacacacact tccagggatg aggagctcac gcagcccctc catgggacag gaagaccctt 23220
cttccatgca gcttgatgtc actctctcac tgggtccagc ccctctgggg cttcaaatct 23280
gtggccccct cagcccttgg cagcctggca gaggtttgca gacaggctga tgttggcttc 23340
ctgtaggagg ctggcgggct gtagaggagg ggtgctggcc cctctgcctg gccctgggga 23400
ctgttggctg ctctcccaag tggcccaggc tgcctgcagc cattgctggg gctctgtgcc 23460
cagtcagcac tttgtgagtg cttgttcagt gagtaagcag ggacaggctg gccggtggac 23520
cacgggagag gaacccgcat tggccgaggg ctccctatgg tgagccacgc ctgtgggttc 23580
accacctcct aggagggtcc agaaaagcag ctccccaagc ctgtgcgcct cgtcctcagc 23640
agatccacct tcttcactat aataaaagcc agtctgggat gctaataagg cctgtgctgg 23700
agtttgtaca caaacctgca gagagaaaac cagtggggtc ctgaaccaca gcgtggtcct 23760
gggacagcca ctgccttcct ctggccccgg agggaagctt tggggaaggg gctggtggga 23820
gttgtttgcc ccaccctggc ctgctctgtg tggaaggcgc actccccaga ggggtgagtg 23880
ccaggcgctg tccgggtgcc ttggcttcac gctgtcacca ggcctgtccg ggaccaccat 23940
gttggtttcc cgtgaggcct ccctctcata agagggccct tcagaagggt cgggacccct 24000
9
CA 02422549 2003-03-14
WO 02/24920 PCT/USO1/29161
cgtagtggac aagctgacat ctgctccctg ctggaggtgg cttgcaccca gggagagcct 24060
cataatgagg tggggggcct gggagaggcc tggaggtccc aactgcagct tttctgtcat 24120
ctcttcaggg aggtggttgc ggttggggga ggattctctg agctcatcca ggaatgtagg 24180
cccctgatgc tggaattgtg cttagtgtag ggggagaggg ggcatatata atttgacgtc 24240
caaatgggga catttttgag agtgaaaggg gaagccatta ataattatgc cagcacggcc 24300
gggtgcggtg gctcacgcct gtaatcccag cactttggga ggccgaggct ggtggatcac 24360
agggtcagga gatcgagacc atcctggcta acacggtgaa accccgtctc tactaaaaat 24420
acaaaaaatc agctgggcgt ggtggcgggc acctggagtc ccagctactc aggaggctga 24480
ggcaggagaa tggcgtgaac ccgggaggca gagcttgcag tgagccaagg tcacgccact 24540
gcactccagc ctgggcgaca gagtgagact ccgtctcaaa aaataataat tattatgcca 24600
gcatggtggc tcatgcctat aatcccagca ctttgggagg ccaaggcagg attgcttgag 24660
gccaggagtt caagaccagc ctgggcaaca tagcaagacc ccatctctaa aaaaaaaaaa 24720
aattagccgg gcgtggtggt gggtgcctgt agtcccagca actcaggagg ctgaggtggg 24780
aggattgctt gagtctggga ggtggaggtt gcagtgagct gagattgcac cactgtactc 24840
cagcctgggt gacagagcca gaccctgtct caaaaaaaaa aaagaaaaaa aagtaataat 24900
aattatgcca ggacagcagg tggacggaca cctggtcctt ctgactcaga gcctgtggtc 24960
cagcaccccc tagtggtgga acaagccaga cacaggataa ggatacattt agtgtctagt 25020
ttgtacctgg caaacagagt gacaagattg ggcttaatac tttccagcta taaaattcta 25080
gaattctgtg acccaagttt aatttggggt agagcttttt aaaaaaaaaa tagagatgga 25140
gtcttgccat gttgcccagg ctggacttaa actcctggcc tcaagccatt tgcccacctt 25200
ggcctccgaa agtgccaggt gattacaggc atgagccacc acacccagcc tccacgttta 25260
actttgaaag aagattttac ttcatcatca agtcccaata tttatccttg atagactgct 25320
ttggtttttt gtttgtttgt tttgagatgg.agtttcactc ttgttgccca ggCtggagtg 25380
cagtggcgca aactcaactc actgcagtct ccgcctctca cattcaagca gttctcttgc 25440
ctcagcctcc caagtagctg ggattacagg tgcatgccac caccacaccg gctaattttt 25500
gtatttttat tagagacggg atttcaccat tttggccagg ctggtctcaa actcctgacc 25560
tcaggtaatc tgcccacctc agcctcccaa agtgctggga ttacaggcgt gagccactgt 25620
gcccggccat agagtttttt atactttggg ataattgtag aaactcagta gtagagttaa 25680
gtggagttgg tcctttttaa agatatcaaa acccatttac tggttatttt aaaaagagac 25740
attttgggag gaaaactaga tatagaaatc tgttgaatat gtgacagaat cccaagactg 25800
atagatggac tctgccctgt gaacaaggca aagaaaaatg caaaatgaaa gcctctctac 25860
ccagatctgc tgggggatga ctgaggtcaa cacagaaggc cctcaggccg ggcacggtgg 25920
ctcacgcctg caatcccaac actttaggag gctgaggtgg atggatcgct tgagcccagg 25980
agtttgagac cagcctgggc aacatggtga aaccctgttt ttatagagat aaaaaaatac 26040
aaaaattagc tgggcgtggt ggcatgtgcc tgtagtctca gctactcagg aggctgaggt 26100
gggaggatcg cttgagcctg gaaggcagag gttgcaatga gctgagattg caccactgca 26160
ctgcagcctg cacgacagag cgagacgctg tctcaaaaca acaacaaaac cacacacaca 26220
gagagaaggc ccttgattag gctgatagtt ggaggatgta gggaagtcag ctgggtcaga 26280
ctgtgagcag ctccagaggc cgtgctggga ggtttagact tcatctctgg tcaatggggg 26340
gccacggagg cgttgcgggc tgagactggg ggctgagaga ccggcaagga gcaactgccg 26400
tgatgtaggg aggccagagg gaggccaagc ttggggcagt gggtgaaggg ggctttgaga 26460
gatgtgggat tcagattcct gtgtgtgtga gggagagtgt ctccctgagt gcatattctg 26520
accctgaggt ccctctgtcc ctggtgtccc ctgaacagga aagaagggtt tggtgaaggg 26580
ggcaagagcc cagagctccc cggcgtccag gaagacgagg ctgcatcctg agcccctgca 26640
tgcacccagg gccacccggc agcacactca tcccgcgcct ccagaggccc accccctcat 26700
gcaacagccg cccccgcagg cagggggctg gggactgcag ccccactccc gcccctcccc 26760
catcgtgctg catgacctcc acgcacgcac gtccagggac agactggaat gtatgtcatt 26820
tggggtcttg ggggcagggc tcccacgagg ccatcctcct cttcttggac ctccttggcc 26880
tgacccattc tgtggggaaa ccgggtgccc atggagcctc agaaatgcca cccggctggt 26940
tggcatggcc tggggcagga ggcagaggca ggagaccaag atggcaggtg gaggccaggc 27000
ttaccacaac ggaagagacc tcccgctggg gccgggcagg cctggctcag ctgccacagg 27060
catatggtgg agaggggggt accctgccca ccttggggtg gtggcaccag agctcttgtc 27120
tattcagacg ctggtatggg ggctcggacc cctcactggg gacagggcca gtgttggaga 27180
attctgattc cttttttgtt gtcttttact tttgttttta acctgggggt tcggggagag 27240
gccctgcttg ggaacatctc acgagctttc ctacatcttc cgtggttccc agcacagccc 27300
aagattattt ggcagccaag tggatggaac taactttcct ggactgtgtt tcgcattcgg 27360
cgttatctgg aaagtggact gaacggaatc aagctctgag cagaggcctg aagcggaagc 27420
accacatcgt ccctgcccat ctcactctct cccttgatga tgcccctaga gctgaggctg 27480
gagaagacac cagggctgac tttgaccgag ggccatggac gcgacaggcc tgtggccctg 27540
cgcatgctga aataactgga acccagcctc tcctcctaca ccggcctacc catctgggcc 27600
caagagctgc actcacactc ctacaacgaa ggacaaactg tccaggtcgg agggatcacg 27660
CA 02422549 2003-03-14
WO 02/24920 PCT/USO1/29161
agacacagaa cctggagggg tgtgcacgct ggcaggtggc ctctgcggca attgcctcac 27720
cctgaggaca tcagcagtca gcctgctcag agcgggggtg ctggagcgcg tgcagacaca 27780
gctcttccgg agcagccttc accttctctc tgggatcagt gtccggctgg ccgacgtggc 27840
atttgctgac cgaatgctca tagaggttga cccccacagg gtcacgcagg actcggacac 27900
tgccctggaa acatggatgg acaagggctt ttggccacag gtgtgggtgt cctgttggag 27960
gagggcttgt ttggagaagg gaggctggct gggggagaaa cccggatccc gctgcatctc 28020
cgcgcctgtg ggtgcatgtc gcgtgctcat ctgttgcaca cagctcactc gtatgtcctg 28080
cactggtaca tgcatctgta atacagtttc tacgtctatt taaggctagg agccgaatgt 28140
gccccattgt cagtgggtcc acgtttctcc ccggctcctc tgggctaagg cagtgtggcc 28200
cgaagcttaa aaagttactc ggtactgttt ttaagaacac ttttatagag ttagtggaag 28260
gcaagttaag agccaatcac tgatccccaa gtgtttcttg agcatctggt ctggggggac 28320
cactttgatc ggacccaccc ttggaaagct caggggtagg cccaggtggg atgctcaccc 28380
tgtcactgag ggttttggtt ggcatcgttg tttttgaatg tagcacaagc gatgagcaaa 28440
ctctataaga gtgttttaaa aattaacttc ccaggaagtg agttaaaaac aataaaagcc 28500
ctttcttgag ttaaaaagaa aaaaaaaagg tttgtgcgta cattttctgc atctggatat 28560
acgttctttc tcagcagctg gaacagctgg ctttgttgaa ttttctggaa gcgtctgagg 28620
caccctaagt ccctgagcag gacagtggtg agaagtggtc ttggcggagg gagggagagg 28680
gaagggctgg ctcaggaggt gaccgggctg cagtccaggg tacagctgag gctcctgggc 28740
gggtccgtgg ccactccttg ggaagaactg cctgtttcac aggggctcag gatgccaagg 28800
tctggtccgg gtaggagcca tagctgctgc ttttggggca gaggtccctg tggtgtcaca 28860
ggagtgcctg tgacaccagc ccagtgacct cccatccccg cttagccttg gacactggta 28920
cagacttttg ggaccccaca cctctgttcc catggtacag ccctccaggg cagcgacgaa 28980
aagagtcatc cttaaggtca cacagccctg agcttgaatc caagctttgc tacttaaaaa 29040
ttgtgtgacc tttggcaggt cattggagga gcctcagttc ccttattgat ttaatgggaa 29100
tgttcccgtg gggtgttttg tttgtttgtt tgagattttt tgagacttgc tctgtcaccc 29160
aggctggagt gcaatggcaa gatctcggct cactgcaacc tcttcctcct gggttcaagc 29220
gattctcctg cctcagcctc ccaagtagct gggactacag gtgcccgcca ccatgcccca 29280
gctaattttt tgtactttta gtagagacgg ggtttcacca tgttggccag gctggtcttg 29340
aactcctgac ctcaggtgat ctgcccacct cggcctccca aagtgctggg attacaggcg 29400
tgagccaccg cgcccacctc cccatggggt ttgaatgcaa acaatgcaaa cgttttcgtc 29460
tgctctcaca ctacaacagt gaacacagaa gacttctgtg accggctggg cgcggtggct 29520
cacgcctgta atcccagcac tttgggaggc tgaggaaggc ggatcatgag gtcggagatc 29580
gagaccatcc tggctgacac ggtgaaaccc cgtctctact aaaaataca 29629
<210> 4
<211> 505
<212> PRT
<213> Rattus norvegicus
<400> 4
Met Glu Arg Ser Pro Ala Va1 Cys Cys Gln Asp Pro Arg Ala Glu Leu
1 5 10 15
Val Glu Arg Val Ala Ala Ile Ser Val A1a His Leu Glu G1u Ala Glu
20 25 30
Glu Gly Pro G1u Pro Ala Ser Asn Gly Val Asp Pro Pro Pro Arg Ala
35 40 45
Arg Ala Ala Ser Val Ile Pro Gly Ser Ala Ser Arg Pro Thr Pro Val
50 55 60
Arg Pro Ser Leu Ser Ala Arg Lys Phe Ser Leu Gln Glu Arg Pro Ala
65 70 75 80
Gly Ser Cys Leu Glu Ala Gln Val Gly Pro Tyr Ser Thr Gly Pro Ala
85 90 95
Ser His Met Ser Pro Arg Ala Trp Arg Arg Pro Thr Ile Glu Ser His
100 105 110
His Val Ala Ile Ser Asp Thr Glu Asp Cys Val Gln Leu Asn Gln Tyr
115 120 125
Lys Leu Gln Ser Glu Ile Gly Lys Gly Ala Tyr Gly Val Val Arg Leu
130 135 140
Ala Tyr Asn G1u Arg Glu Asp Arg His Tyr Ala Met Lys Val Leu Ser
145 150 155 160
Lys Lys Lys Leu Leu Lys Gln Tyr Gly Phe Pro Arg Arg Pro Pro Pro
11
CA 02422549 2003-03-14
WO 02/24920 PCT/USO1/29161
165 170 175
Arg Gly Ser Gln Ala Pro Gln Gly Gly Pro Ala Lys Gln Leu Leu Pro
180 185 190
Leu Glu Arg Val Tyr Gln Glu Ile A1a Ile Leu Lys Lys Leu Asp His
195 200 205
Val Asn Val Val Lys Leu Ile Glu Val Leu Asp Asp Pro Ala Glu Asp
210 215 220
Asn Leu Tyr Leu Val Phe Asp Leu Leu Arg Lys Gly Pro Val Met Glu
225 230 235 240
Val Pro Cys Asp Lys Pro Phe Pro Glu Glu Gln Ala Arg Leu Tyr Leu
245 250 255
Arg Asp Ile Ile Leu Gly Leu Glu Tyr Leu His Cys Gln Lys Ile Val
260 265 270
His Arg Asp Ile Lys Pro Ser Asn Leu Leu Leu G1y Asp Asp Gly His
275 ' 280 285
Val Lys Ile Ala Asp Phe Gly Val Ser Asn G1n Phe Glu Gly Asn Asp
290 295 300
Ala Gln Leu Ser Ser Thr Ala Gly Thr Pro Ala Phe Met Ala Pro Glu
305 310 315 320
Ala Ile Ser Asp Thr Gly Gln Ser Phe Ser Gly Lys Ala Leu Asp Val
325 330 335
Trp Ala Thr Gly Val Thr Leu Tyr Cys Phe Val Tyr Gly Lys Cys Pro
340 345 350
Phe Ile Asp Glu Tyr Ile Leu Ala Leu His Arg Lys I1e Lys Asn Glu
355 360 365
Ala Va1 Va1 Phe Pro Glu Glu Pro G1u Val Ser Glu Glu Leu Lys Asp
370 375 380
Leu Ile Leu Lys Met Leu Asp Lys Asn Pro Glu Thr Arg Ile Gly Val
3'85 390 395 400
Ser Asp Ile Lys Leu His Pro Trp Val Thr Lys His G1y Glu Glu Pro
405 410 415
Leu Pro Ser G1u Glu G1u His Cys Ser Val Val Glu Val Thr Glu G1u
420 425 430
Glu Val Lys Asn Ser Val Lys Leu Ile Pro Ser Trp Thr Thr Val Ile
435 440 445
Leu Val Lys Ser Met Leu Arg Lys Arg Ser Phe Gly Asn Pro Phe Glu
450 455 460
Pro Gln AIa Arg Arg GIu Glu Arg Ser Met Ser A1a Pro Gly Asn Leu
465 470 475 480
Leu Leu Lys Glu G1y Cys Gly Glu Gly Gly Lys Ser Pro Glu Leu Pro
485 490 495
Gly Val Gln Glu Asp Glu Ala Ala Ser
500 505
12
