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CA 02518238 2005-09-06
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ASSOCIATION OF POLYMORPHIC KINASE ANCHOR PROTEINS WITH
CARDIAC PHENOTYPES AND RELATED METHODS
RELATED APPLICATIONS
This application claims the benefit of priority to U.S. provisional
application Serial No. 60/453,215, to Andrews Braun and Stefan
Kammerer entitled "ASSOCIATION OF POLYMORPHIC KINASE ANCHOR
PROTEINS WITH CARDIAC PHENOTYPES AND RELATED METHODS",
filed March 7, 2003, U.S. provisional application Serial No. 60/453,208,
to Andrews Braun and Stefan Kammerer entitled "ASSOCIATION OF
POLYMORPHIC KINASE ANCHOR PROTEINS WITH CARDIAC
PHENOTYPES AND RELATED METHODS", filed March 7, 2003, and U.S.
provisional application Serial No. 50/453,350, to Andrews Braun and
Stefan Kammerer entitled "ASSOCIATION OF POLYMORPHIC KINASE
ANCHOR PROTEINS WITH CARDIAC PHENOTYPES AND RELATED
METHODS", filed March 7, 2003.
Where permitted, the subject matter of each of the above-noted
wpplicwtions is incorporated herein by reference. ~4lso, e~here permitted
the subject mwtter and disclosure of U.S. Application Serial No.
09/834,700 to Andrews Braun entitled "POLYMORPHIC KINASE
ANCHOR PROTEINS AND NUCLEIC ACIDS ENCODING THE SAME", filed
April 12, 2001, and U.S. Application Serial No. 10/428,254 to Andrews
Braun, Charles Cantor, Stefan Kammerer, Susan Taylor, and Lora Burns,
entitled "KINASE ANCHOR PROTEIN MUTEINS, PEPTIDES THEREOF,
AND RELATED METHODS", filed May 1, 2003, are incorporated by
reference.
FIELD OF THE INVENTION
Methods of identifying subjects having or at a risk of developing
disorders of cellular protein phosphorylation and/or signal transduction.
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Methods of determining susceptibility to morbidity and/or increased or
early mortality are also provided.
BACKGROUND OF THE INVENTION
Protein phosphorylation is an important mechanism for enzyme
regulation and the transduction of extracellular signals across the cell
membrane in eukaryotic cells. A wide variety of cellular substrates,
including enzymes, membrane receptors, ion channels and transcription
factors, can be phosphorylated in response to extracellular signals that
interact with cells. A key enzyme in the phosphorylation of cellular
proteins in response to hormones and neurotransmitters is cyclic AMP
(cAMP)-dependent protein kinase (PKA). Upon activation by CAMP, PKA
thus mediates a variety of cellular responses to such extracellular signals.
An array of PI<A isozymes are expressed in mammalian cells. The
PKAs usually exist as inactive tetrameres containing a regulatory (R)
subunifi dimer and two catalytic (C) subunits. Genes encoding three C
subunits (Ca, C,r3 and Cy) and four R subunits (Rla, R1~3, Rlla and RII,~)
have been identified (see Takio e~ al. (1982) Proo. f~la~l. load. Soi, ll.S.
A. ~~:2544-2548; Lee e~ al. ( 1983) Proc. f~la~'7. ~l oad. Sci. (l. S. A,
50:36~8-3612; Jahnsen et al, ( 1996) J. Siol. Chem. 26':12352-12361;
Clegg et al. ( 1988) Proe, l~latl. ~I cad, Sci. U. S. ~l. 55:3703-37~7; and
Scott (1991 ) Pharmacol. Ther. 50:123-145). The type I (RI) a and type II
(R11) a subunits are distributed ubiquitously, whereas RI,~ and R11~3 are
present mainly in brain (see. e.g,, Miki and Eddy (1999) J. Siol. Ohem.
274:29057-29052). The type I PKA holoenzyme (Rla and R1~3) is
predominantly cytoplasmic, whereas the majority of type II PKA (Rlla and
R11~3) associates with cellular structures and organelles (Scott (1991)
Pharmacol. Ther, 50:123-145). Many hormones and other signals act
through receptors to generate cAMP which binds to the R subunits of
PKA and releases and activates the C subunits to phosphorylate proteins.
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Because protein kinases and their substrates are widely distributed
throughout cells, there are mechanisms in place in cells to localize protein
kinase-mediated responses to different signals. One such mechanism
involves subcellular targeting of PKAs through association with anchoring
proteins, referred to as A-kinase anchoring proteins (AKAPs), that place
PKAs in close proximity to specific organelles or cytoskeletal components
and particular substrates thereby providing for more specific PKA
interactions and localized responses (see, e.g., Scott et al. (1990) J. Biol.
Chem. 265:21561-21566; Bregman et al. (1991) J. Biol. Chem.
266:7207-7213; and Miki and Eddy (1999) J. Biol. Chem. 274:29057-
29062). Anchoring not only places the kinase close to preferred
substrates, but also positions the PI<A holoenzyme at sites where it can
optimally respond to fluctuations in the second messenger cAMP
(Mochly-Rosen ( 1995) Science 26B:247-251; Faux and Scott ( 1996)
Trends Biochem. Sci. 2 7:312-315; Hubbard and Cohen ( 1993) Trends
Biochem. Sci. ?B:172-177).
Up to 75~/~ of type II PKs4 is localized to various intracellular sites
through association of the regulatory subunit (R11) with AKAPs (see, e.g.,
Hausken et' al. (1996) J. Biol. Chem. 277:29016-29022). RII subunits of
PKA bind to AI<APs with nanomolar affinity (Carr et al. ( 1992) J. Biol.
Chem. 267:13376-13382), and many AKAP-RII complexes have been
isolated from cell extracts. RI subunits of PKA bind to AKAPs with only
micromolar affinity (Burton et al. (1997) Proc. Nat/, a4cad. Sci. U.S.~I.
94:1 1067-1 1072). Evidence of binding of a PKA RI subunit to an AKAP
has been reported (Miki and Eddy (1998) J. Biol. Chem 273:34384-
34390) in which Rla-specific and Rla/Rlla dual specificity PKA anchoring
domains were identified on FSC1 /AKAP82. Additional dual specific
AKAPs, referred to as D-AKAP1 and D-AKAP2, which interact with the
type I and type II regulatory subunits of PKA have also been reported
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(Huang et al. (1997) J. Biol. Chem. 272:8057-8064; Huang et al, (1997)
Proc. Nat/, Acad. Sci. U.S.A, 94:11184-11189).
More than 20 AKAPs have been reported in different tissues and
species. Complementary DNAs (cDNAs) encoding AKAPs have been
isolated from diverse species, ranging from Caenorhabditis alegans and
Drosophilia to human (see, e.g., Colledge and Scott (1999) Trends Cell
Biol. 9:216-221 ). Regions within AKAPs that mediate association with
RII subunits of PKA have been identified. These regions of approximately
10-18 amino acid residues vary substantially in primary sequence, but
1~ secondary structure predictions indicate that they are likely to form an
amphipathic helix with hydrophobic residues aligned along one face of
the helix and charged residues along the other (Cart et al. (1991 ) J. Biol.
Chem. 256:14188-14192; Carr et al, (1992) J. Biol. Chem. 267:13375-
13382). Hydrophobic amino acids with a long aliphatic side chain, e.g.,
valine, leucine or isoleucine, can participate in binding to RII subunits
(Glantz et al. (1993) J. Biol. Chem. 265:12796-12804).
f~iany AKc~Ps als~ have the ability to bind to multiple proteins,
including ~ther signaling enzymes. For example, AICAP79 binds t~ PKA,
protein kinase C (PI<C) and the protein phosphatase calcineurin (PP2B)
2~ (Coghlan et al. (1995) Science 257:108-1 12 and Klauck et al. (1995)
Science 277:1589-1592). Therefore, the targeting of AKAP79 t~
neuronal postsynaptic membranes brings together enzymes with opposite
catalytic activities in a single complex.
AKAPs thus serve as potential regulatory mechanisms that
increase the selectivity and intensity of a CAMP-mediated response.
There is a need, therefore, to identify and elucidate the structural and
functional properties of AKAPs in order to gain a complete understanding
of the important role these proteins play in the basic functioning of cells.
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SUMMARY
Provided herein are methods for indicating increased susceptibility
of a subject to a disease or disorder. The methods include assessment
of the presence or absence of an allele of the an AKAP gene and other
aspects, including determining EKG features, or methods in which the
AKAP gene is predictive of treatment outcome or response. Methods
provided herein include steps of conducting an EKG examination;
determining the EKG-PR-interval in the subject. If the EKG-PR-interval is
decreased, then identity of an amino acid present in the subject at
position 646 of AKAP10/D-AKAP2 (SEQ ID NO:2) or a nucleotide present
at position corresponding to nucleotide 2073 of SEQ ID N0:1 is
assessed. The presence of Val at position 646 of SEQ ID N0:2 or the
presence of a -G- at nucleotide position 2073 of SECT ID N0:1, indicates
increased susceptibility to a disease or disorder. The disease or disorder
can be selected from among a variety of diseases and discorders,
including, but are not limited to, cardiovascular disorders, cardiac
disease, proliferative disorders, neurological disorders, neurodegenerative
disorders, obesity, diabetes and peripheral refiinopathies.
Also provided are methods for indicating increased susceptibility of
a subject to a disease or disorder associated with the cardiovascular
system, by conducting an EI<G exam; determining the EKG-PR-interval in
the subject, wherein, if the EKG-PR-interval is decreased, then
determining the amino acid present at position 646 of AI<AP10/D-AKAP2
(SEQ ID N0:2) or the nucleotide present at position corresponding to
nucleotide 2073 of SEQ ID N0:1, wherein the presence of Val at position
646 of SEQ ID N0:2 or the presence of a -G- at nucleotide position 2073
of SEQ ID N0:1, indicates increased susceptibility to a disease or
disorder associated with the cardiovascular system. The EKG-PR-interval
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in the subject can be compared to a predetermined age-matched standard
EKG-PR-interval to determine whether it is decreased.
Also provided herein are methods of assessing the susceptibility of
a subject to a disease or disorder associated with the cardiovascular
system, the method comprising determining the amino acid at position
646 of AKAP10/D-AKAP2 (SEQ ID N0:2) or the nucleotide present at
position corresponding to nucleotide 2073 of SEQ ID N0:1, wherein the
presence of Val at position 646 of SEQ ID N0:2 or the presence of a -G-
at nucleotide position 2073 of SEQ ID NO:1, indicates increased
1~ susceptibility to a disease or disorder associated with the cardiovascular
system.
Also provided herein are methods of diagnosing a disease or
disorder associated with the cardiovascular system, comprising detecting
the presence of Val at 646 of D-AI<AP2 (SECT ID N0:2) or the presence
of a G at a nucleotide position corresponding to nucleotide 2073 of SEQ
ID N0:1, wherein the presence of Val at position 646 of SEQ ID NO:2 or
the presence of a -G- at nucleotide position 2073 of SEQ ID f~0:1,
indicates the presence of a disease or disorder associated with the
cardiovascular system. In these methods, fihe disease or disorder can be
2~ from among, but not limited to, one or more of the group consisting of:
atrial fibrillation, sick sinus syndrome, sudden cardiac arrest, ventricular
arrythmia, ventricular fibrillation, ventricular tachycardia, Wolf-Parkinson-
White (WPW) Syndrome, Lown-Ganong-Levin (LGL) Syndrome,
hypertension.
Provided herein are methods for determining responsiveness of a
subject to one or more /3-blocking agents, comprising detecting for
the subject the presence or absence of Val at position 646 of SEQ ID
N0:2 or a -G- nucleotide at a position corresponding to position 2073 of
SEQ ID NO: 1, wherein the presence of a Val at position 646 of SEQ ID
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N0:2 or a -G- at nucleotide 2073 of SEQ ID N0:1, is indicative of an
increased likelihood that a subject has a modulated response to one or
more ~3-blocking agents compared to a subject who does not have the
allelic variant. In one embodiment, the modulated response is a
decreased response to one or more ~3-blocking agents compared to a
subject who does not have the allelic variant. In another embodiment,
the decreased response is a non-response to one or more ~3-blocking
agents compared to a subject who does not have the allelic variant. In
yet another embodiment, the modulated response is an increased
response to one or more ~3-blocking agents compared to a subject who
does not have the allelic variant. The ,r3-blocker can be an antagonist of a
,Q-adrenergic receptor. In another embodiment, the ,~-blocker is an
agonist of a ,Q-adrenergic receptor.
Also provided herein are methods for determining responsiveness
of a subject to one or more ~3-blocking agents, comprising detecting the
presence or absence of Val at position 646 of SEQ ID N0:2 or a -G-
nucleotide at a positi~n corresponding to position 2073 ~f SEA ID f~0: 1,
wherein the presence of a Val at position 646 of SECT ID N0:2 or a -G- at
nucleotide 2073 of SEQ ID N0:1, is indicative of an increased likelihood
that a subject has an increased response to one or more ,~-blocking
agents compared to a subject who does not have the allelic varianfi.
Also provided herein, are methods for determining responsiveness
of a subject to one or more ,~-blocking agents, comprising detecting for
the subject the presence or absence of Val at position 646 of SEQ ID
N0:2 or a -G- nucleotide at a position corresponding to position 2073 of
SEQ ID NO: 1, wherein the presence of a Val at position 646 of SEQ ID
N0:2 or a -G- at nucleotide 2073 of SEQ ID N0:1, is indicative of an
increased likelihood that a subject is non-responsive to one or more ,~-
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_$_
blocking agents compared to a subject who does not have the allelic
variant.
Also provided herein are methods for determining responsiveness
of a subject to one or more ~3-blocking agents, comprising detecting the
presence or absence of Val at position 646 of SEQ ID N0:2 or a -G
nucleotide at a position corresponding to position 2073 of SEQ ID NO: 1,
wherein the presence of a Val at position 646 of SEQ ID N0:2 or a -G- at
nucleotide 2073 of SEQ ID N0:1, is indicative of an increased likelihood
that a subject is hyper-responsive to one or more /3-blocking agents
1 ~ compared to a subject who does not have the allelic variant.
Provided herein are methods for indicating susceptibility of a
subject to acquired long Q-T syndrome, comprising detecting the
presence or absence of Val at position 646 of SEQ ID N0:2 or presence
or absence of a -G- nucleotide at a position corresponding to position
2073 of SEQ ID NO: 1, wherein the presence of a Val at position 646 of
SEQ ID N0:2 or a -G- at nucleotide 2073 of SEQ ID N0:1, is indicative of
increased susceptibility to acquired long Q-T syndrome, compared to the
susceptibility of a subject who does not have the allelic variant. In
accordance with this embodiment, the detecting step can be effected by
a method selected from the group consisting of allele specific
hybridization, primer specific extension, oligonucleotide ligation assay,
restriction enzyme site analysis and single-stranded conformation
polymorphism analysis. Also provided herein, the detecting step can
comprise mass spectrometry. Also provided herein, detection can be
effected by detecting a signal moiety selected from the group consisting
of radioisotopes, enzymes, antigens, antibodies, spectrophotometric
reagents, chemiluminescent reagents, fluorescent reagents and other
light producing reagents.
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Also provided herein, are methods for indicating susceptibility to
morbidity, increased or early mortality, or morbidity and increased or
early mortality of a subject; comprising conducting an EKG exam;
determining the EKG-PR-interval in the subject, wherein if the EKG-PR-
interval is decreased; then determining the amino acid at position 646 of
AKAP10/D-AKAP2 (SEQ ID N0:2) or the nucleotide present at position
corresponding to nucleotide 2073 of SEQ ID N0:1, wherein the presence
of Val at position 646 of SEQ ID N0:2 or the presence of a -G- at
nucleotide position 2073 of SEO. ID N0:1, indicates increased
susceptibility to morbidity, increased or early mortality, or morbidity and
increased or early mortality of a subject.
In one embodiment of each of the methods provided herein, the
subject is heterozygous -GA- at a position corresponding to nucleotide
2073 of SEQ ID N0:1 or heterozygous !!al/Ile at a position corresponding
to position 646 of SEQ ID N0:2. In another embodiment of each of the
methods provided herein, the subject is homozygous -GG- at a position
corresponding to nucleotide 2073 of SEO. ID 10:1 or homozygous
Val/!!al at a position corresponding to position 646 of SECT ID N0:2.
Another non-synonymous D-AKAP2 variation retrieved from dbSNP
2~ has been verified. The G-A transversion in axon 4 results in an Arg to
His substitution at position 249 of SEQ ID N0:2 (R249H; corresponding
to a G to A transversion at nucleotide 883 of SEQ ID N0:1 encoding
human D-AKAP2). The Arg at residue 249 of SEQ ID N0:2 was found to
be in complete linkage disequilibrium with the Ile at position 646 of SEQ
ID N0:646, occurring together in every case, and therefore shows the
same age effect. Accordingly, in the each of the methods provided
herein, where a subject is assayed for the genotype at position 2073 of
SEQ ID N0:1, the subject can also be assayed for the genotype at
position 883 of SEQ ID N0:1. The genotype -G- at position 883 of SEQ
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ID N0:1 corresponds to genotype -A- at nucleotide 2073 of SEQ ID N0:1
and vice versa. Likewise, in the methods provided herein the genotype -
A- at position 883 of SEQ ID N0:1 corresponds to genotype -G- at
nucleotide 2073 of SEQ ID N0:1 and vice versa. For amino acid
residues, the detection of a Ile at residue 646 of SEQ ID N0:2
corresponds to detection of an Arg at residue 249 of SEQ ID N0:2 and
vice versa. Likewise, the detection of a Val at residue 646 of SEQ ID
N0:2 corresponds to detection of an His at residue 249 of SEQ ID N0:2
and vice versa.
Further provided are kits for practicing the methods. The kits can
include reagents for assessing genotype of an AICAP allele and also
reagents and/or components for conducting an EICG and/or a ,~-blocking
agent. For e~:ample, a kit for assessing genotype can include a primer or
probe that specifically hybridizes adjacent to or at a polymorphic region
spanning a position corresponding to position 2073 of SEQ ID NO 1 or 3
of an AICAP10 allele or the complement thereof and a second primer or
probe that specifically hybridizes adjacent to or at a polymorphic region
spanning a position corresponding to positions selected from the group
consisting of position 83587 of SEQ ID NO 13 or 17, position 129600 of
SEQ ID NO 14 or 17, and position 156,277 of SEQ ID NO 18 or 17 of an
AICAP10 allele or the complement thereof. Primers include, but are not
limited to, nucleic acids consisting essentially of the nucleotide sequence
of SEQ ID NO: 8, SEQ ID N0: 15, SEQ ID NO: 19 and SEQ ID NO 20.
Other genotyping components of the kit can include a first primer or
probe that specifically hybridizes adjacent to or at a polymorphic region
spanning a position corresponding to position 883 of SEQ ID NO 1 or 3
of an AKAP10 allele or the complement thereof and a second primer or
probe that specifically hybridizes adjacent to or at a polymorphic region
spanning a position corresponding to positions selected from the group
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consisting of position 83587 of SEQ ID NO 13 or 17, position 129600 of
SEQ ID NO 14 or 17, and position 156,277 of SEQ ID NO 18 or 17 of an
AKAP10 allele or the complement thereof. The kits optionally contain
instructions for performing assays, interpreting results or for aiding in
peforming the methods. The kits also can include at least one
didieoxynucleotide such as ddA, ddC, ddG.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the results of an analysis of covariance that was
conducted t~ test the effect of the genotypes on PR mean levels. Age
was included as a covariate in the model, which was significantly
associated with PR mean up to a third order polynomial. The relationship
between age and PR mean was genotype-dependent, and therefore
interaction terms between genotype and age were included. The
predicted values from the resulting model are shown in Figure 1 for each
genotype.
DETAILED DESCRIPTION
D~fir9iti~r~~
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as is commonly understood by one of skill
in the art to which the inventions) belong. All patents, patent
applications, published applications and publications, GENBANK
sequences, websites and other published materials referred to throughout
the entire disclosure herein, unless noted otherwise, are incorporated by
reference in their entirety. In the event that there are a plurality of
definitions for terms herein, those in this section prevail. Where
reference is made to a URL or other such identifier or address, it is
understood that such identifiers can change and particular information on
the Internet can come and go, but equivalent information is known and
can be readily accessed, such as by searching the Internet and/or
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appropriate databases. Reference thereto evidences the availability and
public dissemination of such information.
As used herein, the phrase "EKG examination" or "ECG
examination" refers to the well-known electrocardiogram examination
that generates an electrical recording of the heart and is conducted on
human subjects to investigate heart function and heart disease.
As used herein, the phrase "PR-interval" or "EKG-PR-interval", in
the context of an electrocardiogram (EKG or ECG) analysis, is the time
(typically expressed herein in units of milliseconds) elapsed between the
beginning of the P wave to the beginning of the next QRS complex. It
corresponds to the time lag from the onset of atrial depolarization to the
onset of ventricular depolarization. This time lag allows atrial systole to
occur, filling the ventricles before ventricular systole. f~iost of the delay
occurs in the Al.~ node. The PR interval is longer with high vagal tone. A
prolonged PR interval corresponds to impaired AV conduction. The
normal range of PR-intervals from about 120 to 200 milliseconds. It is
well-knoe~n that each sguare on a EI~G readout (graph) corresponds to
40 milliseconds.
As used herein the phrase "predetermined standard" refers to an
average of a multiplicity of EKG-PR-intervals that can be empirically
determined from a specifically chosen group of individuals. The group of
individuals can be selected irrespective of disease status, e.g., from a
healthy patient database. In another embodiment, the standard can be
obtained from a group of control age-matched subjects that do not have
a particular disease, such as heart disease. In another embodiment, the
predetermined standard can be obtained from a known age-matched
control that is homozygous -AA- at a position corresponding to
nucleotide 2073 of SEQ ID N0:1 or homozygous Ile/Ile at a position
corresponding to position 646 of SEQ ID N0:2.
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As used herein "age-matched standard EKG-PR-interval" refers to
the average PR-interval (also referred to herein as "PRmean") for a
multiplicity of subjects of the same age. In addition, the average PR-
interval can be obtained from controls having the same genotype, such
as -AA- homozygotes and/or -GA- heterozygotes at a position
corresponding to nucleotide 2073 of SEQ ID N~:1. Accordingly, the
EKG-PR-interval can be stratified by age and/or genotype. For example,
the EKG-PR-interval of the subject being examined can be compared to
PRmean of either: a group of control subjects of the same age having the
-AA- homozygous genotype at a position corresponding to nucleotide
2073 of SEQ ID N0:1; a group of control subjects of the same age
having the -GA- heterozygous genotype at a position corresponding to
nucleotide 2073 of SEA ID N~:1; both groups of control subjects of the
same age having either the -AA- homozygous or the -GA- homozygous
genotype at a position corresponding to nucleotide 2073 of SEQ ID
N0:1. In another embodiment, the EKG-PR-interval of the subject being
e;zamined can be compared to the PRmean of a group of control subjects
of the same age having any genotype at a position corresponding to
nucleotide 2073 of SEQ ID N~:1.
As used herein, the phrase "disease or disorders" is meant to
encompass all genetic or physiological irregularities that can be attributed
to a particular body organ or physiological or cellular system. As used
herein, the phrase "disorder or disease associated with a decreased EKG-
PR-interval" or grammatical variations thereof, refers to any disease or
disorder that exhibits a decreased PR-interval as one of its
characteristics. Exemplary disorders and/or diseases contemplated herein
as associated with decreased EKG-PR-intervals include, but are not
limited to, those involving alterations in cellular protein phosphorylation
and/or signal transduction. Among such disorders and diseases are:
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neurodegeneratives diseases, such as Alzheimer's Disease,
cardiovascular disorders, cardiac disorders, particularly disorders
associated with altered left ventricular function, cardiomyopathies,
proliferative disorders, bipolar disorder and other neurological disorders,
obesity, diabetes and certain peripheral retinopathies, such as retinitis
pigmentosa. As used herein, cardiovascular disorders or cardiac disease
collectively encompass all cardiovascular abnormalities, such as, but not
limited to congenital heart disease, cardiac arrhythmia, brachycardia,
atrial fibrillation, sick sinus syndrome, sudden cardiac arrest, ventricular
1 ~ arrythmia, ventricular fibrillation, ventricular tachycardia, Wolf-
Parkinson-
White (WPW) Syndrome, Lown-Ganong-Levin (LGL) Syndrome,
hypertension, familial cardiac myxomas and Garney complex.
As used herein, a "decreased" EICG-PR-interval or grammatical
variations thereof, refers to a PR-interval that is lower than the average
PR-interval (e.g., PRmean) for subjects of the same age group. As set
forth herein (e.g., Figure 1 ) the average PR-interval (e.g., PRmean)
increases with age for subjects having either a llomozygous -A~4- or
heterozygous -GA- genotype at a position corresponding t~ nucleotide
2073 of SEO. ID N0:1 encoding the AKAP10/D-AKAP2 protein. For
subjects (e.g., humans) having the homozygous -GG- genotype at a
position corresponding to nucleotide 2073 of SEQ ID N0:1, it has been
found herein that the average PR-interval is decreased relative to subjects
of the same age that have either a homozygous -AA- or heterozygous -
GA- genotype. The decreased EICG-PR-interval is evident in subjects
from the age of about 40 up to about 70 years of age.
In addition, although the average PR-interval for the -GA-
heterozygotes increases with age as with the -AA- homozygotes, the
average PR-interval (e.g., PRmean) is always lower for the -GA-
heteroaygotes than for the -AA- homozygotes (see Figure 1 ).
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Accordingly, in one embodiment, the EKG-PR-interval of the subject
being examined is compared to the PRmean (e.g., average PR-interval) of
age-matched -AA- homozygotes. This embodiment serves as a
preliminary screen for subjects that have either 1 or 2 copies of the
1646V variant (e.g., screen for potential -GA- heterozygotes or -AA-
homozygotes at a nucleotide position corresponding to nucleotide 2073
of SEQ ID N0:1 ). In another embodiment, the EKG-PR-interval of the
subject being examined is compared to the PRmean (e.g., average PR-
interval) of age-matched -GA- heterozygotes. This embodiment serves as
preliminary screen for subjects that have 2 copies of the 1646V variant,
e.g., subjects that are -GG- homozygotes.
In the methods provided herein, if the EKG-PR-interval is not
decreased relative to the age-matched average PR-interval (PRmean) for -
AA- homozygotes and/or -GA- heterozygotes at a position corresponding
to nucleotide 2073 of SEQ ID N0:1, then there is no need to determine
the genotype of the subject.
As used herein, a ~-T interval is the time from electrocardiogram Q
wave to the end of the T wave corresponding to electrical systole. This
interval represents the time required for depolarization and repolarization
to occur. In long QT syndrome, the duration of repolarization is longer
than normal. Thus, the QT-interval is prolonged. An interval above 440
milliseconds (msec) is considered prolonged. An interval at or above 430
milliseconds in females or 470 milliseconds in males typically is sufficient
to diagnose a subject as having long QT syndrome.
As used herein, sequencing refers to the process of determining a
nucleotide sequence and can be performed using any method known to
those of skill in the art. For example, if a polymorphism is identified or
known, and it is desired to assess its frequency or presence in nucleic
acid samples taken from the subjects that comprise the database, the
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region of interest from the samples can be isolated, such as by PCR or
restriction fragments, hybridization or other suitable method known to
those of skill in the art, and sequenced. For purposes herein, sequencing
analysis can be carried out using mass spectrometry (see, e.g., U.S.
Patent Nos. 5,547,835, 5,622,824, 5,851,765, and 5,928,906).
Nucleic acids can also be sequenced by hybridization (see, e.g., U.S.
Patent Nos. 5,503,980, 5,631,134, 5,795,714) and including analysis
by mass spectrometry (see, U.S. Application Serial Nos. 08/419,994 and
09/395,409). Alternatively, sequencing can be performed using other
1~ known methods, such as set forth in U.S. Patent Nos. 5,525,464;
5,695,940; 5,834,189; 5,869,242; 5,876,934; 5,908,755; 5,912,118;
5,952,174; 5,976,802; 5,981,186; 5,998,143; 6,004,744; 6,017,702;
6,018,041; 6,025,136; 6,046,005; 6,087,095; 6,1 17,634, 6,013,431,
W0 98/30883; W0 98/56954; W0 99/09218; W~/00/58519, and the
others.
As used herein, "polymorphism" refers to the coexistence of more
than one form of a gene or portion thereof. A portion of a gene of which
there are at least two different forms, i.e., two different nucleotide
sequences, is referred to as a "polymorphic region of a gene". A
polymorphic region can be a single nucleotide, the identity of which
differs in different alleles. A polymorphic region can also be several
nucleotides in length.
As used herein, "polymorphic gene" refers to a gene having at
least one polymorphic region.
As used herein, "allele", which is used interchangeably herein with
"allelic variant" refers to alternative forms of a gene or portions thereof.
Alleles occupy the same locus or position on homologous chromosomes.
When a subject has two identical alleles of a gene, the subject is the to
be homozygous for the gene or allele. When a subject has two different
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alleles of a gene, the subject is the to be heterozygous for the gene.
Alleles of a specific gene can differ from each other in a single
nucleotide, or several nucleotides, and can include substitutions,
deletions, and insertions of nucleotides. An allele of a gene can also be a
form of a gene containing a mutation.
As used herein, "predominant allele" refers to an allele that is
represented in the greatest frequency for a given population. The allele
or alleles that are present in lesser frequency are referred to as allelic
variants.
As used herein, "associated" refers to coincidence with the
development or manifestation of a disease, condition or phenotype.
Association can be due to, but is not limited to, genes rasp~nsible for
housekeeping functions wh~se alteration can provide the foundati~n for a
variety of diseases and conditions, those that are part of a pathway that
is involved in a specific disease, condition or phenotype and those that
indirectly contribute to the manifestation of a disease, condition or
phenotype.
As used herein, the term "subject" refers to mammals and in
particular human beings.
~0 As used herein, the term "gene" or "recombinant gene" refers to a
nucleic acid molecule comprising an open reading frame and including at
least one axon and (optionally) an intron sequence. A gene can be either
RNA or DNA. Genes can include regions preceding and following the
coding region (leader and trailer).
As used herein, "intron" refers to a DNA sequence present in a
given gene which is spliced out during mRNA maturation.
As used herein, "nucleotide sequence complementary to the
nucleotide sequence set forth in SEQ ID NO: x" refers to the nucleotide
sequence of the complementary strand of a nucleic acid strand having
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SEQ ID NO: x. The term "complementary strand" is used herein
interchangeably with the term "complement". The complement of a
nucleic acid strand can be the complement of a coding strand or the
complement of a non-coding strand. When referring to double stranded
nucleic acids, the complement of a nucleic acid having SEQ ID NO: x
refers to the complementary strand of the strand having SEQ ID NO: x or
to any nucleic acid having the nucleotide sequence of the complementary
strand of SEQ ID NO: x. When referring to a single stranded nucleic acid
having the nucleotide sequence SEQ ID NO: x, the complement of this
nucleic acid is a nucleic acid having a nucleotide sequence which is
complementary to that of SEQ ID NO: x.
As used herein, the term "coding sequence" refers to that portion
of a gene that encodes an amino acid sequence of a protein.
As used herein, the term "sense strand" refers to that strand of a
double-stranded nucleic acid molecule that has the sequence of the
mRNA that encodes the amino acid sequence encoded by the double-
stranded nucleic acid molecule.
As used herein, the term "antiasnsa strand" refers to that strand of
a double-stranded nucleic acid molecule that is the complement of the
sequence of the mRNA that encodes the amino acid sequence encoded
by the double-stranded nucleic acid molecule.
As used herein, the amino acids, which occur in the various amino
acid sequences appearing herein, are identified according to their well-
known, three-letter or one-letter abbreviations. The nucleotides, which
occur in the various DNA fragments, are designated with the standard
single-letter designations used routinely in the art (see, Table 1 ).
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As used herein, amino acid residue refers to an amino acid formed
upon chemical digestion (hydrolysis) of a polypeptide at its peptide
linkages. The amino acid residues described herein are typically in the
"L" isomeric form. Residues in the "D" isomeric form can be substituted
for any L-amino acid residue, as long as the desired functional property is
retained by the polypeptide. NH2 refers to the free amino group present
at the amino terminus of a polypeptide. COOH refers to the free carboxy
group present at the carboxyl terminus of a polypeptide. In keeping with
standard polypeptide nomenclature described in J. Bi~l. ahem.~
243:3552-59 (1969) and adopted at 37 C.F.R. ~ ~ 1.321 - 1.822,
abbreviations for amino acid residues are shown in the following Table:
Table 1
Table of Correspondence
SYIl~ 80L
1-Letter 3-Letter Af~ilf~~ ACI~
Y Tyr tyrosine
G Gly glycine
F Phe phenylalanine
IVY f!/let methionine
2~ A Ala alanine
S Ser serine
I Ile isoleucine
L Leu leucine
T Thr threonine
V Val valine
P Pro proline
IC Lys lysine
H His histidine
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SYM BOL
Q Gln glutamine
E Glu glutamic acid
Z Glx Glu and/or Gln
W Trp tryptophan
R Arg arginine
D Asp aspartic acid
N Asn asparagine
B Asx Asn and/or Asp
C Cys cysteine
~0 ?~ Xaa Unknown or other
It should be noted that all amino acid residue sequences
represented herein by formulae have a left to right orientation in the
conventional direction of amino-terminus to carboxyl-terminus. In
'i 5 addition, the phrase °°amino acid residue'° is
broadly defined to include
the amino acids listed in the Table of Correspondence and modified and
unusual amino acids, such as those referred to in 37 C.F.R. ~ ~ 1.821-
1.822, and incorporated herein by reference. Furthermore, it should be
noted that a dash at the beginning or end of an amino acid residue
20 sequence indicates a peptide bond to a further sequence of one or more
amino acid residues or to an amino-terminal group such as NH2 or to a
carboxyl-terminal group such as COOH.
In a peptide or protein, suitable conservative substitutions of
amino acids are known to those of skill in this art and can be made
25 generally without altering the biological activity of the resulting
molecule.
Those of skill in this art recognize that, in general, single amino acid
substitutions in non-essential regions of a polypeptide do not
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substantially alter biological activity (see, e.g., Watson et al. Molecular
Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub.
co., p.224).
Such substitutions are typically made in accordance with those set
forth in TABLE 2 as follows:
TABLE 2
Original residue Conservative substitution
Ala (A) Gly; Ser
Arg (R) Lys
Asn (N) Gln; His
Cys (C) Ser
Gln (Q) Asn
Glu (E) Asp
Gly (G) Ala; Pro
His (H) Asn; Gln
Ile (I) Leu; !/al
Leu (L) Ile; val
Lys (IC) Arg; Gln; Glu
Met (fVl) Leu; Tyr; Ile
Phe (F) Met; Leu; Tyr
Ser (S) Thr
Thr (T) Ser
Trp (VV) Tyr
Tyr (1') Trp; Phe
dal (!/) Ile; Leu
Other substitutions permissible and can be determined
are also
empirically or in accord with known conservative substitutions.
As used herein, a DNA or nucleic acid homolog refers to a nucleic
acid that includes a preselected conserved nucleotide sequence, such as
a sequence encoding a therapeutic polypeptide. By the term
°'substantially homologous°' is meant having at least 80%,
typically at
least 90%, or at least 95% homology therewith or a less percentage of
homology or identity and conserved biological activity or function.
The terms "homology" and "identity" are often used
interchangeably. In this regard, percent homology or identity can be
determined, for example, by comparing sequence information using a
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GAP computer program. The GAP program uses the alignment method of
Needleman and Wunsch (J. Mol. Biol. 48:443 (1970), as revised by
Smith and Waterman (Adv. App/. Math. 2:482 (1981). Briefly, the GAP
program defines similarity as the number of aligned symbols (i.e.,
nucleotides or amino acids) which are similar, divided by the total number
of symbols in the shorter of the two sequences. The default parameters
for the GAP program can include: (1) a unary comparison matrix
(containing a value of 1 for identities and 0 for non-identities) and the
weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.
14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS
~F PR~TElN SEQUENCE AN~ STRUCTURE, National Biomedical
Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each
gap and an additional 0.10 penalty for each symbol in each gap; and (3)
no penalty for
end gaps.
Whether any two nucleic acid molecules have nucleotide
sequences that are at least 80°/~, 85°/~, 90°/~,
95°/~, 95°/~, 9%°/~, 98°/~ or
99°/~ "identical" can be determined using known computer algorithms
such as the "FAST A" program, using for example, the default
parameters as in Pearson and Lipman, Proe. Nat/. Acad. Sci, USA
85:2444 (1988). Alternatively the BLAST function of the National
Center for Biotechnology Information database can be used to determine
identity
In general, sequences are aligned so that the highest order match
is obtained. "Identity" per se has an art-recognized meaning and can be
calculated using published techniques. (See, e.g.: Computational
Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York,
1988; Biocomputing: Informatics and Genome Projects, Smith, D.W.,
ed., Academic Press, New York, 1993; Computer Analysis of Seguence
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Data, Part l, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G.,
Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and
Devereux, J., eds., M Stockton Press, New York, 1991 ). While there
exist a number of methods to measure identity between two
polynucleotide or polypeptide sequences, the term "identity" is well
known to skilled artisans (Carillo, H. & Lipton, D., SIAM ,J Applied Math
4B:1073 (1988)). Methods commonly employed to determine identity or
similarity between two sequences include, but are not limited to, those
disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic
Press, San Diego, 1994, and Carillo, H. ~ Lipton, D., SIAM J Applied
Math 45:1073 (1988). Methods to determine identity and similarity are
codified in computer programs. Typical computer program methods to
determine identity and similarity between two sequences include, but are
not limited to, GCG program package (Devereux, J., et al., Nucleic Acids
Research '2(/J:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S.F., et
a/.° ~d lls~~lec Biol ~~~:403 (1990)).
Therefore, as used herein, the term "identity" represents a
comparison between a test and a reference polypeptide or
polynucleotide. For example, a test polypeptide can be defined as any
polypeptide that is 90°/~ or more identical to a reference polypeptide.
As used herein, the term at least "90°/~ identical to" refers to
percent identities from 90 to 99.99 relative to the reference
polypeptides. Identity at a level of 90% or more is indicative of the fact
that, assuming for exemplification purposes a test and reference
polypeptide length of 100 amino acids are compared. No more than
10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from
that of the reference polypeptides. Similar comparisons can be made
between a test and reference polynucleotides. Such differences can be
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represented as point mutations randomly distributed over the entire
length of an amino acid sequence or they can be clustered in one or
more locations of varying length up to the maximum allowable, e.g.
10/100 amino acid difference (approximately 90% identity). Differences
are defined as nucleic acid or amino acid substitutions, or deletions.
As used herein, stringency conditions refer to the washing
conditions for removing the non-specific probes and conditions that are
equivalent to either high, medium, or low stringency as described below:
1) high stringency: 0.1 x SSPE, 0.1% SDS, 65°C
2) medium stringency: 0.2 x SSPE, 0.1 % SDS, 50°C
3) low stringency: 1.0 x SSPE, 0.1 % SDS, 50°C.
It is understood that equivalent stringencies can be achieved using
alternative buffers, salts and temperatures.
As used herein, "heterologous DNA" is DNA that encodes RNA
and proteins that are not normally produced in vivo by the cell in which it
is expressed or that mediates or encodes mediators that alter expression
of endogenous Df~A by affecting transcription, translation, or otller
regulatable biochemical processes or is not present in the exact
orientation or position as the counterpart DNA in a wildtype cell.
Heterologous DNA can also be referred to as foreign DNA. Any DNA
that one of skill in the art would recognize or consider as heterologous or
foreign to the cell in which is expressed is herein encompassed by
heterologous DNA. Examples of heterologous DNA include, but are not
limited to, DNA that encodes traceable marker proteins, such as a protein
that confers drug resistance, DNA that encodes therapeutically effective
substances, such as anti-cancer agents, enzymes and hormones, and
DNA that encodes other types of proteins, such as antibodies.
Antibodies that are encoded by heterologous DNA can be secreted or
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expressed on the surface of the cell in which the heterologous DNA has
been introduced.
As used herein, isolated with reference to a nucleic acid molecule
or polypeptide or other biomolecule means that the nucleic acid or
polypeptide has separated from the genetic environment from which the
polypeptide or nucleic acid were obtained. It can also mean altered from
the natural state. For example, a polynucleotide or a polypeptide
naturally present in a living animal is not "isolated," but the same
polynucleotide or polypeptide separated from the coexisting materials of
its natural state is "isolated", as the term is employed herein. Thus, a
polypeptide or polynucleotide produced and/or contained within a
recombinant host cell is considered isolated. Also intended as an
"isolated polypeptide'° or an "isolated polynucleotide°' are
polypeptides or
polynucleotides that have been purified, partially or substantially, from a
recombinant host cell or from a native source. For example, a
recombinantly produced version of a compound can be substantially
purified by the one-step method described in Smith and Johnson, ~eoe
~a~:~1-4~ (19~~). The terms isolated and purified are sometimes used
interchangeably.
2~ Thus, by "isolated" is meant that the nucleic acid is free of the
coding sequences of those genes that, in the naturally-occurring genome
of the organism (if any) immediately flank the gene encoding the nucleic
acid of interest. Isolated DNA can be single-stranded or double-stranded,
and can be genomic DNA, cDNA, recombinant hybrid DNA, or synthetic
DNA. It can be identical to a native DNA sequence, or can differ from
such sequence by the deletion, addition, or substitution of one or more
nucleotides.
Isolated or purified as it refers to preparations made from biological
cells or hosts means any cell extract containing the indicated DNA or
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protein including a crude extract of the DNA or protein of interest. For
example, in the case of a protein, a purified preparation can be obtained
following an individual technique or a series of preparative or biochemical
techniques and the DNA or protein of interest can be present at various
degrees of purity in these preparations. The procedures can include for
example, but are not limited to, ammonium sulfate fractionation, gel
filtration, ion exchange change chromatography, affinity chromatography,
density gradient centrifugation and electrophoresis.
A preparation of DNA or protein that is "substantially pure" or
"isolated" should be understood to mean a preparation free from
naturally occurring materials with which such DNA or protein is normally
associated in nature. "Essentially pure" should be understood to mean a
"highly" purified preparation that contains at least 95% of the DNA or
protein of interest.
A cell extract that contains the DNA or protein of interest should
be understood to mean a homogenate preparation or cell-free preparation
obtained from sells that e~epress the protein or contain the DNA of
interest. The term '°cell extract" is intended to include culture
media,
especially spent culture media from which the cells have been removed.
~~ As used herein, receptor refers to a biologically active molecule
that specifically binds to (or with) other molecules. The term
°'receptor
protein" can be used to more specifically indicate the proteinaceous
nature of a specific receptor.
As used herein, recombinant refers to any progeny formed as the
result of genetic engineering.
As used herein, a promoter region refers to the portion of DNA of
a gene that controls transcription of the DNA to which it is operatively
linked. The promoter region includes specific sequences of DNA that
are sufficient for RNA polymerise recognition, binding and transcription
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initiation. This portion of the promoter region is referred to as the
promoter. In addition, the promoter region includes sequences that
modulate this recognition, binding and transcription initiation activity of
the RNA polymerase. These sequences can be cis acting or can be
responsive to traps acting factors. Promoters, depending upon the
nature of the regulation, can be constitutive or regulated.
As used herein, the phrase "operatively linked" generally means
the sequences or segments have been covalently joined into one piece of
DNA, whether in single or double stranded form, whereby control or
regulatory sequences on one segment control or permit expression or
replication or other such control of other segments. The two segments
are not necessarily contiguous. For gene earpression a DNA sequence
and a regulatory sequences) are connected in such a way to control or
permit gene expression when the appropriate molecular, e.g.,
transcriptional activator proteins, are bound to the regulatory
sequences) .
~,s used herein, production by recombinant means by using
recombinant DNA methods means the use of the well known methods of
molecular biology for expressing proteins encoded by cloned DNA,
including cloning expression of genes and methods, such as gene
shuffling and phage display with screening for desired specificities.
As used herein, the term °°conjugated" refers stable
attachment,
such ionic or covalent attachment.
As used herein, a composition refers to any mixture of two or
more products or compounds. It can be a solution, a suspension, liquid,
powder, a paste, aqueous, non-aqueous or any combination thereof.
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As used herein, a combination refers to any association between
two or more items.
As used herein, substantially identical to a product means
sufficiently similar so that the property of interest is sufficiently
unchanged so that the substantially identical product can be used in
place of the product.
As used herein, the term "vector" refers to a nucleic acid molecule
capable of transporting another nucleic acid to which it has been linked.
~ne typical vector is an episome, i.e., a nucleic acid capable of
1 ~ extra-chromosomal replication. Typical vectors are those capable of
auton~mous replication and/or expression of nucleic acids to which they
are linked. Vectors capable of directing the expression of genes t~ which
they are operatively linked are referred to herein as "expression vectors".
In general, expression vectors of utility in recombinant ~f~A techniques
are often in the form of "plasmids" which refer generally to circular
double stranded ~IVA I~ops which, in their vector form are not bound to
the chr~m~some. '°Plasmid" and "vector" are used interchangeably as the
plasmid is the most commonly used f~rm of vector. ~ther such other
forms of expression vectors that serve equivalent functions and that
2~ become known in the art subsequently hereto.
As used herein, "indicating" or "determining" means that the
presence or absence of an allelic variant can be one of many factors that
are considered when a subject's predisposition to a disease or disorder is
evaluated. Thus a predisposition to a disease or disorder is not
necessarily conclusively determined by only ascertaining the presence or
absence of one or more allelic variants, but the presence of one of more
of such variants is among a number of factors considered.
As used herein, "predisposition to develop a disease or disorder"
means that a subject having a particular genotype and/or haplotype has a
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higher likelihood than one not having such a genotype and/or haplotype
for developing a particular disease or disorder.
As used herein, "morbidity" refers to conditions, such as diseases
or disorders, that compromise the health and well-being of an organism,
such as an animal. Morbidity susceptibility or morbidity-associated genes
are genes that, when altered, for example, by a variation in nucleotide
sequence, facilitate the expression of a specific disease clinical
phenotype. Thus, morbidity susceptibility genes have the potential, upon
alteration, of increasing the likelihood or general risk that an organism
will develop a specific disease.
As used herein, "mortality" refers to the statistical likelihood that
an organism, particularly an animal, will not survive a full predicted
lifespan. Hence, a trait or a marker, such as a polymorphism, associated
with increased mortality is observed at a lower frequency in older than
younger segments of a population.
As used herein, "transgenic animal" refers to any animal, typically
a non-human animal, e.~. a mammal, bird or an amphibian, in which one
or more of the cells of the animal contain heterologous nucleic acid
introduced by way of human intervention, such as by transgenic
techniques well known in the art. The nucleic acid is introduced into the
cell, directly or indirectly by introduction into a precursor of the cell, by
way of deliberate genetic manipulation, such as by microinjection or by
infection with a recombinant virus. The term genetic manipulation does
not include classical cross-breeding, or in vitro fertilization, but rather is
directed to the introduction of a recombinant DNA molecule. This
molecule can be integrated within a chromosome, or it can be
extrachromosomally replicating DNA. In the typical transgenic animals
described herein, the transgene causes cells to express a recombinant
form of a protein. Transgenic animals in which the recombinant gene is
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silent are also contemplated, as for example, using the FLP or CRE
recombinase dependent constructs. Moreover, "transgenic animal" also
includes those recombinant animals in which gene disruption of one or
more genes is caused by human intervention, including recombination
and antisense techniques.
As used herein, "target nucleic acid" refers to a nucleic acid
molecule which contains all or a portion of a polymorphic region of a
gene of interest.
As used herein, "signal moiety" refers to any moiety that allows
for the detection of a nucleic acid molecule. Included are moieties
covalently attached to nucleic acids and those that are not.
As used herein, "molecule that modulates or effects the biological
activity of an AICAP10 protein" refers to any drug, small molecule,
nucleic acid (sense and antiasnsa), ribozyme, protein, peptide, lipid,
carbohydrate ate. or combination thereof, that directly or indirectly
changes, alters, abolishes, increases or decreases a biological activity
attributed to AI~AP10 protein.
As used herein, "biological activity of an AI~AP10 protein" refers
to, but is not limited to, binding of AI~AP10 to protein leinase A or its
subunits, localization of AICAP10 protein to a subcellular site, e.g., the
mitochondria, localization of protein lcinase A to the mitochondria and
binding of AICAP10 protein to other proteins including other signaling
enzymes.
As used herein, "combining" refers to contacting the biologically
active agent with a cell or animal such that the agent is introduced into
the cell or animal. For a cell any method that results in an agent
traversing the plasma membrane is useful. For an animal any of the
standard routes of administration of an agent, e.g. oral, rectal,
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transmucosal, intestinal, intravenous, intraperitoneal, intraventricular,
subcutaneous, intramuscular, ete., can be used.
As used herein, a composition refers to any mixture. It can be a
solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or
any combination thereof.
As used herein, a combination refers to any association between
two or among more items.
As used herein, "kit" refers to a package that contains a
combination, such as one or more primers or probes used to amplify or
detect polymorphic regions of AKAP10 genes, optionally including
instructions and/or reagents for their use.
As used herein, "solid support" refers to a support substrate or
matrix, such as silica, polymeric materials or glass. At least one surface
of fibs support can be partially planar. Regions of the support can be
physically separated, for example with trenches, grooves, well or the
like. Some examples of solid supports include slides and beads.
Supports are of such composition so as to allow for the immobilization or
attachment of nucleic acids and other molecules such that these
molecules retain their binding ability.
As used herein, "array" refers to a collection of elements, such as
nucleic acids, containing three or more members. An addressable array
is one in which the members of the array are identifiable, typically by
position on a solid support. Hence, in general the members of the array
will be immobilized to discrete identifiable loci on the surface of a solid
phase.
As used herein, "specifically hybridizes" refers to hybridization of a
probe or primer only to a target sequence preferentially to a non-target
sequence. Those of skill in the art are familiar with parameters that
affect hybridization; such as temperature, probe or primer length and
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composition, buffer composition and salt concentration and can readily
adjust these parameters to achieve specific hybridization of a nucleic acid
to a target sequence.
As used herein "nucleic acid" refers to polynucleotides such as
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term
should also be understood to include, as equivalents, derivatives,
variants and analogs of either RNA or DNA made from nucleotide
analogs, single (sense or antisense) and double-stranded polynucleotides.
Deoxyribonucleotides include deoxyadenosine, deoxycytidine,
deoxyguanosine and deoxythymidine. For RNA, the uracil base is
uridine.
As used herein, "mass spectrometry" encompasses any suitable
mass spectrometric format known to those of skill in the art. Such
formats include, but are not limited to, Matrix-Assisted Laser
Desorption/lonization, Time-of-Flight (MALDI-TOF), Electrospray (ES), IR-
MALDI (see, e.g., published International PCT Application No.
VSO 99/ 57518 and U.S. Patent i~o. 5,1 18,937) Ion Oyclotron Resonance
(10R), Fourier Transform and combinations thereof. I~ALDI, particular UV
and IR, are among the typical formats.
As used herein, "at a position corresponding to" refers to a
position of interest (i.e., base number or residue number) in a nucleic acid
molecule or protein relative to the position in another reference nucleic
acid molecule or protein. Corresponding positions can be determined by
comparing and aligning sequences to maximize the number of matching
nucleotides or residues, for example, such that identity between the
sequences is greater than 95%, greater than 96%, greater than 97%,
greater than 98%, or greater than 99%. The position of interest is then
given the number assigned in the reference nucleic acid molecule. For
example, it is shown herein that a particular polymorphism in AKAP10
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occurs at nucleotide 2073 of SEQ ID No. 1. To identify the
corresponding nucleotide in another allele or isolate, the sequences are
aligned and then the position that lines up with 2073 is identified. Since
various alleles can be of different length, the position designate 2073 can
not be nucleotide 2073, but instead is at a position that "corresponds" to
the position in the reference sequence.
As used herein, "primer" and "probe" refer to a nucleic acid
molecule including DNA, RNA and analogs thereof, including protein
nucleic acids (PNA), and mixtures thereof. Such molecules are typically
of a length such that they are statistically unique (i.e., occur only once)
in the genome of interest. Generally, for a probe or primer to be unique
in the human genome, it contains at least 14, 16 or contiguous
nucleotides of a sequence complementary to or identical to a gene of
interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleic
acids long.
As used herein, "antiasnsa nucleic acid molecule" refers to a
molr~cule encoding a sequence complementary to at least a portion of an
RNA molecule. The sequence is sufficiently complementary to be able to
hybridize with the RNA, typically under moderate or high stringency
conditions to form a stable duplex. The ability to hybridize depends on
the degree of complementarity and the length of the antiasnsa nucleic
acid. Generally, the longer the hybridizing nucleic acid, the more base
mismatches with an RNA it can contain and still form a stable duplex.
One skilled in the art can ascertain a tolerable degree of mismatch by use
of standard procedures to determine the melting point of the hybridized
complex.
As used herein, a "variant protein" refers to a protein encoded by
an allelic variant of a AKAP10 gene which results in a change of an
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amino acid residue at a particular position relative to that position in the
protein encoded by the predominant allele.
As used herein, "signal transduction" refers to the propagation of
a signal. In general, an extracellular signal is transmitted through the cell
membrane to become an intracellular signal. This signal can then
stimulate a cellular response. The term also encompasses signals that
are propagated entirely within a cell. The polypeptide molecules involved
in signal transduction processes are typically receptor and non-receptor
protein kinases, receptor and non-receptor protein phosphatases,
1~ nucleotide exchange factors and transcription factors. One of the key
biochemical mechanisms involved in signal transduction is protein
phosphorylation. AKAP10 proteins are involved in signal transduction as
they bind to protein kinase A (PKA) and are though to anchor the kinase
at a location, e.g., the mitochondria, where PKA acts to phosphorylate a
specific substrate. Thus, an alteration in AKAP10 binding to PKA,
localization to the mitochondria, or phosphorylation by PKA, among other
steps v~rill result in an alteration in signal transduction. Assays including
those that determine phosphorylation by PKA, association of PKA and
AKAP10 and localization of AKAP10 can be used to monitor the state of
2~ signal transduction.
As used herein, "adjacent" refers to a position 5' to the site of a
single nucleotide polymorphism (SNP) such that there could be unpaired
nucleotides between that position and the site of the SNP.
As used herein, "immediately adjacent" refers to a position 5' to
the site of a single nucleotide polymorphism (SNP) such that there are no
unpaired nucleotides between that position and the site of the SNP.
As used herein, "binding to PKA", refers to the interaction of the
PKA binding domain of an AKAP10 protein and the regulatory subunits
RI and/or RII of the protein kinase A holoenzyme.
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B. Methods employing polymorphic AKAPs
Methods herein include a step a identifying the prescence of a
particular allele of an A-kinase anchoring protein (AKAP) genes.
Thus, polymorphic sequences encoding an A-kinase anchoring protein
(AKAP) genes and polymorphic AKAP proteins encoded by polymorphic
AKAP gene sequences are used in methods provided herein. These
polymorphic sequences are based on differences in AKAP genes within
and among different organisms, including humans.
Polymorphisms of the genome can lead to altered gene function,
1~ protein function or mRNA instability. AKAPs provide a mechanism for
regulating ubiquitous cAMP-dependent kinase (PKA) activity by tethering
PKA to specific subcellular locations thereby segregating it with particular
components in a given signaling pathway and contributing to specificity
in cellular responses to extracellular signals. AKAPs thus play a
fundamental role in the basic functioning of cells, the response of cells to
their environment and ultimately in the coordination of vital systems
within an organism. Therefore, polymorphisms in AKAP gene sequences
can affect the proper functioning of cells and systems within organisms
and could be directly linked with certain disorders or could predispose an
2~ organism to a variety of diseases and disorders, especially those
involving alterations in cellular protein phosphorylation and/or signal
transduction. Among such disorders and diseases include, but are not
limited to, neurodegeneratives diseases, such as Alzheimer's Disease,
cardiovascular disorders, cardiac disorders, particularly disorders
associated with altered left ventricular function, cardiomyopathies,
proliferative disorders, bipolar disorder and other neurological disorders,
obesity, diabetes and certain peripheral retinopathies, such as retinitis
pigmentosa. AKAP gene polymorphisms, such as those described herein,
provides for the identification and development of diagnostic and
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prognostic methods, also provided herein, and the development of drug
therapies and treatment regimens. Furthermore, polymorphisms of AKAP
genes aid in the study of AKAP protein structure and function, which
also contributes to the development of diagnostic methods and therapies.
1. AKAP10
The AKAP10 protein is primarily located in mitochondria. The
sequence of a human AKAP10 cDNA (also referred to as D-AKAP2) is
available in the GenBank database, at accession numbers AF037439 and
NM 007202, and is provided in SEQ. ID. N0:1. The AKAP10 gene is
located on chromosome 17.
The sequence of a mouse D-AKAP2 cDNA is also available in the
GenBank database (see accession number AF0~1333). The mouse D-
AKAP2. protein contains an RGS domain near the amino terminus that is
characteristic of proteins that interact with Go° subunits and possess
GTPase activating protein-like activity (Huang et al. (1997) Proc. Nat/.
~Icad. Sci. U.S.~°I. 94:11 1 B4-11 1 B9). The human AKAP10 protein also
has sequences homologous to RGS domains. The carboy-terminal 40
residues of the mouse D-AI~AP~ protein are responsible for the
interaction with the regulatory subunits of PKA. This sequence is fairly
well conserved between the mouse D-AKAP2 and human AKAP10
proteins.
2. P~lym~rphisms ~f the human AKAP10 gene and
p~lym~rphic AKAP10 proteins
Polymorphisms of AKAP genes that alter gene expression,
regulation, protein structure and/or protein function are more likely to
have a significant effect on the regulation of enzyme (particularly PKA)
activity, cellular transduction of signals and responses thereto and on the
basic functioning of cells than polymorphisms that do not alter gene
and/or protein function. Included in the polymorphic AKAPs provided
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herein are human AKAP10 proteins containing differing amino acid
residues at position number 646 of SEQ. ID. No. 2.
Amino acid 646 of the human AKAP10 protein (SEQ. ID. NO: 2) is
located in the carboxy-terminal region of the protein within a segment
that participates in the binding of R-subunits of PKAs. This segment
includes the carboxy-terminal 40 amino acids.
The amino acid residue reported for position 646 of the human
AKAP10 protein is an isoleucine. Polymorphic human AKAP10 proteins
provided herein have the amino acid sequence set forth in SEQ. ID. N0:
2 but contain residues other than isoleucine at amino acid position 646
of the protein. In particular embodiments of the polymorphic human
AKAP10 proteins provided herein, the amino acid at position ~a4G of SEQ.
ID. NO: 2 is a valine (as set forth in SECa. ID. NO: 4), leucine or
phenylalanine residue.
a. An A to G transition at nucleotide 2073 of the human
AKAP10 coding sequence
As described herein, an allelic variant of the human AICR~P10 gene
is at the polymorphic site at position 2073 of the coding sequence (see
SEQ.ID. NO: 3) and encodes a valine at position 646 of the AKAP10
protein. This allelic variant has been found to vary in frequency in DNA
samples from younger and older segments of a healthy population. This
allele has the A at position 2073 of the AKAP10 gene coding sequence
of SEQ. ID. NO: 1 changed to a G, giving rise to the sequence set forth
in SEQ. ID. NO: 3. Consequently, the codon for amino acid 646 changes
from ATT, coding for isoleucine, to GTT, coding for valine.
b. An A to C transversion at nucleotide 2073 of the
human AKAP10 coding sequence
In another human AKAP10 allelic variant, the nucleotide at position
2073 of the coding sequence in SEQ. ID. NO: 1 is changed from an A to
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a C. Thus, changing the codon for amino acid 646 from ATT, coding for
isoleucine, to CTT, coding for leucine.
c. An A to T transversion at nucleotide 2073 of the
human AKAP10 coding sequence
In another human AKAP10 allelic variant, the nucleotide at position
2073 of the coding sequence in SEQ. ID. NO: 1 is changed from an A to
a T. Thus, the codon for amino acid 646 changes from ATT, coding for
isoleucine, to TTT, coding for phenylalanine.
d. Other AKAP10 polymorphisms
TABLE 3
Name GenBank Accession No. SNP Location
10-1 AC005730 T/C 156277
10-7 AC005730 G/A 129600
For AKAP10-1 additional variants are represented by the presence
of A or G at nucleotide position 156277 of SEQ ID NO: 17.
For AI<AP10-7 additional variants are represented by the presence
of C or T at nucleotide position 129600 of SEA ID I~O: 17.
C. Association of AKAP10-5 1646V variant v~ith Cardiac Traits
The SNPs found to be associated with age were analyzed for
association with disease-related quantitative traits in a twin collection.
To identify traits correlated with the observed age association of the
1646V SNP, a cohort of 417 fasting Caucasian twin pairs with extensive
coverage for a variety of disease-related traits was utilized. The analysis
was conducted using a quantitative transmission-disequilibrium test
(QTDT) as described by Abecasis et al., (Am. J. Hum. Genet., 66:279-
292, 2000) to take advantage of the twin-based sample and to control
for admixture and other non-genetic sources of variation. Of the 97
traits analyzed, only the PR-interval from electrocardiogram (EKG or ECG)
analysis was statistically significant at a nominal level of 0.05. The PR
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interval from EKG analysis is referred to herein is "EKG-PR-interval". The
estimate from the QTDT model of the average effect of the G allele (Val
at position 646 of SEQ ID N0:2) was to decrease the PR interval 6.3
units (P = 0.007). The genotype mean values in the subset of 207
informative twin pairs were 157 ~ 23.4, 152 ~ 26.9, and 146 ~ 25.4
(mean ~ standard deviation) for genotypes AA, GA, and GG,
respectively.
An analysis of covariance was also conducted to test the effect of
the genotypes on PR mean levels. Age was included as a covariate in
the model, which was significantly associated with PR mean up to a third
order polynomial as set forth in Figure 1. The relationship between age
and PR mean was genotype-dependent, and therefore interaction terms
between genotype and age were included. The predicted values from the
resulting model are shown in Figure 1 for each genotype. From about
the age of 30 or just beyond and up to about the age of 70, it has been
found that human subjects having the -GG- genotype, on average have a
lower PRmean than human subjects having an -Ad4- hommozygous
genotype at a nucleotide corresponding to position 2073 of SECT ID
N0:1. From about the age of 40, it has also been found that human
2~ subjects having the -GG- genotype, on average have a lower PRmean
than human subjects having either a -GA- heterozygous or -AA-
homozygous genotype at a nucleotide corresponding to position 2073 of
SEQ ID N0:1. Accordingly, on average a human subject about 30 years
or older having a lower PRmean than an age-matched control group of -
AA- homozygous at position 2073 of SEQ ID N0:1 has a higher
likelihood of having either a -GA- heterozygous or a -GG- homozygous
genotype at that position.
Thus, methods of comparing a subjects PR-interval to the PRmean
of an age-matched control group of either one or both of an -AA-
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homozygous or -GA- heterozygous genotype at a nucleotide
corresponding to position 2073 of SEQ ID N0:1 can identify subjects
that have a higher likelihood of possessing a -GG- homozygous genotype
at that position. Once a particular subject is identified as having a lower
EKG-PR-interval than the PRmean from an age-matched control group,
then that subject's particular genotype can be determined using the
methods provided herein at a position corresponding to nucleotide 2073
of SEQ ID N0:1. As set forth herein, those subjects having the -GG-
genotype have an increase susceptibility to a disease or disorder, such as
a cardiovascular disease or disorder.
As also evident from Figure 1, on average a human subject at any
age having a lower PRmean than an age-matched control group of an -
AA- homozygous at a nucleotide corresponding to position 2073 of SEQ
ID NO:1 has a higher likelihood of having a -GA- heterozygous genotype
than a -AA- homozygous genotype. Thus, for embodiments where it
would be useful to identify subjects that can contain at least one "G"
allele at a nucleotide corresponding to position 2073 of SEO ID ~t0:1,
methods of comparing a subjects PR-interval to the PRmean of an age-
matched control group of an -AA- homozygous genotype at a nucleotide
corresponding to position 2073 of SEA ID N0:1 can identify subjects
that have a higher likelihood of possessing at least one -G- allele at that
position.
SNPs in the D-AKAP2 gene have been identified herein that are
associated with morbidity using a genome-wide association study from
an age-stratified healthy population and 6,500 gene-based SNPs. The
combined genetic and biochemical evidence points to the Ile/Val variant
as the functional polymorphism. The Val variant is contemplated to be
the deleterious allele in a Caucasian-American cohort, and this finding is
replicated in Hispanic-Americans. The variant maps to the conserved
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AKB domain of the D-AKAP2 gene. It has been found that the 1646V
variation impacts the binding to PKA in an isoform-specific manner both
in vitro and in cells. The Ile variant have been found to bind three-fold
weaker to the Rla isoform than to the Val variant. At the cellular level,
this affinity difference results in a dramatic decrease in
compartmentalization of Rla for the Ile variant. The Ile/Val variant has
been found to be critical for binding to only the Rla isoform of PKA.
Increasing evidence suggests that the RI and RII isoforms of PKA
have distinct functions. The RI isoform has been implicated in a variety
of biological functions such as cell proliferation, tumor suppression,
immune regulation, and embryonic levelopment. In addition, the Rla
isoform plays a significant r~le in maintaining cAll~P-regulation of PI<A as
evidenced by the embryonic lethality of mice deficient in the gene.
Interestingly, these mice have defects in cardiac morphogenesis.
The identified correlation of the 1646V variant (e.g., -G- at position
2073 of SEQ ID IV0:1 ) with the EKG PR interval measurement is
contemplated herein to indicate that the I~a4BV polymorphism is a
predisposing factor for a disease or disorder, such as a cardiovascular
disease or disorder manifesting a cardiac phenotype. For example,
2~ individuals homozygous for the Val variant exhibit shorter depolarization
intervals of the atrium (PR) as compared to individuals homozygous for
Ile. This phenotypic correlation combined with reports in the literature
supporting a role for AKAP mediated PKA signaling in normal cardiac
function suggest a lead into the pathogenesis coded for by this functional
variant. AKAP-mediated targeting of PKA in cardiac myocytes has been
implicated in regulating cell contractility (Fink et al., Cir. /3es., 83:291-
297, 2001 ). Stimulation of the f3-adrenergic signaling pathway in cardiac
myocytes results in activation of PKA and phosphorylation of a variety of
PKA substrates, including the sarcolemmal L-type Ca2+ channel, the
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ryanodine receptor (RyR), phospholamban (PLB) of the sarcoplasmic
reticulum (SR), the myofibrillar proteins troponin I (Tnl) and myosin
binding protein C (MBP-C) (Holroyde et al., Biochim. Biophys. Acta,
586:63-69, 1979; Kranias et al., Nature, 298:182-184, 1982; Brum et
al., Pflugers Arch., 401:1 1 1-1 18, 1984; Garvey et al., Biochem. J.,
249:709-714, 1988; Marx et al., Cell, 101:365-376, 1999).
Phosphorylation of these substrates acts in concert to generate both
enhanced contractility and accelerated relaxation in response to (3-
adrenergic stimulation.
Although PKA has broad substrate specificity, it can be highly
selective by targeting of PKA to distinct subcellular locations via
interaction with AI<APs (Colledge et al., Treads Cell Bi~l., 19:216-221,
1999). Three AKAPs have been shown to interact with PI<A in cardiac
myocytes, muscle-selective AKAP (mAKAP), AKAP18 and Yotiao.
mAKAP targets PKA to the perinuclear region of differentiated myocytes,
c~ordinating both PKA and phosphodiesterase activity in a single
complex (l~apiloff ct al., J. Call Sci., 1 14:3167-3175, 2001 ). AKAP18
couples PKA fio L-type Ca2 + channels, which enhances Ca2 + influx
through the channel following ,Q-adrenergic stimulati~n (Gray et al., ,l.
Bi~l. Chem., 272:6297-6302, 1997). Yotiao, previously ass~ciated with
NM~A receptors, has been shown to interact with the KCNQ1-KCNE1
K+ channel subunits in human hearts (Marx et al., Science, 295:496-
499, 2002). This channel is responsible for the slow delayed rectifier
current that repolarizes the myocyte membrane and controls action
potential duration. Mutations in this channel associated with hereditary
long QT syndrome abolish Yotiao interactions with the channel, thereby
attenuating PI<A regulation (Marx et al., 2002). Additionally, in cardiac
myocytes the Rla subunit is the predominant isoform associated with the
sarcolemma (Robinson et al., Arch. Biochem. Biophys., 330:181-187,
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1996; Reinitz et al., Arch. Biochem. Biophys., 348:391-402; 1997).
Mutations in RI are associated with both familial cardiac myxomas and
Carney complex, implicating this isoform in cardiac growth and
differentiation (Casey et al., J. Clin. Invest., 106:831-38, 2000;
Kirschner et al., Nat. Genet., 26:89-92, 2000). Direct involvement of
PKA in heart disease was also recently reported in a transgenic mice
study (Antos et al., Circ. Res., 89:997-1004, 2001 ). The transgenic
mice that overexpressed the catalytic subunit of PKA developed dilated
cardiomyopathy with reduced cardiac contractility and increased risk of
arrhythmias. These cardiac abnormalities correlated with PKA-mediated
hyperphosphorylation of the ryanodine receptor and Ca2 + release from
the sarcoplasmic reticulum (SR) and phospholamban, which regulates the
activity of the SR Ca2+ -ATPase (Autos et al., 2001 ).
D-AI<AP2 1AKAP10-5) contains a PD2 binding motif (TKL) at the
C-terminus (Fig. 3a), which is contemplated herein to serve as a targeting
domain to membrane-bound receptors or ion-channels (Harris et al., J.
Cell Sci., 1 14:3219-3231, 2001 ), and two RGS domains, which are
contemplated herein to coordinate upstream G alpha signaling with
downstream PKA signaling. It is contemplated herein that D-AKAP2 is
2~ part of a signaling complex associated with a cardiac ion-channel. The
D-AKAP2 variants is contemplated herein to impact the phosphorylation
state of the ion-channel by recruiting different amounts of PKA-Rla and
thereby modulate heart contraction. This model is in agreement with the
observed association with an EKG phenotype. The shorter depolarization
intervals for Val/Val homozygous individuals is contemplated herein to be
due to increased activation of ion-channels in cardiac myocytes.
In summary, through the analysis of significant morbidity markers
in a well-phenotyped healthy twin population, cardiac phenotypes have
been associated to particular D-AKAP2 genotypes.
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1. Method for indicating increased susceptibility of a subject to
a disease or disorder
Accordingly, provided herein are methods for indicating increased
susceptibility of a subject to a disease or disorder, comprising:
conducting an EKG examination;
determining the EKG-PR-interval in the subject, wherein, if the
EKG-PR-interval is decreased, then
determining the amino acid present in the subject at position 646
of AKAP10/D-AKAP2 (SEQ ID N0:2) or the nucleotide present at position
1~ corresponding to nucleotide 2073 of SEQ ID N0:1, wherein the presence
of Val at position 646 of SEQ ID NO:2 or the presence of a -G- at
nucleotide position 2073 of SEC2 ID NO:1, indicates increased
susceptibility to a disease or disorder. The disease or disorder can be
selected from among cardiovascular disorders, cardiac disease,
proliferative disorders, neurological disorders, neurodegenerative
disorders, obesity, diabetes and peripheral retinopathies.
In one embodiment, the EKG-PR-interval in the subject is compared
to a predetermined age-matched standard EI~G-PR-interval. The
predetermined standard EKG-PR-interval can be obtained from a known
2~ age-matched control group that is homozygous -AA- at a position
corresponding to nucleotide 2073 of SEQ ID N0:1 or homozygous Ile/Ile
at a position corresponding to position 646 of SEQ ID N0:2. The
predetermined standard EKG-PR-interval can be obtained from a known
age-matched control group that is heterozygous -GA- at a position
corresponding to nucleotide 2073 of SEQ ID N0:1 or heterozygous
Val/Ile at a position corresponding to position 646 of SEQ ID N0:2. The
predetermined standard EKG-PR-interval can be obtained from a known
age-matched control group that is selected from either homozygous -AA-
at a position corresponding to nucleotide 2073 of SEQ ID N0:1 or
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homozygous Ile/Ile at a position corresponding to position 646 of SEQ ID
N0:2; or heterozygous -GA- at a position corresponding to nucleotide
2073 of SEQ ID N0:1 or heterozygous Val/Ile at a position corresponding
to position 646 of SEQ ID N0:2. In another embodiment, the
predetermined standard EKG-PR-interval is obtained from a control age-
matched subject without heart disease.
In one embodiment, the subject is heterozygous -GA- at a position
corresponding to nucleotide 2073 of SEQ ID N0:1 or heterozygous
Val/Ile at a position corresponding to position 646 of SEQ ID N0:2. In
another embodiment, the subject is homozyg~us -GG- at a position
corresponding to nucleotide 2073 of SEQ ID N0:1 or homozygous
Val/Val at a position corresponding to position 646 of SEA ID N0:2.
Also provided are methods for indicating increased susceptibility of
a subject to a disease or disorder associated with the cardiovascular
system, comprising:
conducting an EKG eacam;
determining the EI<G-PR-interval in the subject, wherein, if the
EKG-PR-interval is decreased, then
determining the amino acid present at position 646 of AKAP10/D-
AKAP2 (SEO. ID N0:2) or the nucleotide present at position
corresponding to nucleotide 2073 of SEQ ID N0:1, wherein the presence
of Val at position 646 of SEQ ID N0:2 or the presence of a -G- at
nucleotide position 2073 of SEQ ID N0:1, indicates increased
susceptibility to a disease or disorder associated with the cardiovascular
system. The EKG-PR-interval in the subject can be compared to a
predetermined age-matched standard EKG-PR-interval to determine
whether it is decreased. The predetermined standard EKG-PR-interval
can be obtained from a known age-matched control that is homozygous -
AA- at a position corresponding to nucleotide 2073 of SEQ ID N0:1 or
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homozygous Ile/Ile at a position corresponding to position 646 of SEQ ID
N0:2. The predetermined standard EKG-PR-interval can be obtained from
a known age-matched control group that is heterozygous -GA- at a
position corresponding to nucleotide 2073 of SEQ ID N0:1 or
heterozygous Val/Ile at a position corresponding to position 646 of SEQ
ID N0:2. The predetermined standard EKG-PR-interval can be obtained
from a known age-matched control group that is selected from either
homozygous -AA- at a position corresponding to nucleotide 2073 of SEQ
ID N0:1 or homozygous Ilellle at a position corresponding to position
646 of SEQ ID N0:2; or heterozygous -GA- at a position corresponding
to nucleotide 2073 of SEQ ID N0:1 or heterozygous Valllle at a position
corresponding to position 546 of SEQ ID N0:2. In another embodiment,
the predetermined standard EKG-PR-interval can be obtained from a
control age-matched subject without heart disease.
In particular embodiments of the various methods provided herein,
in the conteact of determining whether the EKG-PR-interval is decreased, a
~9ecreased EKG-PR-interval is less than 150 for a subject 40 or more year
old. In another embodiment, a decreased EI~G-PR-interval is less than
155 for a subject 50 or more year old. In another embodiment, a
decreased EI<G-PR-interval is less than 150 for a subject 50 or more year
old. In another embodiment, a decreased EKG-PR-interval is less than
160 for a subject 60 or more year old. In another embodiment, a
decreased EKG-PR-interval is less than 155 for a subject 60 or more year
old. In another embodiment, a decreased EKG-PR-interval is less than
150 for a subject 60 or more year old.
In yet other embodiments, a decreased EKG-PR-interval for the
subject is less than 146. In another embodiment, a decreased EKG-PR-
interval for the subject is less than 130. In another embodiment, a
decreased EKG-PR-interval for the subject is less than 120.
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The disease or disorder can be selected from one or more of the
group consisting of: atrial fibrillation, sick sinus syndrome, sudden
cardiac arrest, ventricular arrythmia, ventricular fibrillation, ventricular
tachycardia, Wolf-Parkinson-White (WPW) Syndrome, Lown-Ganong-
Levin (LGL) Syndrome, hypertension. In one embodiment, the methods
can further comprise monitoring the subject for cardiovascular disease.
In one embodiment, the subject is heterozygous -GA- at a position
corresponding to nucleotide 2073 of SEQ ID N0:1 or heterozygous
Val/Ile at a position corresponding to position 646 of SEQ ID NO:2. In
1~ another embodiment, the subject is homozygous -GG- at a position
corresponding to nucleotide 2073 of SEQ ID NO:1 or homozygous
Val/Val at a position corresponding to position 045 of SECT ID NO:2.
In another embodiment, the methods can further comprise
administering to the subject prophylactic steps. For ea:ample, in view of
developments in genetics and technology as well as epidemiology, the
methods provided herein permit the determination of the probability and
risk assessment for the development of disease, in particular heart
disease in an individual. lJsing the genetic screening methods herein
and/or family health histories, it is possible to predict the probability a
particular individual has for developing any one of several types of
disease, such as heart disease. Those individuals identified as being
predisposed to developing a particular form of disease by using the
methods provided herein, can take prophylactic steps towards reducing
the risk of the particular disease, such as a heart disease. Accordingly,
high-risk individuals identified herein can take one or more of the well-
known prophylactic steps against the form of disease that they have a
predisposition to develop.
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a. Methods of assessing the susceptibility of a subject
to a disease or disorder associated with the
cardiovascular system
Also provided herein are methods of assessing the susceptibility of
a subject to a disease or disorder associated with the cardiovascular
system, the method comprising determining the amino acid at position
646 of AKAP10/D-AKAP2 (SEQ fD N0:2) or the nucleotide present at
position corresponding to nucleotide 2073 of SEQ ID N0:1, wherein the
presence of Val at position 646 of SEQ ID N0:2 or the presence of a -G-
at nucleotide position 2073 of SEQ ID N0:1, indicates increased
susceptibility to a disease or disorder associated with the cardiovascular
system.
Also provided herein are methods of diagnosing a disease or
disorder associated with the cardiovascular system, comprising detecting
the presence of Val at 646 of D-AKAP2 (SEQ ID N0:2) or the presence
of a G at a nucleotide position corresponding to nucleotide 2073 of SEQ
ID f~0:1, wherein the presence of Val at position ~4.~ of SECT ID X10:2 or
the presence of a -G- at nucleotide position 2073 of SEQ ID N0:1,
indicates the presence of a disease or disorder associated with the
cardiovascular system. In these methods, the disease or disorder can be
from among one or more of the group consisting of: atrial fibrillation, sick
sinus syndrome, sudden cardiac arrest, ventricular arrythmia, ventricular
fibrillation, ventricular tachycardia, Wolf-Parkinson-White (WPW)
Syndrome, Lown-Ganong-Levin (LGL) Syndrome, hypertension.
In one embodiment, the subject is heterozygous -GA- at a position
corresponding to nucleotide 2073 of SEQ ID N0:1 or heterozygous
Val/Ile at a position corresponding to position 646 of SEQ ID N0:2. In
another embodiment, the subject is homozygous -GG- at a position
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corresponding to nucleotide 2073 of SEQ ID N0:1 or homozygous
VallVal at a position corresponding to position 646 of SEQ ID N0:2.
2. Methods for determining responsiveness of a subject to one
or more ,l3-blocking agents
The presence of the 1646V variant in a subject is contemplated
herein to affect the D-AICAP2-mediated ~3-adrenergic signaling pathway.
For example, it is contemplated herein that the presence of 1 or 2 copies
of the 1646V variant (e.g., a -g- at nucleotide 2073 of SEQ ID NO:1 ) in a
subject renders the subject resistant to treatment with, or the effects of,
1 ~ the well-known ~3-blockers. Beta-blockers (,~-adrenergic blocking drugs)
"block" the effects of adrenaline on the body's beta receptors. This
slows the nerve impulses that travel through the heart. As a result, the
heart does not have to work as hard because it needs less blood and
oxygen. Beta-blockers also block the impulses that can cause an
arrhythmia. In one embodiment, the heterozygous presence of the 1646V
variant (e.g., a -GA- heterozygous genotpye at a nucleotide
corresponding t~ p~sition 2003 of SEQ ID 10:1 ) is contemplated herein
to produce a ,~-blocker resistance phen~type. In another embodiment,
the homozygous presence of the 1646V variant (e.g., a -GG-
2~ heterozygous genotpye at a nucleotide corresponding to position 2073 of
SEQ ID N0:1 ) is contemplated herein to produce the ~3-blocker resistance
phenotype.
Accordingly, provided herein are methods for determining
responsiveness of a subject to one or more (~-blocking agents,
comprising:
detecting for the subject the presence or absence of Val at
position 646 of SEQ ID N0:2 or a -G- nucleotide at a position
corresponding to position 2073 of SEQ ID NO: 1, wherein the presence
of a Val at position 646 of SEQ ID N0:2 or a -G- at nucleotide 2073 of
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SEQ ID N0:1, is indicative of an increased likelihood that a subject has a
modulated response to one or more ~3-blocking agents compared to a
subject who does not have the allelic variant. In one embodiment, the
modulated response is a decreased response to one or more ~3-blocking
agents compared to a subject who does not have the allelic variant. In
another embodiment, the decreased response is a non-response to one or
more ,~-blocking agents compared to a subject who does not have the
allelic variant. In yet another embodiment, the modulated response is an
increased response to one or more ,~-blocking agents compared to a
subject who does not have the allelic variant. The ,~-blocker ican bean
antagonist of a ~3-adrenergic receptor. In another embodiment, the ~3-
blocker is an agonist of a ~3-adrenergic receptor.
Also provided herein are methods for determining responsiveness
of a subject to one or more ,Q-blocking agents, comprising:
detecting the presence or absence of Val at position 646 of SEQ
ID N0:2 or a -G- nucleotide at a position corresponding to position 2073
of SEA ID i~~10: 1, wherein the presence of a Val at position 046 of SEr~
ID N0:2 or a -G- at nucleotide 2073 of SEQ ID N0:1, is indicative of an
increased likelihood that a subject has an increased response to one or
more ,Q-blocking agents compared to a subject who does not have the
allelic variant. In one embodiment, the ~3-blocker is an antagonist of a ,~-
adrenergic receptor. In another embodiment, the ,Q-blocker is an agonist
of a /3-adrenergic receptor.
Also provided herein, are methods for determining responsiveness
of a subject to one or more ~3-blocking agents, comprising:
detecting for the subject the presence or absence of Val at
position 646 of SEQ ID N0:2 or a -G- nucleotide at a position
corresponding to position 2073 of SEQ ID NO: 1, wherein the presence
of a Val at position 646 of SEQ ID N0:2 or a -G- at nucleotide 2073 of
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SEQ ID N0:1, is indicative of an increased likelihood that a subject is
non-responsive to one or more ~i-blocking agents compared to a subject
who does not have the allelic variant. In one embodiment, the ~3-blocker
is an antagonist of a ~3-adrenergic receptor. In another embodiment, the
~3-blocker is an agonist of a ~3-adrenergic receptor.
Also provided herein are methods for determining responsiveness
of a subject to one or more ~3-blocking agents, comprising:
detecting the presence or absence of Val at position 646 of SEQ
ID N0:2 or a -G- nucleotide at a position corresponding to position 2073
1 ~ of SEQ ID NO: 1, wherein the presence of a Val at position 646 of SEQ
ID N0:2 or a -G- at nucleotide 2073 of SEQ ID N0:1, is indicative of an
increased likelihood that a subject is hyper-responsive to one or more ~-
blocking agents compared to a subject who does not have the allelic
variant. The ,~-blocker can be an antagonist of a ~3-adrenergic receptor.
In another embodiment, the ~3-blocker is an agonist of a ~3-adrenergic
receptor.
Ea;emplary ,~-blockers well known in the art include, but are not
limited to, 64cebutolol, atenolol, I3etae~olol, 6isoprolol, Oarteolol,
Garbedilol, Esmolol, Labetolol, IVletoprolol, Nadolol, Penbutolol, Pindolol,
2~ Propranolol and Timolol. These ,Q-blocking agents are used to treat: high
blood pressure, angina, abnormal heart rythms, hypertrophic
cardiomyopathy, heart failure, vasovagal fainting, migraines, essential
tremor, bleeding from esophageal varices, stage fright, glaucoma and to
prolong survival of heart attack patients. Thus, the methods provided
herein are useful to identify subjects that require or would benefit from a
different treatment regimen than the use of ~3-blockers for their respective
disease or disorder.
In one embodiment, the subject is heterozygous -GA- at a position
corresponding to nucleotide 2073 of SEQ ID N0:1 or heterozygous
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Valllle at a position corresponding to position 646 of SEQ ID N0:2. In
another embodiment, the subject is homozygous -GG- at a position
corresponding to nucleotide 2073 of SEQ ID N0:1 or homozygous
Val/Val at a position corresponding to position 646 of SEQ ID N0:2.
3. Method for indicating susceptibility of a subject to acquired
long Q-T syndrome
The long QT syndrome (LQTS) is an abnormality of the heart's
electrical system. The mechanical function of the heart is entirely
normal. The electrical problem is due to defects in heart muscle cell
structures called ion channels. These electrical defects predispose
affected persons to a very fast heart rhythm (arrhythmia) called torsade
de pointer which leads t~ sudden loss of consciousness (syncope) and
can cause sudden cardiac death. The syndrome can be inherited (the
genetic form) or acquired. The inherited long OT Syndrome was first
clearly described in 1957. There are two variants, the autosomal
dominant Romano-Ward type and the autosomal recessive Jervell and
Lunge f~ielren type. Even though LOTS eras described alm~rt 40 years
ago, t~~ many physicians are unaware of it. Vi~hereas acquired long OT
syndrome is m~rt often due to the administration of medication. These
2~ medications are contraindicated in patients with the long OT syndrome.
There are a number of drugs which are known to prol~ng the OT-
interval and to cause heart rhythm abnormalities, particularly in patients
with the long OT syndrome (LQTS). Accordingly, patients with LQTS
should always inquire their physician or other health care provider about
the risk of any medication suggested or prescribed for them. In addition,
LQTS-patients should always inform their doctors) and dentists) about
their disease and make sure they know there are many medications
which are contraindicated in this condition. For example, the department
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of pharmacology at Georgetown university provides a complete and up-
to-date list of drugs that prolong the QT-interval (external link).
The frequency is unknown but long Q-T syndrome appears to be a
common cause of sudden and unexplained death in children and young
adults. It is certainly much more common than previously thought. It
can be as frequent as 1 in 5,000, and can cause 3,000-4,000 sudden
deaths in children and young adults each year in the United States. The
Jervell and Lange Nielsen form is associated with congenital deafness
and is rare, but the Romano-Ward variant, with normal hearing, is being
1~ recognized with increasing frequency.
The usual symptoms are syncope (sudden loss of consciousness)
or sudden death, typically occurring during physical activity or emotional
upset. These most commonly begin in preteen to teenage years, but can
present from a few days of age to middle age. The syncopal episodes
are often misdiagnosed as the common faint (vasovagal event) or a
seizure. Actual seizures are uncommon in long QT syndrome, but
epilepsy is one of the comm~n errors in diagnosis. Sudden loss of
consciousness during physical exertion or during emotional excitement
should strongly raise the possibility of the long QT syndrome. A family
2~ history of unexplained syncope or sudden death in young people should
also raise suspicion. Importantly, about one third of individuals who have
the long QT syndrome never exhibit symptoms, and therefore, the lack of
symptoms does not exclude a person or family from having LQTS. Any
young person that has an unexplained cardiac arrest should be
considered for LQTS, as well as those with unexplained syncope.
Since the electrocardiographic Q-T intervals vary in a given
individual from day to day, and since Q-T prolongation in affected
individuals can be mild, the diagnosis can be missed even if an EKG is
performed. Therefore, because a patients can not receive an existing
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effective treatment in time because the condition which can kill quickly is
sometimes very hard to diagnose, other methods of diagnosing long Q-T
or a predisposition for long Q-T syndrome would be very useful. It is
contemplated herein that the 1646V variant in D-AKAP2 is indicative of a
predisposition to the acquired form of Long Q-T syndrome. Accordingly,
provided herein are methods that identify subjects who are predisposed
or susceptible to acquired long Q-T syndrome. These methods are useful
in identifying the class of subjects who should avoid taking particular
medications, such as the well-documented group of medications that
1~ those diagnosed with long Q-T syndrome should avoid.
For example, provided herein are methods for indicating
susceptibility of a subject to acquired long Q-T syndrome, comprising:
detecting the presence or absence of Val at position 646 of SEQ
ID N0:2 or presence or absence of a -Ca- nucleotide at a position
corresponding to position 2073 of SEQ ID N0: 1, wherein the presence
of a Val at position 646 of SEQ ID N0:2 or a -G- at nucleotide 2073 of
SEA ID 10:1, is indicative of increased susceptibility to acquired long Q-
T syndrome, compared to the susceptibility of a subject who does not
have the allelic variant. The detecting step can be effected by a method
2~ selected from the group consisting of allele specific hybridization, primer
specific extension, oligonucleotide ligation assay, restriction enzyme site
analysis and single-stranded conformation polymorphism analysis. In
addition, the detecting step can comprise mass spectrometry. The
detection can be effected by detecting a signal moiety selected from the
group consisting of radioisotopes, enzymes, antigens, antibodies,
spectrophotometric reagents, chemiluminescent reagents, fluorescent
reagents and other light producing reagents.
In one embodiment, the subject is heterozygous -GA- at a position
corresponding to nucleotide 2073 of SEQ ID N0:1 or heterozygous
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Val/Ile at a position corresponding to position 646 of SEQ ID N0:2. In
another embodiment, the subject is homozygous -GG- at a position
corresponding to nucleotide 2073 of SEQ ID N0:1 or homozygous
Val/Val at a position corresponding to position 646 of SEQ ID N0:2.
4. Methods for indicating susceptibility to morbidity, increased
or early mortality, or morbidity and increased or early
mortality of a subject
Also provided herein, are methods for indicating susceptibility to
morbidity, increased or early mortality, or morbidity and increased or
1~ early mortality of a subject; comprising:
conducting an EKG exam;
determining the EKG-PR-interval in the subject, wherein if the EKG-
PR-interval is decreased; then
determining the amino acid at position 64C of AKAP10/D-AKAP2
(SEQ ID NO:2) or the nucleotide present at position corresponding to
nucleotide 2073 of SEQ ID N0:1, wherein the presence of Val at position
~a45 of SEO ID i~0:2 or the presence of a -G- at nucleotide position 2073
of SEQ ID N0:1, indicates increased susceptibility to morbidity, increased
or early mortality, or morbidity and increased or early mortality of a
2~ subject. The EKG-PR-interval in the subject can be compared to a
predetermined standard EKG-PR-interval. The predetermined standard
EKG-PR-interval can be obtained from a known age-matched control that
is homozygous -AA- at a position corresponding to nucleotide 2073 of
SEQ ID N0:1 or homozygous Ile/Ile at a position corresponding to
position 646 of SEQ ID N0:2. The detecting step can be effected by a
method selected from the group consisting of allele specific hybridization,
primer specific extension, oligonucleotide ligation assay, restriction
enzyme site analysis and single-stranded conformation polymorphism
analysis. The detecting step can comprise mass spectrometry. The
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detection step can be effected by detecting a signal moiety selected from
the group consisting of radioisotopes, enzymes, antigens, antibodies,
spectrophotometric reagents, chemiluminescent reagents, fluorescent
reagents and other light producing reagents.
In one embodiment, the subject is heterozygous -GA- at a position
corresponding to nucleotide 2073 of SEQ ID N0:1 or heterozygous
Val/Ile at a position corresponding to position 646 of SEQ ID N0:2. In
another embodiment, the subject is homozygous -GG- at a position
corresponding to nucleotide 2073 of SEQ ID N0:1 or homozygous
Val/Val at a position corresponding to position 646 of SEQ ID N0:2.
D. Detecti~n of Polymorphisms in Human AKAP10 Genes
I~ilethods of determining the presence or absence of allelic variants
of a human AKAP10 gene are also provided. In particular methods, the
detection or identification of a G, C, or T nucleotide at position 2073 of
the sense strand of the human AKAP10 gene coding sequence (see SEO.
ID N0: 1 ), or the detection or identification of a C, G or A nucleotide at
the same position in the antiasnsa strand of the human AI~AP10 gene
coding sequence, indicates the presence of an allelic variant. In these
particular methods, the detection or identification of an A nucleotide at
position 2073 of the sense strand of the human AKAP10 gene coding
sequence, or the detection or identification of a T nucleotide at the same
position in the antisense strand of the human AKAP10 gene coding
sequence, indicates the absence of polymorphism.
Other methods for determining the presence or absence of an
allelic variant of the AKAP10 gene detect or identify a nucleotide other
than a C at position 83587 of the SEQ ID NO: 17 or a nucleotide other
than a G on the complementary strand, a nucleotide other than a G at
position 129600 of the SEQ ID NO: 17 or a nucleotide other than a C on
the complementary strand or a nucleotide other than T at position
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156,277 of SEQ ID N0: 17 or a nucleotide other than A on the
complementary strand.
1. Nucleic acid detection methods
Generally, these methods are based in sequence-specific
polynucleotides, oligonucleotides, probes and primers. Any method
known to those of skill in the art for detecting a specific nucleotide
within a nucleic acid sequence or for determining the identity of a
specific nucleotide in a nucleic acid sequence is applicable to the
methods of determining the presence or absence of an allelic variant of
the AKAP10 gene. Such methods include, but are not limited to,
techniques utilizing nucleic acid hybridization of sequence-specific
probes, nucleic acid sequencing, selective amplification, analysis of
restriction enzyme digests of the nucleic acid, cleavage of mismatched
heteroduplexes of nucleic acid and probe, alterations of electrophoretic
mobility, primer specific extension, oligonucleotide ligation assay and
single-stranded conformation polymorphism analysis. In particular,
primer ea~tension reactions that specifically terminate by incorporating a
dideooynuoleotide are useful for detection. Several such general nucleic
acid detection assays are known (see, e.g.,U.S. Patent No. 6,030,778).
2~ a. Primer extension-based methods
Several primer extension-based methods for determining the
identity of a particular nucleotide in a nucleic acid sequence have been
reported (see, e.g., PCT Application Nos. PCT/US96/03651
(W096/29431 ), PCT/US97/20444 (WO 98/20166), PCT/US97/20194
(WO 98120019), PCT/US91 /00046 (W091 /13075), and U.S. Patent Nos.
5,547,835, 5,605,798, 5,622,824, 5,691,141, 5,872,003, 5,851,765,
5,856,092, 5,900,481, 6,043,031, 6,133,436 and 6,197,498.) In
general, a primer is prepared that specifically hybridizes adjacent to a
polymorphic site in a particular nucleic acid molecule. The primer is then
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extended in the presence of one or more dideoxynucleotides, typically
with at least one of the dideoxynucleotides being the complement of the
nucleotide that is polymorphic at the site. The primer and/or the
dideoxynucleotides can be labeled to facilitate a determination of primer
extension and identity of the extended nucleotide.
In an exemplary method, primer extension and/or the identity of
the extended nucleotides) are determined by mass spectrometry (see,
e.g., PCT Application Nos. PCT/US96/03651 (WO96/29431 ), PCT
Application No. PCT/US97/20444 (WO 98/20166), PCT Application No.
1 ~ PCT/US97/20194 (WO 98/20019), PCT Application No.
PCT/US91 /00046 (W091 /13075), and U.S. Patent Nos. 5,605,798,
5,622,824, 5,856,092.
b. P~lyrrr~rphisrr~-specific pr~be hybridizati~n
A typical detection method is allele specific hybridization using
probes overlapping the polymorphic site and having about 5, 10, 15, 20,
25, or 30 nucleotides around the polymorphic region. The probes can
contain naturally occurring or modified nucleotides (see U.S. Patent No.
6,156,501). For example, oligonucleotide probes can be prepared in
which the known polymorphic nucleotide is placed centrally (allele-
2~ specific probes) and then hybridized to target DNA under conditions
which permit hybridization only if a perfect match is found (Saiki et al.
(1986) Nature 324:163; Saiki et al. (1989) Proc. Natl Acad. Sci USA
86:6230; and Wallace et al. (1979) Nucl. Acids Res. 6:3543). Such
allele specific oligonucleotide hybridization techniques can be used for
the simultaneous detection of several nucleotide changes in different
polymorphic regions. For example, oligonucleotides having nucleotide
sequences of specific allelic variants are attached to a hybridizing
membrane and this membrane is then hybridized with labeled sample
nucleic acid. Analysis of the hybridization signal will then reveal the
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identity of the nucleotides of the sample nucleic acid. In one
embodiment, several probes capable of hybridizing specifically to allelic
variants are attached to a solid phase support, e.g., a "chip".
Oligonucleotides can be bound to a solid support by a variety of
processes, including lithography. For example a chip can hold up to
250,000 oligonucleotides (GeneChip, Affymetrix, Santa Clara, CA).
Mutation detection analysis using these chips comprising
oligonucleotides, also termed "DNA probe arrays" is described e.g., in
Cronin et al. (1996) Human Mutation 7:244 and in Kozal et al. (1996)
Nature Medicine 2:753. In one embodiment, a chip includes all the allelic
variants of at least one polymorphic region of a gene. The solid phase
support is then contacted with a test nucleic acid and hybridization to the
specific probes is detected. Accordingly, the identity of numerous allelic
variants of one or more genes can be identified in a simple hybridization
experiment.
c. Nucleic acid amplification-based methods
In other detectican methods, it is necessary to first amplify at least
a portion of an AKAP gene prior to identifying the allelic variant.
Amplification can be performed, e.g., by PCR and/or LCR, according to
methods known in the art. In one embodiment, genomic DNA of a cell is
exposed to two PCR primers and amplification is performed for a number
of cycles sufficient to produce the required amount of amplified DNA. In
typical embodiments, the primers are located between 150 and 350 base
pairs apart.
Alternative amplification methods include: self sustained sequence
replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. U.S.A.
87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,
1989, Proc. Natl. Acad. Sci. U.S.A. 86:1173-1177), Q-Beta Replicase
(Lizardi, P. M. et al., 1988, Bio/Technology 6:1 197), or any other nucleic
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acid amplification method, followed by the detection of the amplified
molecules using techniques well known to those of skill in the art. These
detection schemes are especially useful for the detection of nucleic acid
molecules if such molecules are present in very low numbers.
Alternatively, allele specific amplification technology, which
depends on selective PCR amplification can be used in conjunction with
the alleles provided herein. Oligonucleotides used as primers for specific
amplification can carry the allelic variant of interest in the center of the
molecule (so that amplification depends on differential hybridization)
(Gibbs et al. ( 1989) Nucleic Acids Res. 17:2437-2448) or at the extreme
3° end of one primer where, under appropriate conditions, mismatch can
prevent, or reduce polymerise extension (Prossner (1993) Tibtech
1 1:38; Newton e~ a/. (1989) Nucl. Acids Res. 17:2503). In addition it
can be desirable to introduce a restriction site in the region of the
mutation to create cleavage-based detection (Gasparini et al. (1992) Mol.
Cell Probes 6:1 ).
d. h~l~cl~ic ~~ia7 ~~a~~~n~ing-5a~~d r~~tC-r~~s
In one embodiment, any of a variety of sequencing reactions
known in the art can be used to directly sequence at least a portion of an
AKAP gene and fio detect allelic variants, e.g., mutations, by comparing
the sequence of the sample sequence with the corresponding wild-type
(control) sequence. Exemplary sequencing reactions include those based
on techniques developed by Maxim and Gilbert (Proc. Natl. Acid. Sci.
USA (1977) 74:560) or Singer (Singer et al. (1977) Proc. Natl. Acid.
Sci 74:5463). It is also contemplated that any of a variety of automated
sequencing procedures can be used when performing the subject assays
(Biotechniques (1995) 19:448), including sequencing by mass
spectrometry (see, for example, U.S. Patent Nos. 5,547,835, 5,691,141,
and International PCT Application No. PCT/US94/00193 (WO 94/16101 ),
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entitled "DNA Sequencing by Mass Spectrometry" by H. Koster; U.S.
Patent Nos. 5,547,835, 5,622,824, 5,851,765, 5,872,003, 6,074,823,
6,140,053 and International PCT Application No. PCT/US94/02938
(WO 94/21822), entitled "DNA Sequencing by Mass Spectrometry Via
Exonuclease Degradation" by H. Koster), and U.S. Pat. Nos. 5,605,798,
6,043,031, 6,197,498, and International Patent Application No.
PCT/US96/03651 (WO 96/29431 ) entitled "DNA Diagnostics Based on
Mass Spectrometry" by H. Koster; Cohen et al. (1996) Adv
Chromatography 36:127-162; and Griffin et al. (1993) Appl Biochem
1~ Biotechnol 38:147-159). It will be evident to one skilled in the art that,
for certain embodiments, the occurrence of only one, two or three of the
nucleic acid bases need be determined in the sequencing reaction. For
instance, A-track sequencing or an equivalent, e.g., where only one
nucleotide is detected, can be carried out. Other sequencing methods
are known (see, e.g., in U.S. Patent No. 5,580,732 entitled "Method of
DNA sequencing employing a mixed DNA-polymer chain probe" and U.S.
Patent fro. 5,5/1,575 entitled "f~lr~thod for mismatch-directed i~a ~i~r~
DNA sequencing"). .
e. Restriction enzyme digest analysis
2~ In some cases, the presence of a specific allele in nucleic acid,
particularly DNA, from a subject can be shown by restriction enzyme
analysis. For example, a specific nucleotide polymorphism can result in a
nucleotide sequence containing a restriction site which is absent from the
nucleotide sequence of another allelic variant.
f. Mismatch Cleavage
Protection from cleavage agents, such as, but not limited to, a
nuclease, hydroxylamine or osmium tetroxide and with piperidine, can be
used to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNA
heteroduplexes (Myers, et al. (1985) Science 230:1242). In general, the
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technique of "mismatch cleavage" starts by providing heteroduplexes
formed by hybridizing a control nucleic acid, which is optionally labeled,
e.g., RNA or DNA, comprising a nucleotide sequence of an allelic variant
with a sample nucleic acid, e.g, RNA or DNA, obtained from a tissue
sample. The double-stranded duplexes are treated with an agent, which
cleaves single-stranded regions of the duplex such as duplexes formed
based on basepair mismatches between the control and sample strands.
For instance, RNA/DNA duplexes can be treated with RNase and
DNA/DNA hybrids treated with S1 nuclease to enzymatically digest the
1~ mismatched regions.
In other embodiments, either DNA/DNA or RNA/DNA duplexes can
be treated with hydroxylamine or osmium tetroxide and with piperidine in
order to digest mismatched regions. After digestion of the mismatched
regions, the resulting material is then separated by size on denaturing
polyacrylamide gels to determine whether the control and sample nucleic
acids have an identical nucleotide sequence or in which nucleotides they
differ (see, for eazample, C~tton e~ ~/. (1988) Proc. i~atl Acad Sci USA
85:4397; Saleeba e~ e/. 11992) Methods Enzymod. 217:286-295). The
control or sample nucleic acid is labeled for detection.
g. Electrophoretic mobility alterations
In other embodiments, alteration in electrophoretic mobility is used
to identify the type of allelic variant in an AICAP gene. For example,
single-strand conformation polymorphism (SSCP) can be used to detect
differences in electrophoretic mobility between mutant and wild type
nucleic acids (Orita et al. ( 1989) Proc. Natl. Acad. Sci. USA 86:2766,
see also Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992)
Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments of
sample and control nucleic acids are denatured and allowed to renature.
The secondary structure of single-stranded nucleic acids varies according
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to sequence, the resulting alteration in electrophoretic mobility enables
the detection of even a single base change. The DNA fragments can be
labeled or detected with labeled probes. The sensitivity of the assay can
be enhanced by using RNA (rather than DNA), in which the secondary
structure is more sensitive to a change in sequence. In another
embodiment, the subject method uses heteroduplex analysis to separate
double stranded heteroduplex molecules on the basis of changes in
electrophoretic mobility (Keen et al. (1991 ) Trends Genet 7:5).
h. Polyacrylamide Gel Electrophoresis
In yet another embodiment, the identity of an allelic variant of a
polymorphic region of an AKAP gene is obtained by analyzing the
movement of a nucleic acid comprising the polymorphic region in
polyacrylamide gels containing a gradient of denaturant is assayed using
denaturing gradient gel electrophoresis (DGGE) (~ilyers e~ a/. (1985)
Nature 313:495). When DGGE is used as the method of analysis, DNA
will be modified to ensure that it does not completely denature, for
example by adding a GO clamp of appro~zimately 4.0 by of high-melting
GG-rich DNA by POR. In a further embodiment, a temperature gradient is
used in place of a denaturing agent gradient to identify differences in the
mobility of control and sample DNA (Rosenbaum and Reissner (1987)
Biophys Chem 265:1275).
i. ~ligonucleotide ligation assay (~LA)
In another embodiment, identification of the allelic variant is
carried out using an oligonucleotide ligation assay (OLA), as described,
e.g., in U.S. Patent No. 4,998,617 and in Landegren, U. et al., Science
241:1077-1080 (1988). The OLA protocol uses two oligonucleotides
which are designed to be capable of hybridizing to abutting sequences of
a single strand of a target. One of the oligonucleotides is linked to a
separation marker, e.g,. biotinylated, and the other is detestably labeled.
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If the precise complementary sequence is found in a target molecule, the
oligonucleotides will hybridize such that their termini abut, and create a
ligation substrate. Ligation then permits the labeled oligonucleotide to be
recovered using avidin, or another biotin ligand. Nickerson, D. A. et al.
have described a nucleic acid detection assay that combines attributes of
PCR and OLA (Nickerson, D. A. et al., Proc. Natl. Acad. Sci. (U.S.A.)
87:8923-8927 (1990). In this method, PCR is used to achieve the
exponential amplification of target DNA, which is then detected using
OLA.
Several techniques based on this OLA method have been
developed and can be used to detect specific allelic variants of a
polymorphic region of a gene. For example, U.S. Pat. No. 5,593,826
discloses an OLA using an oligonucleotide having 3'-amino group and a
5'- phosphorylated oligonucleotide to form a conjugate having a
phosphoramidate linkage. In another variation of OLA described in Tobe
et al. (1996) Nucl. Acids Res. 24: 3728), OLA combined with PCR
permits typing of te~so alleles in a single microtiter sell. By marking each
of the allele-specific primers with a unique hapten, i.e. digo~;igenin and
fluorescein, each OLA reaction can be detected by using hapten specific
antibodies that are labeled with different enzyme reporters, alkaline
phosphatase or horseradish peroxidase. This system permits the
detection of the two alleles using a high throughput format that leads to
the production of two different colors.
j. SNP detection methods
Also provided are methods for detecting single nucleotide
polymorphisms. Because single nucleotide polymorphisms constitute sites
of variation flanked by regions of invariant sequence, their analysis
requires no more than the determination of the identity of the single
nucleotide present at the site of variation and it is unnecessary to
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determine a complete gene sequence for each patient. Several methods
have been developed to facilitate the analysis of such single nucleotide
polymorphisms.
In one embodiment, the single base polymorphism can be detected
by using a specialized exonuclease-resistant nucleotide, as disclosed,
e.g., in Mundy, C. R. (U.S. Patent No. 4,656,127). According to the
method, a primer complementary to the allelic sequence immediately 3'
to the polymorphic site is permitted to hybridize to a target molecule
obtained from a particular animal or human. If the poiymorphic site on
the target molecule contains a nucleotide that is complementary to the
particular exonuclease-resistant nucleotide derivative present, then that
derivative will be incorporated onto the end of the hybridized primer.
Such incorporation renders the primer resistant to exonuclease, and
thereby permits its detection. Since the identity of the
exonuclease-resistant derivative of the sample is known, a finding that
the primer has become resistant to exonucleases reveals that the
nucleotide present in the polymorphic site of the target molecule was
complementary to that of the nucleotide derivative used in the reaction.
This method has the advantage that it does not require the determination
of large amounts of extraneous sequence data.
In another embodiment, a solution-based method for determining
the identity of the nucleotide of a polymorphic site is employed (Cohen,
~. et al. (French Patent 2,65~,S4~; PCT Application No. W091 /Q2~i37)).
As in the Mundy method of U.S. Patent No. 4,656,127, a primer is
employed that is complementary to allelic sequences immediately 3' to a
polymorphic site. The method determines the identity of the nucleotide of
that site using labeled dideoxynucleotide derivatives, which, if
complementary to the nucleotide of the polymorphic site will become
incorporated onto the terminus of the primer.
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k. Genetic Bit Analysis
An alternative method, known as Genetic Bit Analysis or GBATM is
described by Goelet, et al. (U.S. Patent No. 6,004,744, PCT Application
No. 92/15712). The method ofi Goelet, et al. uses mixtures of labeled
terminators and a primer that is complementary to the sequence 3' to a
polymorphic site. The labeled terminator that is incorporated is thus
determined by, and complementary to, the nucleotide present in the
polymorphic site of the target molecule being evaluated. In contrast to
the method of Cohen et al. (French Patent 2,650,840; PCT Application
1 ~ No. W091 /02087), the method of Goelet, et al. is typically a
heterogeneous phase assay, in which the primer or the target molecule is
immobilised to a solid phase.
I. Other primer-guided nucle~tide inc~rp~rati~n
pr~cedures
Other primer-guided nucleotide incorporation procedures for
assaying polymorphic sites in DNA have been described (fComher, J. S.
et al., Nucl. Acids Res. 17:7779-7784- (1989); S~k~lov, B. P., f~l~acl.
Acids Res. 18:3561 (1990); Syvanen, A. C., anal., Genomics 8:584-692
(1990), I~uppuswamy, 1!/l. N. e~'a/., Proc. Natl. Acad. Sci. (U.S.A.)
2~ 88:1143-1147 (1991); Prezant, T. R. etal., Hum. nllutat. 1:159-164
(1992); Ugozzoli, L, et al., GATA 9:107-1 12 (1992); Nyren, P. et al.,
Anal. Biochem. 208:171-175 (1993)). These methods diffier from GBATM
in that they all rely on the incorporation of labeled deoxynucleotides to
discriminate between bases at a polymorphie site. In such a format, since
the signal is proportional to the number of deoxynucleotides
incorporated, polymorphisms that occur in runs of the same nucleotide
can result in signals that are proportional to the length of the run
(Syvanen, A. C., et al., Amer. J. Hum. Genet. 52:46-59 (1993)).
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For determining the identity of the allelic variant of a polymorphic
region located in the coding region of a gene, yet other methods than
those described above can be used. For example, identification of an
allelic variant which encodes a mutated protein can be performed by
using an antibody specifically recognizing the mutant protein in, e.g.,
immunohistochemistry or immunoprecipitation. Binding assays are
known in the art and involve, e.g., obtaining cells from a subject, and
performing binding experiments with a labeled lipid, to determine whether
binding to the mutated form of the protein differs from binding t~ the
1 ~ wild-type protein.
m. Molecular structure determination
If a polymorphic region is located in an axon, either in a coding or
non-coding region of the gene, the identity of the allelic variant can be
determined by determining the molecular structure of the mRNA,
pre-mRNA, or cDNA. The molecular structure can be determined using
any of the above described methods for determining the molecular
structure ~f the genomic Di~A, e.g., sequencing and SSCP.
n. l~7as~ spec~:r~metric methoe~s
Nucleic acids can also be analyzed by detection methods and
2~ protocols, particularly those that rely on mass spectrometry (see, e. g.,
U.S. Patent Nos. 5,605,798, 6,043,031, 6,197,498, and International
Patent Application No. WO 96/29431, allowed co-pending U.S.
Application Serial No. 08/617,256, allowed co-pending U.S. Application
Serial No. 08/744,481, U.S. Application Serial No. 08/990,851,
International PCT Application No. WO 98/20019). These methods can
be automated (see, e.g., co-pending U.S. Application Serial No.
09/285,481, which describes an automated process line). Among the
methods of analysis herein are those involving the primer oligo base
extension (PROBE) reaction with mass spectrometry for detection (see
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e.g., U.S. Patent Nos. 6,043,031 and 6,197,498, Patent Application
Serial Nos. 09/287,681, 09/287,682, and 09/287,679, allowed co-
pending U.S. Application Serial No. 081744,481, International PCT
Application No. PCT/US97/20444 (WO 98/20166), and based upon U.S.
Patent Nos. 5,900,481, 6,024,925, 6,074,823, Application Serial Nos.
08/746,055, 08/786,988, 08/933,792, 08/746,055, and 08/786,988;
see, also U.S. Application Serial No. 09/074,936, and published
International PCT Application No. PCT/US97/20195 (WO 98/20020)).
A typical format for performing the analyses is a chip based format
in which the biopolymer is linked to a solid support, such as a silicon or
silicon-coated substrate, for example, in the form of an array. More
typically, when analyses are performed using mass spectrometry,
particularly MALDI, nanoliter volumes of sample are loaded on, such that
the resulting spot is about, or smaller than, the size of the laser spot. It
has been found that when this is achieved, the results from the mass
spectrometric analysis are quantitative. The area under the peaks in the
resulting mass spectra are proportional t~ concentration (when
normalized and corrected for background). Methods for preparing and
using such chips are described in U.S. Patent No. 6,024,925, co-pending
U.S. Application Serial Nos. 08/786,988, 09/364,774, 09/371,150 and
09/297,575; see, also PCT Application No. PCT/US97/20195
(WO 98/20020). Chips and kits for performing these analyses are
commercially available from SEQUENOM under the trademark
MassARRAYT"". MassARRAYT"" relies on the fidelity of the enzymatic
primer extension reactions combined with the miniaturized array and
MALDI-TOF (Matrix-Assisted Laser Desorption Ionization-Time of Flight)
mass spectrometry to deliver results rapidly. It accurately distinguishes
single base changes in the size of DNA fragments relating to genetic
variants without tags.
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Multiplex methods allow for the simultaneous detection of more
than one polymorphic region in a particular gene. This is the typical
method for carrying out haplotype analysis of allelic variants of the
AICAP10 gene.
Multiplexing can be achieved by several different methodologies.
For example, several mutations can be simultaneously detected on one
target sequence by employing corresponding detector (probe) molecules
(e.g., oligonucleotides or oligonucleotide mimetics). The molecular
weight differences between the detector oligonucleotides must be large
enough so that simultaneous detection (multiplexing) is possible. This
can be achieved either by the sequence itself (composition or length) or
by the introduction of mass-modifying functionalities into the detector
oligonucleotides (see below).
Mass modifying moieties can be attached, for instance, to either
the 5'-end of the oligonucleotide, to the nucleobase (or bases), to the
phosphate backbone, and to the 2'-position of the nucleoside
(nucleosiales) and/or to the terminal 8°-position. Examples ~f mass
modifying moieties include, for eacample, a halogen, an azido, or of the
type, XR, wherein X is a linking group and R is a mass-modifying
functionality. The mass-modifying functionality can thus be used to
introduce defined mass increments into the oligonucleotide molecule.
The mass-modifying functionality can be located at different
positions within the nucleotide moiety (see, e.g., U.S. Patent No.
5,547,835 and International PCT Application No. WO 94/21822). For
example, the mass-modifying moiety, M, can be attached either to the
nucleobase, (in case of the c' -deazanucleosides also to C-7), to the
triphosphate group at the alpha phosphate or to the 2'-position of the
sugar ring of the nucleoside triphosphate. Modifications introduced at
the phosphodiester bond, such as with alpha-thio nucleoside
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triphosphates, have the advantage that these modifications do not
interfere with accurate Watson-Crick base-pairing and additionally allow
for the one-step post-synthetic site-specific modification of the complete
nucleic acid molecule e.g., via alkylation reactions (see, e.g., Nakamaye
et al. (1988) Nucl. Acids Res. 16:9947-59). Typical mass-modifying
functionalities are boron-modified nucleic acids since they are better
incorporated into nucleic acids by polymerases (see, e.g., Porter et al.
(1995) Biochemistry 34:1 1963-1 1969; Hasan et al. (1996) Nucleic Acids
Res. 24:2150-2157; Li et al. (1995) Nucl. Acids Res. 23:4495-4501 ).
Furthermore, the mass-modifying functionality can be added so as
to affect chain termination, such as by attaching it to the 3'-position of
the sugar ring in the nucleoside triphosphate. For those skilled in the art,
it is clear that many combinations can be used in the methods provided
herein. In the same way, those skilled in the art will recognize that
chain-elongating nucleoside triphosphates can also be mass-modified in a
similar fashion with numerous variations and combinations in
functionality and attachment positions.
For example, without being bound to any particular theory, the
mass-modification can be introduced for X in ?(R as well as using
oligo-/polyethylene glycol derivatives for R. The mass-modifying
increment (m) in this case is 44, i.e. five different mass-modified species
can be generated by just changing m from 0 to 4 thus adding mass units
of 45 (m = 0), 89 (m = 1 ), 133 (m = 2), 177 (m = 3) and 221 (m = 4) to the
nucleic acid molecule (e. g., detector oligonucleotide (D) or the nucleoside
triphosphates, respectively). The oligo/polyethylene glycols can also be
monoalkylated by a lower alkyl such as, but are not limited to, methyl,
ethyl, propyl, isopropyl and t-butyl. Other chemistries can be used in the
mass-modified compounds (see, e.g., those described in Oligonucleotides
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and Analogues, A Practical Approach, F. Eckstein, editor, IRL Press,
Oxford, 1991 ).
In yet another embodiment, various mass-modifying functionalities,
R, other than oligo/polyethylene glycols, can be selected and attached via
appropriate linking chemistries, X. A simple mass-modification can be
achieved by substituting H for halogens, such as F, CI, Br and/or I, or
pseudohalogens such as CN, SCN, NCS, or by using different alkyl, aryl
or aralkyl moieties such as methyl, ethyl, propyl, isopropyl, t-butyl, hexyl,
phenyl, substituted phenyl, benzyl, or functional groups such as CHEF,
CHF2, CF3, Si(CH3)3, Si(CH3)2(C~HS), Si(CH3)(C2H5)2, SI(C~H5)3. Yet
another mass-modification can be obtained by attaching homo- or
heteropeptides through the nucleic acid molecule (e.g., detector (D)) or
nucleoside triphosphates). One example, useful in generating mass-
modified species with a mass increment of 57, is the attachment of
oligoglycines (m) to nucleic acid molecules (r), e.g., mass-modifications
of 74 (r=1, m=~), 131 (r=1, m=1), 18B (r=1, m=2), 245 (r=1,
m = 3) are achieved. Simple oligoamides also can be used, e.~.,
mass-modifications of 74 (r =1, m = ~), 33 (r = ~, m = ~), 102 (r = 3,
m=~), 116(r=4, m=~), ete. are obtainable. Variations in additions to
2~ those set forth herein will be apparent to the skilled artisan.
Different mass-modified detector oligonucleotides can be used to
simultaneously detect all possible variants/mutants simultaneously.
Alternatively, all four base permutations at the site of a mutation can be
detected by designing and positioning a detector oligonucleotide, so that
it serves as a primer for a DNA/RNA polymerase with varying
combinations of elongating and terminating nucleoside triphosphates.
For example, mass modifications also can be incorporated during the
amplification process.
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A different multiplex detection format is one in which
differentiation is accomplished by employing different specific capture
sequences which are position-specifically immobilized on a flat surface
(e.g., a 'chip array'). If different target sequences T1-Tn are present,
their target capture sites TCS1-TCSn will specifically interact with
complementary immobilized capture sequences C1-Cn. Detection is
achieved by employing appropriately mass differentiated detector
oligonucleotides D1-Dn, which are mass modifying functionalities
M1-Mn.
0. ~ther methods
Additional methods of analyzing nucleic acids include
amplification- based methods including polymerase chain reaction (PCR),
ligase chain reaction (LCR), mini-PCR, rolling circle amplification,
autocatalytic methods, such as those using ~J replicase, TAS, SSR, and
any other suitable method known to those of skill in the art.
Other methods for analysis and identification and detection of
polymorphisms, include but are not limited to, allele specific probes,
Southern analyses, and other such analyses.
2. Primers, Iprobes and antiasnsa nucleic acid molecules
Primers refer to nucleic acids which are capable of specifically
hybridizing to a nucleic acid sequence which is adjacent to a polymorphic
region of interest or to a polymorphic region and are extended. A primer
can be used alone in a detection method, or a primer can be used
together with at least one other primer or probe in a detection method.
Primers can also be used to amplify at least a portion of a nucleic acid.
For amplifying at least a portion of a nucleic acid, a forward primer (i.e.,
5' primer) and a reverse primer (i.e., 3' primer) will typically be used.
Forward and reverse primers hybridize to complementary stands of a
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double stranded nucleic acid, such that upon extension from each primer,
a double stranded nucleic acid is amplified.
Probes refer to nucleic acids which hybridize to the region of
interest and which are not further extended. For example, a probe is a
nucleic acid which hybridizes adjacent to or at a polymorphic region of an
AICAP gene and which by hybridization or absence of hybridization to the
DNA of a subject will be indicative of the identity of the allelic variant of
the polymorphic region of the gene. Typical probes have a number of
nucleotides sufficient to allow specific hybridization to the target
nucleotide sequence. Where the target nucleotide sequence is present in
a large fragment of DNA, such as a genomic DNA fragment of several
tens or hundreds of kilobases, the size of a probe can have to be longer
to provide sufficiently specific hybridization, as compared to a probe
which is used to detect a target sequence which is present in a shorter
fragment of DNA. For example, in some diagnostic methods, a portion of
an AICAP gene can first be amplified and thus isolated from the rest of
the chromosomal Df~A and then hybridized to a probe. In sucll a
situation, a shorter probe will likely provide sufficient specificity of
hybridization. For example, a probe having a nucleotide sequence of
~0 about 1 ~ nucleotides can be sufficient.
Primers and probes (RNA, DNA (single-stranded or double-
stranded), PNA and their analogs) described herein can be labeled with
any detectable reporter or signal moiety including, but not limited to
radioisotopes, enzymes, antigens, antibodies, spectrophotometric
reagents, chemiluminescent reagents, fluorescent and any other light
producing chemicals. Additionally, these probes can be modified without
changing the substance of their purpose by terminal addition of
nucleotides designed to incorporate restriction sites or other useful
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sequences, proteins, signal generating ligands such as acridinium esters,
and/or paramagnetic particles.
These probes can also be modified by the addition of a capture
moiety (including, but not limited to para-magnetic particles, biotin,
fluorescein, dioxigenin, antigens, antibodies) or attached to the walls of
microtiter trays to assist in the solid phase capture and purification of
these probes and any DNA or RNA hybridized to these probes.
Fluorescein can be used as a signal moiety as well as a capture moiety,
the latter by interacting with an anti-fluorescein antibody.
Any probe, primer or antisense molecule can be prepared
according to methods well known in the art and described, e.g., in
Sambrook, J. Fritsch, E.F., and Maniatis, T. (1989( M~lecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. For example, discrete fragments of the DNA can be
prepared and cloned using restriction enzymes. Alternatively, probes and
primers can be prepared using the Polymerase Chain Reaction (PCR)
using primers having an appropriate sequence.
0ligonucleotides can be synthesized by standard methods known
in the art, e. g. by use of an automated DNA synthesizer (such as are
commercially available from 8iosearch (Novato, CA); Applied 8iosystems
(Foster City, CA) and other methods). As examples, phosphorothioate
oligonucleotides can be synthesized by the method of Stein et al. (1988,
Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides, for
example, can be prepared by use of controlled pore glass polymer
supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-
7451 ) .
Probes and primers used in the methods of detecting allelic
variants in human AICAP10 genes are of sufficient length to specifically
hybridize to portions of AICAP10 gene at polymorphic sites. Typically
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such lengths depend upon the complexity of the source organism
genome. For humans such lengths are at least 14-16 nucleotides, and
typically can be 20, 30, 50, 100 or more nucleotides.
The methods of detecting polymorphisms in human AKAP10 genes
provided herein, probes and primers include the following:
(1) at least 14 or 16 contiguous nucleotides of the AKAP10 allele
or complement thereof, wherein nucleic acid molecule includes at least 5
contiguous nucleotides from nucleotide 2069 to nucleotide 2077 of SEQ.
ID. N~: 3;
(2) at least 14 or 16 contiguous nucleotides of the AKAP10
allele or complement thereof, wherein the nucleic acid includes the
nucleotide at position 2073 of SE(~ ID No. 1 replaced with G, G or T.
(3) at least 14 or 16 contiguous nucleotides of the AKAP10
allele or complement thereof, wherein the nucleic acid molecule includes
at least 5 contiguous nucleotides from nucleotide 129556 to nucleotide
129604 of SEQ. ID. N~: 14;
(4) at least 14 or 16 contiguous nucleotides of the AICd4P10
allele or complement thereof, wherein the nucleic acid includes the
nucleotide at position 129600 of SEQ ID No. 17 replaced with A, G or T;
2~ (5) at least 14 or 16 contiguous nucleotides of the AKAP10
allele or complement thereof, wherein the nucleic acid molecule includes
at least 5 contiguous nucleotides from nucleotide 83583 to nucleotide
83591 of SEQ.. ID. N~: 13;
(6) at least 14 or 16 contiguous nucleotides of the AKAP10
allele or complement thereof, wherein the nucleic acid includes the
nucleotide at position 83587 of SEQ ID No. 17 replaced with G, A or T;
(7) at least 14 or 16 contiguous nucleotides of the AKAP10
allele or complement thereof, wherein the nucleic acid molecule includes
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at least 5 contiguous nucleotides from nucleotide 156,273 to nucleotide
156281 of SEQ. ID. NO: 18;
(8) at least 14 or 16 contiguous nucleotides of the AfCAP10
allele or complement thereof, wherein the nucleic acid includes the
nucleotide at position 156277 of SEQ ID No. 17 replaced with C, A or G;
With respect to each of the above described probes and primers,
they have fewer nucleotides than the sequence of nucleotides 138 to
2126 of SEQ. ID. NO: 1 or fewer nucleotides than the sequence of
nucleotides 83,580 to 156,577 of SEQ ID N0: 17.
1 ~ Antisense compounds can be conveniently and routinely made
through the well-known technique of solid phase synthesis. Equipment
for such synthesis is sold by several vendors including, for example,
Applied 8iosystems (Foster City, Calif.). Any other means for such
synthesis known in the art can additionally or alternatively be employed.
It is well known to use similar techniques to prepare oligonucleotides
sash as the phosphorothioates and alkylated derivatives.
Antisense c~mpounds are typically 8 to 30 nucleotides in length
complementary to a targeted to a nucleic acid molecule and modulates its
expression. The targeted nucleic acid molecule represents the coding
2~ strand. For example, for the AiCAP10-5 alleleic variant an antisense
compound is an antisense oligonucleotide which comprises the
complement of at least an 8 nucleotide segment of SEQ ID N0: 3
including the nucleotide at position 2073 of SEQ ID N0: 3.
An antisense compound can contain at least one modified
nucleotide which can confer nuclease resistance or increase the binding
of the antisense compound with the target nucleotide. The antisense
compound can containing at least one internucleoside linkage wherein
the modified internucleoside linkage of the antisense oligonucleotide can
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be a phosphorothioate linkage, a morpholino linkage or a peptide-nucleic
acid linkage.
Typical modified oligonucleotide backbones that do not include a
phosphorus atom therein have backbones that are formed by short chain
alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl
or cycloalkyl internucleoside linkages, or one or more short chain
heteroatomic or heterocyclic internucleoside linkages. These include
those having morpholino linkages (formed in part from the sugar portion
of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone
backbones; formacetyl and thioformacetyl backbones; methylene
formacetyl and thioformacetyl backbones; alkene containing backbones;
sulfamate backbones; methyleneimino and methylenehydra~ino
backbones; sulfonate and sulfonamide backbones; amide backbones; and
others having mixed N, O, S and GHQ component parts.
Representative United States patents that teach the preparation of
the above oligonucleosides include, but are not limited to, U.S. Pat. Nos.:
5,034,505; 5,156,315; 5,185,444; 5,214,134; 5,215,141; 5,235,~33;
5,264,562; 5,264,564; 5,4~5,938; 5,434,257; 5,466,677; 5,47~,967;
5,489,677; 5,541,3~7; 5,561,225; 5,596,~86; 5,602,240; 5,61~,289;
5, 6Q2, 24~; 5, 6~8,~46; 5, 61 ~, 289; 5, 618, 704; 5, 623,~70; 5, 663, 312;
5,633,36~; 5,677,437; and 5,677,439, each of which is herein
incorporated by reference.
An antisense compound can contain at least one least one
modified sugar moiety wherein the modified sugar moiety of the
antisense oligonucleotide is a 2'-O-methoxyethyl sugar moiety or a 2'-
dimethylaminooxyethoxy sugar moiety.
Modified oligonucleotides can also contain one or more substituted
sugar moieties. Typical oligonucleotides comprise one of the following at
the 2' position: OH; F; O--, S--, or N-alkyl; O--, S--, or N-alkenyl; O--, S--
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or N-alkynyl; or 0-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can
be substituted or unsubstituted C~ to Coo alkyl or C2 to Coo alkenyl and
alkynyl. Exemplary oligonucleotides contain are O[(CH2)n O]m CH3,
O(CHa)" OCH3, O(CH2)" NHS, O(CHz)~ CH3, O(CH2)~ NHS, and O(CH2)
ON[(CH2)~ CH3)]~, where n and m are from 1 to about 10. Other
oligonucleotides comprise one of the following at the 2' position: C, to
C~~ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-
aralkyl, SH, SCH3, OCN, CI, 8r, CN, CF3, OCF3, SOCH3, SO~ CH3, ONO,
N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a reporter
group, an intercalator, a group for improving the pharmacokinetic
properties of an oligonucleotide, or a group for impr~ving the
pharmacodynamic properties of an oligonucleotide, and ~ther
substituents having similar properties. A typical modification includes an
alkoxyalkoxy group, 2'-methoxyethoxy (2'-O-CH2 CHI OCH3, also known
as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 488-504). Another exemplary modificati~n includes 2'-
dimethylaminooxyethoxy, i.e., a O(CH~)~ ON(CH3)2 group, also known as
2'-DMAOE.
Other modifications include 2'-methoxy (2'-0-CH3), 2°-
aminopropoxy (2'-OCH~ CH2 CHI NHz) and 2'-fluoro (2°-F). Similar
modifications can also be made at other positions on the oligonucleotide,
particularly the 3' position of the sugar on the 3' terminal nucleotide or in
2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
Oligonucleotides can also have sugar mimetics such as cyclobutyl
moieties in place of the pentofuranosyl sugar. Representative United
States patents that teach the reparation of such modified sugar
structures include, but are not limited to, U.S. Pat. Nos.: 4,981,957;
5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;
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5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;
5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633;
and 5,700,920, each of which is herein incorporated by reference.
An antisense compound can contain at least one modified
nucleobase. Oligonucleotides can also include nucleobase (often referred
to in the art simply as "base") modifications or substitutions. As used
herein, "unmodified" or "natural" nucleobases include the purine bases
adenine (A) and guanine (G), and the pyrimidine bases thymine (T),
cytosine (C) and uracil (lJ). Modified nucleobases include other synthetic
and natural nucleobases such as 5-methylcytosine (5-me-C), 5-
hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-
methyl and other alkyl derivatives of adenine and guanine, 2-propyl and
other alkyl deri~atieres of adenine and guanine, 2-thiouracil, 2-thiothymine
and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and
cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-
thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-
substituted adenines and guanines, 5-halo particularly 5-bromo, 5-
trifluoromethyl and other 5-substituted uracils and cytosines, 7-
methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-
deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-
deazaadenine.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte
Chemie, International Edition, 1991, 30, 613, and those disclosed by
Sanghvi, Y. S., Crooke, S. T., and Lebleu, B. eds., Antisense Research
and Applications, CRC Press, Boca Raton, 1993, pp. 289-302. Certain of
these nucleobases are particularly useful for increasing the binding
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affinity of the oligomeric compounds of the invention. These include 5-
substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6
substituted purines, including 2-aminopropyl-adenine, 5-propynyluracil
and 5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi,
Y. S., Crooks, S. T. and Lebleu, B., ads., Antisense Research and
Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are
typical base substitutions, even more particularly when combined with
2'-O-methoxyethyl sugar modifications.
The antisense compound can be a chimeric oligonucleotide.
Chimeric antiasnsa compounds can be formed as composite structures of
two or more oligonucleotides, modified oligonucleotides, oligonucleosides
and/or oligonucleotide mimetics as described above. Such compounds
have also been referred to in the art as hybrids or gapmers.
Representative United States patents that teach the preparation of such
hybrid structures include, but are not limited to, U.S. Pat. Nos.:
5,013,830; 5,149,79'x; 5,220,~~~'; 5,255,%75; 5,305,878; 5,403,711;
5,491,133; 5,555,350; 5,623,055; 5,552,355; 5,552,356; and
5,700,922, sash of which is herein incorporated by reference.
E. nBleasuring and Electr~cardi~gram
Devices that can be used to measure an electrocardiogram are
referred to as electrocardiographs. A variety of electrocardiographs are
well known in the art and include, for example, those disclosed is U.S.
Pat. Nos. 4,377,813, 4,483,346, 4,98,479, 4,840,183, 4,974599. Any
known electrocardiograph can be used in the methods provided herein.
Methods of using electrocardiographs to determine P-R interval and Q-T
interval also are well known in the art, and any such method can be used
in the methods provided herein.
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F. Association of AKAP10 Allelic Variants with Morbidity or Increased
Mortality
Polymorphisms of the genome can lead to altered gene function,
protein function or mRNA instability. To identify those polymorphisms
that have clinical relevance is the goal of a world-wide scientific effort.
Discovery of such polymorphisms will have a fundamental impact on the
identification and development of diagnostics and drug discovery. The
strategy to identify valuable polymorphisms is cumbersome and
dependent upon the availability of many large patient and control cohorts
to show disease association. Furthermore, genes, and their associated
polymorphisms, that cause a general risk of the population to suffer from
any disease will escape these case/control studies entirely.
A morbidity susceptibility gene could be a gene that is expressed
in many different cell types or tissues (housekeeping gene) and its altered
function can facilitate the expression of a clinical phenotype caused by a
disease-specific susceptibility gene that is involved in a pathway specific
for this disorder. In other words, morbidity susceptibility genes might
predispose people to develop a distinct disease according to their genetic
make-up for this disease. Candidates for these genes can involve basic
cellular processes such as: transcription, translation, heat-shook proteins,
protein trafficking, DNA repair, assembly systems for subcellular struc-
tures (e.g., mitochondria, peroxysomes and other cellular microbodies),
receptor signaling cascades, immunology, etc. Those pathways control
the quality of life at the cellular level as well as for the entire organism.
Mutations/polymorphisms located in genes encoding proteins for those
pathways can reduce the fitness of cells and make the organism more
susceptible to express the clinical phenotype caused by the action of a
disease-specific susceptibility gene. Therefore, these morbidity
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susceptibility genes can be potentially involved in a whole variety of
different complex diseases if not in all.
An example of possible candidate morbidity susceptibility genes
are mutants of the A kinase anchoring protein (AKAP) genes. Protein
phosphorylation is an important mechanism for enzyme regulation and
signal transduction in eukaryotic cells. cAMP dependent protein kinase
(PKA) mediates a variety of hormonal and neurotransmitter responses by
phosphorylating a wide variety of substrates including enzymes,
membrane receptors, ion channels and transcription factors. AKAPs
1~ direct the subcellular localization of CAMP-dependent protein kinase by
binding to its regulatory subunits and therefore plays a role in G-protein
mediated receptor-signaling pathways. (Huang et al. Proc. Natl. Acad.
Sci., USA 94:111 S4, 1997). AKAPs have a PI<A binding region located
in their C~~H-terminal portion.
Polymorphic AKAP genes, such as those provided herein, serve as
markers for detecting predisposition to disease and various conditions.
~,Is~, the AI~c~P alleles and gene products, especially the AKAP10-5 gene
product should be suitable pharmaceutical targets and gene therapy
targets.
2~ In a further allele, designated AKAP10-7 contains a single
nucleotide polymorphism (SNP), a G-to-A transition, at nucleotide
position 129,600 of the human chromosome 17 sequence (also referred
to herein as SNP "1n10"). This SNP is located four bases 3' to the exon
10lintron 10 boundary of AKAP10 gene. Another identified SNP,
AKAP10-1 is an allelic variant with a T to C transversion at nucleotide
position 156,277 of the AKAP10 genomic clone which is located in the
3' untranslated region of the gene (also referred to herein as SNP "3'
UTR").
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Utilizing a healthy patient database, the frequency of occurrence of
two allelic variants of the AKAP10 gene, AKAP10-5 and AKAP10-1, in
such a population were found to decrease with age. The AKAP10-5 and
AKAP10-1 alleles are useful markers for predicting susceptibility to
morbidity and/or increased or early mortality. The methods provided
herein can be used for predicting susceptibility to morbidity, increased or
early mortality, or morbidity and increased mortality, by detecting the
presence of the various AKAP10 allelic variants known in the art or
dislcosed herein, individually, or in combination with other AKAP10 allelic
variants, in an organism, particularly an animal and particularly a human.
AKAP10-5 and other allelic variants of the AKAP10 gene known in the
art or dislcosed herein are potential functional variants of a morbidity
susceptibility gene and/or of a gene involved in increased mortality and/or
a gene related to an alteration in signal transduction and associated
disorders and thus is useful for screening for potential therapeutics.
G. Effect ~f Allelic !lariants
The effect of an allelic variant on a AKAP10 gene etr~pression
(amount of mRNA, mRNA stability) and AKAP protein (amount, stability,
intracellular localization, activity) can be determined according to
methods known in the art. Allelic variants of AKAP genes can be
assayed individually or in combination.
In general, any method known to those skilled in the art of
determining the presence or absence of a specific messenger RNA
transcript or a specific translated protein can be used to presence of
absence of a polymorphic protein or a polymorphism in the genetic
sequence.
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1. RNA Analysis
a. Northern Blot Detection of RNA
The northern blot technique is used to identify a RNA fragment of
a specific size from a complex population of RNA using gel
electrophoresis and nucleic acid hybridization. Northern blotting is a well-
known technique in the art. Northern blot analysis is commonly used to
detect specific RNA transcripts expressed in a variety of biological
samples and have been described in Sambrook, J. et al. (Molecular
Cloning, Brd Edition, Cold Spring Harbor Press).
Briefly, total RNA is isolated from any biological sample by the
method of Chomczynski and Sacchi (Anal. Biochem. (1987) 162, 156-
159). Poly-adenylated mRNA is purified from total RNA using mini-oligo
(dT) cellulose spin column kit with methods as outlined by the suppliers
(Invitrogen, Carlsbad CA.). ~enatured RNA is electrophoresed through a
denaturing 1.5% agarose gel and transferred onto a nitrocellulose or
nylon based matrix. The mRNAs are detected by hybridization of a
radiolabeled or biotinylated oligonucleotide probe specific to the
polymorphic regions as disclosed herein.
b. Dot Blot/Slot Blot
Specific RNA transcripts can be detected using dot and slot blot
assays to evaluate the presence of a specific nucleic acid sequence in a
c~mplex mix of nucleic acids. Specific RNA transcripts can be detected
by adding the RNA mixture to a prepared nitrocellulose or nylon
membrane. RNA is detected by the hybridization of a radiolabeled or
biotinylated oligonucleotide probe complementary to the AKAP
sequences as disclosed herein.
c. RT-PCR
The RT-PCR reaction can be performed, as described by K.-Q. Hu
et al., Virology 181:721-726 ( 1991 ), as follows: the extracted mRNA is
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transcribed in a reaction mixture 1 micromolar antisense primer, and 25
U AMV (avian myeloblastosis virus) or MMLV (Moloney murine leukemia
virus) reverse transcriptase. Reverse transcription is performed and the
cDNA is amplified in a PCR reaction volume with Tap polymerase.
Optimal conditions for cDNA synthesis and thermal cycling can be readily
determined by those skilled in the art.
2. Protein and Polypeptide Detection
a. Expression of Protein in a Cell Line
Using the disclosed nucleic acids AICAP10 proteins can be
1Q expressed in a recombinantly engineered cell such as bacteria, yeast,
insect, mammalian, or plant cells. Those of skill in the art are
knowledgeable in the numerous expression systems available for
expression of a nucleic acid encoding proteins such as polymorphic
human AI<AP10 proteins.
b. Expression of AKAP Protein
The isolated nucleic acid encoding a full-length polymorphic human
AI~AP10 protein, or a portion thereof, such as a fragment containing the
site of the polymorphism, can be introduced into a vector for transfer
into host cells. Fragments of the polymorphic human AICAP10 proteins
can be produced by those skilled in the art, without undue
experimentation, by eliminating portions of the coding sequence from the
isolated nucleic acids encoding the full-length proteins.
Expression vectors are used expression of the protein in the host
cell is desired. An expression vector includes vectors capable of
expressing nucleic acids that are operatively linked with regulatory
sequences, such as promoter regions, that are capable of effecting
expression of such nucleic acids. Thus, an expression vector refers to a
recombinant DNA or RNA construct, such as a plasmid, a phage,
recombinant virus or other vector that, upon introduction into an
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appropriate host cell, results in expression of the cloned DNA.
Appropriate expression vectors are well known to those of skill in the art
and include those that are replicable in eukaryotic cells and/or prokaryotic
cells and those that remain episomal or those which integrate into the
host cell genome. Such plasmids for expression of polymorphic human
AICAP10-encoding nucleic acids in eukaryotic host cells, particularly
mammalian cells, include cytomegalovirus (CMV) promoter-containing
vectors, such as pCMV5, the pSV2dhfr expression vectors, which
contain the SV40 early promoter, mouse dhfr gene, SV40
polyadenylation and splice sites and sequences necessary for maintaining
the vector in bacteria, and MMTV promoter-based vectors.
The nucleic acids encoding polymorphic human AKAP10 proteins,
and vectors and cells containing the nucleic acids as provided herein
permit production of the polymorphic proteins, as well as antibodies to
the proteins. This provides a means to prepare synthetic or recombinant
polymorphic human AKAP10 proteins and fragments thereof that are
substantially free of contamination from other AKR~Ps and proteins in
general, the presence of which can interfere with analysis of the
polymorphic proteins. In addition, the polymorphic proteins can be
expressed in combination with selected other proteins that AKAP10 can
associate with in cells. The ability to selectively express the polymorphic
AICAP10 proteins alone or in combination with other selected proteins
makes it possible to observe the functioning of the recombinant
polymorphic proteins within the environment of a cell. The expression of
isolated nucleic acids encoding an AICAP protein will typically be
achieved by operably linking, for example, the DNA or cDNA to a
promoter (which is either constitutive or regulatable), followed by
incorporation into an expression vector. The vectors can be suitable for
replication and integration in either prokaryotes or eukaryotes. Typical
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expression vectors contain transcription and translation terminators,
initiation sequences, and promoters useful for regulation of the
expression of the DNA encoding a protein. To obtain high level
expression of a cloned gene, it is desirable to construct expression
vectors which contain, a strong promoter to direct transcription, a
ribosome binding site for translational initiation, and a
transcription/translation terminator. One of skill in the art would
recognize that modifications can be made to an AKAP10 protein without
diminishing its biological activity. Some modifications can be made to
1~ facilitate the cloning, expression, or incorporation of the targeting
molecule into a fusion protein. Such modifications are well known to
those of skill in the art and include, for example, a methionine added at
the amino terminus to provide an initiation site, or additional amino acids
(e.g., poly His) placed on either terminus to create conveniently located
purification sequences. Restriction sites or termination codons can also
be introduced. There are expression vectors that specifically allow the
expression ~f fu~,ctional proteins. One such vector, Plasmid 5~~,
described in IJ.S. Pat. No. 6,0~0,12~ and incorporated herein by
reference, has been constructed for the expression of secreted antigens
2~ in a permanent cell line. This plasmid contains the following DNA
segments: (a) a fragment of pBR322 containing bacterial beta-lactamase
and origin of DNA replication; (b) a cassette directing expression of a
neomycin resistance gene under control of HSV-1 thymidine kinase
promoter and poly-A addition signals; (c) a cassette directing expression
of a dihydrofolate reductase gene under the control of a SV-40 promoter
and poly-A addition signals; (d) cassette directing expression of a rabbit
immunoglobulin heavy chain signal sequence fused to a modified
hepatitis C virus (HCV) E2 protein under the control of the Simian Virus
40 T-Ag promoter and transcription enhancer, the hepatitis B virus
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surface antigen (HBsAg) enhancer I followed by a fragment of Herpes
Simplex Virus-1 (HSV-1 ) genome providing poly-A addition signals; and
(e) a fragment of Simian Virus 40 genome late region of no function in
this plasmid. All of the segments of the vector were assembled by
standard methods known to those skilled in the art of molecular biology.
Plasmids for the expression of secreted AKAP proteins can be
constructed by replacing the hepatitis C virus E2 protein coding sequence
in plasmid 577 with a AKAP sequence of SEQ ID N0: 3 or a fragment
thereof. The resulting plasmid is transfected into CHO/dhfr-cells (D?CB-
1 1 1 ) (Uriacio, et al., PNAS 77, 4451-4460; 1980); these cells are
available from the A.T.C.C., 12301 Parklawn Drive, Rockville, Md.
20852, under Accession No. CRL 9096), using the cationic liposome-
mediated procedure (P. L. Felgner et al., PNAS 84:7413-7417 (1987).
Proteins are secreted into the cell culture media.
Incorporation of cloned DNA into a suitable expression vector,
transfection of cells with a plasmid vector or a combination of plasmid
vectors, each encoding one or more distinct proteins or with linear DNA,
and selection of transfected cells are well known in the art (see, e.g.,
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory Press). Heterologous nucleic acid
can be introduced into host cells by any method known to those of skill
in the art, such as transfection with a vector encoding the heterologous
nucleic acid by CaP04 precipitation (see, e.g., Wigler et al. (1979) Pr~e.
Nat/. Acad. Sci. USA 76:1373-1376) or lipofectamine (GIBCO BRL
#18324-012). Recombinant cells can then be cultured under conditions
whereby the polymorphic human AKAP10 protein encoded by the nucleic
acid is expressed. Suitable host cells include mammalian cells (e.g.,
HEK293, including but are not limited to, those described in U.S. Patent
No. 5,024,939 to Gorman (see, also, Stillman et al. (1985) Mol. Cell.
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Biol. 5:2051-2060); also, HEK293 cells available from ATCC under
accession #CRL 1573), CHO, COS, BHKBI and Ltk- cells, mouse
monocyte macrophage P388D1 and J774A-1 cells (available from ATCC,
Rockville, MD) and others known to those of skill in this art), yeast cells,
including, but are not limited to, Pichia pastoris, Saccharomyces
cerevisiae, Candida tropicalis, Hansenula polymorpha, human cells and
bacterial cells, including, but are not limited to, Escherichia coli.
~Cenopus oocytes can also be used for expression of in ~ritro RNA
transcripts of the DNA.
Heterologous nucleic acid can be stably incorporated into cells or
can be transiently expressed using methods known in the art. Stably
transfected mammalian cells can be prepared by transfecting cells with
an expression vector having a selectable marker gene (such as, for
example, the gene for thymidine kinase, dihydrofolate reductase,
neomycin resistance, and the like), and growing the transfected cells
under conditions selective for cells expressing the marker gene. To
prepare transient transfectants, mammalian cells are transfected with a
reporter gene (such as the E. coli f3-galactosidase gene) to monitor
transfection efficiency. Selectable marker genes are not included in the
transient transfections because the transfectants are typically not grown
under selective conditions, and are usually analyzed within a few days
after transfection.
Heterologous nucleic acid can be maintained in the cell as an
episomal element or can be integrated into chromosomal DNA of the cell.
The resulting recombinant cells can then be cultured or subcultured (or
passaged, in the case of mammalian cells) from such a culture or a
subculture thereof. Methods for transfection, injection and culturing
recombinant cells are known to the skilled artisan. Similarly, the
polymorphic human AKAP10 proteins or fragments thereof can be
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purified using protein purification methods known to those of skill in the
art. For example, antibodies or other ligands that specifically bind to the
proteins can be used for affinity purification and immunoprecipitation of
the proteins.
b. Protein Purification
The AKAP10 proteins can be purified by standard techniques well
known to those of skill in the art. Recombinantly produced proteins can
be directly expressed or expressed as a fusion protein. The recombinant
protein is purified by a combination of cell lysis (e.g., sonication, French
press) and affinity chromatography. The proteins, recombinant or
synthetic, can be purified to substantial purity by standard techniques
well known in the art, including detergent solubili~ation, selective
precipitation with such substances as ammonium sulfate, column
chromatography, immunopurification methods, and others. (See, for
example, R. Scopes, Protein Purification: Principles and Practice,
Springer-Verlag: New York (1952); Deutscher, Guide to Protein
Purification, Academic Press (199~)). For ea;ample, antibodies can be
raised to the proteins as described herein. Purification from E. coli can be
achieved following procedures described in U.S. Pat. No. 4,511,5~3. The
protein can then be isolated from cells expressing the protein and further
purified by standard protein chemistry techniques as described herein.
Detection of the expressed protein is achieved by methods known in the
art and include, for example, radioimmunoassays, Western blotting
techniques or immunoprecipitation.
3. Immunodetection of the AKAP10 protein.
Generally, the AICAP proteins, when presented as an immunogen,
should elicit production of a specifically reactive antibody.
Immunoassays for determining binding are well known to those of skill in
the art, as are methods of making and assaying for antibody binding
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specificity/affinity. Exemplary immunoassay formats include ELISA,
competitive immunoassays, radioimmunoassays, Western blots, indirect
immunofluorescent assays, in vivo expression or immunization protocols
with purified protein preparations. In general, the detection of
immunocomplex formation is well known in the art and can be achieved
by methods generally based upon the detection of a label or marker, such
as any of the radioactive, fluorescent, biological or enzymatic tags.
Labels are well known to those skilled in the art (see U.S. Pat. Nos.
3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149
and 4,366,241, each incorporated herein by reference). 0f course, one
can find additional advantages through the use of a secondary binding
ligand such as a second antibody or a biotin/avidin ligand binding
arrangement, as is known in the art.
Production of P~lyclonal Antisera Against AKAP
Antibodies can be raised to AKAP proteins, including individual,
allelic, strain, or species variants, and fragments thereof, both in their
naturally occurring (full-length) forms and ir, recombinant forms.
Additionally, antibodies are raised to these proteins in either their native
configurations or in non-native configurations. Anti-idiotypic antibodies
can also be generated. A variety of analytic methods are available to
generate a hydrophilicity profile of proteins. Such methods can be used
to guide the artisan in the selection of peptides for use in the generation
or selection of antibodies which are specifically reactive, under
immunogenic conditions. See, e.g., J. Janin, Nature, 277 (1979) 491-
492; Wolfenden, et al., Biochemistry 20(1981 ) 849-855; Kyte and
Doolite, J. Mol. Biol. 157 (1982) 105-132; Rose, et al., Science 229
( 1985) 834-838.
A number of immunogens can be used to produce antibodies
specifically reactive with AKAP proteins. Isolated recombinant,
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synthetic, or native polypeptides are typical immunogens (antigen) for the
production of monoclonal or polyclonal antibodies. Polypeptides are
typically denatured, and optionally reduced, prior to formation of
antibodies for screening expression libraries or other assays in which a
putative AKAP protein is expressed or denatured in a non-native
secondary, tertiary, or quartenary structure.
The AKAP protein (SEQ ID NO: 4, or a portion thereof) is injected
into an animal capable of producing antibodies. Either monoclonal or
polyclonal antibodies can be generated for subsequent use in
immunoassays to measure the presence and quantity of the protein.
Methods of producing polyclonal antibodies are known to those of skill in
the art. In brief, an immunogen (antigen), typically a purified protein, a
protein coupled to an appropriate carrier (e.g., GST, keyhole limpet
hemanocyanin, etc.), or a protein incorporated into an immunization
vector such as a recombinant vaccinia virus (see, U.S. Pat. No.
4,722.,848) is mixed with an adjuvant and animals are immunized with
the mixture. The animates immune response to the immunogen
preparation is monitored by taking test bleeds and determining the titer of
reactivity to the proteiri of interest. When appropriately high titers of
antibody to the immunogen are obtained, blood is collected from the
animal and antisera are prepared. Further fractionation of the antisera to
enrich for antibodies reactive to the protein is performed where desired
(See, e.g., Coligan, Current Protocols in Immunology, Wiley/Greene, NY
(1991); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Press, NY (1989)).
b. Western Blotting of Tissue Samples for the AKAP protein
Biological samples are homogenized in SDS-PAGE sample buffer
(50 mM Tris-HCI, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1
bromophenol blue, 10% glycerol), heated at 100 degrees Celsius for 10
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min and run on a 14% SDS-PAGE with a 25 mM Tris-HCI, pH 8.3, 250
mM Glycine, 0.1 % SDS running buffer. The proteins are
electrophoretically transferred to nitrocellulose in a transfer buffer
containing 39 mM glycine, 48 mM Tris-HCI, pH 8.3, 0.037% SDS, 20%
methanol. The nitrocellulose is dried at room temperature for 60 min and
then blocked with a PBS solution containing either bovine serum albumin
or 5% nonfat dried milk for 2 hours at 4 degrees Celsius.
The filter is placed in a heat-sealable plastic bag containing a
solution of 5% nonfat dried milk in PBS with a 1:100 to 1:2000 dilution
1~ of affinity purified anti-AKAP peptide antibodies, incubated at 4 degrees
Celsius for 2 hours, followed by three 10 min washes in PBS. An alkaline
phosphatase conjugated secondary antibody (i.e., anti-mouse/rabbit IgG),
is added at a 1:200 to 1:2000 dilution to the filter in a 150 mM iVaCl, 50
mM Tris-HCI, pH 7.5 buffer and incubated for 1 h at room temperature.
The bands are visualized upon the addition and development of a
chromogenic substrate such as 5-bromo-4-chloro-3-indolyl
phosphal:e/nitro blue tetrazoliun, (BCIP/i~BT). The filter is incubated in the
solution at room temperature until the bands develop to the desired
intensity. Molecular mass determination is made based upon the mobility
2~ ~f pre-stained molecular weight standards (Rainbow markers, Amersham,
Arlington Heights, III.).
c. Microparticle Enzyme Immun~assay (MEIA)
AKAP10 proteins and peptides are detected using a standard
commercialized antigen competition EIA assay or polyclonal antibody
sandwich EIA assay on the IMx.RTM Analyzer (Abbott Laboratories,
Abbott Park, III.). Samples containing the AKAP10 protein are incubated
in the presence of anti-AKAP10 coated microparticles . The
microparticles are washed and secondary polyclonal anti-AKAP10
antibodies conjugated with detectable entities (i.e., alkaline phosphatase)
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are added and incubated with the microparticles. The microparticles are
washed and the bound antibody/antigen/antibody complexes are
detected by adding a substrate (i.e. 4-methyl umbelliferyl phosphate)
(MUP) that will react with the secondary conjugated antibody to generate
a detectable signal.
d. Immunocytochemistry
Intracellular localization of the AICAP10 protein can be determined
by a variety of in situ hybridization techniques. In one method cells are
fixed with fixed in 4% paraformaldehyde in 0.1 M phosphate buffered
1~ saline (PBS; pH7.4) for 5 min., rinsed in PBS for 2 min., dilapidated and
dehydrated in an ethanol series (50, 70 and 95°/~) (5 min, each and
stored in 95°/~ ethanol at 4 degrees Celsius).
The cells are stained with the primary anti-AICAP10 antibody and a
mixture of secondary antibodies used for detection. Laser-scanning
confocal microscopy is performed to localize the AICAP10 protein.
4. Binding Assays
Assays to measure the interaction between AICAP10 a~,d the
regulatory subunits RI and/or RII of the Protein ICinase A holoenzyme
include immobilized binding assays, solution binding assays and the like.
2~ In some instances, it can be desirable to monitor binding between
AICAP10 and PICA. In other instances, it can be desirable to specifically
monitor the binding between AICAP10 and a cellular component (other
than PICA) to which it binds. Assays can be performed in a variety of
formats, including cell-based assays, such as di-hybrid screening or
complementation assays as described in U.S. Pat. No. 5,283,173 and
Patent Cooperation Treaty (PCT) Publication No. W0 91 /16457,
respectively. Assays of this type are particularly useful for assessing
intracellular efficacy of test compounds. Non-cell-based assays include
scintillation proximity assays, CAMP competition assays, ELISA assays,
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radioimmunoassays, chemiluminescent assays, and the like. Such assay
procedures are well known in the art and generally described, e.g., in
Boudet et al., J. Immunol. Meth., 142:73-82 (1991 ); Ngai et al., J.
Immunol. Meth., 158:267-276 (1993); Pruslin et al., J. Immunol. Meth.,
137:27-35 (1991 ); Udenfriend et al., Proc. Natl. Acad. Sci. USA,
82:8672-8676 (1985); Udenfriend et al., Anal. Biochem., 161:494-500
(1987); Bosworth and Towers, Nature, 341:167-168 (1989); Gilman,
Proc. Natl. Acad. Sci. USA, 67:305-312 (1970); and U.S. Pat. No.
4, 568, 649.
a. In vitr~ binding assay
Huang et al. Proc. Natl. Acad. Sci. USA, 272:8057-8064 ( 1997);
Protein preparations containing AI~AP10 are incubated with glutathione
resin in PBS for 2 hours at 4 degrees Celsius with 0.1 ~f~ Triton ?~-100,
1 mM phenylmethylsulfonyl fluoride, 1 mM E~TA, 5mM benzamidine, and
5mM B-mercapthoethanol and washed extensively with the same buffer.
200 micrograms of PICA regulatory subunit RII and/or RI were added to
the resin and incubated at 4 degrees Celsius. Proteins associated with
the AI~AP10 are eluted and analyzed by Laemmli electrophoresis. The
proteins were visualized by Coomassie Staining. PICA proteins can be
radiolabeled or labeled with a flurophore to allow detection.
b. PICA phosphorylation of protein substrate
Cyclic AMP-dependent protein kinase (PICA) catalyzes the transfer
of gamma phosphate from adenosine triphosphate (ATP) to a serine or
threonine residue in a protein substrate. A short synthetic peptide
(Leucine-Arg-Arg-Alanine-Serine-Leucine-Glycine or LRRASLG) is used as
a substrate to assay the specific type of PICA activity as described in
Pearson et. al., Methods of Enzymology 200, 62-81 (1991 ).
The PICA assay is typically carried out in a reaction of the enzyme
with a peptide substrate and gamma 32P-ATP followed by separation of
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the 32P-peptide product from the unreacted gamma 32P-ATP on a
phosphocellulose membrane. This method requires at least one basic
amino acid residue in the peptide substrate. The peptide substrate can be
tagged with a biotin group so that the biotinylated 32P-peptide product
consistently binds to a streptavidin membrane in a manner independent
of the peptide sequence as described in Goueli et al Analytical
Biochemistry 225, 10-17, (1995). The separation of the 32P-peptide
product from the free gamma 32P-ATP using affinity binding and
ultrafiltration separation to analyze a mixture sample as described in U.S.
Patent No. 5,869,275.
If the mutation is located in an intron, the effect of the mutation
can be determined, e.g., by producing transgenic animals in which the
allelic variant has been introduced and in which the wild-type gene or
predominant allele can have been knocked out. Comparison of the level
of expression of the protein in the mice transgenic for the allelic variant
with mice transgenic for the predominant allele will reveal whether the
mutation results in inci°eased or decreased synthesis of the associated
protein and/or aberrant tissue distribution or intracellular localization of
the associated protein. Such analysis could also be performed in
cultured cells, in which the human variant allele gene is introduced and,
e.g., replaces the endogenous gene in the cell. For mutant AI~AP
proteins binding to signaling enzymes such as PICA is also examined.
Thus, depending on the effect of the alteration a specific treatment can
be administered to a subject having such a mutation. Accordingly, if the
mutation results in decreased production of AICAP protein, the subject
can be treated by administration of a compound which increases
synthesis, such as by increasing AKAP gene expression, and wherein the
compound acts at a regulatory element different from the one which is
mutated. Alternatively, if the mutation results in increased AICAP
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protein, the subject can be treated by administration of a compound
which reduces protein production, e.g., by reducing AKAP gene
expression or a compound which inhibits or reduces the activity of AKAP
protein.
H. Diagnostic and Prognostic Assays
Typically, an individual allelic variant that associates with morbidity
and/or mortality and/or an alteration in signal transduction will not be
used in isolation as a prognosticator. An allelic variant typically will be
one of a plurality of indicators that are used. The other indicators can be
the manifestation of other risk factors for morbidity and/or mortality and
other evidence of altered signal transduction.
Useful combinations of allelic variants of the AKAP10 gene can be
determined. Variants can be assayed individually or assayed
simultaneously using multiplexing methods as described above or any
other labelling method that allows different variants to be identified. In
particular, variants of the AKAP10 gene can be assayed using kits (see
below) or any of a variety microarrays known to those in the art. For
example, oligonucleotide probes comprising the polymorphic regions
surrounding any polymorphism in the AKAP10 gene can be designed and
2~ fabricated using methods such as those described in U.S. Patent Nos.
5,492,806; 5,525,464; 5,695,940; 6,018,041; 6,025,136; WO
98/30883; WO 98/56954; W099/09218; WO 00/58516;
WO 00/58519, or references cited therein.
I. Databases
Use of databases containing sets of parameters associated with
subjects in populations selected on the basis of apparent good health,
not manifesting detectable disease (i.e., an unbiased population not
selected for any disease state), allows for identification of such morbidity
susceptibility genes tsee, U.S. Provisional Application Serial No.
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60/159,176 filed October 13, 1999, U.S. Provisional Application Serial
No. 60/217,658 filed on July 10, 2000 and U.S. Application Serial No.
09/687,483 filed October 13, 2000).
For example, in a method for determining susceptibility to
morbidity, increased or early mortality, or morbidity and increased or
early mortality in a human being, provided herein, exemplary steps
include detecting the presence or absence of an allele of the human
AKAP10 containing other than an A at position 2073 of the coding
sequence of the AKAP10 gene; wherein the presence of an allele
containing other than an A at position 2073 is indicative of increased
susceptibility to morbidity, increased or early mortality, or morbidity and
increased or early mortality as compared to the susceptibility of a human
being who does not comprise an allele containing other than an A at
position 2073 of the AKAP10 gene coding sequence.
As noted above, using a healthy patient database (see, U.S.
Provisional Application Serial No. 60/159,176, U.S. Provisional
~4pplication Serial fro. 50/217,558 and U.S. Application Serial l~o.
09/587,483 filed October 13, 2000), the frequency of occurrence of the
AKAP10-5 SNP in such a population was found to decrease with age,
thus making the allele a potential morbidity susceptibility gene, a gene
associated with increased mortality or both. Using the healthy database,
it was found that the homozygote GG genotype drops in the elderly
population (over > 60 years), by a statistically significant amount,
p = 0.02.
J. Isolation of Polymorphic AKAP10 Gene Sequences
Exemplary nucleic acid sequences encoding polymorphic human
AKAP10 proteins are represented by nucleotides which encode the amino
acid sequence as set forth in SEQ. ID. NO: 3. Such polymorphic
nucleotide sequences can encode variant amino acid sequences, such as
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the sequence set forth in SEQ. ID. NO: 4 in which amino acid 646 has
been replaced with a valine; other amino acid sequence variants at amino
acid 646 include leucine or phenylalanine.
Other exemplary nucleic acid sequences represent allelic variants
of the AKAP10 gene which are not located in protein coding regions.
Such as set forth in nucleotide position 83,580 to position 156,577 of
SEQ ID N0: 13, 14 and 18.
Nucleic acid encoding polymorphic human AKAP10 proteins and
genes provided herein can be isolated by screening suitable human cDNA
or human genomic libraries under suitable hybridization conditions with
nucleic acids such as those provided in SEQ. ID. NOS: 1, 3, 13, 14, 17
and 18. Suitable libraries can be prepared from human tissue and cell
samples. In order to isolate cDNA encoding a polymorphic human
AKAP10 libraries prepared from different tissues can be screend since
the allele can not be expressed in all tissues or at similar levels in
different tissues. The library can be screened with a portion of DNA
including substantially the entire human AKAP10 or polymorphic
AKAP10 protein-encoding sequence as set forth in SECO. ID. NOS. 1, 3,
13, 14, 17 and 18, or the library can be screened with a suitable probe.
After screening the library, positive clones are identified by
detecting a hybridization signal; the identified clones are characterized by
restriction enzyme mapping and/or DNA sequence analysis, and then
examined, by comparison with the sequences set forth herein to
ascertain whether they include DNA encoding a complete polymorphic
human AKAP10 protein (i.e., if they include translation initiation and
termination codons). If the selected clones are incomplete, they can be
used to rescreen the same or a different library to obtain overlapping
clones. If the library is genomic, then the overlapping clones can include
exons and introns. If the library is a cDNA library, then the overlapping
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clones will include an open reading frame. In both instances, clones can
be identified by comparison with the DNA and encoded proteins provided
herein.
In an alternative method, oligonucleotides based on the human
AKAP10 or polymorphic AKAP10 protein-encoding sequence as set forth
in SEQ. ID. NOS. 1, 3, 13, 14, 17 and 18, can be used to amplify
fragments of the protein coding region of the AKAP10 gene from human
cDNA or genomic sequence.
The isolated nucleic acid sequences can be incorporated into
vectors for further manipulation. As used herein, vector (or plasmid)
refers to discrete elements that are used to introduce heterologous DNA
into cells for either eacpression or replication thereof. Selection and use
of such vehicles are well within the skill of the artisan,
K. Transgenic Animals
Methods for making transgenic animals using a variety of
transgenes have been described in Wagner et al., Proc. Nat. Acad. Sc.
Cd.S.A., Vol. 78, p. 50158 1981; Stewart e~ ~/., Science, Vol. 217, p,
1045, 1982; Constantini a~ al., Nature, Vol. 294, p. 92, 1981; Lacy et
a/., Cell, Vol, 34, p. 343, 1983; McKnight et al., Cell, Vol. 34., p. 335,
1983; 8rinstar et al., Nature, Vol. 306, p. 332, 1983; Palmiter e~ al.,
Nature, Vol. 300, p. 611, 1982; Palmiter eta/.,Cell, Vol. 29, p. 701,
1982 and Palmiter et al., Science, Vol. 222, p. 809, 1983. Such
methods are described in U.S. Patent Nos. 5,175,057; 6,180,849 and
6,133,502.
The term "firansgene" is used herein to describe genetic material
that has been or is about to be artificially inserted into the genome of a
mammalian cell, particularly a mammalian cell of a living animal. The
transgene is used to transform a cell, meaning that a permanent or
transient genetic change, typically a permanent genetic change, is
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induced in a cell following incorporation of exogenous DNA. A
permanent genetic change is generally achieved by introduction of the
DNA into the genome of the cell. Vectors for stable integration include,
but are not limited to, plasmids, retroviruses and other animal viruses and
YACS. Of interest are transgenic mammals, including, but are not limited
to, cows, pigs, goats, horses and others, and particularly rodents,
including rats and mice. Typically, the transgenic-animals are mice.
Transgenic animals contain an exogenous nucleic acid sequence
present as an extrachromosomal element or stably integrated in all or a
portion of its cells, especially germ cells. Unless otherwise indicated, it
will be assumed that a transgenic animal comprises stable changes to the
germline sequence. During the initial construction of the animal,
"chimeras" or "chimeric animals" are generated, in which only a subset
of cells have the altered genome. Chimeras are primarily used for
breeding purposes in order to generate the desired transgenic animal.
Animals having a heterozygous alteration are generated by breeding of
chimeras. f~iale and female hetero~ygotes are typically bred to generate
homozygous animals.
The exogenous gene is usually either from a different species than
the animal host, or is otherwise altered in its coding or non-coding
sequence. The introduced gene can be a wild-type gene, naturally
occurring polymorphism or a genetically manipulated sequence, for
example having deletions, substitutions or insertions in the coding or
non-coding regions. When the introduced gene is a coding sequence, it
is usually operably linleed to a promoter, which can be constitutive or
inducible, and other regulatory sequences required for expression in the
host animal.
Transgenic animals can comprise other genetic alterations in
addition to the presence of alleles of AKAP genes. For example, the
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genome can be altered to affect the function of the endogenous genes,
contain marker genes, or contain other genetic alterations (e.g., alleles of
other genes associated with cardiovascular disease).
A "knock-out" of a gene means an alteration in the sequence of
the gene that results in a decrease of function of the target gene,
typically such that target gene expression is undetectable or insignificant.
A knock-out of an endogenous AKAP gene means that function of the
gene has been substantially decreased so that expression is not
detectable or only present at insignificant levels. "Knock-out"
transgenics can be transgenic animals having a heterozygous knock-out
of an AKAP gene or a homozygous knock-out. "Knock-outs" also include
conditional knock-outs, where alteration of the target gene can occur
upon, for example, exposure of the animal to a substance that promotes
target gene alteration, introduction of an enzyme that promotes
recombination at the target gene site (e.g., Cre in the Cre-lox system), or
other method for directing the target gene alteration postnatally.
A "knock-in" of a target gene means an alteration in a host cell
genome that results in altered expression (e.g., increased (including
ectopic)) of the target gene, e.g., by introduction of an additional copy of
the target gene, or by operatively inserting a regulatory sequence that
provides for enhanced expression of an endogenous copy of the target
gene. "Knock-in" transgenics of interest can be transgenic animals
having a knock-in of an AKAP gene. Such transgenics can be
heterozygous or homozygous for the knock-in gene. "Knock-ins" also
encompass conditional knock-ins.
A construct is suitable for use in the generation of transgenic
animals if it allows the desired level of expression of an AKAP encoding
sequence or the encoding sequence of another gene associated with
cardiovascular disease. Methods of isolating and cloning a desired
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sequence, as well as suitable constructs for expression of a selected
sequence in a host animal, are well known in the art and are described
below.
For the introduction of a gene into the subject animal, it is
generally advantageous to use the gene as a gene construct wherein the
gene is ligated downstream of a promoter capable of and operably linked
to expressing the gene in the subject animal cells. Specifically, a
transgenic non-human mammal showing high expression of the desired
gene can be created by microinjecting a vector ligated with the gene into
a fertilized egg of the subject non-human mammal (e.g., rat fertilized egg)
downstream of various promoters capable of expressing the protein
and/or the corresponding protein derived from various mammals (rabbits,
dogs, cats, guinea pigs, hamsters, rats, mice etc., typicall rats ete.)
Useful vectors include Escherichia coli-derived plasmids, Sacillus
subtilis-derived plasmids, yeast-derived plasmids, bacteriophages such as
lambda, phage, retroviruses such as I~lloloney leukemia virus, and animal
viruses s~acll as vaccinia virus or baculovirus.
Useful promoters for such gene expression regulation include, for
example, promoters for genes derived from viruses (cytomegalovirus,
Moloney leukemia virus, JC virus, breast cancer virus etc.), and
promoters for genes derived from various mammals (humans, rabbits,
dogs, cats, guinea pigs, hamsters, rats, mice ete.) and birds (chickens
etc.) (e. g., genes for albumin, insulin II, erythropoietin, endothelin,
osteocalcin, muscular creatine kinase, platelet-derived growth factor
beta, keratins K1, K10 and K14, collagen types I and II, atrial natriuretic
factor, dopamine beta-hydroxylase, endothelial receptor tyrosine kinase
(generally abbreviated Tie2), sodium-potassium adenosine
triphosphorylase (generally abbreviated Na,K-ATPase), neurofilament light
chain, metallothioneins I and IIA, metalloproteinase I tissue inhibitor,
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MHC class I antigen (generally abbreviated H-2L), smooth muscle alpha
actin, polypeptide chain elongation factor 1 alpha (EF-1 alpha), beta
actin, alpha and beta myosin heavy chains, myosin light chains 1 and 2,
myelin base protein, serum amyloid component, myoglobin, renin etc.).
In one embodiment, the above-mentioned vectors have a sequence
for terminating the transcription of the desired messenger RNA in the
transgenic animal (generally referred to as terminator); for example, gene
expression can be manipulated using a sequence with such function
contained in various genes derived from viruses, mammals and birds. In
one example, the simian virus SV40 terminator etc. are commonly used.
Additionally, for the purpose of increasing the expression of the desired
gene, the splicing signal and enhancer region of each gene, a portion of
the intron of a eukaryotic organism gene can be ligated 5' upstream of
the promoter region, or between the promoter region and the
translational region, or 3' downstream of the translational region as
desired.
A translational region for a protein of interest can be obtained
using the entire or portion of genomic DNA of blood, kidney or fibroblast
origin from various mammals (humans, rabbits, dogs, cats, guinea pigs,
hamsters, rats, mice etc.) or of various commercially available genomic
DNA libraries, as a starting material, or using complementary DNA
prepared by a known method from RNA of blood, kidney or fibroblast
origin as a starting material. Also, an exogenous gene can be obtained
using complementary DNA prepared by a known method from RNA of
human fibroblast origin as a starting material. All these translational
regions can be used in transgenic animals.
To obtain the translational region, it is possible to prepare DNA
incorporating an exogenous gene encoding the protein of interest in
which the gene is ligated downstream of the above-mentioned promoter
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(typically upstream of the translation termination site) as a gene
construct capable of being expressed in the transgenic animal.
DNA constructs for random integration need not include regions of
homology to mediate recombination. Where homologous recombination
is desired, the DNA constructs will comprise at least a portion of the
target gene with the desired genetic modification, and will include
regions of homology to the target locus. Conveniently, markers for
positive and negative selection are included. Methods for generating
cells having targeted gene modifications through homologous
recombination are known in the art. For various techniques for
transfecting mammalian cells, see ICeown et al. (1 J90) Methods in
Enzymology 155:57-537.
The transgenic animal can be created by introducing an AI~AP
gene construct into, for example, an unfertilized egg, a fertilized egg, a
spermatozoon or a germinal cell containing a primordial germinal cell
thereof, typically in the embryogenic stage in the development of a non-
human mammal (more typically in the single-cell or fertilized cell stage
and generally before the S-cell phase), by standard means, such as the
calcium phosphate method, the electric pulse method, the lipofection
method, the agglutination method, the microinjection method, the particle
gun method, the DEAE-dextran method and other such method. Also, it
is possible to introduce a desired AICAP gene into a somatic cell, a living
organ, a tissue cell or other cell, by gene transformation methods, and
use it for cell culture, tissue culture and any other method of
propagation. Furthermore, these cells can be fused with the above-
described germinal cell by a commonly known cell fusion method to
create a transgenic animal.
For embryonic stem (ES) cells, an ES cell line can be employed, or
embryonic cells can be obtained freshly from a host, e.g. mouse, rat,
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guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder
layer or grown in the presence of appropriate growth factors, such as
leukemia inhibiting factor (LIF). When ES cells have been transformed,
they can be used to produce transgenic animals. After transformation,
the cells are plated onto a feeder layer in an appropriate medium. Cells
containing the construct can be detected by employing a selective
medium. After sufficient time for colonies to grow, they are picked and
analyzed for the occurrence of homologous recombination or integration
of the construct. Those colonies that are positive can then be used for
embryo manipulation and blastocyst injection. Blastocysts are obtained
from 4 to 6 week old superovulated females. The ES cells are
trypsinized, and the modified cells are injected into the blastocoel of the
blastocyst. After injection, the blastocysts are returned to each uterine
horn of pseudopregnant females. Females are then allowed to go t~ term
and the resulting litters screened for mutant cells having the construct.
Sy providing for a different phenotype of the blastocyst and the ES cells,
chimeric pr~geny can be readily detracted. The chimeric animals are
screened f~r the presence of the m~dified gene and males and females
having the modification are mated to produce homozygous progeny. If
the gene alterations cause lethality at some point in development, tissues
or organs can be maintained as allogeneic or congenic grafts or
transplants, or in in ~ritr~ culture.
Animals containing more than one transgene, such as allelic
variants of AICAP genes and/or other genes associated with morbidity
and/or mortality can be made by sepuentially introducing individual alleles
into an animal in order to produce the desired phenotype (manifestation
of morbidity and/or predisposition to early mortality).
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L. Screening assays for modulators
Modulators of AKAP10 biological activities can be identified by
using any of the disclosed methods related to AKAP10 binding to PKA,
AKAP10 localization in the mitochondria, binding to other signaling
enzymes and phosphorylation by PKA.
In particular, once a variant protein such as AKAP10-5 is
contacted with a potential modulating molecule the effect of the
molecule on the binding between AKAP protein and PKA can be
determined by using the assays disclosed in the section entitled " Effect
of Allelic Variants". For example mitochondria can be isolated from cells
exposed to the potential modulating molecule. PKA protein can then be
isolated and quantitated or phosphor,<lation can be determined using the
disclosed PKA assay. An increase in the amount of PICA protein in the
mitochondria or the quantity of test peptide phosphorylated by
mitochondrial isolated PKA would indicate a positive effect of the test
molecule. Binding of AKAP10 protein and PKA could be directly
assessed using an in vitro binding assay, or rather disclosed binding
assays, or by immunoassays such as immunoprecipitati~n.
For allelic variants that d~ not alter the AKAP10 protein the effect
of a potential modulating molecule can be assayed by examining PKA
RNA using the various methods disclosed for RNA analysis.
M. Ribozymes
A ribozyme targets the RNA genome and RNA transcripts and
copies thereof. Each ribozyme molecule contains a catalytically active
segment capable of cleaving the plus or minus strand of RNA, and further
comprises flanking sequences having a nucleotide sequence
complementary to portions of the target RNA. The flanking sequences
serve to anneal the ribozyme to the RNA in a site-specific manner.
Absolute complementarity of the flanking sequences to the target
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sequence is not necessary, however, as only an amount of
complementarity sufficient to form a duplex with the target RNA and to
allow the catalytically active segment of the ribozyme to cleave at the
target sites is necessary. Thus, only sufficient complementarity to permit
the ribozyme to be hybridizable with the target RNA is required.
In some embodiments of the present invention the enzymatic RNA
molecule is formed in a hammerhead motif but the ribozyme can also be
formed in the motif of a hairpin, hepatitis delta virus, group I intron or
RNAse P RNA (in association with an RNA guide sequence). Examples of
hammerhead motifs are described by Rossi et al., AIDS Res. Hum.
Retrovir. 8:183 ( 1992), hairpin motifs are described by Hampel et al.,
f3iochem. 28:4929 (1989) and Hampel et al., Nucl. Acids Res. 18:299
(1990), the hepatitis delta virus motif is exemplified in Perrotta and Been,
l3iochem. 31:16 (1992), an RNAseP motif is described in Cueerier-
Takada et al., Cell 35:849 (1983), and examples of the group I intron
motif are described in Cech et al., U.S. Pat. No. 4,987,071, each of the
foreg~ing disclosures being incorporated herein by reference.
Ribozymes can be prepared by chemical synthesis or produced by
recombinant vectors according to methods established for the synthesis
of RNA molecules. See, e.g., Sambrook et al., Molecular Cloning, A
Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. (1989), incorporated herein by reference. The
ribozyme sequence can be synthesized, for example, using RNA
polymerases such as T7 or SP6. The ribozymes can be prepared from a
corresponding DNA sequence (DNA which on transcription yields a
ribozyme) operably linked to an RNA polymerase promoter such as the
promoter for T7 RNA polymerase or SP6 RNA polymerase. A DNA
sequence corresponding to a ribozyme can be ligated in to a DNA vector,
such as a plasmid, bacteriophage or other virus. Where the transfer
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vector contains an RNA polymerase promoter operably linked to DNA
corresponding to a ribozyme, the ribozyme can be conveniently produced
upon incubation with an RNA polymerase. Ribozymes can therefore be
produced in vitro by incubation of RNA polymerase with an RNA
polymerase promoter operably linked to DNA corresponding to a
ribozyme, in the presence of ribonucleotides. In vivo, procaryotic or
eucaryotic cells (including mammalian cells) can be transfected with an
appropriate vector containing genetic material corresponding to a
ribozyme, operably linked to an RNA polymerase promoter such that the
ribozyme is transcribed in the host cell. Ribozymes can be directly
transcribed in vivo from a transfer vector, or alternatively, can be
transcribed as part of a larger RNA molecule. For example, DNA
corresponding to ribozyme sequence can be ligated into the 3' end of a
carrier gene, for example, after a translation stop signal. Larger RNA
molecules can help to stabilize the ribozyme molecules against nuclease
digestion within the cells. On translation the carrier gene can give rise to
a protein, e~hose presence can be directly assayed if desired, for
example, by enzymatic reaction when the carrier gene encodes an
enzyme.
Those of skill in the art based on the above description and the
sequences disclosed herein can design ribozymes to target RNA
representing the allelic variants of the AffAP1 ~ gene. For example, the
sequence of anti-AICAP10-5 hammerhead ribozyme is
5-UCCA CUGANGAGCCUGGACGAAACU-3'(SEQID
NO: 25). The sequence UGCA is complementary to target RNA with C
hybridizing to the G nucleotide at position 2073 of SEQ ID NO: 3 of the
AICAP1O-5 allelic variant. The simplest hammerhead ribozyme must have
UG at the 5' end of the substrate binding site.
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N. Kits
Kits can be used to indicate whether a subject is at risk of
increased susceptibility to morbidity and/or predisposition for premature
or increased or early mortality. The kits can also be used to determine if
a subject has a genetic predisposition to a disorder related to signal
transduction. This information could be used, e.g., to optimize treatment
of such individuals as a particular genotype can be associated with drug
response.
The kits comprise a probe or primer which is capable of hybridizing
adjacent to or at a polymorphic region of AKAP1 O and thereby identifying
whether the AKAP1 ~ gene contains an allelic variant which is associated
with increased susceptibility to morbidity and/or predisposition for
premature or increased or early mortality or a genetic predisposition to a
disorder related to signal transduction and/or protein phosphorylation.
The kits further comprise instructions for use in carrying out assays,
interpreting results and diagnosing a subject as having increased
susceptibility to morbidity an~9/or predisposition for premature or
increased or early mortality or a genetic predisposition to a disorder
related to signal transduction and/or protein phosphorylation.
Kits for amplifying a region of AKAP1 ~ gene or other genes
associated with morbidity and/or mortality and/or signal transduction
comprise two primers which flank a polymorphic region of the gene of
interest. For example primers can comprise the sequences of SEQ ID
NOs.:S, 6, 7, 10, 12 and 16. For other assays, primers or probes
hybridize to a polymorphic region or 5' or 3' to a polymorphic region
depending on which strand of the target nucleic acid is used. For
example, specific probes and primers comprise sequences designated as
SEQ ID NOs: 8, 15, 19 and 20. Those of skill in the art can synthesize
primers and probes which hybridize adjacent to or at the polymorphic
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regions described herein and other SNPs in genes associated with
morbidity and/or mortality and/or signal transduction
Yet other kits comprise at least one reagent necessary to perform
an assay. For example, the kit can comprise an enzyme, such as a
nucleic acid polymerise. Alternatively the kit can comprise a buffer or
any other necessary reagent.
Yet other kits comprise microarrays of probes to detect allelic
variants of the AKAP10 gene. The kits further comprise instructions for
their use and interpreting the results.
The following examples are included for illustrative purposes only
and are not intended to limit the scope of the invention. The practice of
methods and development of the products provided herein employ,
unless otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology, recombinant
DNA, and immunology, which are within the skill of the art. Such
techniques are explained fully in the literature. See, for example,
i~iolecular Cloning A Laboratory f~lanual, end Ed., ed. by Sambrook,
Fritsch and lifiianiatis (Cold Spring Harbor Laboratory Press: 1989); DNA
Cloning, Volumes I and II (D.N. Glover ed., 1985); ~ligonucleotide
2~ Synthesis (M.J. Gait ed., 1984); Mullis era/, IJ.S. Patent No. 4,683,195;
Nucleic Acid Hybridizatiion (B.D. Names 8~ S.J. Higgins eds. 1984);
Transcription and Translation (B.D. Hames ~c S.J. Higgins eds. 1984);
Culture of Animal Cells (R.1. Freshney, Alan R. Liss, Inc., 19871;
Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical
Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology
(Academic Press, Inc., New York); Gene Transfer Vectors For Mammalian
Cells (J.H. Miller and M.P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al, eds.,
Immunochemical Methods In Cell and Molecular Biology (Mayer and
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Walker, eds., Academic Press, London, 1987); Handbook of Experimental
Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986);
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1986).
The following examples are included for illustrative purposes only
and are not intended to limit the scope of the invention.
EXAMPLES
EXAMPLE 1
Isolation of DNA from blood samples of a healthy donor population
1 ~ The results are provided of a screen comparing allele frequencies
of 6,5~~ SNPs located in approximately 5,0~~ genes between a sample
of young and elderly healthy individuals. This resulted in the
identification of a gene encoding a functional variant with an impact on
morbidity that can be inv~Ived in the etiology of cardiac dysfunction.
All subjects involved in the studies signed a written informed
consent and the institutional ethics committees of participating
institutions appr~ved the e~zperimental protocols. Subjects for the
disease susceptibility genome screen were part of a sample thafi was
recruited during a routine blood donation from private blood collection
2~ centers in San Bernardino and Rancho Mirage, California, IJSA. The staff
of the blood collection agencies invited all healthy blood donors to
participate, and helped the subjects fill out a consent form and a simple
personal data collection form prior t~ sample collection. The data
collection form included information about age, sex, body size, personal
and family disease history, and ethnic background of both parents.
Information about the identity of the study participants was not recorded.
Subjects would be excluded if they failed to meet the blood donation
eligibility guidelines established by the American Red Cross. Ethnicity
was defined for each subject if they identified both parents as having the
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same ethnic/geographic bacleground, otherwise they are indicated as
"Other". For the purpose of identifying disease susceptibility associated
SNPs, derived from this collection was a discovery cohort consisting of
male and female Caucasian-Americans divided into young (18-39 years)
and old ( > 60 years) groups. These groups and others used in this study
are shown in Table 4.
TABLE 4
Group Gender N Age Range Mean Age (S.D.)
CA-YF Female 276 18-39 27.0 (6.68)
1 ~ CA-YM Male 276 18-39 27.1 (6.65)
CA-OF Female 184 60-69 64.1 (2.84)
CA-OM Male 367 60-79 66.7 (4.78)
HI-YF Female 359 18-39 29.0 (6.42)
HI-YM Male 173 18-39 28.9 (6.67)
HI-OF Female 61 50-78 55.6 (5.66)
HI-OM Male 64 50-89 57.9 (7.49)
~RF Female/il~lale 97/97 18-76 37.4 (11.49)
AS Female/Male 62/64 18-65 34.4 ( 12.18)
Table 4.. Composition of age-, gender-, and ethnicity-stratified groups.
Abbreviations:
2~ CA, Caucasian-American; HI, Hispanic-American; AF, African-American; AS,
Asian-
American; YF, Young Female; YM, Young Male; ~F, Old Female; OM, Old Male;
S.D.,
Standard Deviation.
A follow-up study of the SNPs significantly associated with age in
the genome scan was carried out in a sample of 417 Caucasian twin
pairs from the adult twin registry at St Thomas Hospital, London, United
Kingdom. Participants in this collection were enrolled without regard to
health status as previously described (Andrew et al., 2001, Twin Res.,
4:464-477). For this study, 97 traits were selected to explore possible
disease associations. The selected traits have connections to many
disease areas, including cardiovascular diseases, diabetes, hypertension,
obesity, and osteoporosis.
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Practically a healthy subject, when human, is defined as human
donor who passes blood bank criteria to donate blood for eventual use in
the general population. These criteria are as follows: free of detectable
viral, bacterial, mycoplasma, and parasitic infections; not anemic; and
then further selected based upon a questionnaire regarding history.
Thus, a healthy population represents an unbiased population of
sufficient health to donate blood according to blood bank criteria, and not
further selected for any disease state. Typically such individuals are not
taking any medications.
Blood was obtained from a donor by venous puncture and
preserved with 1 mM EDTA pH 8Ø Ten milliliters of whole blood from
each donor was centrifuged at 2000x g. One milliliter of the buffy coat
was added to 9 milliliters of 155mM NH4,C1, 1 OmM I<HC03, and 0.1 mM
Na2EDTA, incubated 10 minutes at room temperature and centrifuged for
10 minutes at 2000x g. The supernatant was removed and the white
cell pellet was washed in 155mM NH4C1, 1 OmM I~HC03, and 0.1 mM
f~la~EDTRa and resuspended in 4.5 milliliters of 50m1~i Tris, 5mf~i EDTA,
and 1 % 3DS. Proteins were precipitated from the cell lysate by 5M
ammonium acetate pH 7.3 and separated from the nucleic acid by
centrifugation at 3000x g. The nucleic acid was recovered from the
supernatant by the addition of an equal volume of 100% isopropanol and
centrifugation at 2000x g. The dried nucleic acid pellet was hydrated in
IOmM Tris pH 7.6 and 1 mM Na~EDTA and stored at 4°C.
Statistical Analysis
Estimates of allele frequencies derived from pooled DNA were
based on independent mass spectrometry measurements of four analyte
aliquots derived from a single PCR reaction. The median standard
deviation for these values was approximately 0.01. For comparing allele
frequencies between the young and old pools, females and males were
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analyzed separately. The statistic used to test the difference in allele
frequencies between pools was of the form:
py ' po
__________________________________________________
(py(~-py)/2ny+(po)/2o+ey+Eo)~iz
which follows a standard normal distribution. Here py and p0 are the
allele frequency estimates and Ey and Eo are estimates of measurement
variability calculated from measurement replicates in the young and old
pools, respectively. In this study, no correction was made for additional
1~ sources of variation or for multiple testing. Rather, SNPs were identified
that had p-values less than 0.05 among all measured SNPs, followed by
a second, independent measurement of all significant SNPs based on
three separate PGRs of each DNA pool. The results of the second round
of measurement were analyzed in a manner similar to the first round, and
were compared for consistency. SNPs that showed statistically
significant differences between young and old groups from pooled DNA
analyses were individually genotyped for final validation.
Estimates of allele frequencies using individual genotype data were
found using the gene counting method. Comparisons of allele
2~ frequencies as well as genotype frequencies between groups were
carried out using a chi-squared test of independence.
The SNPs found to be associated with age were further analyzed
for association with disease-related quantitative traits in the twin
collection. The analysis was conducted using a quantitative
transmission-disequilibrium test (QTDT) as described by Abecasis et al.,
2000, Am J Hum Genet., 66:279-292 to take advantage of the twin-
based sample and to control for admixture and other non-genetic sources
of variation. The form of the test was implemented that does not require
the estimation of variance components. Formal statistical procedures to
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account for multiple testing were not use, but the distribution of the
resulting p-values is reported.
Results of Genome-wide SNP Analysis for Morbidity Gene Discovery
A strategy was pursued that utilizes estimates of allele frequencies
in DNA pools to screen large numbers of SNPs. To apply this to disease
susceptibility gene discovery, the Caucasian-American individuals were
divided by age (under 40 and over 60 years) and by sex (Table 4). The
fraction of included subjects reporting any health problem was only 1.8%
and 2.9% in young females and males, and 3.8% and 3.5~/o in old
females and males, respectively. In this study, a collection was used of
6,500 exonic SNPs located in approximately 5,000 genes or expressed
sequence tag (EST) clusters. The majority of assays for these SNPs,
originally identified in an in silico discovery project, were developed in
collaboration with the National Cancer Institute (NCI).
Identification of D-AKAP2 as a Candidate Gene
More than 50 markers were identified out of the 6,500 tested
markers that show a reproducibly significant allele frequency change
between the two age groups in at least one gender (P G 0.05). The SNP
that demonstrated the strongest association with age in both genders is
located within the D-AKAP2 gene.
D-AI<AP2 codes for dual-specific A-kinase anchor protein 2, which
is part of a family of scaffold proteins known as A-kinase anchoring
proteins (AKAPs). AKAPs bind the regulatory subunit of cAMP-
dependent Protein Kinase (PKA), and target the kinase to various
intracellular locations, localizing CAMP-mediated activation of the kinase.
PKA is a broad specificity kinase and phosphorylates numerous proteins
that function in many essential cellular processes such as metabolism,
gene transcription, cell division, and neuronal transmission. In the
inactive state, PKA is a tetramer consisting of two catalytic (C) and two
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regulatory (R) subunits. The dual specificity of D-AKAP2 is defined by its
ability to bind both the RI and RII isoforms of PKA.
An A>G polymorphism in the 3' untranslated region (3'UTR) of D-
AKAP2 showed a significant decrease of about 8% (P < 0.01 ) of the G
allele in the older sample of both genders (Table 5). The marker was
individually genotyped and the frequency differences between young and
old individuals calculated from the genotypes were very similar to the
pool results. There was a slight skewing of frequencies in the pools
likely resulting from uneven PCR amplification of the two alleles. This led
to an underestimation of the G allele frequency in all pools but did not
impact the significance of the differences between young and old (Table
5).
TABLE 5
Allelle Frequency
Young Old Difference P-Value
P~~I Date
3' UTR
Caucasian Female 0.322 0.240 0.082 0.007
Caucasian Male 0.362 0.232 0.076 0.002
Genotype ~ata
3' UTR
Caucasian Female 0.274 0.212 0.062 0.034
Caucasian Male 0.304 0.232 0.072 0.004
1646V
Caucasian Female0.402 0.318 0.084 0.009
Causian Male 0.429 0.369 0.060 0.030
Hispanic Female 0.445 0.316 0.129 0.008
Hispanic Male 0.436 0.375 0.061 0.229
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In10
Caucasian Female 0.877 0.871 0.006 0.147
Caucasian Male 0.880 0.865 0.015 0.360
Table 5. Comparison of allele frequencies between young and old groups.
Sample sizes are the same as those of the corresponding groups in Table
2. Allele frequencies for each SNP are given for the G allele.
To identify common polymorphisms in the D-AICAP2 gene, the 15
axons and 100-200 by of the flanking sequence were sequences. The
analysis of 36 chromosomes revealed only two additional
polymorphisms: An A > G SNP in intron 10, six nucleotides downstream
from axon 10 (1n10), and an A>G SNP in axon 14 (corresponding to
AI<AP10-5 at nucleotide 2073 of SE(~ ID N0:1 ), leading to an amino acid
substitution Ile to Val at p~sition 646 (1646V). Individual genotyping of
the Caucasian-American samples showed that the intron 10 SNP exhibits
no morbidity-association. The 1646V polymorphism, however, was found
to be significantly different between y~~ane~ and ~1d in both males (P =
0.03) and females (P = 0.009) (Table 5). There was n~ significant
difference between young males and females and between old males and
females. The Bayesian, coalescent theory-based method (Stephens et
al., 2001, e4m ,l Hum Genet. 68:978-989) was applied to construct
haplotypes at these three tightly linked sites for each subject. The
estimates of the disequilibrium (D') between the 3'UTR and the 1646V
and In10 SNPs were 0.991 and 0.255 (r2 = 0.55 and 0.03),
respectively. The distance between the markers in strong disequilibrium,
3'UTR and 1646V which both showed association, is about 4 kb, while
the intron 10 SNP is located approximately 23 kb upstream of 1646V.
As expected, the changes in genotype frequencies between age
groups for the three sites showed a similar level of statistical significance
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as the changes in allele frequencies. At the 3'UTR and the 1646V
variable sites, GG homozygotes were reduced and AA homozygotes
increased in the older sample population of both genders. This further
supports the hypothesis that the G allele, which determines the Val allele
at 1646V, is associated with a negative health impact.
Hispanic-American samples were also genotyped for the 1646V
variation. Since there were only a small number of Hispanic-American
individuals over 60 years, the older sample population was extended to
all individuals older than 50 years of age (Table 4). While females
showed a statistically significant allele frequency difference between old
and young (P = 0.008), the males did not, which can be due to the
relatively small number of male individuals (Table 5). The allele
frequency differences in both genders were comparable to those
observed in Caucasian-Americans. The frequency of the G allele
decreased by 0.129 in females and 0.061 in males, the GG homozygote
by 0.087 in females and 0.072 in males. These results further support
the association of the Val allele with morbidity and/or mortality, and
therefore the involvement of this gene in one or several disease
processes. Another non-synonymous ~-AKAP2 variation retrieved from
dbSNP has been verified. The G-A transversion in exon 4 results in an
Arg to His substitution at position 249 (R249H; corresponding to a G to
A transversion at nucleotide 883 of SEQ I~ N0:1 encoding human ~-
AKAP2). The Arg was found to be in complete linkage disequilibrium
with the Ile at position 646, occurring together in every case, and
therefore shows the same age effect.
Association of D-AKAP2 Genotypes with a Cardiac Trait
In an effort to identify traits correlated with the observed age
association of the 1646V SNP, we utilized a cohort of 417 fasting
Caucasian twin pairs with extensive coverage for a variety of disease-
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related traits. Of the 97 traits analyzed, only the PR interval was
statistically significant at a nominal level of 0.05. The estimate from the
QTDT model of the average effect of the G allele (Val) was to decrease
the PR interval 6.3 units (P = 0.007). The genotype means in the
subset of 207 informative twin pairs were 157 ~ 23.4, 152 ~ 26.9,
and 146 ~ 25.4 (mean ~ standard deviation) for genotypes AA, GA,
and GG, respectively at a position corresponding to nucleotide 2073 of
SEQ ID N0:1.
EXAMPLE 2
Detection of AI(AP10-1 by MassEXTENDT"~ Assay ~etection Methods
AKAP10-1 is an allele of the AI~AP10 gene with a single
nucleotide polymorphism at nucleotide number 156277 (based on the
sequence of a genomic clone of the Af~AP10 gene, GenSank Accession
No. AC005730). The single nucleotide polymorphism is a T to C
transversion located in the 3'non-translated region of the gene encoding
AICAP10. PCR primers were synthesized by OPERON (Alameda, CA)
using phosphoramidite chemistry. Amplification of the s41GAP10 target
sequence was carried out in single 50~u1 PCR reaction with 25ng of
human genomic DNA obtained from samples as described in Eacample 1.
Each reaction containing IX PCR buffer (Qiagen, Valencia, CA), 200~M
dNTPs, 1 U Hotstar Taq polymerase (Qiagen, Valencia, CA), 4mM MgCl2,
and 25 pmols of the forward primer containing the universal primer
sequence and the target specific sequence
5'-TCTCAATCATGTGCATTGAGG-3' (SEQ ID NO: 5) 2 pmoles of the
reverse primer
5'-AGCGGATAACAATTTCACACAGGGATCACACAGCCATCAGCAG-3'
(SEQ ID NO: 6) and IOpmoles of a biotinylated universal primer
complementary to the 5' end of the PCR amplicon
5'-AGCGGATAACAATTTCACACAGG-3' (SEQ ID NO: 7). Alternatively,
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the biotinylated universal primer could be 5'-GGCGCACGCCTCCACG-3'
(SEQ ID NO: 16). After an initial round of amplification of the target with
the specific forward and reverse primer, the 5' biotinylated universal
primer was hybridized and acted as a reverse primer thereby introducing
a 3' biotin capture moiety into the molecule. The amplification protocol
resulted in a 5'-biotinylated double stranded DNA amplicon, which
dramatically reduces the cost of high throughput genotyping by
eliminating the need to 5' biotin label each forward primer used in a
genotyping. Thermal cycling was performed in 0.2mL tubes or 96 well
plate using an MJ Research Thermal Cycler (Waltham, MA) (calculated
temperature) with the following cycling parameters: 94°C for 5 min; 45
cycles: 94°C for 20 sec, 56°C for 30 sec, 72°C for 60
sec; 72°C 3
min.
Immobilization of ~i~A
The 50~u1 PCR reaction was added to 25,1 of streptavidin coated
magnetic bead (Dynal) prewashed three times and resuspended in 1 M
i~H4,Cl, O.OGi~I i~Hq,OH. The PCR amplicons were allowed to bind to the
beads for 15 minutes at room temperature. The beads were then
collected with a magnet and the supernatant containing unbound DNA
was removed. The unbound strand was release from the double
stranded amplicons by incubation in 100mM NaOH and washing of the
beads three times with 10mM Tris pH B.O.
Genotyping
Genotyping was carried out using the MassEXTENDTM assay and
MALDI-TOF. The SNP identified at position 156277 of AICAP10 in the
GenBank sequence is represented as a T to C transversion. The
MassEXTENDTM assay detected the sequence of the complementary
strand at the polymorphic position, thus the primer extension product
incorporated either a T or a C. The DNA coated magnetic beads were
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resuspended in 26 mM Tris-HCL pH 9.5, 6.5 mM MgCl2 and 50 mM
each of dTTPs and 50 mM each of ddCTP, ddATP, ddGTP, 2.5U of a
thermostable DNA polymerase (Amersham Pharmacia Biotech,
Piscataway, NJ) and 20 pmoles of a template specific oligonucleotide
primer 5'-CTGGCGCCCACGTGGTCAA-3' (SEQ ID NO: 8) (Operon,
Alameda, CA). Primer extension occurs with three cycles of
oligonucleotide primer was hybridization and extension. The extension
products were analyzed after denaturation from the template with 50 mM
NH~CI and transfer of 150 n1 each sample to a silicon chip preloaded with
150 n1 of H3PA (3-hydroxy picolinic acid) (Sigma Aldrich, St. Louis, MO)
matrix material. The sample material was allowed to crystallize and
analyzed by MALDI-TOF (Broker Daltonics, Billerica, MA, PerSeptive,
Foster City, CA). The mass of the primer used in the MassE~TENDT""
reaction was 5500.6 daltons. The allelic variant results in the addition
of ddC to the primer to produce an extension product having a mass of
5773.8 daltons. The predominant allele is extended by the addition of
dT and ddG to tile primer to produce an ea~tension product having a mass
of 5101 daltons.
The SNP that is present in AI~AP10-1 is a T to C transversion at
nucleotide number 156277 of the sequence of a genomic clone of the
AICAP10 gene (GenBank Accession No. AC005730) (SEQ ID N0: 17).
SEO. ID N0:17 represents the nucleotide sequence of human
chromosome 17, which contains the genomic nucleotide sequence of the
human AICAP10 gene at approximately nucleotide 83,580 to nucleotide
156,577. SEQ ID NO: 18 represents the nucleotide sequence of human
chromosome 17, which contains the genomic nucleotide sequence of the
human AICAP10-1 allele.
The frequency of the AI~AP10-1 allelic variant was measured in a
population of age selected healthy individuals. Five hundred fifty-two
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(552) individuals between the ages of 18-39 years (276 females, 276
males) and 552 individuals between the ages of 60-79 ( 184 females
between the ages of 60-69, 368 males between the age of 60-79) were
tested for the presence of the allelic variant localized in the
non-translated 3' region of AICAP 10. Differences in the frequency of
this variant with increasing age groups were observed among healthy
individuals. Statistical analysis showed that the significance level for
differences in the allelic frequency for alleles between the "younger" and
the "older" populations was p = 0.0009 and for genotypes was
1~ p=0.003. Differences between age groups are significant. For the total
population allele significance is p = 0.0009, and genotype significance is
p = 0.003.
The young and old populations were in Hardy-l~Veinberg equilibrium. A
preferential change of one particular genotype was not seen.
The polymorphism is localized in the non-translated 3'-region of
the gene encoding the human protein kinase A anchoring protein
(AI<AP10). The gene is located on chromosome 17. Its structure
includes 15 exzons and 14 intervening sequences (introns). The encoded
protein is responsible for the sub-cellular localization of the cAMP-
dependent protein kinase and, therefore, plays a key role in the G-protein
mediated receptor-signaling pathway (Huang et al. PNAS (1007)
94:1 1 184-1 1 189). Since its localization is outside the coding region,
this polymorphism is most likely in linkage disequilibrium (LD) with other
non-synonymous polymorphisms that could cause amino acid
substitutions and subsequently alter the function of the protein.
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EXAMPLE 3
Discovery of AKAP10-5 Allele
G.enomic DNA was isolated from blood (see Example 1 ) of
seventeen (17) individuals with a genotype CC at the AKAP10-1 gene
locus and a single heterozygous individual (CT) (as described in Example
2). A target sequence in the AKAP10-1 gene which encodes the C-
terminal PKA binding domain was amplified using the polymerase chain
reaction. PCR primers were synthesized by OPERON (Alameda, CA)
using phosphoramidite chemistry. Amplification of the AKAP10-1 target
sequence was carried out in individual 50,u1 PCR reaction with 25ng of
human genomic DNA templates. Each reaction containing I X PCR buffer
(O.iagen, !lalencia, CA), 200~1~ dNTPs, IU Hotstar Taq polymerase
(C~iagen, l~alencia, CA), 4. mfVl l~lgCl~, and 25 pmols of the forward
primer containing the universal primer sequence and the target specific
sequence 5'-TCC CAA AGT GCT GGA ATT AC-3' (SEQ ID N0: 9),
2pmoles of the reverse primer 5'-GTC CAA TAT ATG CAA ACA GTT
G-S'(SEQ ID i~0:10). Thermal cycling was performed in 0.2 mL tubes or
96 well plate using an fi~IJ Research Thermal Cycler (ii~iJ Research,
Waltham, MA) (calculated temperature) with the following cycling
parameters: 94°C fior 5 min; 45 cycles; 94°C for 20 sec,
56°C for 30
sec, 72°C for 60 sec; 72°C 3min. After amplification the
amplicons
were purified by chromatography (Mo Bio Laboratories (Solana Beach,
CA) .
The sequence of the 1 B amplicons, representing the target region,
was determined using a standard Sanger cycle sequencing method with
25 nmoles of the PCR amplicon, 3.2 ,uM DNA sequencing primer 5'-CCC
ACA GCA GTT AAT CCT TC-3' (SEQ ID N0:1 1 ) and chain terminating
dRhodamine labeled 2', 3' dideoxynucleotides (PE Biosystems, Foster
City, CA) using the following cycling parameters: 96°C for 15 sec,
25
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cycles: 55°C for 15 sec, 60°C for 4 min. The sequencing products
were precipitated by 0.3M NaOAc and ethanol, the precipitate was
centrifuged and dried. The pellets were resuspended in deionized
formamide and separated on a on a 5% polyacrylamide gel. The
sequence was determined using the "Sequencher" software (Gene
Codes, Ann Arbor, MI).
The sequence of all 17 of the amplicons which are homozygous
for the AKAP10-1 SNP revealed a polymorphism at nucleotide position
152171 (numbering for GenBank Accession No. AC005730 for AICAP10
genomic clone) with A replaced by G. This SNP can also be designated
as located at nucleotide 2073 of a cDNA clone of the wildtype AICAP10
(SEQ ID N0:1 ) (GenBank Accession No. AF037439). This single
nucleotide polymorphism was designated as AI~AP10-5 (SEQ ID N0:3)
and results in a substitution of a valine for an isoleucine residue at amino
acid position 646 (SEQ ID N0:4).
E~Ce4IVIPLE 4.
~~6~ ~r~pllflcatl~n and I~a~~E~'~Eb~~rM ~~~ay d~t~c$i~n ~f ~1~~9~'i ~-5 in a
healthy d~n~r p~pulati~n
A healthy population stratified by age is a very efficient and a
universal screening tool for morbidity associated genes by allowing for
the detection of changes of allelic frequencies in the young compared to
the old population. Individual samples of this healthy population base
can be pooled to further increase the throughput.
Healthy samples were obtained through the blood bank of San
Bernardino, CA. Both parents of the blood donors were of Caucasian
origin. Practically a healthy subject, when human, is defined as human
donor who passes blood bank criteria to donate blood for eventual use in
the general population. These criteria are as follows: free of detectable
viral, bacterial, mycoplasma, and parasitic infections; not anemic; and
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then further selected based upon a questionnaire regarding history.
Thus, a healthy population represents an unbiased population of
sufficient health to donate blood according to blood bank criteria, and not
further selected for any disease state. Typically such individuals are not
taking any medications.
PCR primers were synthesized by OPERON (Alameda, CA) using
phosphoramidite chemistry. Amplification of the AICAP10 target
sequence was carried out in single 50~u1 PCR reaction with 1 OOng- 1 ug of
pooled human genomic DNAs in a 50,u1 PCR reaction. Individual DNA
1 ~ concentrations within the pooled samples were present in equal
concentration with the final concentration ranging from 1-25ng. Each
reaction contained 1 ~ PCR buffer (Oiagen, !lalencia, CA), 200,ciM dNTPs,
1lJ Hotstar Taq polyrtierase (~iagen, ~/alencia, CA), 4 mM MgCl2, and 25
pmols of the forward primer containing the universal primer sequence
and the target specific sequence
5'-AGCGGATAACAATTTCACACAGGGAGCTAGCTTGGAAGATTGC-3'
(SEf~ ID i~0:12), 2pmoles of the reverse primer
5'-GTCCAATATATGCAAACAGTTG-~' (SEQ ID NO: 10) and 10 pmoles
of a biotinylated universal primer complementary to the 5' end of the
2~ PCR amplicon 510:5'-AGCGGATAACAATTTCACACAGG-3' (SEO. ID NO:
7). After an initial round of amplification with the target with the specific
forward and reverse primer, the 5' biotinylated universal primer can then
hybridized and acted as a forward primer thereby introducing a 5' biotin
capture moiety into the molecule. The amplification protocol resulted in
a 5'-biotinylated double stranded DNA amplicon and dramatically reduces
the cost of high throughput genotyping by eliminating the need to 5'
biotin label every forward primer used in a genotyping.
Thermal cycling was performed in 0.2 mL tubes or 96 well plate
using an MJ Research Thermal Cycler (Waltham, MA) (calculated
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-127-
temperature) with the following cycling parameters: 94°C for 5 min; 45
cycles: 94°C for 20 sec, 56°C for 30 sec; 72°C for 60
sec; 72°C 3
min.
Immobilization of DNA
The 50,u1 PCR reaction was added to 25~u1 of streptavidin coated
magnetic beads (Dynal, Oslo, Norway) (Lake Success, NY), which were
prewashed three times and resuspended in 1 M NH~.CI, 0.06M NH40H.
The 5' end of one strand of the double stranded PCR amplicons were
allowed to bind to the beads for 15 minutes at room temperature. The
1~ beads were then collected with a magnet and the supernatant containing
unbound DNA was removed. The hybridized but unbound strand was
released from the double stranded amplicons by incubation in 100mM
NaOH and washing of the beads three times with 10mM Tris pH ~Ø
Gr~notyping
The identity of the nucleotide present at the polymorphic site of
AICAP 10-5 was determined by using the MassE?CTENDTM assay and
f~iALDI-TOF (see, U.S. Patent f~lo. 5,043,031 ). The I~iassE~TENDT""
assay is a primer extension assay that utilizes a primer that hybridizes
adjacent to the polymorphic region and which is extended in the
presence of one or more ddNTPs. Extension is stopped by the
incorporation of a dideoxy nucleotide. At a polymorphic site the different
alleles produce different length extension products, which are
distinguishable by mass spectrometry.
The MassE?CTENDTM assay detected the sepuence of the sense
strand and resulted in the incorporation of either T or C into the
extension product. The DNA coated magnetic beads were suspended in
26mM Tris-HCL pH 9.5; 6.5 mM, MgCl2 and 50mM each of dTTPs and
50mM each of ddCTP, ddATP, ddGTP, 2.5U of a thermostable DNA
polymerase (Amersham Pharmacia Biotech, Piscataway NJ) and 20
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-128-
pmoles of a template specific oligonucleotide primer
5'-ACTGAGCCTGCTGCATAA-3' (SEQ ID N0:15) (Operon) (Alameda,
CA). Primer extension occurs with three cycles of oligonucleotide primer
hybridization and extension. The extension products were analyzed after
denaturation from the template with 50 mM NH4C1 and transfer of 150 n1
each sample to a silicon chip preloaded with 150 n1 of H3PA (3-hydroxy
picolinic acid) (Sigma Aldrich, St. Louis, MO) matrix material. The
sample material was allowed to crystallize and analyzed by MALDI-TOF
(Bruker Daltonics, Billerica, MA, PerSeptive, Foster City, CA). The primer
had a mass of 5483.6 daltons. The allelic variant resulted in the addition
of a ddC to the primer to produce an extension product having a mass of
5756.8 daltons. The predominant allele resulted in the addition a T and
ddG to the primer giving an extension product with a mass of 6101
daltons.
EXAMPLE 5
Discovery of AKAP10-7
Genomic DNA isolation, amplification of the target regions and
sequencing of amplicons was carried out as in Example 3. lJsing the
sequence of the cDNA for AKAP10, chromosome 17 was BLAST
searched to identify the number of axons. Sanger sequencing of the
regions around and containing the axons was performed and resulted in
the discovery of AKAP10-7 polymorphic region. For AfCAP10-7 the
forward sequencing primer was CACTGCACCCAGCCTTATG (SEO. ID
N0: 23) and the reverse sequencing primer was
CTGGGATGTGAAGGAAAGGA (SEQ ID NO: 24).
EXAMPLE 6
MassEXTENDT"~ assay detection of AKAP10-7
Samples are obtained and amplified as in Example 4.
CA 02518238 2005-09-06
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-129-
The identity of the nucleotide present at the polymorphic site of
AKAP 10-7 is determined by using the MassEXTENDTM assay and MALDI-
TOF (see, U.S. Patent No. 6,043,031 ). The MassEXTENDTM assay
detects the sequence of the complementary strand and resulted in the
incorporation of either G or A into the extension product. Reactions are
carried out as in Example 4. The template specific oligonucleotide
primer 5'-CTCTGCGTCTCAGGTATT-3' (SEQ ID NO: 20). (Operon,
Alameda, CA). The primer has a mass of 5456.6 daltons. The allelic
variant results in the addition of a ddA to the primers to produce an
extension product having a mass of 5753.6 daltons. The predominant
allele results in the addition a G and ddA to the primer giving an
extension product v~rith a mass of 6033.0 daltons.
Since modifications veil) be apparent to those of skill in this art, it
is intended that this invention be limited only by the scope of the
appended claims.
CA 02518238 2005-09-06
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-1-
SEQUENCE LISTING
<110> SEQUENOM, INC.
Braun, Andreas
ICammerer, Stefan
<120> ASSOCIATION OF POLYMORPHIC ICINASE ANCHOR PROTEINS WITH CARDIAC
PHENOTYPES AND RELATED METHODS
<130> 24736-2074PC
<140> Not Yet Assigned
<141> Herewith
<150> US 60/453,215
<151> 2003-03-07
<150> US 60/453,208
<151> 2003-03-07
<150> US 60/453,350
<151> 2003-03-07
<160> 25
<170> FastSEQ for Windows Versi~n 4.0
<210> 1
<211> 2363
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (138)...(2126)
<223> Wild Type ATCAP-10
<300>
<308> GenBank AF037439
<309> 1997-12-21
<400>
1
gcggcttgtt gataatatgg tgcctgggca tcccgaggag
gcggtggggc60
cggctggagc
ccactcccgg aagaagggtc ggcccctctg gacccggaag120
ccttttcgcg
ctagtgcagc
tccgggccgg ttgctga ccc ccg cag tcc 170
atg tcc cgc
agg
gga
gcc
ggg
Met Arg Gly Pro Pro Gln Ser
Ala Gly Ser Arg
1 5 10
CCC aCCCtC Cgt CCC gac ggc gccatgtCC ttC cgg 218
CgC ccg ccc ttC
Pro ThrLeu Arg Pro Asp Gly AlaMetSer Phe Arg
Arg Pro Pro Phe
15 20 25
cgg gtgaaa ggc aaa gaa gag acctcagat aag tcc 266
aaa caa aag gtg
Arg ValLys Gly Lys Glu Glu ThrSerAsp Lys Ser
Lys Gln Lys Val
30 35 40
att gettca ata tcc gta tcc caaaaaagc aaa aat 314
aaa cat cca act
Ile AlaSer Ile Ser Val Ser GlnLysSer Lys Asn
Lys His Pro Thr
45 50 55
cat ttgctg gag get gca cca catgttgca aat gcc 362
gcc gga agt atc
His LeuLeu Glu Ala Ala Pro HisValAla Asn Ala
Ala Gly Ser Ile
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
_2_
60 65 70 75
atttctgcc aacatggac tccttttca agtagcagg acagccaca ctt 410
IleSerAla AsnMetAsp SerPheSer SerSerArg ThrAlaThr Leu
80 85 90
aagaagcag ccaagccac atggagget getcatttt ggtgacctg ggc 458
LysLysGln ProSerHis MetGluAla AlaHisPhe GlyAspLeu Gly
95 100 105
agatcttgt ctggactac cagactcaa gagaccaaa tcaagcctt tct 506
ArgSerCys LeuAspTyr GlnThrGln GluThrLys SerSerLeu Ser
110 115 120
aagaccctt gaacaagtc ttgcacgac actattgtc ctcccttac ttc 554
LysThrLeu GluGlnVal LeuHisAsp ThrIleVal LeuProTyr Phe
125 130 135
attcaattc atggaactt cggcgaatg gagcatttg gtgaaattt tgg 602
IleGlnPhe MetGluLeu ArgArgMet GluHisLeu ValLysPhe Trp
140 145 150 155
ttagagget gaaagtttt cattcaaca acttggtcg cgaataaga gca 650
LeuGluAla GluSerPhe HisSerThr ThrTrpSer ArgIleArg Ala
160 165 170
cacagtcta aacacaatg aagcagagc teactgget gagcctgtc tct 698
HisSerLeu AsnThrMet LysGlnSer SerLeuAla GluProVal Ser
175 180 185
ccatctaaa aagcatgaa actacagcg tctttttta actgattct ctt 746
ProSerLys LysHisGlu ThrThrAla SerPheLeu ThrAspSer Leu
190 195 200
gataagaga ttggaggat tctggctca gcacagttg tttatgact cat 794
AspLysArg LeuGluAsp SerGlySer AlaGlnLeu PheMetThr His
205 210 215
tcagaagga attgacctg aataataga actaacagc actcagaat cac 842
SerGluGly IleAspLeu AsnAsnArg ThrAsnSer ThrGlnAsn His
220 225 230 235
ttgctgctt teecaggaa tgtgacagt gcccattct ctccgtctt gaa 890
LeuLeuLeu SerGlnGlu CysAspSer AlaHisSer LeuArgLeu Glu
240 245 250
atggccaga gcaggaact caccaagtt tccatggaa acccaagaa tct 938
MetAlaArg AlaGlyThr HisGlnVal SerMetGlu ThrGlnGlu Ser
255 260 265
tcctctaca cttacagta gccagtaga aatagtccc gettctcca cta 986
SerSerThr LeuThrVal AlaSerArg AsnSerPro AlaSerPro Leu
270 275 280
aaagaattg tcaggaaaa ctaatgaaa agtatagaa caagatgca gtg 1034
LysGluLeu SerGlyLys LeuMetLys SerIleGlu GlnAspAla Val
285 290 295
aatactttt accaaatat atatctcca gatgetget aaaccaata cca 1082
AsnThrPhe ThrLysTyr IleSerPro AspAlaAla LysProIle Pro
300 305 310 315
attacagaa gcaatgaga aatgacatc atagcaagg atttgtgga gaa 1130
IleThrGlu AlaMetArg AsnAspIle IleAlaArg IleCysGly Glu
320 325 330
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-3-
gatggacaggtg gatcoc aactgtttcgtt ttggcacag tccatagtc 1178
AspGlyGlnVal AspPro AsnCysPheVal LeuAlaGln SerIleVal
335 340 345
tttagtgcaatg gagoaa gagoactttagt gagtttctg cgaagtcac 1226
PheSerAlaMet GluGln GluHisPheSer GluPheLeu ArgSerHis
350 355 360
catttctgtaaa taccag attgaagtgctg accagtgga actgtttac 1274
HisPheCysLys TyrGln IleGluValLeu ThrSerGly ThrValTyr
365 370 375
ctggetgacatt ctottc tgtgagtcagcc ctcttttat ttctctgag 1322
LeuAlaAspIle LeuPhe CysGluSerAla LeuPheTyr PheSerGlu
380 385 390 395
tacatggaaaaa gaggat gcagtgaatatc ttacaattc tggttggca 1370
TyrMetGluLys GluAsp AlaValAsnIle LeuGlnPhe TrpLeuAla
400 405 410
gcagataactto cagtct oagcttgetgcc aaaaagggg caatatgat 1418
AlaAspAsnPhe GlnSer GlnLeuAlaAla LysLysGly GlnTyrAsp
415 420 425
ggacaggaggca cagaat gatgocatgatt ttatatgac aagtactto 1466
GlyGlnGluAla GlnAsn AspAlaMetIle LeuTyrAsp LysTyrPhe
430 435 440
tccctocaagoc acacat octottggattt gatgatgtt gtaogatta 1514
SerLeuGlnAla ThrHis ProLeuGlyPhe AspAspVal ValArgLeu
445 450 455
gaaattgaatco aatatc tgcagggaaggt gggccactc cccaactgt 1562
GluIleGluSer AsnIle CysArgGluGly GlyProLeu ProAsnCys
460 465 470 475
ttcaoaactcoa ttaCgt oaggcotggaoa aooatggag aaggtcttt 1610
PheThrThrPro LeuArg GlnAlaTrpThr ThrMetGlu LysValPhe
480 485 490
ttgCCtggCttt ctgtcc agcaatctttat tataaatat ttgaatgat 1658
LeuProGlyPhe LeuSer SerAsnLeuTyr TyrLysTyr LeuAsnAsp
495 500 505
ctcatccattcg gttcga ggagatgaattt ctgggcggg aaogtgtcg 1706
LeuIleHisSer ValArg GlyAspGluPhe LeuGlyGly AsnValSer
510 515 520
CCgaCtgetCCt ggCtot gttggccctCCt gatgagtCt CaCCCaggg 1754
ProThrAlaPro GlySer ValGlyProPro AspGluSer HisProGly
525 530 535
agttotgaoagc tctgog totoagtcoagt gtgaaaaaa gooagtatt 1802
SerSerAspSer SerAla SerGlnSerSer ValLysLys AlaSerIle
540 545 550 555
aaaatactgaaa aatttt gatgaagcgata attgtggat goggoaagt 1850
LysIleLeuLys AsnPhe AspGluAlaIle IleValAsp AlaAlaSer
560 565 570
ctggatccagaa tottta tatoaacggaca tatgooggg aagatgaca 1898
LeuAspProGlu SerLeu TyrGlnArgThr TyrAlaGly LysMetThr
575 580 585
ttt gga aga gtg agt gac ttg ggg caa tto atc cgg gaa tct gag cct 1946
CA 02518238 2005-09-06
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-4-
Phe Gly Val Asp GlyGln PheIleArg Glu Ser Pro
Arg Ser Leu Glu
590 595 600
gaa cct gta aaa aaagga tccatgttc tca caa atg 1994
gat agg tca get
Glu Pro Val Lys LysGly SerMetPhe Ser Gln Met
Asp Arg Ser Ala
605 610 615
aag aaa gtg gga actgat gaggcccag gaa gag get 2042
tgg caa aat cta
Lys Lys Val Gly ThrAsp GluAlaGln Glu Glu Ala
Trp Gln Asn Leu
620 625 630 635
tgg aag get atg gtcagt gacattatg cag cag cag 2090
att aaa ata get
Trp Lys Ala Met ValSer AspIleMet Gln Gln Gln
Ile Lys Ile Ala
640 645 650
tat gat ccg gag tctaca aagttatga ctcaaaactt 2136
caa tta aaa
Tyr Asp Pro Glu SerThr LysLeu
Gln Leu Lys
655 660
gagataaaggaaatctgctt tttcccttgg 2196
gtgaaaaata ttggattctt
agagaacttt
caacacagccaatgaaaaca cactgttgtt 2256
gcactatatt tccagggaga
tctgatctgt
gaatggggagacaatcctag tacctgtagg 2316
gacttccacc gcataattgg
ctaatgcagt
atggcacatgatgtttcaca ttaccaa 2363
cagtgaggag
tctttaaagg
<210>
2
<211>
662
<212>
PRT
<213> Sapiens
Homo
<400> 2
Met Arg Gly Ala Gly Pro Ser Pro Arg Gln Ser Pro Arg Thr Leu Arg
1 5 10 15
Pro Asp Pro Gly Pro Ala Met Ser Phe Phe Arg Arg Lys Val Lys Gly
20 25 30
Lys Glu Gln Glu Lys Thr Ser Asp Val Lys Ser Ile Lys Ala Ser Ile
35 40 45
Ser Val His Ser Pro Gln Lys Ser Thr Lys Asn His Ala Leu Leu Glu
50 55 60
Ala Ala Gly Pro Ser His Val Ala Ile Asn Ala Ile Ser Ala Asn Met
65 70 75 80
Asp Ser Phe Ser Ser Ser Arg Thr Ala Thr Leu Lys Lys Gln Pro Ser
85 90 95
His Met Glu Ala Ala His Phe Gly Asp Leu Gly Arg Ser Cys Leu Asp
100 105 110
Tyr Gln Thr Gln Glu Thr Lys Ser Ser Leu Ser Lys Thr Leu Glu Gln
115 120 125
Val Leu His Asp Thr Ile Val Leu Pro Tyr Phe Ile Gln Phe Met Glu
130 135 140
Leu Arg Arg Met Glu His Leu Val Lys Phe Trp Leu Glu Ala Glu Ser
l45 150 155 160
Phe His Ser Thr Thr Trp Ser Arg Ile Arg Ala His Ser Leu Asn Thr
165 170 175
Met Lys Gln Ser Ser Leu Ala Glu Pro Val Ser Pro Ser Lys Lys His
180 185 190
Glu Thr Thr Ala Ser Phe Leu Thr Asp Ser Leu Asp Lys Arg Leu Glu
195 200 205
Asp Ser Gly Ser Ala Gln Leu Phe Met Thr His Ser Glu Gly Ile Asp
210 215 220
Leu Asn Asn Arg Thr Asn Ser Thr Gln Asn His Leu Leu Leu Ser Gln
225 230 235 240
Glu Cys Asp Ser Ala His Ser Leu Arg Leu Glu Met Ala Arg Ala Gly
245 250 255
Thr His Gln Val Ser Met Glu Thr Gln Glu Ser Ser Ser Thr Leu Thr
260 265 270
Val Ala Ser Arg Asn Ser Pro Ala Ser Pro Leu Lys Glu Leu Ser Gly
CA 02518238 2005-09-06
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275 280 285
Lys Leu Met Lys Ser Ile Glu Gln Asp Ala Val Asn Thr Phe Thr Lys
290 295 300
Tyr Ile Ser Pro Asp Ala Ala Lys Pro Ile Pro Ile Thr Glu Ala Met
305 310 315 320
Arg Asn Asp Ile Ile Ala Arg Ile Cys Gly Glu Asp Gly Gln Val Asp
325 330 335
Pro Asn Cys Phe Val Leu Ala Gln Ser Ile Val Phe Ser Ala Met Glu
340 345 350
Gln Glu His Phe Ser Glu Phe Leu Arg Ser His His Phe Cys Lys Tyr
355 360 365
Gln Ile Glu Val Leu Thr Ser Gly Thr Val Tyr Leu Ala Asp Ile Leu
370 375 380
Phe Cys Glu Ser Ala Leu Phe Tyr Phe Ser Glu Tyr Met Glu Lys Glu
385 390 395 400
Asp Ala Val Asn Ile Leu Gln Phe Trp Leu Ala Ala Asp Asn Phe Gln
405 410 415
Ser Gln Leu Ala Ala Lys Lys Gly Gln Tyr Asp Gly Gln Glu Ala Gln
420 425 430
Asn Asp Ala Met Ile Leu Tyr Asp Lys Tyr Phe Ser Leu Gln Ala Thr
435 440 445
His Pro Leu Gly Phe Asp Asp Val Val Arg Leu Glu Ile Glu Ser Asn
450 455 460
Ile Cys Arg Glu Gly Gly Pro Leu Pro Asn Cys Phe Thr Thr Pro Leu
465 470 475 480
Arg Gln Ala Trp Thr Thr Met Glu Lys Val Phe Leu Pro Gly Phe Leu
485 490 495
Ser Ser Asn Leu Tyr Tyr Lys Tyr Leu Asn Asp Leu Ile His Ser Val
500 505 510
Arg Gly Asp Glu Phe Leu Gly Gly Asn Val Ser Pro Thr Ala Pro Gly
515 520 525
Ser Val Gly Pro Pro Asp Glu Ser His Pro Gly Ser Ser Asp Ser Ser
530 535 540
Ala Ser Gln Ser Ser Val Lys Lys Ala Ser Ile Lys Ile Leu Lys Asn
545 550 555 560
Phe Asp Glu Ala Ile Ile Va_1 Asp Ala Ala Ser Leu Asp Pro Glu Ser
565 570 575
Leu Tyr Gln Arg Thr Tyr Ala Gly Lys Met Thr Phe Gly Arg Val Ser
580 585 590
Asp Leu Gly Gln Phe Ile Arg Glu Ser Glu Pro Glu Pro Asp Val Arg
595 600 605
Lys Ser Lys Gly Ser Met Phe Ser Gln Ala Met Lys Lys Trp Val Gln
610 615 620
Gly Asn Thr Asp Glu Ala Gln Glu Glu Leu Ala Trp Lys Ile Ala Lys
625 630 635 640
Met Ile Val Ser Asp Ile Met Gln Gln Ala Gln Tyr Asp Gln Pro Leu
645 650 655
Glu Lys Ser Thr Lys Leu
660
<210> 3
<211> 2363
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (138) . . . (2126)
<223> AICAP-10-5
<221> allele
<222> 2073
<223> Single Nucleotide Polymorphism: A to G
<400> 3
CA 02518238 2005-09-06
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-6-
gcggcttgtt tgcctgggca tcccgaggag 60
gataatatgg gcggtggggc
cggctggagc
ccactcccgg ggcccctctg 120
aagaagggtc gacccggaag
ccttttcgcg
ctagtgcagc
tccgggccgg 170
ttgctga
atg
agg
gga
gcc
ggg
ccc
tcc
ccg
cgc
cag
tcc
Met
Arg
Gly
Ala
Gly
Pro
Ser
Pro
Arg
Gln
Ser
1 5 10
ccccgcacc ctccgtccc gacccg ggccccgccatg tccttcttc cgg 218
ProArgThr LeuArgPro AspPro GlyProAlaMet SerPhePhe Arg
15 20 25
cggaaagtg aaaggcaaa gaacaa gagaagacctca gatgtgaag tcc 266
ArgLysVal LysGlyLys GluGln GluLysThrSer AspValLys Ser
30 35 40
attaaaget tcaatatcc gtacat tccccacaaaaa agcactaaa aat 314
IleLysAla SerIleSer ValHis SerProGlnLys SerThrLys Asn
45 50 55
catgccttg ctggagget gcagga ccaagtcatgtt gcaatcaat gcc 362
HisAlaLeu LeuGluAla AlaGly ProSerHisVal AlaIleAsn Ala
60 65 70 75
atttctgcc aacatggac tccttt tcaagtagcagg acagccaca ctt 410
IleSerAla AsnMetAsp SerPhe SerSerSerArg ThrAlaThr Leu
80 85 90
aagaagcag ccaagccac atggag getgetcatttt ggtgacctg ggc 458
LysLysGln ProSerHis MetGlu AlaAlaHisPhe GlyAspLeu Gly
95 100 105
agatcttgt ctggactac cagact caagagaccaaa tcaagcCtt tct 506
ArgSerCys LeuAspTyr GlnThr GlnGluThrLys SerSerLeu Ser
110 115 120
aagaccctt gaacaagtc ttgcac gacactattgtc ctcccttac ttc 554
LysThrLeu GluGlnVal LeuHis AspThrIleVal LeuProTyr Phe
125 130 135
attcaattc atggaactt cggcga atggagcatttg gtgaaattt tgg 602
IleGlnPhe MetGluLeu ArgArg MetGluHisLeu ValLysPhe Trp
140 145 150 155
ttagagget gaaagtttt cattca acaacttggtog cgaataaga gca 650
LeuGluAla GluSerPhe HisSer ThrThrTrpSer ArgIleArg Ala
160 165 170
cacagtcta aacacaatg aagcag agctcactgget gagCCtgtC tct 698
HisSerLeu AsnThrMet LysGln SerSerLeuAla GluProVal Ser
175 180 185
ccatctaaa aagcatgaa actaca gcgtctttttta actgattct ctt 746
ProSerLys LysHisGlu ThrThr AlaSerPheLeu ThrAspSer Leu
190 195 200
gataagaga ttggaggat tctggc tcagcacagttg tttatgact cat 794
AspLysArg LeuGluAsp SerGly SerAlaGlnLeu PheMetThr His
205 210 215
tcagaagga attgacctg aataat agaactaacagc actcagaat cac 842
SerGluGly IleAspLeu AsnAsn ArgThrAsnSer ThrGlnAsn His
220 225 230 235
ttgctgctt tcccaggaa tgtgac agtgcccattct ctccgtctt gaa 890
LeuLeuLeu SerGlnGlu CysAsp SerAlaHisSer LeuArgLeu Glu
240 245 250
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_7_
atggccaga gcaggaact caccaagtt tccatggaa acccaagaa tct 938
MetAlaArg AlaGlyThr HisGlnVal SerMetGlu ThrGlnGlu Ser
255 260 265
tcctctaca cttacagta gccagtaga aatagtccc gettctcca cta 986
SerSerThr LeuThrVal AlaSerArg AsnSerPro AlaSerPro Leu
270 275 280
aaagaattg tcaggaaaa ctaatgaaa agtatagaa caagatgca gtg 1034
LysGluLeu SerGlyLys LeuMetLys SerIleGlu GlnAspAla Val
285 290 295
aatactttt accaaatat atatctcca gatgetget aaaccaata cca 1082
AsnThrPhe ThrLysTyr IleSerPro AspAlaAla LysProIle Pro
300 305 310 315
attacagaa gcaatgaga aatgacatc atagcaagg atttgtgga gaa 1130
IleThrGlu AlaMetArg AsnAspIle IleAlaArg IleCysGly Glu
320 325 330
gatggacag gtggatccc aactgtttc gttttggca cagtccata gtc 1178
AspGlyGln ValAspPro AsnCysPhe ValLeuAla GlnSerIle Val
335 340 345
tttagtgca atggagcaa gagcacttt agtgagttt ctgcgaagt cac 1226
PheSerAla MetGluGln GluHisPhe SerGluPhe LeuArgSer His
350 355 360
oatttctgt aaataccag attgaagtg ctgaccagt ggaactgtt tac 1274
HisPheCys LysTyrGln IleGluVal LeuThrSer GlyThrVal Tyr
365 370 375
ctggetgac attctcttc tgtgagtca gccctcttt tatttctct gag 1322
LeuAlaAsp IleLeuPhe CysGluSer AlaLeuPhe TyrPheSer Glu
380 385 390 395
tacatggaa aaagaggat gcagtgaat atcttacaa ttctggttg gca 1370
TyrMetGlu LysGluAsp AlaValAsn IleLeuGln PheTrpLeu Ala
400 405 410
gcagataac ttccagtct cagcttget gccaaaaag gggcaatat gat 1418
AlaAspAsn PheGlnSer GlnLeuAla AlaLysLys GlyGlnTyr Asp
415 420 425
ggacaggag gcacagaat gatgccatg attttatat gacaagtac ttc 1466
GlyGlnGlu AlaGlnAsn AspAlaMet IleLeuTyr AspLysTyr Phe
430 435 440
tccctccaa gccacacat cctcttgga tttgatgat gttgtacga tta 1514
SerLeuGln AlaThrHis ProLeuGly PheAspAsp ValValArg Leu
445 450 455
gaaattgaa tccaatatc tgcagggaa ggtgggcca ctccccaac tgt 1562
GluIleGlu SerAsnIle CysArgGlu GlyGlyPro LeuProAsn Cys
460 465 470 475
ttcacaact ccattacgt caggcctgg acaaccatg gagaaggtc ttt 1610
PheThrThr ProLeuArg GlnAlaTrp ThrThrMet GluLysVal Phe
480 485 490
ttgCCtggC tttctgtcc agcaatctt tattataaa tatttgaat gat 1658
LeuProGly PheLeuSer SerAsnLeu TyrTyrLys TyrLeuAsn Asp
495 500 505
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_g_
ctcatccat tcggttcga ggagatgaa tttctgggc gggaacgtg tcg 1706
LeuIleHis SerValArg GlyAspGlu PheLeuGly GlyAsnVal Ser
510 515 520
ccgactget CCtggCtct gttggccct cctgatgag tctcaccca ggg 1754
ProThrAla ProGlySer ValGlyPro ProAspGlu SerHisPro Gly
525 530 535
agttctgac agctctgcg tctcagtcc agtgtgaaa aaagccagt att 1802
SerSerAsp SerSerAla SerGlnSer SerValLys LysAlaSer Ile
540 545 550 555
aaaatactg aaaaatttt gatgaagcg ataattgtg gatgcggca agt 1850
LysIleLeu LysAsnPhe AspGluAla IleIleVal AspAlaAla Ser
560 565 570
ctggatcca gaatcttta tatcaacgg acatatgcc gggaagatg aca 1898
LeuAspPro GluSerLeu TyrGlnArg ThrTyrAla GlyLysMet Thr
575 580 585
tttggaaga gtgagtgac ttggggcaa ttcatccgg gaatctgag cct 1946
PheGlyArg ValSerAsp LeuGlyGln PheIleArg GluSerGlu Pro
590 595 600
gaacctgat gtaaggaaa tcaaaagga tccatgttc tcacaaget atg 1994
GluProAsp ValArgLys SerLysGly SerMetPhe SerGlnAla Met
605 610 615
aagaaatgg gtgcaagga aatactgat gaggcccag gaagagcta get 2042
LysLysTrp ValGlnGly AsnThrAsp GluAlaGln GluGluLeu Ala
620 625 630 635
tggaagatt getaaaatg atagtcagt gacgttatg cagcagget cag 2090
TrpLysIle AlaLysMet IleValSer AspValMet GlnGlnAla Gln
640 645 650
tatgatcaa ocgttagag aaatctaca aagttatga ctcaaaactt 2136
TyrAspGln ProLeuGlu LysSerThr LysLeu
655 660
gagataaagg tttcccttgg 2196
aaatctgctt ttggattctt
gtgaaaaata
agagaacttt
caacacagcc cactgttgtt 2256
aatgaaaaca tccagggaga
gcactatatt
tctgatctgt
gaatggggag tacctgtagg 2316
acaatcctag gcataattgg
gacttccacc
ctaatgcagt
atggcacatg ttaccaa 2363
atgtttcaca
cagtgaggag
tctttaaagg
<210>
4
<211>
662
<212>
PRT
<213>
Homo
Sapiens
<400>
4
MetArgGly AlaGlyPro SerProArg GlnSerPro ArgThrLeu Arg
1 5 10 15
ProAspPro GlyProAla MetSerPhe PheArgArg LysValLys Gly
20 25 30
LysGluGln GluLysThr SerAspVal LysSerIle LysAlaSer Ile
35 40 45
SerValHis SerProGln LysSerThr LysAsnHis AlaLeuLeu Glu
50 55 60
AlaAlaGly ProSerHis ValAlaIle AsnAlaIle SerAlaAsn Met
65 70 75 80
AspSerPhe SerSerSer ArgThrAla ThrLeuLys LysGlnPro Ser
85 90 95
HisMetGlu AlaAlaHis PheGlyAsp LeuGlyArg SerCysLeu Asp
100 105 110
CA 02518238 2005-09-06
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_g_
Tyr Gln Thr Gln Glu Thr Lys Ser Ser Leu Ser Lys Thr Leu Glu Gln
115 120 125
Val Leu His Asp Thr Ile Val Leu Pro Tyr Phe Ile Gln Phe Met Glu
130 135 140
Leu Arg Arg Met Glu His Leu Val Lys Phe Trp Leu Glu Ala Glu Ser
145 150 155 160
Phe His Ser Thr Thr Trp Ser Arg Ile Arg Ala His Ser Leu Asn Thr
165 170 ' 175
Met Lys Gln Ser Ser Leu Ala Glu Pro Val Ser Pro Ser Lys Lys His
180 185 190
Glu Thr Thr Ala Ser Phe Leu Thr Asp Ser Leu Asp Lys Arg Leu Glu
195 200 205
Asp Ser Gly Ser Ala Gln Leu Phe Met Thr His Ser Glu Gly Ile Asp
210 215 220
Leu Asn Asn Arg Thr Asn Ser Thr Gln Asn His Leu Leu Leu Ser Gln
225 230 235 240
Glu Cys Asp Ser Ala His Ser Leu Arg Leu Glu Met Ala Arg Ala Gly
245 250 255
Thr His Gln Val Ser Met Glu Thr Gln Glu Ser Ser Ser Thr Leu Thr
260 265 270
Val Ala Ser Arg Asn Ser Pro Ala Ser Pro Leu Lys Glu Leu Ser Gly
275 280 285
Lys Leu Met Lys Ser Ile Glu Gln Asp Ala Val Asn Thr Phe Thr Lys
290 295 300
Tyr Ile Ser Pro Asp Ala'Ala Lys Pro Ile Pro Ile Thr Glu Ala Met
305 310 315 320
Arg Asn Asp Ile Ile Ala Arg Ile Cys Gly Glu Asp Gly Gln Val Asp
325 330 335
Pro Asn Cys Phe Val Leu Ala Gln Ser Ile Val Phe Ser Ala Met Glu
340 345 350
Gln Glu His Phe Ser Glu Phe Leu Arg Ser His His Phe Cys Lys Tyr
355 360 365
Gln Ile Glu Val Leu Thr Ser Gly Thr Val Tyr Leu Ala Asp Ile Leu
370 375 380
Phe Cys Glu Ser Ala Leu Phe Tyr Phe Ser Glu Tyr Met Glu Lys Glu
385 390 395 400
Asp Ala Val Asn Ile Leu Gln Phe Trp Leu Ala Ala Asp Asn Phe Gln
a_.05 410 415
Ser Gln Leu Ala Ala Lys Lys Gly Gln Tyr Asp Gly Gln Glu Ala Gln
420 425 430
Asn Asp Ala Met Ile Leu Tyr Asp Lys Tyr Phe Ser Leu Gln Ala Thr
435 440 445
His Pro Leu Gly Phe Asp Asp Val Val Arg Leu Glu Ile Glu Ser Asn
450 455 460
Ile Cys Arg Glu Gly Gly Pro Leu Pro Asn Cys Phe Thr Thr Pro Leu
465 470 475 480
Arg Gln Ala Trp Thr Thr Met Glu Lys Val Phe Leu Pro Gly Phe Leu
485 490 495
Ser Ser Asn Leu Tyr Tyr Lys Tyr Leu Asn Asp Leu Ile His Ser Val
500 505 510
Arg Gly Asp Glu Phe Leu Gly Gly Asn Val Ser Pro Thr Ala Pro Gly
515 520 525
Ser Val Gly Pro Pro Asp Glu Ser His Pro Gly Ser Ser Asp Ser Ser
530 535 540
Ala Ser Gln Ser Ser Val Lys Lys Ala Ser Ile Lys Ile Leu Lys Asn
545 550 555 560
Phe Asp Glu Ala Ile Ile Val Asp Ala Ala Ser Leu Asp Pro Glu Ser
565 570 575
Leu Tyr Gln Arg Thr Tyr Ala Gly Lys Met Thr Phe Gly Arg Val Ser
580 585 590
Asp Leu Gly Gln Phe Ile Arg Glu Ser Glu Pro Glu Pro Asp Val Arg
595 600 605
Lys Ser Lys Gly Ser Met Phe Ser Gln Ala Met Lys Lys Trp Val Gln
610 615 620
Gly Asn Thr Asp Glu Ala Gln Glu Glu Leu Ala Trp Lys Ile Ala Lys
CA 02518238 2005-09-06
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-10-
625 630 635 640
Met Ile Val Ser Asp Val Met Gln Gln Ala Gln Tyr Asp Gln Pro Leu
645 650 655
Glu Lys Ser Thr Lys Leu
660
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> 0ligonucleotide Primer
<400> 5
tctcaatcat gtgcattgag g 21
<210> 6
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide Primer
<400> 6
agcggataac aatttcacac agggatcaca cagccatcag cag 43
<210> 7
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide Primer
<400> 7
agcggataac aatttcacac agg 23
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> 0ligonucleotide Primer
<400> 8
ctggcgccca cgtggtcaa 19
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> 0ligonucleotide Primer
<400> 9
tcccaaagtg ctggaattac 20
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence
CA 02518238 2005-09-06
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-11-
<220>
<223> Oligonucleotide Primer
<400> 10
gtccaatata tgcaaacagt t 21
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide Primer
<400> 11
cccacagcag ttaatccttc 20
<210> 12
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> 0ligonucleotide Primer
<400> 12
agcggataac aatttcacac agggagctag cttggaagat tgc 43
<210> 13
<211> 162025
<212> DNA
<213> Homo Sapien
<220>
<221> allele
<222> 83587
<223> Nucleotide sequence of Chromosome 17 with genomic
sequence of the allelic variant AICAP10-6
<400> 13
gaattcctat ttcaaaagaa acaaatgggc caagtatggt ggctcatacc tgtaatccca 60
gcactttggg aggccgaggt gagtgggtca cttgaggtca ggagttccag gccagtctgg 120
ccaacatggt gaaacactgt ctctactaaa aatacaaaaa ttagccgggc gtggtggcgg 180
gcacctgtaa tcccagctac tcaggaggct gaggcaggag aattgcttga acctgggaga 240
tggaggttgc agtgagccga gatcgcgcca ctgctctcca gcctgggtgg cagagtgaga 300
ctctgtctca aaaagaaaca aagaaataaa tgaaacaatt ttgttcacat atatttcaca 360
aatttgaaat gttaaaggta ttatggtcac tgatatcctg tttcattctt tatataatca 420
ttaagtttga aatgtatact tgcactacta acacagtagt taatcttagt cctacaagtt 480
actgctttta cacaatatat tttcgtaata tgtatgcact ggtgtttatg tacgtgttta 540
tgtttatatc tgttaaaatt agcagtttcc atctttttct attttgtacc atcacatcag 600
ttcagaagga ttgacagagc aaaatgattt gatgaagtat aaaagtcaca tggtgagtgg 660
cataaataca actctgaaca attaggaggc tcactattga ctggaactaa actgcaagcc 720
agaaagacac atatcctata tgtcaagaga tgtaccaccc aggcagttaa agaagggaag 780
tacacataga aagcacaatg gtgaataatt aaaaaattgg aatttatcag acactggatt 840
catttgctcc taaagtcaga gtcctctatt gtttttttgt ttttgtgggt ttctttttaa 900
atttttttat tttttgtaga gtcggagtct cactgtgtta cccgggctgg tctagaactc 960
ctggcctcaa acaaacctcc tgcctcagct tcccaaagca ttgggattac agacatgagc 1020
cactgagccc agcccagacg ctttagcatt tatgaagctt ctgaaatagt tgtagaaacc 1080
gcataagctt tccatgtcac tttcaaagtt tgatggtctc tttagtaaac caaccaagtt 1140
attcctcaag ggcaaaataa catttctcag tgcaaaactg atgcacttca ttaccaaaag 1200
gaaaagacca caactataga ggcgtcattg aaagctgcac tcttcagagg ccaaaaaaaa 1260
aggtacaaac acatactaat ggaacattct ttagaagagc cccaaagtta atgataaaca 1320
ttttcatcaa agagaaaaga gaacaaggtg ttagcaaatt cctctatcaa ataacactaa 1380
acatcaagga acatcaatgg catgccatgt ggaagaggaa gtgctagctc atgtacaaac 1440
cagtagataa tttcaacttg ctgccgaatg aaacctcttt gcaaggtatg aatcagcact 1500
CA 02518238 2005-09-06
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_12_
tctcatgtttgttttgctttgttttgttttgtttttagagacaggcccttgctctgtcac 1560
acaggctggagtgcagtggcacgatcagagctcactgcaacctgaaactcctgggctcaa 1620
gggatCCtCCtgccttagcctcccaagtagCtgggaCtaCaggCCCaCCatgcccagcta 1680
attttttaaattttctatagagatgggatctcactagcacctttcatgtttgatgttcat 1740
atacaacgaccaaggtacaatgtggaaaagggtctcagggatctaaagtgaaggaggacc 1800
agaaagaaaaggggttgctacatagagtagaagaagttgcacttcatgccagtctacaac 1860
actgctgttttcctcagagcagagttgatgatctaaatcaggggtccccaacccccagtt 1920
catagcctgttaggaaccgggccacacagcaggaggtgagcaataggcaagcgagcatta 1980
ccacctgggcttcacctcccgtcagatcagtgatgtcattagattctcataggaccatga 2040
accctattgtgaactgagcatgcaagggatgtaggttttccgctctttatgagactctaa 2100
tgccggaagatctgtcactgtcttccatcaccctgagatgggaacatctagttgcaggaa 2160
aacaacctcagggctcccattgattctatattacagtgagttgtatcattatttcattct 2220
atattacaat gtaataataatagaaataaaggcacaataggccaggcgtggtggctcaca 2280
cctgtaatcccagcacttcgggaggccaaggcaggcggatcacgaggtcaggagatcgag 2340
accatcctggctaaaacggtgaaaccccgtctactaaaaattcaaaaaaaaattagccgg 2400
gtgtggtggtgggcacctgtagtcccagctactcgagaggctgaggcaggagaatggtgt 2460
gaacctgggaggcagagcttgaggtaagccgagatcacgccactgcactccagcctgggc 2520
gacagagcgatactctgtctcaaaaaaaaaaaaaaaaaaaaaagaaataaagtgaacaat 2580
aaatgtaatgtggctgaatcattccaaaacaatccccccaccccagttcacggaaaaatt 2640
ctcccacaaaaccagtccctggtgccaaaaaggttggggaccgctaatctaaataatcta 2700
atcttcattcaatgctaaaaaatgaataaacttttttttaaatacacggtctcactttgt 2760
tgcccaggctggagtacggtggcatgatcacagctcactgtagcctcaatcacccaggcc 2820
ccagcgatcctcccacctaaacttcctgagtagctgggactacaggcacgcacc'accatg2880
cccagctaatttttaaattttttatagagatgggggtctcaccatgttgcccagactggt 2940
CtCaaaCCCtgggCtCaagtgatCCtCCCtcaaactcctggactcaagtgatcctccttc 3000
cttggcctcccaaagtgctgggattacaagcatgagccactgtacccagctggataaaca 3060
ttttaagtcgCa.CtaCagtCatggaCaatCaggCttttCaaCatgCagtatggacagtga 3120
gtcccagggtctgcttttccatactgaaatacatgtgatactaaggagaaaggtgctcgc 3180
aaggatatttaaaatgaagaatatttaaaatgaggaaaaaactgtttcttcatgactttg 3240
ataaggctgataaagaccatttctgtgatctcaggtgattcactcaagtagtatatttca 3300
gtaatcattatctggaacagCCtgaatCttaaccaaaataccatgattttttaatgctgt 3360
tatgataccttgatgatatgaccaaactgcaatgtaggcagctaaatctccacgagtttg 3420
acttccccgagagttgacagttttcttcacaaattaaagaaatatattttttgatacatg 3480
attggcatatttaaaaactacactgaaatgctgcaaaatgatataaagaaacattttcca 3540
gaatcaaatgcaatcaaagagtggattaggaatctactcaccattatcaactaaatagaa 3600
acacttggactgggtgtggtggctcacatctgtaatctcagcactttgggaggccaaggc 3660
aggtggattgcttgaggccaggagctcaagaccagcctgagcaacatagcaaaactctgt 3720
ctctacaaaaaaaaaaaaaaattaaccaggcatggtggcagatgcttgtaatcccagcta 3780
CtCtggaagCtgaagtaggaggaCtgCttgagCCCaggagatCaagaCtgCagtgagCCg 3840
tggtCatgCtgCgCCaCagCCtgagtgaCagagagagaCCCtgtCtCaaaaaCaaaaaCa 3900
aacaaaaaacacttaaccttcctgttttttgctgttgttgttgttgtttgtttgttttga 3960
gatggagtctCaCtCtgttgcccaggctggagtgcagtggcgtgatcttggctcactgca 4020
agCtCtgCCtCCCgggttCaCgCCattCtCCtgCCtCagCCtCCCgagtagCtgggaCta 4080
taggcgcccgCCaCCaCgCCCggCtaCttttttgCatttttagtagagatggggtttCaC 4140
cgtgttagccaggatggtcttgatCtCCtgaCCtCgtgatCCaCCtgCCtCggCCtCCCa 4200
aagtgctgggattacaggcatgagccaccgcacccggccaacctttctgttttttagttt 4260
gatatgcttgttaactcagcagctgaaagaatgctgaaagtggccttcagtaaaaaaatt 4320
tcactagaatctctacatccatatttaatctgaatgcatatccagattgatcagttagag 4380
caaaaacactcatcatcattcctgatgacctctaattctggtttcggctttctatttcaa 4440
tggaaacagaataaggaaagaaatggaagggctctggaaatttgtcctgggctatagata 4500
ctatcaaagatcaccaacaataagatctctcctataaatataaaacaagtataattaatt 4560
ttttaattatttttttctcttcagaggattttatttcaagataaaacataacttctaccc 4620
atactattgattccaaaggttagaaaaagtgtttttcctcatcttatccttcaaagaggt 4680
cacagcaatgcaaacatctataaaatgcctctgcataattgtcagaagctatagtccaga 4740
aatcattgaaaatgcttttccattttaagcttaggtgaggtgtcttaggaaacctctatg 4800
acaacttactctatttattgggaggtaaactcccagactctcccagggtctcctgtattg 4860
atctcattttttaggcttcctaatcccttgaagcacaatcgaaaaagccctggatctctt 4920
ttctgcacatatcatcgcggaattcattcggcttccagcaagctgacactccatgataca 4980
agcggcctcgcccttctccggacgccagtccttgctgcggttagctaggatgaggggttt 5040
gctgggcttcagtgcaggcttctgcgggttcccaagccgcaccaggtggcctcacaggct 5100
ggatgtcaccattgcacactgagctcctggcaggctgtaccaattttttaattatttaat 5160
atttatttttaaaattatggtgaatattttggtattctgctctaaaataggcccataaat 5220
gcacagcagatatctcttggaacccacagctttccactggaagaactaagtatttttctt 5280
ttaaagatgctactaagtctctgaaaagtccagatcctctacctctttccatcccaaact 5340
aagacttggaatttatgagagatctagctaacagaaatcccagacacatcattggttctt 5400
CA 02518238 2005-09-06
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-13-
cccagagtgcagtcctcctaaagaggctcagccctaagcaggcccctgcaccaggagggt 5460
gggtctgagacccacatagcacttcccaaggtgcatgctccagagaggcactgaaacagc 5520
tgagcacaagcctgcaagcctggagaactctcacagtcagaacggagggggcccagtggg 5580
actaacataaagagaaaagggaacacagagaaatggatggcaccaacaaccagcaaagcc 5640
ttcatggccaatgaaagcatcagtgacggggccagaaccctcatccccaaagactcttca 5700
ctgcctttagtgaaaaacaatggctagagagtgaagttatgatcatgtatagagaggtaa 5760
agttacatttttatattctgactctgctaatgtgaaattccctatctgctagactaaaag 5820
tttcagacaccctgttcaaatatcccattagttgctagagacttaaaatgaacagaacgc 5880
acattgtcaggatgactattaccaaaaaatcaaaagacagcaagtattggtgaggatgta 5940
gagaaactggaacttttgtgcactgtttatgagaatgtaaaatggagcagctgctgtgga 6000
aaagagtatgcaggttcctcaaagagtaaaaccaagatgtggaaacaactaaatgcccat 6060
cagtggatgaaggggtagacaatatgtggtatatacataccatggagtactattcagcct 6120
ctaaaaaaaaaaaaggaaattctataacatgcaacagcatggatgaatcttgaggacatt 6180
ttgctaatgaaataaggcagtcatagaaagacaaatactgcacgactccacttatatgag 6240
ataccaaaaatagacaaattcatagaatcaaagagtacaatggaggttacctggagctgc 6300
agggcgggaaacgaggagttactaatcaacgaacataacgttgcagttaagtaagatgaa 6360
taagctctcaagatcagctgtacaacactgtacctagagtcaacaataatgtattgtaca 6420
cttaaaaatttgttaagggtagattaacaaatgtagtagatccacaaatgtggttaagtg 6480
ttcttaccacagtaaaataaaaaaagaatatcaagcccaggagttcgagactagcctggg 6540
taacatggtgaaaccctgtctctacagaaaatacaaaaattagccagctgtggaggtgca 6600
ctcctagggaggctgaggtgggaggcttgcttgagcccaggaggtcaaggctgcagtgag 6660
ccatgattgcaccactgtactccagcccagatgacagagcaagacaccaccccccccaaa 6720
aaaagaaaaagaatatcaaacattttaaaagatcagatacgcaagaacaacaacaaaaaa 6780
gagatgaacagagcatcgaccctcatctagtgggattcttggtctaactgaaaaacagac 6840
attgagagacaaacaatgacagtgatgtgatcacagcaattacacaggtatcccctgggg 6900
actgcagaagaaaggaggaatgcctaactttcagaaaatagagaaagcgtcaaacagttg 6960
gtgaaagccttccaaaactagagagaactgcacacaccaaatcacagaaagaagaaaagc 7020
cgtgggagattctgggacccaccggctatttttgatggctgaacaccctgctgcaggaga 7080
gacaggagctggaaagcatggtgggatgaaacctcaaacagctttgcctgcattgcttaa 7140
gatgactgggcttgattaactctagtcaatggggacaattcaatcaaagaagaaagatgc 7200
tcaaattcacattttagaatgattttttatggcagtatggggaatagattaaaagagagt 7260
gaagctggaggcaagaaacttgttaagaggCaaCtgaaaCagtCtagatgataaataata 7320
aactgacagagtgactagaaaaatcagaacaggctgaatcaacagatacctagatgaaaa 7380
taacaggacttgatcaccagttgtatcttggagaggaagg,agttgtttccttgctttccc 7440
tacgactgggaatacggaaggtttgccgtgtgtattggttatatactggtgtgtagccaa 7500
tcactgacaaecatttagcagcttaaaacacaaaggcttatctcccagtttctgtgggcc 7560
aggaatctaagataggcttagctggctggttctggctcagagtttctcaagaggttgcaa 7620
tcaagatgtcagctggggttgcatcatctgaaggctcaactggggccggagggtccactt 7680
ccaaggagttcactcacctgcctgacaaggcagtgctggttgttggcaggagatctcaat 7740
tcattgccaagtgagcctctctatagcattgctggaacatcctccccatctggcagttgg 7800
cttctctcagcatgagtgatctgagagagagagcaaggaggaagccacagtgttcttcct 7860
aCtCCtaCtCCtaaCaCtatggacctactcCtaaCaCtCtCa.CttCtgCCttattCCatt 7920
agttagaaagggaactaagctccacctcttgaaataagaagtgtcaaagaatttgtggat 7980
atatttaaaaatcatcacactgtggaagtggatagggggttcaattaatgctgaacttga 8040
aatgcctgagacattcaaatgtccaacaggcaatgaacatacccatagatggtcatgact 8100
ttagcaagaatagaggaagatcacagaattaaggaggaattgaaaggtaaaagaagtgga 8160
gtcagattccccctgaaaagtgagccatgaaaggaactttaactattgagttagaggtca 8220
gagtaggaaatttcggtggaattcttttttaaagaaaggaaccatataagcatgttttga 8280
ggtagagggagaataaatcagtagacagggagaggtaaaaaacataaatgataggggata 8340
gttgacaaaggtcttggcagaatcccttacccattgacttggggccaagagagggacact 8400
tctttgtttgagggataaggaaaataagaaagaatgggtgctatttagtgtggtcctgtc 8460
tctagggcaaacgcataggtaacaaactgtgtgtgttaggaatatagatgtgacctcaca 8520
ttgagattctcacctcaaatccattttgttgttacctgtaccttcctaccttctcttttt 8580
gctacatgcagactgctgttttgtcttcctggcctgttccaggtttcagcattctggcat 8640
atctgctaccctgttcccaaacctctctagagtccatgctccttccttggatagtgtttg 8700
attgggccacgtatctaagaagtgatgccttcagttaggcctgagaacctcctctatgga 8760
aatctccatcagtgaccctgacagacttggtatcttggagatgtcactgctcccagcctg 8820
tggtctaggagaatctcagcctgggcctctagtagtatggataaggcgttaaggtatctt 8880
tgaaccagagtctgtcatattcctcaatgtgggacagataaaacagtggtagtgctggtg 8940
tttctgagctagaactctggtttttggtctagattctttgatgtatgacctttcagaggt 9000
attaaaatttgttctaatacaatgttcaatacaaatgtagttccttttctgttaggacct 9060
caacaaaacatgaccaactgtagatgaacattaaactatgacaattcatggaaatgaata 9120
cagtaatacctgcggttcccccattttagcagtcactatggtgacatttggcacaaatgg 9180
ctatttaagggtgcttttgttaaaacctaccatcttactaggcacatgatattgaaacta 9240
atgaaataatggagaaacttcttaaaaacttttaatgaataaagtgatgaagtgataata 9300
CA 02518238 2005-09-06
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-14-
ttttagctgc tatttataaa gtgactatta caggtcaaac attcttctag ggtttttttg 9360
ttgaagttgt cacatttaat ccttaataac ccactatgag tcaggtattc ttctctcccc 9420
tttggacagt tggggaaatg ggggtcagag aggttaggta atttgctcag ggccacacaa 9480
cctgcatgta gaaaatctga gatttgtaca ggaacgtatc aaactctgaa gtccatgctt 9540
ctattttccc atgctgcctt tctaataaaa ggtaactaat gctactggat gctgccccca 9600
aagtgagtca ctttcacccc accctacttg attttctcca taaaactaat cacatcctga 9660
caacttattt attgctgatc tcccccaCta gattataaac tcaataaaag caagatcctt 9720
gtctgctgaa tatcagtacc taaaacgctg tctagcacag agcaagtaat taatatttgt 9780
tgaatgaaca aataaaggaa aaaaattcaa aggaagaaaa agccctaaaa cagatgttta 9840
cctaaacata cattttaaaa gaaagcatat aacaaattca ggacagaatt taaatttgat 9900
tttttaaaga aataaccaag tgctagctgg gcacagtggc tcacacctgt aatcctagca 9960
ctctgggagg ccgaggcagg cagatcactt gaggtcaaga gttcaagacc agcctggcca 10020
acatggtgaa acctgtctct actaaaaata cagaaattat ccaggcatgg tggcaggtcc 10080
ctgtaacccc agctactcag gaggctgagt caggagaatt gcttgaaccc aggaggcaga 10140
ggttgcagtg ggccaagatt gcaccactgc actccagcct gagtaacaaa gcaagactct 10200
gtctgaagga gaaggaaaga aagaaggaaa gaaggaaaga aggaaagaag gaaagaagga 10260
aagaaagaaa gaaagaaaga aagaaagaaa gaaagaaaga aagaaagaaa gaaagaaaga 10320
aagaaagaaa aagaaagaaa gaaagaaaga accaagtgct tatttgggac ctactatgct 10380
atgtttttcc atgcacgcta ttttcagtaa agcagttagc aaacttgcaa gatcataaca 10440
acaaatatat gcttctataa ctctaaaatt gtgctttaag aagttcctct ttaccagctc 10500
atgtatgcat tagttttcta agagttacta gtaacttttt ccctggagaa tatccacagc 10560
cagtttattt aaccaaagga ggatgcttac taacatgaag ttatcaaatg tgagcctaag 10620
ttgggccagt tcatgttaat atactccaga acaaaaacca tcctactgtc ctctgacaat 10680
tttacctgaa aattcatttt ccacattacc aaggagccag ggtaggagaa tatagaaaga 10740
CCaCCCaaga atCCttaCtt ctttcagcaa aatcaattca aagtaggtaa ctaaacacat 10800
gccctaacaa tgaatagcag attgtgctca gaagaatgat ctacaacatc ttactgtgaa 10860
ggaactactg aaatattcca ataagacttc tctccaaaat gattttattg aatttgcatt 10920
ttaaaaaata ttttaagcct aaattttaaa aggtttgata ttggtacatg aatagaCaaa 10980
cagacatgga ctagaccaag aattaggttc aaacatatac aggaatttaa tatacgataa 11040
atctagtatt ccaaaggaac caacaaatgg tgttcagaca gcaggatagg catcaggaaa 11100
aacacagttg ggcaccctac cttactccta acaccaggag taactgaagg agcaccaaat 11160
atttatttat tttaattata gttttaagtt ctagggtacg tgtgcacaac atgcaggttt 11220
attacatagg tatacatgtg ccatgttggt gaggagcacc aaatatttaa aagaaaaaaa 11280
ttggccaggg gcggtggctc acacctgtaa tcccagcact ttgggaggcc aaggtgggca 11340
gatcacctga ggtcgggagt tcgagaccag cctgagcaac atggagaaac cccatctcta 11400
ctaaaaatac aaaattagcc aggcatggtg gcacatgcct gtaatcccag ctacttggga 11460
ggctgaggca ggagaatagc tttaatctgg gaggcacagg ttgcggtgag ctgagatatt 11520
gcactcCagc ctgggcaaca agagcaaaac ttcaactcaa aaaaattaat aaataaataa 11580
aaataaagaa agaaaagaaa aaaatgaaaa tagtataatt agcagaagaa aacaccgtag 11640
aatcctcgga ctcttaggat ggggaatgcc tata_atataa aaaccctgaa gttataaaag 11700
agaaaatcac ctacatacaa accaaatctt tctacatgcc taaaacatag cacaaacaca 11760
gctaaataat catagctgaa tgaactggga aaacaaaact tgactcatat ccagacagag 11820
ttaattttcc tacacataaa gagtacctat ataaacccaa caaaaaaacc accactaacc 11880
caaaataaaa atgtgacagg taatgaacag gtagttcaca gagaatacaa atggctcttc 11940
ggcacataag atgctcagac tgacttttac ttatttattt tttgagagac agggtctcac 12000
gatgttgccc aggttaggct caaactcctg ggctcaaatg atagtaccag gactacaggt 12060
gtgccccacc gcacctggct cctcaaccac ctgtattaac aggaaatgca aaataaaact 12120
ttcaaatcta ttttacctat tagaatggca aaaatttgaa aaacttcaaa catcatcatg 12180
ttggtgagaa tgtgaggaga ctggcactct cattttttgc tgatagcata tatatactga 12240
tggcttctat ggaaagcaat ctggcagcgt ctatcaaatg tacaagtgca tatatccttt 12300
gacaaagcaa ttccactcta ggaatgtgtt ctatatggtt gtgcttcctg gggctgggaa 12360
ctgggagcta agggacaggg gcagaagata atcttctttt ccctccttcc ccgttaaaca 12420
tgttgaattt tatatactgt aatatattat ttttcacaaa agataatttt taagcgatat 12480
gtctgggaat tttttttttt cttttctgag acagggtctc actctgtcat ccaggctgga 12540
atgccatggt atgatctcag ctgactgcag cctcgacctc ctgggttcaa gcaatcctcc 12600
cacctcagcc tcctgagtag ctgggactac aggcacgtgc catcatgcta atttttgtat 12660
atacagggtc tcactatgtt gcccaggcta atgtcaaact cctaggctca agcaatccac 12720
ccacctcagg ctccaaagtg ctgggattac aggcgtgagc caccgcgcct ggccctggga 12780
attcttacaa aagaaaaaat atctactctc cccttctatt aaagtcaaaa cagagaagga 12840
aattcaacct ataatgaaag tagagaaggg cctcaaccct gagcaacaaa cacaaaggct 12900
atttctgaga caggaatttg ctgaacaaaa tcgagggaag atgacaagaa tcaagactca 12960
cttctcggct gggcgcagtg gctcacacct gtaatcccag cactttggga ggccgaggcg 13020
gacagatcac gaggtcagga gattgagacc atactggcta acacagtgaa acccagtctc 13080
tactaaaaat acaaaaaatt agccgggcgt ggtggcaggt gcctgtagtc ccagctactt 13140
gggaagctga ggcaggagaa tggcgtgaac ccaggaagcg gagcttgcag tgagccgaga 13200
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-15-
tcacgccactgcactccagcctgggtgacagagcaagactctgtctcaaaaaaaaaaaaa13260
aagactcatttctctagatcttgagccgtattcaaatttatctcagcttagtgagaggtt13320
aaagcaaggaatatccttccctgtgggccctgctccttactgaaggaaggtaacggatga13380
gtcaaggacaccaatggagaaaagcactaacaccattatctgatgaacattacgtgaaga13440
agggtaagaagtgaagtggaattgctgaagaagtcagtgaaagcggacattcatttgggg13500
aaatggaatataggaaatccataaaagtgattaaaaagatgttagaggctgaggcggggg13560
gaccacagggtcaggagatcgagaccatcctggctaacacggtgaaaccccatctctact13620
aaaaatacaaaaaattagccaggcgtggtggcaggcacctgtagtcccaactactcggga13680
gactgaggcaggagaatggcatgaacctgggagacggagcttgcagtgagccgagatcac13740
gccactgcactccagcctgggtgacagagtgagactccatctcaaaaaaaaaagttagat13800
acgagagataaagatccaacagacacacaactgctaattctgaacagaacaaaacaaatg13860
gcacaggaaaagaaaatttaagatataacaccggaaaactttcctgaaattgagtaactg13920
aatctatagcttgaaagggtttagcatatgccaagaaaaatcagtagagtccaaccagca13980
caagacacatctagcaaggctggtgattctaccaacacagagaaagaagtgggtgaccca14040
taatgcggaaaaaggcagaccatctgcagtcttctccagaacactggagtctgaagacaa14100
aagaatgctgcctactgagccagaagggagagaaagtgacccaacacatctttaccaagt14160
tagaatgtcacgcattatttaaaggctgcaaaagccatgaaagacatgaaagaacacaag14220
catttacaacatgaaagaacacaagcattctcatactcaagaatccttaagaaaaatgta14280
gtcctaatccagcccactgaaagttaaatgtacttaatgtgctcattaatgggaacttca14340
tagcttcaaatcagtctggtcccatctaccaacatctctcgcccggctttcctgcaatag14400
tCagCaCCtttCCCtCCtCCCagtCttgtCCCCtggagtCtgctctcagcatagcagagt14460
gaccacatcaacacccaagtcagagccctccagtgcgcactggtctacaaagcccttccc14520
aCCCCCCa.CCCCaCgtgCCCtccggatccttgtgacgtgtCtCCtgCataCCCtagcagC14580
CCtggCCtCCtCaCtgCCCCtCCtgtaCatcaggaaggcgactccttgagtcttggctct14640
ggccgcctcctccacctgcagtgagttaactcccttacctactctaggtcattgctcaaa14700
tgtCagCatCtCaatggggCCCtCCCtgaCtaCCCtatttaaattCtaCataCtCCCCtt14760
gaCCCCatggaCCtCa.CtCaCCCtattCCaCttttattCttaCaatttagCaCttgttCt1482~
CttCtaaCgtattCtaagaCttaCrCatttattaCattgtttgCCaCCCCCtCtagtaCa14880
taaactccagaggggcagggatttctgtctatttattcatttctttatccctaggacata14940
gaacagggcatagttcagagtattcaatgttatcaatgaatgaactagcagtagtaccag15000
ttccagttaggcacagaattaaatctaaatagaattaaatctcatggtctgggttaacta15060
tggatagaaaattagatataattttaagaagcctagaaagaaaaaattaataatgtaaaa15120
ataatattaatttgataataataacaaaaactctgccaggcactgtggctcaaatctgca15180
atcccagctactcaggaggctgaggtggaaggatcacttgagaccagagttcaagactca15240
gcctaggcaacacggcaagaaactgtctctaaaaaaattaaaacttaaatttttaaaaaa15300
gaattctcaaagcgtcacaaaaactggagattaaggtacaggaagtgtgaagtaatatta15360
ctatgctaatggttttttttttttttagaaaggtataaccaa_aagatttctttctcaagt15420
cgataaactgagaaagataagcatatcttccaattaacagagggggaggaaaagccagat15480
acaacaaaataagatataaattagtttccagttgaaaacaagagtaggagttattttgca15540
tcacctcacctgtgacctcccccagcccaaaaaacactactgataaacagggtagaaaag15600
catcatctcagataaagcaggaaaaactgccacagtctcaaaccacaaactataagcaca15660
cacctggccaaccctgccaagtctgggctcagtaggaggaacgtgctgagagctaggatg15720
taccaacttagacattctgtgggatacagatgtccctggaagggtcacaccatctcaaag15780
gcacctgtaatgCCCaCtgattacagccaccatatgtgagagagaaactcagggcactta15840
gagagtataacaagaaccttatgtcatctgagatgaggaatcctcagccctgcaaattaa15900
ccaactctttagaacaactggcaaaacataaatatccacaacttttgtttcagtaattcc15960
actcttagatatcaatccaaagtacatgagacagcagatacacacacaaaatggtattta16020
ctgcagcattgtttataatagcaaaaaacaagaaataatccatatgtctcaataggatac16080
tgggtacatgagggtatgtacccatcattcaaccatcaaaaagagtgatatggatgtcca16140
cagatggacataaaaagctgtgtgttacgtgaaaacaaactcaagcagcagcaggatggg16200
cttatgatagtcagtatgagctaatttctggaaaaaaaaatctagtgtgtgcacagaaaa16260
catctgaaagaacagaaacaaaactatcagcagaatattgagatgttttactaagttgta16320
tatctatactgcttgtaatttttaccccaagcaagaattactttttggaaaaagaaaatt16380
caggaaataaagcatttctttaaacttcatgtttaaacaaatggtgatggaataaaagag16440
ttcttattcatcataaacacacacagcacacatgcacgcatgtgcgtgagcacacccttt16500
acttgataaataccatgttgaatattttagtctttccttttaggttctatcccttcactc16560
aaaatgcggttataaataaatgtacttttcatgtgccttctgcctaaacccactttaata16620
taactttacagtcccattatcattatagtctcaaagctagactcagcctgaaactaccct16680
ttcatttggaacccttattaaaatgccacatacagctccttcaaataaaaacaaacccta16740
ggacctgacactaggcttcctttgttgctactcataatggccaagttctgtgcttataat16800
acatcttctttcattttattgctacatatccaagggttttatatgtttttcttattatat16860
cttaattcaaaacaccatcacgctcttttccagatgaaaataaggaaaagaaattgagca16920
actgactgacttaaaggtcataaaactatatagtagcagagtcagcaaaagaagaaacac16980
acatctcccaagtagaggctgaaaaccagtaccattcacctccagggtgagctatataca17040
gattacaaagtcaccttctctaaatgttcaaactgaatcccatacccatactttaccact17100
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-16-
acctcgtaagaacagcctcagatcttgttatagccttttttttagcatgctgaagccaat17160
aaaatgcttcccattcagcaagagaaacaagttctgaaacactgaataatctgcccaggg17220
cctatgaacatttccactgtgagaaatgttctccactgtgtggagaagatccttactctt17280
ctccacacaggcagaacattagaaaaattcttggattctatgatgcacagcttaggagtc17340
tgtttagcacaatttaagtccaaatagttattaaatcctcctctgttccagaaacagtgc17400
taaatactgtgaatataaaaattgaaaagatactctcctggctcccaagaaagtcagcca17460
gatagaggagacacaggcacacaaatcactgtcacatgaagctctacctccctaacttca17520
aacgagggcctaagtcaccaagaatacagtagcagtt'gtgactacgagtaactactataa17580
ttcaatactttatcttcccttagaaaactcttctcccttggaaatttatttgcatttcta17640
aataccattccttactaaaaggaagcagggctccttggggaaatagctgattctaggtgt17700
ggactatgaaatgaaaatggtgagtctgggacatcccatgttgcccagaaatcaaggaac17760
tgcccaaagattaacagagtcatgttaaatggacctaagagtgaaccagaaggagctcac17820
tttgccccgcgtggaacaatttcaagaaaaacatgacagtaatgaattataaaacatgaa17880
ttaaaatacatattggtactaaaaagagaacaaaaggatgtggctttggataaagctctt17940
cttcatggaagaataccagctaataaatgtaaaggaaatgagagaattagaaaaattatc18000
attttgtaaaccttaatatattcacctagacatgctaaaaccactgagtaaaaggctgct18060
tgggaagaggatgctcacatgatctcagagtttcacaccacagataatttattagataca18120
ggaaggaagatgtgatcaagCttCCtgtgaCCCCCagCCaggccccacaacactatgtgc18180
ctccttgtgatgtgggagctacacagcatcgcccacacagcttctcgccaaaactgtttg18240
aagctaatcacaagggaagaactggacagcttctgaccatgagacgctccaccagacaac18300
ttgcttggcctctccaaagaaacttgcttggcctctccaaagaaaactcagtttcattta18360
aaaacaaaactaattatttaaaaacaaacgaaaagcaagttgtggacttgagctccaggg18420
acagagcagacatacttttccctgttcttcccagtaagtggtaataaaaaccctcaacac18480
tagatataaaacaaatataagaaggttctggaaggggaagaggaggcagactatccaggt18540
gccttgaggcccacagaacaacccagtgatgggttcactgggtcttctttttgcttcatt18600
atctcagacttggagctgaagcagcaggcaacttcaaaacaccaaggggcacagattgaa18660
aagccccaagaaaagCCtgCCCtCtCtagCCaaaggaCCaggaaggagacagtctaatga18720
gatggaacacatttagacagtaactgcccatttaccagcaataactgagcagggagccta18780
gacttccagtcttgtgaggacgtaccaaggtacccaacacccccaccaaggctgagtaag18840
gactgcgacttttatccctgcatggcagtagtaaggagcccatccctcacccgccagcag18900
tgtcaggggaacctggacttccactcccacccaggagtgatgaggccctccctgctgggg18960
tcatgtcagaggaggcctagtggagattcagtgacttaaccttttcccagagataatgag19020
gCCa.CCtttCCtCCCtCttCCCCCatggtgacagtgaaagcactgtggcaagcagtaggc19080
aCtCCtaCCCCtCCtagCCagggaggtatcagggaggccaagtagggaaccagaataccc19140
acaaccacccagcagcaacaggggtCCCCCaCCCCattgggtgtCaatggaagcagagcg19200
gaaagcctggatatttacccccatctagaagtaacaagctgatgtcccccttcttctact19260
acaatggtgttcaaaacaggtttaaataaggtctagagtctgataacgtaatacccaaat19320
cgttgaagttttcattgaggatcatttataccaagagtcaggaagatcccaaactgaaag19380
agagaaaagacaattgacagacactagcactaagagagcacagatattagaactacctga19440
aaggatgttaaagcacatatcataagcctcaacaggctgggcgcggtggctcacgcctgt19500
aaccccagcactttgggaggccgaggcaggtggatcacaagatcaggagatcgagaccat19560
cctggctaacacggtgaaaccccgtctctactaaaaatacaaaaaaaaatagcaaggcat19620
ggtggtgggCaCCtgtagtCCCagCtaCtCgggagcctgaggcaggagaatggcatgaac19680
ctgggaagaggagcagtgagCCgagatCgCaCCaCCgCaCtCCagCCtgggCaaCagagC19740
aagacttcgtcccaaaaaaaaaaaaaaaaaaaaaaaaagcctcaacaaacaactacaaac19800
gtgcttgaaacaaatgaaaaaaaaatcttggcaaagaaataaaagatatatattttggcc19860
aggtgcagtggctcacagcctgtaatCCCtgCa.Ctttgggaggctgaggcaggcggatca19920
CCtgaggtCaggagtttgagaCCagCCtgaCCaaCatggagaaaCCCCgtCtCtaCtaaa19980
aatacaaaattagccagtcatggtggcacatgcctgtaatcctagctactcaggaggccg20040
aggcaggagaatcgcttgaactcaggaggtggaggttgcggtgagccgagatcccgccat20100
tgcacattgcactccagcctgggcaacaagagcaaaactccatctcaaaaaaatagatac20160
atattttaatggaaattttagaattgaaaaatacagtaaccaaattgaatggaaagacaa20220
catagaatggagggggcagacaaaataatcagtgaacttcaacagaaaataatagaaatt20280
acccaatatgaagaacagaaagaaaatagactggccaaaaaataaagaagaaaaaagagg20340
agcagcaggaggaatgatggaaaaagagaaaggaaggaaggaagggaaggagggagggaa20400
ggagtgagggagaaagtctcaaagacctctgagactaaaataaaagatctaacacttgtc20460
atcagggtccaggaaagagacaaagatggcacagctggaaacgtattcaaaaaataatag20520
ctgaaaacttcccaaatttggcaagagacataaacctatagattcgaaatgctgaacccc20580
aaataaaaagcccaataaaatccacaccaaaatacatcatagtcaaacttctgaaaagac20640
gaaaagagaaaacgtcttgaaagcagtgagtgaaacaacacttcatgtataagggaaaaa20700
caattcaagtaacagatttcttacagaaattaaggaagccagaaggaaatgacacaatgg20760
ttttcaagtgctgaaagaaaagaagtgtcaacacaaaattctagattcagtaaaaatatc20820
cttcaagaatcaatgggaaatcaagacagtctcagataaagcaaaataagagaatatgtt20880
gccagcagatctcccctaaaggaatggcaaaaggaagatcatgcaacagaccaaaaaatg20940
atgaaagaaggaatccagaaacatcaagaagaaagaaataacatagtaagcaaaaataca21000
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-17-
tgtaattacaataaaatttctatctcctcttaagacttctaaattatattgatggttgaa21060
gcaaaaattataaccctgtctgaagtgcttctactaaatgtatgcagagaattataaatg21120
gggaaagtataggtttctatacctcattgaagtggtaaaatgacaacactgtgaaaagtt21180
acatacacacacacacgtaagtatatataaatatatgtgtgtatatgtgtgtgtatatat21240
atatatacatataatgtaatacagcaaccactaacaacactatacaaagagataataacc21300
aaaaacaatttagataaattgaaatggaattctaaaaaatattcaaatactctacaggaa21360
gacaagacaaaaagagaaaaaaagaggaggacaaactaaattttttaaaaacataaataa21420
aatggtagacttaagccctaacttatcaataattacataaatgtaaatgatctaattata21480
tcaattaaaagacagagatagcagagttaatttaaaaacatagctataagaaacctgctt21540
tgggctgagtgcagtgactcacacttgtaatcccagcacttcgggaggccaaggcgggtg21600
gatcacctgaggtcaggagttccagaccagcctggacaacatggtaataccccatctcta21660
ctaaaaatacaaaaaaattagccaggcatggtggcacacgcctgtagtcccaactactca21720
ggaggctgcgacacaagaactgcttgaacccgggcagcagaggtagcagtgggccaagat21780
tgcgccactccagcctgaacgacagagtgagactccacctcagttgaaaaacaaaaaaga21840
aacctgctttaaatataccaacatatgttggttgaaattaaaagaataaaatatatcatg21900
aaaacattaatcaaaagaaaggagtggctatattaataacataaaatagacttcagagaa21960
aagaaaatttcaagagacaggaataaaaggatcaagaaaagatcctgaaagaaaagcagg22020
caaatcaatcattctgcttggagattcaacaccctctcttaacaactgatagaacaacta22080
gacaaaaaaatcagcatggagttgagaagaacttaacaccactgaacaacaggatctaat22140
agacatttacggaacactctacccaacaatagcaaaataaacattcttttcaagtattca22200
ctgaacatatccttagaccctaccctgggccataaaacaaagctcactagtgattgccga22260
aggcttggatggacagtggaagagctgcatggggagggagaaggtgacagttaaagagtg22320
taggatttctttttgggataatgaaaatgttccaaaattgattgtggtgatgttggcgca22380
actctacaaatataaaaaaggccattgaattgtacgttttaagtgggtgaaacatatggt22440
atgtggattatatctaacgctttttaaaaacttaacacatttcaaagaatagaagtcata22500
cagagtgtgctctactggaatcaaactagaaagaggtaactggaggataacgagaaaagc22560
ctccaaatacttgaaaactggacagcacatttctaaaatcatccgtgggtcaaagatatt22620
catttctgatattcatttttattgtttaatgtatttttaaaaatttcttaagggaaataa22680
aCtgaCtaaaaatgaatatggCtgggtgCggtggCtCaCgCCtgtgatCCCagCaCtttg22740
ggaggCCgaggCtggtggatcacaagatcaggagttcgagaccagcctggccaagatggt22800
gaaaCCCCgtCtCaaCtaaaaaactacaaaaagtagccaagCgCagtggCgggagCCtgt2286~
ggtcccagctacttgggaggctgaggtaggagaatcgcttgaacacaggcagcagaggtt22920
gcagtgagccaagattgtgccactgcacgccagcctgggcgacagagactgcctcaaaaa22980
aaaaaaaaaaaaaaagaatatcaaaatttgtgggacatagttaaagcaatgctgagaggg23040
aaatttataacactaaatgtttacattagaaaagagaaaaagtttcaaatcaatagtctc23100
cactcccatctcaagaacacagaagatgaagagcaaaataaacccaaagcaagcaaaaga23160
aagaaaatataaaaataaatcagtaaaattgaaaacagaaacacaataaagaaaatcagt23220
gaaacaaagtactgattcttcgaaagattaataaaattgacaaacctctagcaaggctaa23280
caaacaaaaaagaaagaagacacggattaccagttattagaatgaaagcataattagaaa23340
caactctacacattataaatttgacaatgtagatgaaatggaetaattactgaaaaaaca23400
caaattaccacaactcacccaatatgaaatagataattgggatagcctgataactactga23460
gaaaattgaatttgtaattttaacactcttaaaacagaaacattaaacttaatattttat23520
aaatattagataaggtaattatacccttccttaacaaataaaaacgacaaattattttgc23580
agctaaagagatgtatgtactgtgaaaaatatcttcagaaaaatagaactttgtttgaag23640
.
aataaggatttaaaaaatgtttttaactctcaagaagcaaatatctgggcccagatggtt23700
tcactgaagaattctaccaaatgtttaatgaagaattaccaccaactctacatagcatct23760
ttgagaaaactgaagagaagggaacatctcccagttcattttatgaagtgggtgttactc23820
tgatactagaactgtataaggacagctactcttgacacactgcctatgggtagctctgct23880
ctgcaggaacagtcagaaaaaaaaaaaaaagaagcactggacaagggcagtataaaaaaa23940
gaaaactgggccaggtgcagtggctcacacctgtaatctcagcactttgggaggctgacg24000
ctggtggatcacctgaggtcaggagtttgagaCtagCCtggccaacatggtaaaaccctg24060
tctctactaaaatacaaaaattagccaggcagggtggtggggaaaataaaaaggaaaaaa24120
aaacaaaaataaactgcagaccaatatccttcatgagtatagacacaaaactccttaaac24180
tccttaacaaaatattagcaagtagaagcaatatataaaaataattatacaccatgatca24240
agtgggacttattccagaaacgcaagtctggttcaacatttgaaaacaaggtaacccact24300
atatgaacgtactaaagaggaaaactacataatcacatcaatcaatgcagaaaaaagcat24360
ttgccaaaatccaatatccattcatgatactctaataagaaaaataagaataaaggggaa24420
attccttgacttgataaagcttacaaaagactacaaaagcttacagctaacctatactta24480
atggtgaaaaactaaatgctttcccctacgatcaggaacaaagcaaggatgttcactctc24540
attgctcttatttaacatagccctgaagttctaacttgtgcaaaacgataagaaagggaa24600
atgaaagacctgcagattggcaaagaagaaataaaactgttcctgtttgcagatgacatg24660
attgtctcatagaaaatgtaaagcaactaggggtaggggggcagtggagacacgctggtc24720
aaaggataccaaatttcagttaggaggagtaagttcaagatacctattgcacaacatggt24780
aactatacttaatatattgtattcttgaaaatactaaaagagtgggtgttaagcgttctc24840
accacaaaaatgataactatgtgaagtaatgcatacgttaattagcacaacgtatattac24900
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-18-
tccaaaacat catgttgtac atgataaata cacacaattt tatctgtcag tttaaaaaca 24960
catgattttg gccaggcaca gtggctcata cctgtaatcc cagcatttta ggaggctgag 25020
gcgagcagaa aacttgaggt cgggagtttg agaccagaat ggtcaacata gtgaaatccc 25080
gtctccacta ataatacaaa aattagcagg atgtggtggc gtgcacctgt agacccagct 25140
acttgggagg ctgaggcacg agaattgctt gaacaaggga ggcagaggtt gcagtgagct 25200
gggtgccact gcattccagc ctggtgacag agtgagactc catctcaaaa aaaataaaat 25260
aaagcatgac ttttcttaaa tgcaaagcag ccaagcgcag tggctcatgc ctgtaatccc 25320
accactttgg gaggccgagg caggcagatc acaaggtcag gagtttgaga ccagcctgac 25380
caacatggtg aaaccccatc tctactaaaa aatatataaa ttagccaggc atgtgtagtc 25440
tcagctactc aggaggctga ggcaggagaa tcacttgaac ccggaggcag aggttgcagt 25500
gttgagccac cgcactccag cctgggtgag agaacgagac tccgtctcaa aaaaaaaaag 25560
caaaataacc taattttaaa aacactaaaa ctactaagtg aattcagtaa gtctttagga 25620
ttcaggatat atgatgaaca tacaaaaatc aattgagctg gacaaaggag gattgtttta 25680
ggtcagtagt ttgaggctgt aatgcacaat gattgtgcct gtgaatagct gctgtgctcc 25740
agcctgagca gcataatgag accacatctc tatttaaaaa aaaaaaaatt gtatctctat 25800
gtactagcaa taagcacatg ggtactaaaa ttaaaaacat aataaatac't gtttttaatt 25860
gcctgaaaaa aatgaaatac ttacatataa atctaacaaa atgtgcagga cttgtgtgct 25920
gaaaactaca aaacgctgat aaaagaaatc aaagaagact taaatagcgt gaaatatacc 25980
atgcttatag gttggaaaac ttaatatagt aaagatgcca attttatcca aattattaca 26040
caggataaca ttattactac caaaatccca gaaaaatttt acatagatat agacaagatc 26100
atacaaaaat gtatacggaa atatgcaaag gaactagagt agctaaaaca aatttgaaaa 26160
agaaaaataa agtgggaaga atcagtctat ccagtttcaa gacttacata gctacagtaa 26220
tcaagactgt gatattgaca gagggacagc tatagatcaa tgcaaccaaa tagagaacta 26280
agaaagaagc acacacaaat atgcccaaat gatttctgac aaaggtgtta aaacacttca 26340
acgggggaag atatgtctct cattaaaggg tgtagagtca ttgcacatct ataggcaaaa 26400
agatgaacct gaacctcaca ccctacagaa aaattaactc aaaatgactc aaggactaaa 26460
cataagatat acatctataa aacatttaga aaaaggccac gcacggtggc tcacgctcgt 26520
aatcccagca ctttgggagg ccaaggcagg tggatcacct aaggtcagga gtttgagacc 26580
agccggatca acatggagaa gccccatctc tactaaaaat acaaaattag ctggacgtgg 26640
tggcacatgc ctgtaatccc agctacttgg gaggctgagg catgagaatc gcttgaaccc 26700
ggggggcaga ggttgcggtg agccaagatc acaccattgc actccagcct gggcaacaag 26760
agcaaaactc caactcaaaa aaaaaaaaaa aaaggaaaaa tagaaaatct ttgggatgta 26820
aggcgaggta aagaattctt acacttgatg ccaaactaag atctataagg ccagtcgtgg 26880
tggctcatgc ctgtaattcc agcactttgg tcaactagat gaaaggtata tgggaattca 26940
ctgtattatt ctttcaactt ttctgtaggt ttgacatttt tttagtaaaa aattggggga 27000
aagacctgac gCagtggCtC aCaCCtgtaa tcccagcact ttgggaggcc ggggcaggtg 27060
gatcacacgg tcaggagttc gagaccagcc tggccaacat ggtgaaaccc cgtctctacc 27120
aaaaatataa aaaattagcc gggtgtcatg gtgcatgcct gtaatcccag ctactgagga 27180
ggctgaggca ggagaatcac ttgaacctgg gaggtggaag ttgcagtgag ccgagattgt 27240
gccactgcac tccagccttg ggtgacagag cgagactccg tctcaaaaga aaaaaaaaaa 27300
aaagaatatc aaacgcttac tttagaaact atttaaagga gccagaattt aattgtatta 27360
gtatttagag caatttttat gctccatggc attgttaaat agagcaacca gctaacaatt 27420
agtggagttc aacagctgtt aaatttgcta actgtttagg aagagagccc tatcaatatc 27480
actgtcattt gaggctgaca ataagcacac ccaaagctgt aectccttga ggagcaacat 27540
aaggggttta accctgttag ggtgttaatg gtttggatat ggtttgtttg gccccaccga 27600
gtctcatgtt gaaatttgtt ccccagtact ggaggtgggg ccttattgga aggtgtctga 27660
gtcatggggg tggcatatcc ctcctgaatg gtttggtgcc attcttgcag gaatgagtga 27720
gttcttactc ttagttCCCa CaaCaaCtgg ttattaaaaa CagCCtggCa CtttCCCCCa 27780
tCtCtCgCtt CCtCtCtcaC catgtgatct CaCtggttCC CCttCCCttt atgcaatgag 27840
tggaagcagc ctgaagccct cgccagaagc agatagtgat gccatgcttc ttgtacagcc 27900
tacaaaacca tgagcccaat aaaccttttt tctttataaa ttatccagcc tcaggtattc 27960
ctttatagca agacaaatga accaagacag ggggaaatca acttcattaa aataatctat 28020
gcagtcacta aacaaataag aacaagaggc tccagaagtg ggaagccaat acccagagtt 28080
cctacaatac agtatctgaa aagtccagtt tccaaccaaa aaatatatat atacaggccg 28140
gacatggtag cttatgtctg taatcccagc actttgggat gctgaggcgg gcagatcacc 28200
ctaggtcagg agttcgagac cagcctggcc aatatggcaa aaccccgtct ctactaaaaa 28260
tacaaaaatt agccaggcat ggtggtggat gcctgtaatc ccagctactc gggaggctga 28320
ggcagggaat cacttgaacc caggaggcag aggttgcagt gagccgagat cacgccactg 28380
aactccagcc tgggcaacaa agtgagactc cacctcaaaa aaaaaaaaaa tatacatata 28440
tatatgtgtg tgtgtgtgtg tgcgcgcgtg tgtgtatata cacatacaca tatatacata 28500
tatacagaca cacatatata tatgaagcat gaaaagaaac aaggaagtat gaaccatact 28560
ttctgtggtt atgataggat ggggtatcac gggggaagta gacaagggaa actgcaagtg 28620
agagcaaaca gttatcagat ttaacagaaa aagactttgg agtaaccatt ataaatatgt 28680
ccacagaatt aaagaaaagc gtgattaaaa aaggaaagga aagtatcata acaatattac 28740
tccaaataga gaatatcaat aaaggcatag aaattataaa atataataca atggaaattc 28800
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-19-
cggagttgaa aggtagaata actaaaattt aaaattcact agagaaggtt caacactata 28860
tttgaactgg cagaagaaaa atttagtgag acaaatatac ttcaatagac attattcaaa 28920
tgaaaaataa aaagaaaaaa gaatgaagaa aaataaacag aatctcagca aaatgtggca 28980
caccattaat cacattaaca tatgcatact gagagtaccg gaagcagatg agaaagagga 29040
agaaaaaata ttcaaatgat ggccagtaac ttcctagatt tttgttttaa agcaataacc 29100
tatacaatca agaaactcaa tgaattccaa gtaggataaa tacaaaaaga accacaaaca 29160
gatacaccat ggtaaaaatg ctgtaagtca aaaacagaga aaatattgaa agcagctaga 29220
ggaaaactta taagagaacc tcacttacaa aagaacatca cttataaaag aaccacaata 29280
atagaaacag ttgacctctc atcagaaaca atgaatgata acatatttga agtgctcaaa 29340
gaaaaaaaat aaagattcct atatacgaca aagctgtctt tcaaaaatat acatccaaaa 29400
ggattgaaac cagggtcttg aagagttatt tgtacatcca tgttcatagc agcattattc 29460
acaatagcca aaaggtagaa gcaacccaag ggtccatcga caaataaata aaatgtggta 29520
tatgtataca caatggaatt tattcagtat taaaaaggaa tgaaattctg acacatgcta 29580
caacatggct aaaccttgag aacactatgc taagtgaaat aagccagcca caaaaggaca 29640
aataccatat tacttcactt gtatgaaata cctagggtag tcaaattcag agatagaaag 29700
taaaacagtg gttgccaagg gctgagggag ggagtaacgt ggagttattg ttgaatgggt 29760
acagaatttc agttttgcaa gataaaaaga gttctggaga cagatggtgg tgagggtggt 29820
acaacaatac aaatatactt tatactactg aacagtatac ttaaaaatga ttaacatggt 29880
gaaaccccgt ctctactaaa aatacaaaaa aattagctgg gtgtggtggc gggcacctgt 29940
aatcccagct acttgggagg ctgaggcagc agaattgctt gaaaccagaa ggcggaggtt 30000
gcagtgagct gagattgcgc caccgcactc tagcctgggc aataagagca aaactccgtc 30060
tcaaaaaata aaaaataaaa aaaatttaaa aatgattaag caggaggcca ggcacggtgg 30120
ctcacaccta taatgccagc actttgggag gccgaggcag gcgatcactt gagaccagga 30180
gtttgagacc agcctggcca acatggcaaa accctgtctc tgctaaaaat acaaaaatta 30240
gccaggcatg gtggcatata cttataatcc cagctactgg tgagactgag acacgagaat 30300
tgcttgaacc caggaggcag agattgcagt gagtcgagat cgcgccactg aattccagcc 30360
tgggcgacag agcaagattc tgtctcgaaa aaacaaaaac aaaaacaaaa agcaaaacca 30420
aaaaataatt aagcaggaaa cgagattgct gctgaggagg agaaagatgt gcaggaccaa 30480
ggctcatgag agcacaaaac ttttcaaaaa atgtttaatg attaaaatgg taaattttat 30540
atgtatctta ccacaaaaaa aagggctggg gggcaggaaa tgaaggtgaa ataaagacat 30600
cccagagaaa caaaagtaga gaatttgttg ccttagaaga aacaccacag gaagttcttc 30660
aggctgaaaa caagtgaccc cagagggtaa tctgaattct cacagaaaat tgaagcatag 30720
cagtaaaggt tattctgtaa ctatgacact aacaatgcat attttttcct ttcttctctg 30780
aaatgattta aaaagcaatt gcataaaata ttatatataa agcctattgt tgaacctata 30840
acatatatag aaatatactt gtaatatatt tgcaaataac tgcacaaaag agagttggaa 30900
caaagctgtt actaggctaa agaaattact acagatagta aagtaatata acagggaact 30960
taaaaataaa attttaaaaa atttaaaaat aataattaca acaataatat ggttgggttt 31020
gtaatattaa tagacataat acaaaa_atac cacaaaaagg gaagaagaca atagaactac 31080
ataggaataa cattttggta tctaactaga attaaattat aaatatgaag tatattctgg 31140
taagttaaga cacacatgtt aaaccctaga tactaaaaag taactcacat aaatacagta 31200
aaaaaataaa taaaataatt aaaatgtttg tattagtttc ctcagggtac agtaacaaac 31260
taccacaaat tgagtggctt aacacaactt aaatgtattt tctcccagtt ctggaggcta 31320
aacacctgca atcaaggtga gtacagggcc atgctccctg tgaaggctct aggaaagaat 31380
CCtCCCttgt CtCttCCagC ttCCagtggt tctcagtaac CCtaagtgCt CCttggCttg 31440
tagctatatc attcctagca accagaaaga agaaaataat aaagattatg gcaaaaaata 31500
atgaaatcaa aaggagaaaa atggaaaaaa ataaataaaa ccaaaagcta gttctttgaa 31560
aagatcaacc aagttaacaa accttttaac tagactgaca aaaaggaggt aagactcaaa 31620
ttactagaat cagaaataaa agaggggaca ttactaatga gggattagaa aagaatacta 31680
cgaacaaatg tgtgccaaca aattagaaaa cttagatgaa atggacaggt tcctaggaca 31740
acatcaacta ccaaaattta ctcaagaaga aagagacaat ttgaatgagc tataacaagg 31800
gaagagactg aattgacaac caagaaacta tccacaaaga aaatcccagg cccagaagat 31860
ttcactgtga aattctttca aacttataaa tataaattaa catcagttct tcacaaactc 31920
ctccaaaaaa aagaacagat ctctatttac aggcgatacg atctttagaa aatcctaagg 31980
gaactactaa gacactatga taactgataa acaagttcag caaggctgca ggatagaaaa 32040
ccaatataca aaaatctatt atatttctat acacttgcag tgaacaaccc aaaaatgaga 32100
ttaagaaaat aattcaattt acaataacat caaaaagaat aaaaacactc aaaaataaat 32160
ttattcaagt aagtgcaaaa cttatactct agaagctaca aaacactgtt aaaagaaatt 32220
aaaggtttac ataaatgaaa aactatccca tgttcatgga tcaaaagact tattactggc 32280
aatgctctcc aaattgatct ataaattcaa caaaatcctt atcaaaatcc cagatgaggc 32340
tgggggtggc ggttcatgcc tgtaatccca gcactttggg aggctgaggc acgcagatta 32400
cctgaggtcg ggagctcgag atcagcctga ccaacatgga gaaaccctat ctcttctaaa 32460
aatacaaaat tagtcaggcg tggtggcaca tgcctataat cccagctact cgggaagctg 32520
aggcaggaga atcgcttgaa cccaggaggc agaggttgca gtgagccaag atcgtgccat 32580
tgcactccag cctgggcaac aagagcaaaa ttccatctca aaaaaaaaaa aaaaaaaatc 32640
ccagatgact tcactgttga aattgaaaag attattctaa aattcacatg gaattgcaag 32700
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-20-
accttgagaatagccaaaacaaacttgaaaaacacgaacaaaatataggatgactcactt32760
gccaattgcaaatgttacgacacagcaacagtaatcaagactgtgtggtactggcaaaag32820
acacatacatacatacatatcaatggaatataattgagagtacagaaacaagcctaaaca32880
tctatggtaagtgcttttctatttttttctttttttttttcttttttgtagagatagaat32940
ctcaccatgttgcccaggctggtcttcaacttctgggctcaagcaatcctcccactgtgg33000
cctcccaaagtgctgggataactggcatgagccaccacatccagcccagatgattttcaa33060
aaaagtcaacaagaccattcttttcaacaaataggtctgggatgatcagatagtcacatg33120
aaaaaaaaaatgaagttggaccctccatcacactaaagtgctgcgattataggcatcagc33180
caccacatccagcccaaatgattttcaaaaaggtcaacaagaccattcttttcaacaaat33240
aggtctgggataatcagatagtcacatgaaaaaaaaaatgaagttggaccctccatcaca33300
ccatatgcaaaaattaattcaaaaatgaattgatgacttaaacgtaagagttacgactgt33360
aaaactcttagaaggaaacatacgggtaaatcttaaagacgttaggtttgacaaagaatt33420
cttagacatgacaccaaaagcatgaccaactaaggtaaaatagggtaaattgtacctacc33480
aaaatgaaaaacctttgtgctggaaaggacaccatcaagaaatggaaagccaaaatagcc33540
aaggcaatattaagcaaaaagaacaaagctggaggcatcatactacctgacttcaaagca33600
acagtaaccaaaacagcatggtactagtagaaaaacagacacatagaccaatggaacaga33660
ataaagaacccaaaaataaatccacatatttatagtcaactgatttttgacaatgacacc33720
ccttcaataaatgatactaggaaaactggatatcgatatgcagaagaataaaactagacc33780
cctatctctcaccatatagaaaaatcaactcagactgaattaaagacttgaatgtaagac33840
ccaaaactataaaactactggtagaaaacataaggaaaaacgcttcaggacattggtcca33900
ggcaaagatcttatggctaaaacctcaaaaacacaggcaacaaaaacaaaaatggaaaaa33960
tagcactttattaaactaaaaagctcctgcacagcaaaggaaacaacagaatgaaaagac34020
aacctgtagaatgggagaaaatatttgcaaactatccatccatcaagggactagtatcca34080
gaacacacaagtgactaaaaCaactcaacagcaaaaaagcaaataatctggtttttatat34140
gggcaaaagatctgaataaacattctcaaaggaagacatacaaatgtcactatcattctg34200
ccagtaccacactgtcttgattacttgttagtgtataaatttttaaattgggaagtgtga34260
gtcatcctacactttgttcttgtttttcaagtttgttttggctattctgggagccttgca34320
agtataaaatagccaacaagtatgaaaaaatgctcaccatcactaatcatcagagaaata34380
aaaatcaagaccactatgagatatcctctcactccagttagaatggctactatcaaaaag34440
acaaaatataatggatgctggcaaagatttggagaaaggggaactcctatacactgtggg34500
tagggatgcaaattggtaatggccattatggaaaataatactgaggtttttcaaaaaact34560
gaaaatagaactaccatatgatccagcaaccctactactgggtatttatccaaaggaaag34620
aagtcagtatactgaagaaatatatgcactctcatgttaattgcaacactgttcacaaca34680
gccaagacagggaataaatctaaatgtgcatcaacagatgaatggataaagaaaatgtgg34740
catatacactcaatagaatactattcagccattaaagaagaatgaaatcctgtcatccca34800
gcaacatggatgaacctggaggacattatatttaatgaaataagtaaagcacaaaaagat34860
aaacagtacatgttctcactcagacatgggtgctaaaaagaaaatggggtcacagaatta34920
gaaggggaggcttgggaaaagttaatggataaaaatttacagctatgtaagaagaataag34980
ttttagtgttctatagaactgtagggcgagtatagttaccaataacttattgtacatgtt35040
caaaaagctagaagagattttggatgttcccagcacaaaggaatgataaatgtttgtgat35100
gatggatatcctaattaccctgattcaatcattacacattgcatacatgtatcaaattat35160
cactctgtacctcataaatatgtataattattacgtcaacaaaaaaaggaaaaaaaagaa35220
aattaagaeaacccacataatggaagaaataaaatatctgcaaattatatatatctgata35280
aatatttaatatttataatatataaagaactcctacaactcaagaacaacaacaaaacaa35340
cccaattcaaaaatgggtaaaagccttgaatatacacttatctaaagactatatacaatt35400
ggccaataaagacacgaaaagatgctcaacatcactagtcatcagggaaatataaatcaa35460
aaccacaatgtagaatgtagacaccacttcatatgcactaggatggctagaataaaaagg35520
taataacaaatgttggtaaggatgtgaaaaaatcagaaacctcattcgctgctgttggga35580
atgtaaagtgatgcagccactttggaaaacagtctggcagctcctcaaattattaaatac35640
agagttaccgtatgacccaggaatattcctcctgggtctataaccaaaaaaatgaaaaca35700
tatatccacataaaaacttgtacatgggcatttatagcaacattattcataacagcaaag35760
gtggtaagaacccatatgcccatcatctgatgaacaggtaaataacatgcggtattatcc35820
atacactagaatattatctgcccatacaaggagtgacatccagctacatgctacaaggat35880
gaatctcggaaaccttatgctaagtgaaagaagccagtcacaaatgaccacagattatga35940
ttccatgcatcggaaatgaccagaatagggaaatctatagagacagaaagtagattagtg36000
gttgggtggggctgggaggacaggtagtacactactttcccagaactactggaacaaagt36060
accacaaactggggagcttaaacatagaaattgatttcctcacagttctggagactagga36120
ctctgagatcaaggtgtcagcagagctggttctttctgagggccctgaggcaaggctctg36180
tcccaggcctctctccttggctggcaggtggccatcttctCCCtgCgtCttCaCatCatC36240
ttttctctgtgtgtgcccatgtccaaattttgattggctcattctgggtcatggccaatt36300
gctatgcacaaagtgaagtctacttccaaaagaagggaagagggaacactgactaggcta36360
aacttatagtcattttaatgtccgcttttcctatgagattgtgaacacacagaagtaggg36420
tttttatctacattgtgcaaagtttaataagaaaaatagaattcaagagaagcagttcaa36480
tagcaggaatttaatatgggaactaattacaaggtttagggcaggactaaaaagccagtt36540
gggatggtgagccaacccagagattagcaacagtgggaccccatctacctaccacccatg36600
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
_21_
aagctggaag gataaaggag gggctattat cagagtccac aagccagtgt cagagtcctt 36660
ggctggagct gggaccaccc tagagacact gtgcaaagca gaaaacaagg gggaaaaacc 36720
ctgacttctc ccttcctccc acctttcaat ctcccactag tgcttcctac tagccatact 36780
tggccagaga cagtgacaag gaacactgca aaatgaagtt tgtaggaatc atctccctct 36840
gagacagaga aatatggaag ggtagaaaat gaatcagagg ataaagagaa aaaaccctga 36900
gtactatctt atttatcttt gtatctccag tgcctaatct gtctctcaaa aaaggaaagc 36960
aattgagaga aactgaaaac tccaattgaa atgaaagaat ggagaattac tggactagaa 37020
gagaagagaa aaatttattc cgcatagagt aaacaagaat ggattcacaa aggacgtgat 37080
gaatgaaaag ctataatcag caaagatttg ccagagaaat taaaaagtgg taaactcagc 37140
cacgctgtac aacctgaagg cacaatgcat gaaaacgttt caagaaatga caagatttga 37200
agtcaaattc taagtgcttt tccagaatct ctcaagacga ttatatagct accccatttt 37260
attaaataaa atggaaactt actaaacttt ccccttgtat taaactaaca tatgtcctaa 37320
tagcaaacga ttctggaatt cctagagtaa aatatatttc gtcaaagtgt attgctcttt 37380
taatattctg ctgacctcct tttgctattt aggatatttg tatacacatc acacgtaaat 37440
ttggtctata gtttacatct acgggcttat actgttcttt ttttcatttt tttaaaattt 37500
ccaaccccca gtatccatat actgctctct atcagggtta ttttaacttt gtaaaatcag 37560
ctgagatgct ttccatgttt ttttttttta ttttctgcca catttgaata gcataggagt 37620
taccaccatc aaccttggat tatttaagca ttcacgattc cacgtgtgga ttttttattc 37680
agagtctttc ttgtcattcc tgctatcagc acagaaccca atctcagctt tccagctata 37740
ctctcacccc atggaatttg cagatgaagt tcaaaaggac ctttgcatta tcctgcctcg 37800
ccctcttccc ccttcattta gacatcacct tcttctagaa cgtcttacct gacatgccct 37860
gctcccaacc cctgctgccc aattgtgtgc tctcccgtgt cctggcctgc catcctcttt 37920
agtaattgcc tgctccctca tctgtctccc cacccagaca ttaagctgaa tagactggat 37980
ttgtgtcttg tccatcacta taatctcagc acctagtacc tagtaggtac ttaccatgta 38040
ttcattagca aaatgttatg tataaccttg caccttaaaa acaagagaag gaagacaaaa 38100
ttaagtctta agactatggt ttagaacatg gatcagaaac tacagtctgc agcccaaatc 38160
cagaccaaat gaagagacca tgttcattta catacaacct atagcagctt tcacactaca 38220
ggagcagagc taagtagttc caagggaaca cacggccctg caaagcctaa aatatttact 38280
ctatagctct tcacagaaaa agttttcaga tccctcgttt agaactcttg ttcatatgca 38340
atttcactaa accatagttt tttgggtttg tttggttttt tttggcaaaa aggaatgagc 38400
cgatccagaa aaggttgaaa agaatgaatc attactgctg aaagaatgtg cacacagtcc 38460
gtcagtattc tgctgccatg CtgaCaCCCa tccaatagtg tcatgagatg cagcagctac 38520
tactgtgttc tcaatgccga gtccacccac tccataacca tgtccaagca atcttgggaa 38580
catcatcacc atgcttgttt atccttaagg tattgcctca catacagcag tggctggtca 38640
taaagtcaaa tgacactagt ggccaggagg tcaagagaat gagtgaggac aggtgggtag 38700
gCagCCCagg ccctagcaac agcaggagct cacccctcag tcactctagc caggactgaa 38760
atacttttca ccctttcaag agagactagg aatctggatt tttatgtgaa atatcttgat 38820
tactaaatgt tgtcaacaga catgtcaaaa ggtaaaacta agtaagttca tggggcagat 38880
tgactattca ggttatagaa ttaaggattc ttatccaaca cagataccaa ccaaaaagct 38940
gacgtataac atattaggag aaactatgtg cactgtcgaa acatcaacaa ggggctaatg 39000
tctaaaatag tctatattgg attccagttg aaacatgggg aaaggacatg aacaggcaac 39060
ttatgtcaat ggaaactcaa aaagataaca agcatatata aaagcattct caaattcagt 39120
agtaaacaga cagatgcaaa taaaaagagg gaaactgctg ccgggcacag tggctcacac 39180
ctgtaatccc agcactttgg gaggccgagg cgggcggatc atgaagtcag gagatcgaga 39240
ccatcctggc taacatggtg aaaccccgtc tctactgaaa acacaaaaaa ttagccaggc 39300
gtagtggtgg gcaccagtag tcccagctac tcaggaggtt gaggcaggag aatggcatga 39360
acccaggagg cggagattgc agtgagccga gaccatgcca ctgcactcca gcctgggcga 39420
ctgagtgaaa ctccatctca aaaaatataa taataattat aattataata ataataaata 39480
gtaaataaat aaaaagagag agactgctaa agtctagaaa gttgaatgat gccaagcgca 39540
tgcaaagatc agggccttgg gatggccggg tgcagtggct cacgcctgta atcccaccac 39600
tttgggaggc caaggcgggc ggatcatgag gtcaagagat caagaccatc ctggccgaca 39660
cagtgaaacc cggtctctac taaaagtaca aaaaaatata tatatatata tatattatta 39720
tattatatat atatatatca gagccttggg aatccttgtg tgctgctggg gaaggtagtg 39780
gtgcagccac ccttgacagc aatctggcag tacttggtta tattaagtat aggcacacac 39840
cacgaccagg cagtcctact cctgggtcta aatcccaaag aattctcaca caagtccata 39900
aggagacatg tacgaggctc attcagcatt actgggagtg ggaatcaacc tgggtgtcca 39960
tctacaggag acgagatgga caaaatgtgg tggatattaa gaccagaatc accaagtaac 40020
agagatgggt ggtgagtgac aatcctaaga tacagaataa aggctagaac atgatgccat 40080
tcatgtaaat taaaaataga tgcacacaaa gcagtatacg cgtgaccctt gaatagcaca 40140
ggtttgaact gcctgtgtcc acttacatgt ggattttctt ccacttctgc tacccccaag 40200
acagcaagac caacccctct tcttcctcct ccccctcagc ctactcaaca tgaagatgac 40260
aaggatgaag acttttatga taatccaatt ccaaggaact aatgaaaagt atattttctc 40320
ttccttatga ttttctttat ctctagctta cattattcta agaatatggt acataataca 40380
catcacacgc aaaataaatg ttaattgact gtttatatta tgggtaaggc ttccactcaa 40440
cagtaggctg tcagtagtta agttttggga gtcaaaagtt atacacagat tttcaactgt 40500
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-22-
gcaggcaatc agttcccctg accccctcat tgttcacggg tcaactgtat atacacaaaa 40560
gtattatatg aacctcatta gaatagctgt ctatagggag aagagaatga gagtgggata 40620
aaacggaatg aacaaataaa ccaacaaatg cattaacaag caaaacaaca gaggggcttg 40680
catgggccag tgatgataaa gggctaagaa tgagaatata attaattcaa ttcctcacac 40740
ctgaggtcta aaaccaagga aagggagggc caggcgtgga ggctcacgcc tgtaatccca 40800
gcactttggg aggctgaggc gggcggatca caagattagg agtttgagat cagcctggcc 40860
aacacagtga aagcccatct ctacaaaaaa tacaagaatt acccaggtgt ggtggcacat 40920
gcctgtagtt agctactctg gaggctgagg caggagaatc acttgaaccc aggaggcgga 40980
ggttgcaggg agccgagatc acaccattgc actccagcct gggtgacaga gtaagactct 41040
gtctcaaaaa aataaaaaaa ataaaaaaac agagaaaggg aggaaactag atccaggctg 41100
actagataca gcctttagag ttagaaaaga tgatttgaca atctaagccc acactcagat 41160
tgaatgaaat tgaaaagcct ttcaaactaa aacatttaat tacaccatct gctgcagaca 41220
gaactcagac aactcaaaca ggtaatgtca gcgtggtgtt ttatatcacc accctcaaca 41280
cagaataaaa atcagctgca tgtgaagcag tgactagaat gaagaaaagg ctgcttctta 41340
cttccttcta gtggttcttt ccgaaaacat taataggcac cagctctatg catgtcaccc 41400
tgcagggaga catggggtat ataactatga cttactgttc attcctcaag gaattcccaa 41460
tcttgtggaa gattatacac aatgaggcaa caaaaactat ccaataaaac cacggaaaag 41520
aagccagtga caaagaagcc agtgatgaaa ggccctgtga gcagagctga tggccatttg 41580
gggaagaaag accaacatgg atgggggtga tcagggtggc tccgtgggaa agctggaaga 41640
gaagtggcag atctctgagc tggatgatgg gccactacca tctgtatatg gctaattaaa 41700
gaccatgtgt ggatttttta ttcagctctt tcgtgtcatt cctgctatca gcacagaacc 41760
caatctcaac tttccagcta tattgagcta aacttctcac ctcatggaat ttgcagataa 41820
agttcaaaag gatccttgcc ttttcaaaat aattttgaat ggttgagtag tccctctgtg 41880
ctctctcact gacaccctct caaggctgct gagcacgtgc catgctatgg ctttctccaa 41940
catcaggaaa tgttctccac tcagtttcac cttaatacaa atgtgttctc tcttcagaga 42000
aggcaaaaaa attcatgacc atctgactgg gagaagtcat ttctaggtaa agtgtccatc 42060
tttttctgag gaacacagga ggaaaatctt acagaaaaga gttaacacag caggcctaag 42120
actgcttttt aaaataaata aataaataaa taaataaata aataaataaa taaataaata 42180
aataaatgaa tgatagggtc ttctgtattg gccaggctag tctcaaattc ctggcttcaa 42240
gagatcctcc caecttggtc tcccacagtg ttgggattat agacatgagc cattgtgctt 42300
ggcccaagac tgttattctt aaaaagtetc ataaaaagca tggttaatcc ttggctggca 42360
cctgggaact tagatttcag aagggttccc accatccaaC ctggaaagag ggactcactg 42420
tgcctaaatt attgtgtggt ttatgctgaa ctcctgcttt tcttcaggta gcgtggaatg 42480
tggtatgtgc tgggcaaagg gggcctgcat gaccagcccc caataaaaac cctgggtgtt 42540
gggtctctag tgagtttccc tggtagacag CatttCaCat gCgttgtCa.C agCt CCttCC 42600
tcggggagtt aagcacatac atcctgtgtg actgcactgg gagaggatgc ttggaagctt 42660
gtgcctggct tcctttggac ttggccccat gcacctttcc ctttgctgat tgtgctttgt 42720
atcctttcac tgtaataaat tacagccgtg agtacaccac atgctgagtc ttccaagtga 42780
accaccagat Ctgagcatgg tcctgggggc ccccaacaca gaaataaatt ataaaagacc 42840
aaggactggg catggtggcc catgccggta atctcagcgc tttgggaggc cgaggcagga 42900
ggaccagtta agcccaaaag ttcaaagtta cagtgaccta tgactgcgcc aatgcactct 42960
aacctgggag acagagcaag accctgtccc caaaacaata aactaaacac atacttctgc 43020
cttccaagtg tcttaaaatt caatggaatg gtagaaacat ttttaaaaca ctaaatcaaa 43080
agaaacctgg aaaacaagag tgccgatggc caactaaaat gtctaggaaa tttctgaaaa 43140
gtaaaaagta ctcagaacca gattacctga gcaaaccata gcccaataca agcttgggag 43200
gaggctgtta tgcagaagga aatggtaaca ggtttccagg aacagacttg taacagcaga 43260
tagaacagca gaggtagaac ctgacaaggt gattacctgg ggaactgcag tctgaatgac 43320
caggactgtt ggacccttcc cctcacatgg aatacacacg ccactcagca gcacaccaca 43380
gctcttcaac aatcacagga ggcacgctac gcctagtaag acaggaaaaa aggaattctc 43440
aaacttcgaa gatgaacaca taaagaatca ccaagttttt attcagtatg atgaaacagg 43500
gacactgaat caacagaaca caaacccaag caaagataat tactagagca catagaagaa 43560
attattagat attcttggga agacctaagg ggacattata aagagcaagc agttggtatg 43620
tgacgatctt tgtgatatac caagaaataa aaacacagga tgaagaccag atagagaata 43680
atgctactat ttgtgcaaaa aaggagaaat ggagaatctg attcatattt gcttgtattt 43740
gcatgaagaa actttggaag gtacataagt aactaacaac aatggttacc tacttgtaag 43800
gcgagagaag taagaggaca ggaatggtgg gaacaccttt tgtgtccgga attggtgggt 43860
tcttggtctg acttggagaa tgaagccgtg gaccctcgcg gtgagcgtaa cagttcttaa 43920
aggcggtgtg tctggagttt gttccttctg atgtttggat gtgttcggag tttcttcctt 43980
ctggtgggtt cgtagtctcg ctgactcagg agtgaagctg cagaccttcg cggcgagtgt 44040
tacagctctt aagggggcgc atctagagtt gttcgttcct cctggtgagt tcgtggtctc 44100
gctagcttca ggagtgaagc tgcagacctt cgaggtgtgt gttgcagctc atatagacag 44160
tgcagaccca aagagtgagc agtaataaga acgcattcca aacatcaaaa ggacaaacct 44220
tcagcagcgc ggaatgcgac cgcagcacgt taccactctt ggctcgggca gcctgctttt 44280
attctcttat ctggccacac ccatatcctg ctgattggtc cattttacag agagccgact 44340
gctccatttt acagagaacc gattggtcca tttttcagag agctgattgg tccattttga 44400
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-23-
cagagtgctgattggtgcgtttacaatccctgagctagacacagggtgctgactggtgta44460
tttacaatcccttagctagacataaaggttctcaagtccccaccagactcaggagcccag44520
ctggcttcacccagtggatccggcatcagtgccacaggtggagctgcctgccagtcccgc44580
gccctgcgcccgcactcctcagccctctggtggtcgatgggactgggcgccgtggagcag44640
ggggtggtgctgtcagggaggctcgggccgcacaggagcccaggaggtgggggtggctca44700
ggcatggcgggccgcaggtcatgagcgctgccccgcagggaggcagctaaggcccagcga44760
gaaatcgggcacagcagctgctggcccaggtgctaagcccctcactgcctggggccgttg44820
gggccggctggccggccgctcccagtgcggggcccgccaagcccacgcccaccgggaact44880
cacgctggcccgcaagcaccgcgtacagccccggttcccgcccgcgcctctCCCtCCaCa44940
cctccctgcaaagctgagggagctggctccagccttggccagcccagaaaggggctccca45000
cagtgcagcggtgggctgaagggctcctcaagcgcggccagagtgggcactaaggctgag45060
gaggcaccgagagcgagcgaggactgccagcacgctgtcacctctcactttcatttatgc45120
ctttttaatacagtctggttttgaacactgattatcttacctattttttttttttttttt45180
tgagatggagtcgctctctgtcgcccagactggagtgcagtggtgccatcctggctcact45240
gcaagctccgcctcccgggttcacaccattctcctgcctcaacctcctgagtagctggga45300
ctacaggcaatcgccaccacgcccagctaattttttattttattttttttttagtagaag45360
cggagtttcaccatgttagccagatggtctcaatctcctgacctcgtgatccatccgcct45420
cggcctcccaaagtgctgggattacagacgtgagccactgcgccctgcctatcttaccta45480
tttcaaaagttaaactttaagaagtagaaacccgtggccaggcgtggtggctcacgcctg45540
taaccccagcactttgggaggccgaggcgggcggatcacgaggtcaggagatcgagatca45600
tcctggttaacacagtgaaaccccgtcgctactaaaaatacaaaaaattagccgggcgtg45660
gtggtgggcaccggcagtcctcgctactggggaggctgaggcaggagaatggcgtgaacc45720
tgggaggcagagcttgcagtgagccgagatagtgccattgCCttCCagCCtgggcgacag45780
agcgagactccacctcaaaaaaaaaaaaaaaaaatagagacccggaaagttaaaaatatg45840
ataatcaatatttaaaaacactcaagagatgggctaaagagttgacggaacaaatctaaa45900
tattagattggtgacctgcaaaaccagcccaaggaacatcccagaatgcagcccataaag45960
ataaagagagcatttccgctgggcacagtggtatggcaggggaattgcctgagtccaaga46020
gttgcaggtcacattgaaccacaccattgcactccaggcctgggcaacacagcaatactc46080
tgtctcaaaaaaaaaaaaaattaaattaaaaaagacagaatatttgagagaaaaaaatgc46140
ttatttcaagaaacatgaaagataaatcaagatattctaattcccaagtaagaataattc46200
cagaagcagaaaatagaatagaggcaaggaaacactcaaaacttctccagtgccatagaa46260
atgtgtattaatctttagaatgaaacggactaccaaatgctgagcaggaagaacaaaaga46320
gatccactcttaagccagtgtggtgcccaagcgcagtggctcatgcctgtaatcccagca46380
ctttgggaggccgaggcaggtggatcacctgaggtcaggagtttgagatcagtcaggcca46440
acatggtgaaaccctgtctgtactaaaaatacaaacattagctgggtatggtggtgcaca46500
tCtgtaatCCCaaCtaCttgggaggCtaaggCaggagaatcacttgaaaccaggaggtgg46560
aggttgtagtgagccgagatcatgccacactcccagcctgggtgacagagcaagattcca46620
tctcaaaaaaaaaatccactcctagacaaataatagttaaattttagaacaccaaggaga46680
aagaaaaaaaattgtaaagcttcagagaaaataaacattaactacaaagaaacgagagtc46740
agacgcgtgcacttcttcctagataccagcagataaagcaatatctccaaaattcagaag46800
gttttaacgtagaatcctatacccagtcaagaatattcacatggaaaagtgaaataaaaa46860
acattgtttaaacatgcaagggttcagaaagtttaccattcacagaatccctgaaaacaa46920
aaccaaataatcacttaaggactcattaagaaaacaaatgaaataaaagcaccaatgatg46980
agtaaataatcagaaaaatttacagtttacctaaataactgtttatgcataatgtatgaa47040
aacccaaaaatttaatatgggacagaattaaaatcatgataagattcttttttgctttac47100
tcatggagagttcacataaacagattatcttttaatagcaagagaaaaaaatgtttagat47160
atgtgtgaaaaactaagggtaccaaaacagtgcaaattcatttatcatcaggaaaatcca47220
aattaaaaccacagtatccaccagaataactaaaaggtaaaagacagaaattaccaagag47280
ttggcaagaatgtggagcaaccacatatacttctggggtaaataagttggtgcaaccggt47340
actgaaaactgtttgctagtatctactaaaaccgagcacatgcacagactacaaccaagc47400
agttccactcccagatacacactcaacagaaatgcacacactcactcaacaaaagacgtg47460
tactagagtgttcatgtacttactattcataatagtccaaaaatgcaaacaaccaactgc47520
caatcaaagtcaaatgtatatctatattagggatatatacaatggcatatacacagcaat47580
gagaatgaaatgaaccagctcggcacagtggttcatgcctgtaatctcagcactttgggc47640
gggtaaggcaggcagatcacttgaggtcagaaatttgagactagcctggccaacacggtt47700
aaaacctgtccccactaaaaacacaaaaattagccgggcatagtggttgcaggcctgtaa47760
ttccagctactcgggaggctgggttgggagaatcgtttgaacccgaaagccggaggtcgc47820
agtgagcggagatcgtgccactgcactccagcctggacgatagagcaagactccgtctca47880
aaaaaggaaatcaaaaatataaaataagatgacaggaataatccgcaaaagatcagtaat47940
caaaataaatataaatgggctaaagctacctattaaaagacaaagatttcacacccataa48000
ggatagctactatcaaaaaaagagagagaataacagatgttagcaaggatgtatggaaac48060
tgaaattctcacgcattgctggtgagaatataaaatggttcagcctctgcggaaaacact48120
atgctgggtcatcaaaaaattaaaaatagaagtactacttgatccaacaattctacttct48180
gggtatatacccaaataactgaaagcagggtcttgaagagatatttgtacacccatgatc48240
atggcagcattattcataatagctatgatgtggaaccaacataaatatcctttgataaat48300
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-24-
atatggataagcaaaatgtggtgtatacattcaatggaatattaattagcaataaaaatg48360
aagaaaattctgacacatgctacaacatggatgaaccttgagggcattacattaaatgaa48420
ataagccagttataaaaagacaaatactatatgaggtactatattagatactcatgcaag48480
gtacctaaaataggcaaattcatagagacaaaaagcagaatggtggttgccaggggctgc48540
ggtaatggatacagagcttcaattttgtaagatgaaaaaattctggagattggttgcata48600
acaatgtgcacacacttaacactggggaactgtaaacttaaaagtagtaaatggtaaaaa48660
taaaaataataaataataaattttatgttattttaccacaatatttattaaaagacaaag48720
attaactaattaaacaaaatccagccataagctaatggtaagagtaacaattaaagaaga48780
cacagaaaattgaaaatcagtgactagaaaaagatattccatataaatgctaacaaaaag48840
caagtacagcaatataaagagaatgaacaaaaaaaaaattaaataagatggctcgtttat48900
tcccaaaaggtacaattcaccaagaagatacaagaattgtgaacctttaagcacataaaa48960
cagcttcaaaaatacaacatttaaagaaaaatatatattaaacatagaaatagtacaaaa49020
acccctacaagaatcataatgggagtcttcaatacaactctccatatcaacaggtcaaac49080
agagaaaaaaaataagttaaggatgcagaaaacctgaattaccatcaataaacttgagat49140
taatatagaactgtatacccaatatactaagagttcagggaacagtcgtgactgacagtg49200
gactgcaaattaatctgttcttaatctttgtttttctttcagcactgtggcagaatagag49260
atcctaaaaaccttccagctacaaaacatctttttaaaaatataaaaaaatacaaaaata49320
actctgaaatcaatagaagacacatggtgaaaccaaaattctagaatacagggagaataa49380
aggcattttcagatattacaaaaacagaaaattgatcattgctgaagtaatttctaaaga49440
atgtacttgagggagaagaaaaatgttccaaagaaaagtatctgtgatacaagaaggaat49500
ggaaagtgaagaaatggtaaacaggtagataaagctaataaatgttgacctagaaaataa49560
caaaaacaatagcaataatgtctcgttggaagggttgaagtaaaaatacaattaaggcca49620
aatgtgaggtaagtggaatgaaagaattagaagtccttgccttgttcacaggactgatta49680
aataaatgagccaggttttccattcaaacagttaaaacttgaacaaaataaactcaaatt49740
aagtagaaagataaaaaacagaaattaatgtcatagaaaaataaaaaatcaatagaatta49800
atcaataaatcctggttaataaaagctggttctttgaaaggattaataaaataatcatta49860
agcaagtctgatcaaaaaaaaagagaaaaggtaccaaaaaaagtactgtatcagaaagag49920
aacatacagatacatacagatatgtaagagtctgttttcttacaccagaatactatatac49980
aacattatgctagcatatattaaatttcaataatgttaatgattttctaggaaaacagaa50040
aatattaaatttactttgaagaaacagaaaaactgagaaaaataaatgatcatgaaaaaa50100
atgaaaaggtaattaaatactgatattaactgcctaaacaacaecagcagcagcccaggc50160
agtctgcagtcaagttctgccaaacttgagggaacagataattcttctattccagagcat50220
agaaaatgatggaaagtttcccaatttaatcagagaggacagcctgatccttgttatgaa50280
cacagataaaaatggggtaaactatatgccaaactcagataccaaaaccctaaataagat50340
gctagcttattgatgtgaacaatccaaaagtgcattttaaattagcccagggttttagag50400
aaagaaaatctagcaatgtgaCCa.CCaCttatgttaacaattttaagacgaaaatctaca50460
tgatcatatcaatgcatgctacacaaaagcatttgggcaaaaaacccaacacccaccctt50520
gactttttaaactcttagtaattaggcataaacagaaatgtacttaatgtgatagaatac50580
actcggtgaagatacagagggaatgctccctaaaaccaagcccaagacaaagattcctat50640
ttaacctcaatagtcaacactgcagcgagagtaatctatggaagacaaggaaaaaagtaa50700
aaacatgagagacatctgttgtttaacagacaataagatcacctacttggaagaggcaaa50760
cgaatcaagcgaaaaactattaaaactgagacaggctttagtatggaggctcagcttcag50820
ctgtagtttgggctaccaaattcaactcgcttgcttggagagttaatcctgcaaagctaa50880
tttctgttgaggtattaggattgacaagcctgtgCtCCtCCCtCCtCCCCCatCttCaaC50940
actgaaataacacggtgtttggaactggataacagaatcttccaaaaacaaaaattgtcc51000
tgaagggctgaCttgtgCCCttaCtCaaaaaaCaCtttatCtgCtgCCtgCagCtCCtaC51060
agttgctggtggataagcctgccaaccagctcggcgtaattcttcctgcagagggcaagg51120
aagagcactttcacaggaaaatttttttccgaactgtatgccgcttattacataaactta51180
cgtgctggcaaatggagctccagcaaaataagatattcagagtcaaacttccttaggaaa51240
aaaaaaaaaaaaaagcaagcacataacactaatttccttgcatgggcactggggaaggag51300
gtCgttaCttCCgCaCgCCCgCaggtCCgCaCCa.CCgggaaaCCCaCgggcaccgcgcgc51360
tgcccccgggccttccaggtgcactgcgccgcggcgccccagctgacccgggatgcgcag51420
ccctagcccttcccctgtcaccccggccaggaaggggcgggagcgcggcggacgccgagg51480
gcgaagggcttctcggtcctctgcaccacgcagcacccccaaggcacaacagggagggtg51540
cgggaggctcccgagacccaggagccggggccgggcgtgcccgcgcacctgtcccactgc51600
ggcgagggctggggtcgcctccagggccgcagctgtcgggagccacctggctctcagtcc51660
cgggtccctgcgacaaccctcgggcccggaggggaggaggcggccacctgccgctgccac51720
ctgcggcaccggtcccaccgctccgggccgggcaggacaggccaggacgtccctcctggg51780
ctggggacaggacacgcgacgaggggaccggggcccccgcggcgaagacgcagcacgcct51840
tcccagaaaggcagtcccgtgcccccacgacggactgccggacccccgcgctcgcccgcc51900
catcccttcagaccacgcggctgaggcgcaaagagceggccggcgggcgggctggcggcg51960
cggctagtactcaccggccccgctggctcagcgccgccgcaacccccagcggccacggct52020
ccgggcgctcactgatgctcaggagagggacccgcgctccgccggcgcctccagccatcg52080
ccgccagggggcgagcgcgagccgcgcggggctcgctgggagatgtagtacccggaccgc52140
cgcctgcgccgtcctccttcagccggcggccgggggccccctctctcccagctctcagtg52200
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
_25_
tctcatctccctatctgctcatcctctggtcgcacataatcgatgtttgggcgtcccaag52260
ccagatgtggaccccatttccgcactctacactggaggttttctaagggtggtgcccgga52320
ccagcagcttcagcctcatctgggaacttgagaaaatgcagattctccgtcccacccagc52380
ctattcggtttttcctgcactaaaaccatgaaggtggggcccagcagtccacattctcgc52440
aagcccgtcaagtgattctgaggcgccctccagtttgagagctatgctcacggcctcacc52500
tccgccccgcaaggagcccggtcttgcctgtggcgctagccgcacacggacacctcatcc52560
tgcggggcccgcccccccgctgcaccctcaccgcccaacgcctcctccgggatgcagcgg52620
aggcgcctggaagtcggcaaggtcaacatccccctcagcatCttCCCtaCCCtCaCggCt52680
CCtCCtCCaggggtgCCtCatggccaggggttagaaagagccactgtgtttcttgacatg52740
gaagtggcctaagaccttaatgaaaactgcaggagtggaatgacagaacctttggtcata52800
cttgagggcgtgaagctcaaatgaggaggaaggaaaggatccagggagaataaccaaccc52860
tggcaagttgtggcgcccaggtagaggggcgagcctaggctagcggttctcgaccagggc52920
cggtgttgcccctcctcgccgccccgcgtacatttggggaggtctggagacatttttggt52980
tgtcatgatgcgggagttgctactgttgcctaagtgggtagacacgagggtgctcctcaa53040
catcctacctgaaggacaggactgccccacaaggaagaatgatccggccccaaataagaa53100
accctgggctggtcagcaacaacccctttgttctgagaagagaggaggaaagaataaaag53160
aagtggggtgaagttttggtttggtagaggaaacttgaagacattttcactggaaaggaa53220
gagaggaagaggagggagatgtctgtaaggacgagcaaaccgggtgacagctgatttcct53280
catattgaagtaatgagtcctagttataataaattcctaataaaaacccagtttatccct53340
gcaataaacttgtcttttttttttaaatatactgcttgattctgtttgctaatattttat53400
ttacaggctttgcattgatatgcaaaaatgagatgggcaataattttctttttgaatgtc53460
taatgttgtttggtttcagaatcaatgttatgctcacatcataaaaaatttggaaccgag53520
gcaggaggagtgcttgaggccagaagttcgagaccagtctaggaaacacagtgagacccc53580
cccatctctacaaaaaaaaaaaaagaaaaaaaaatgggcatgtttgctttttccttttac53640
tctgaacaatttaaggagcattaaaattatctattctttgaggtttgatcatttcccagt53700
taaaaatgttcctcccagcctgatgctttctttggggagggtaaatcttttaaggctaga53760
aaagtttcttctgtggcaattttattatttacattttaaaaattattctagagttaattt53820
tgataaagcatgtatttcttaaaacaaattatcctttttttccagatgttcaagtgtatt53880
tgcataaagttgaggaaagtagtcttttgtgaatcttttaacttctcccaaatatcttat53940
tttgtgtatttttgcttetttattttgttaacttttaaaagtgtatttttttttcaaaga54000
atcagctcttaggtttatgtttttggttatactggagcttttttcttcttctttttaaaa54060
tattttttctcctttattttttagacgtattttgatctaacgtaatcggaagaaggtaaa54120
ttagaatcttttgttactattgtgtttttatttctccttatttctctgaagtcctgcttt54180
ataaatagtaccatgttatttgtgcataaatattcatttgtcttatattcttgggaattt54240
tcccacttcatcataaaatgaccttccttgtctcatttaatgtgttcaaactttgccctg54300
aatttaaCtttgtCtgatattttaCCatCCtgctgaattttgtttgttaccccaaacaac54360
ctttgctgttttcgtcttttctgaaccctttattttaggtaatcccttgaattagagcac54420
taagttttgctttgtgattaaatctgaaaatctttatcttgccatagatgagttgagccc544.80
tattcatgtgacagctatattatgctgtttcatagcccttttggtccttttttcactctt54540
gcattgcatattttgtgtttattgtgttttgtgtttcttctgataatttggaaggtttgt54600
atttttattcagggagttgccttataatcatactccgcaatacacatcgtcctcagtttc54660
ttcagactgtctgttaactccctattctgaataaaaatgacattgtaatttccctctttt54720
ttctttaccccttttcttctcctcacctaatgtaaatgattttatccttctttagtattt54780
gcttttttaattaactacatttataaatatctttatcacttgatttttaaatcagctttg54840
aatgagatatttggattcctagatataaaagatgttaattataccatttccacgttagta54900
ggtttataaaatcatacattctgctgtgtaaccataatcccacgtttgttttagttccac54960
tcctacagttaaaagattcagaagtattattaacagttattttgccatagttttttcccc55020
aacccattttgtggtaagttatgatcctgctttagtttcttaagaataatttatagagca55080
gagtgtggtggctcacgtttgtaatcccagcactttgggagacaagaggtagaaggatcg55140
cttgaagccagcagttcaagaccaccctgagcaacatagtgagaccttgtctctacaaaa55200
aattttaaaatttagccagacgtagtggcgtgtgcctatagtCCCagCtaCt CaggaggC55260
tgaggcaagaggattgctagagcccagaagtttgaggctgcagtgacctctgattgtgcc55320
actgcaccccagtctgggcaagaaagtgagaacctatctctttaaaataacaataataac55380
ttatgaaaattatattccctgagtttttcatgtttaaaaatatttgttgcctttatcctg55440
taaaagtttgagtataaattcttgggttatactttatttattgaagaatgtataagtatt55500
gtcttctagaattgagtgttgctgtaatgaaaccagaagtcagcctggtttatttttcct55560
cagaaatgaggtaattgccggccggacaccgtggctcatgcctgtaatcccaacactttg55620
ggaggccgagacaggtggatcacgaggtcaggagattgagaccatcctggctaacatggt55680
gaaaccccggctctactaaaagtacaaaaagttagctgggcatggtggtggacgcctgta55740
atcccagctacccgggaggctgaggcaggagaatggcgtgaacctgggaggaggagcttg55800
cagagagctgagatcgcgccactgcactccagcctgggcgacagagtgagactccgtctc55860
aaaaaaacaaaaaaaaaacaaagaagtgaagtaattgccatgatgctccaagaattatct55920
ctttgtctatgaaatccagaaatctcactgttatacattttggaattattattctgggcc55980
aatatttcctgggacacaatagattgactctatagatttaattttttttttttttttgag56040
acagagtctcactgcaatctcagcttactgcaacctctgcctcacgggttcaagcaattc56100
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-26-
tcctgcctca gcctcccaag tagctgggac tacaggcgcg tggcaccatg cctggctaat 56160
ttttgtcttt ttagtagaga cagggtttca ccatgttggc caggctggtc ttgaacgcct 56220
aacctcaagt gatccacctg cctcagcctc ccaaagtgct gggattacag gcgtgagcca 56280
ccatgcccag cctcaattcc tctttctatc tggtaatttt tctgaagttg aaaacatttg 56340
ttctaatacg ttatttcagt gttcttctaa gatgtgtaaa gcaccctatt cccaggtcag 56400
cccccatctt gctagtgagc tcggctggtt cttcacaaga gctctggttt tctcctgctt 56460
aatctcaagt acctctgtca gcctccacct ggtttatgat ttggagtttt tggtttttg 56520
ttttttgttt ttgacagagt cttactctgt cacccaggct ggagagcagt ggcataatct 56580
cagctcactg caacctctgt ctcccaggtt tgagcgattc tcctgcctca gcctactgag 56640
tagctgggat tacaggcgcg tgccaccaca cccggctaat ttttgtattt ttagtagaga 56700
tggggtttca ccatgttggc cagggtggtc ttgaactcct gacctcaggt aatccacctg 56760
cctcagcctc ccaaagtgct gagattacag gcgtgagcca ccgcgcctgg catggtttgg 56820
agttttaatc tgtagtttta ataaagatag tgcttatgtt tgtgtttctt atatttcttg 56880
gtactcttgg gtaatttgta agatccccat atctacacaa gaagtccatt ttcaattctt 56940
ttcttcagac tgtttatttt attttatttt attttatttt tatgtttgag atggagtctc 57000
gctgtgtcac ttctggaggc tggagtgcag tggcgcgatc tcaggtcact gcaacctccg 57060
tctcccgggt tcaagcaatt ctcctgcctc agcctcccga gtagctggga ttacaggcac 57120
ctgccacttt ttaatttttt tagagacaga gtctcgcttt gttgaccagg ctggagtgcg 57180
gtggtgcaat catggctgac tataacctcc aaatcctggg ctcaagtgat cctcctgcct 57240
cagcctcctg agtagctggg actacaggca catgccacca tgcccagtta attttaattt 57300
ttttgtagag acagggtctc catatgttgc ccaggctggc ctcctactcc tggcctcaag 57360
taatcctcct acctcagcct cccaaattac taggattata agcatgagcc accatgccca 57420
gccttgttct actactttaa tttcatatgt taggtgacca tgtaattgat catccaaacc 57480
aggatactgt aagaatgaaa gaggctgaca gtagtatgat gctgggacta gcattgtgca 57540
ctgagattat ttctgggaaa gcaggagata cggtcaccct acttatagtg tgcttgtctt 57600
tggattgttg aatttggagt ttctatttgc aggcttattt caactgggca gccttgatcc 57660
gCCCtgCCCa gCaatgCtaC CgttCtCtCC aCCgggtCtC tgggaCCCCt tCagtCaCta 57720
taCttagCtC agttCCCCaC CCtCCCaCtC CCtaaaagCg taaCCaggaa tCCtgCCtCa 57780
ggtctactgc cgtcttccgt gggctgtttc agttcctatt acccagagtc aaactcccag 57840
cattccctac ctgattccag aCttggagtC CagagCttta aCCtCttCag gCCaaCtCCC 57900
CaCtttgCat ttCtgtCCCt atatCttagt CCatggagat aCatttcatg tCtttgagtC 57960
tacttacaaa gtaaattttg ctgtttttta attttttttt tgagatggag tcttgccctg 58020
tcacccaggc tgtggtgcaa tgacgccatc tcggctcact gcaacctccg cctcctgggt 58080
tcaagcgatt catctgcctc agcctcccaa gtagctgtga ttacagacag gcaccaccac 58140
gcccagctaa ttttttttat cttttagtag agacagggtt tcaccatgtt ggccaggctg 58200
gtCttgaatt CCtgaCCtCg tgatCtgCCC atCtCggCCt CCCaaagtgC tgagattaCa 58260
ggcgtgagcc actgtgccca gccaattttg ctttttttat atttcattgc tatatgttta 58320
gaggataagt ttacagtgct atatgcattc ecaaatatta gaccaaaaaa atctecaaaa 58380
aattagaaag aaaatccaaa aaatctcaaa aaataccaaa aagcaacaat ctcacagacc 58440
atactcactg acccccaata aaataaaatt agaaattaac Cacaacttaa caaaataaag 585OO
tactcaagtc agagaggaaa gaggaaataa acatcaaaat tacaaagtct aggcggtggc 58560
tcacgcctgt aatcccagca ctttgggagg ccaaggcggg cagatcacaa ggtcaggaat 58620
tcgagaccag cctggccaat atggtgaaac cccgtttcca ctaaaaatac aaaaattagc 58680
caggcatagt gatgtgtgcc tgtaatccag ccacttggga ggctgaggca ggagaatcac 58740
tgaacccagg gagacgaaga ttgcagtgag ccaaaatcgt gccactgcac ttcggcctgg 58800
gtgacaaagc gagactccat ctcaaaaaaa aaaaaattac aaactcttta gatagaaatt 58860
ttggtgtttt tttttgagac ggagtctcac tctgtcgcag aggctggagt gcagtgggac 58920
tatgtcagct caccgcaacc tCCatCtCCt ggattCaagC aattCtCCtg tCtCagCCtC 58980
ccaagtagct aggattaCag gCgCCCaCCa CCagaCCCag ctagttttta tatttttagt 59040
agagatggtg tttcaccatg ttggccaggc tggtctcaaa ctcctgacct caagtgatcc 59100
aCCtgCttca gCCtCCCaaa gtgCtCagat taCaggCgtg agCCaCCgCa CCCCaCCtag 59160
atagaaattt caacatgagg ccgggcacaa tggctcacgc ctgtaatctc agcacttcag 59220
gaggctgagg cgtgggagga tcacttgggc ccaggagttc aggaccagca tgggtgacag 59280
agacagaccc tgtctctatt tatttgaaaa aaaaaaaaaa aaagagagag agaaagaaat 59340
ttcaacatga aaagtatctc tcaaaccctt cgagatgttg gcaaaaagcg actcaaagga 59400
aaatgtatta ctgtgtgtga atttgcttga aaataagaaa gaggccgggt gtggtggcta 59460
acacctgtaa tcccaacact ctgggagtcc gaatcaagtg gatcatgagg tcaggagatc 59520
gagaccatcc tggctaacat ggtgaaaccc tgtctctact aaaaatacaa aaaattagct 59580
aggcgcggtg gctcatgcct gtaatcccag cactttggga ggctgaggca ggtggatcac 59640
ctgaggtcag gggtttgaga ccagcctggc ctacatggtg aaacctcgtc tcttctacaa 59700
atacaaaaat tagctgggcg tggtggtggg tgcctgtaat cccagctact cagaggctga 59760
ggcaggagaa tcgcttgaac ccgggaggcg gaggttgcgg tgagccgaga tcgcaccact 59820
acactccagc ctgggcaaca gcctgggtga cacagtgaga ctccatctca aaaaatacaa 59880
aaaattagct gggtgtggtg gcctgcgcct gtagtcccag ctacccggga ggctgaggca 59940
ggagaatgga gtgaacctgg gaggaggagc ttgcagtgag ccgagatccc accactgcac 60000
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-27-
tccagcctgggcgacagagcaagactcttgtctcaaaaaaaagaaaaaaaaaggaaaaaa60060
gaaccctgataataaagaaaccaaatgttcaactctcaaagctcggacactttaaagaaa60120
taattaataaaggcagaagttaaagggaggatgataaagcaattttttttgttggttttt60180
ttgagatggagtcttgctctgtcacccaggctggagtgcagtgatgcgatcttggctcac60240
tgcaacctctgcctcccgggttcaagcaattctcctgcctcagcctcctgagtagctggt60300
actacaggtgcgcgccacctggcccagctaatttttgtatttttattagagacggggttt60360
caccatatttgttaggctggtctcaaactcctgatctcaggtaatctgcccacctcggcc60420
tctcaaagtgctgggattacaggcaggcgccaccgcgcctggcctaaagcaaaatattgg60480
ttctgtgcaaaaggtcaataaaaagagcaaacgtttacaaactggagccagcacccattc60540
agctcagtgtgtctggagaaaaaacaatctcgcttcagaattcatgattacgcagccctt60600
tttgcttcctaaaaatcctactatgttgctgttgaccattctctctctttctctctctct60660
tgctttctctccagaaaagctattcagacattCtCCtCtttCCtCaaaCCtccaacactt60720
cctcctccatccttagcctcagctgctgacctcacttctaatcattgagaaaccaggaga60780
agcatttaagagtgaaCCtCCgCCtCCCCgcacgggcaaaaccacccacccacagaattg60840
tgccccaattctgcgtcctctcctctcaccatggatggacggtccaggctccgagccaaa60900
gccaggcctcccctggagctctggatccaccacctgcagcttctcaggcagggccccagc60960
agctcccctgCtCCCttgtaCCatCaatCCCtCCCCtCaCtgggtcactcccaacaatat61020
atatatttagtgatgtttctcccatgtggtaaaatcacttagcctctctcctcccccagc61080
tactatcctatttgtttctttccattctctgcaaaacttctcaaagcattgtgtctatgt61140
gCtgaCtCCatttatCttCtCCCgttCtCtgctgagtccttCCCaCagaCtCtCaCCCCa61200
gttactccatgaaatgacctctgcactgccacatccaatggtgaatgttcagttcttaat61260
tttattcagtctttcagcagcatttgacctggccgatcactccctcttcttaaaaatact61320
tttctcagccaggcgtgatggctcacacctgtaatcccaacactttgggaggccaaggcg61380
ggaggatcatgagagcccaggagttcaagatcagcctgggcaacatggcaagaccctatc61440
tctacaaaaactaaaaagtagccagtgtgatggcatgcacctgtagtcccatctacttag61500
gaggctgaggcagtaggatgacttgagcctgggaaatcaaggctgcagtgagccatgatt61560
gC~CCa.CtgCaCtCCagCCtgagtgaCagCgagaCCCtgtCtCaaaaagaCaaaatagga61620
aacttttctcagcatattcctctgattctcctgctgcttctgtctgcacagattcagtct61680
CCtttgCCggttCttCCtCatCCtCCtgatCtCttgaCCttgaagtgCCCCagagtaCag61740
tctttttttttttttttgagacgcagtctcgtctgtcacccaagctggagtgcaatggcg61800
aggtCtCagCtCatgCaaCCtCtgCCtCCtgggttcaagcgattCtCCtgCCtCagCCtC61860
CCaagtagCCaggaCtaCaggCaCatgCCaCCatgCCCagCaaattgttgtatttttagt61920
agagacagggttttactatattggccacgctggtctcaaactcctgaactcgtgaaccac61980
ccgcctcggcctcccaaagtgctgagattacaggcatgagccaccacacccggcccagag62040
tacagtctttagacggcctctCtaCCtataCttgCtCCCCtCataaaCtCCtCCtgCCtC62100
atggCtttaaataCCatCggtagaCtgatgaCtCCCatatttCtCttttttttttggaga62160
cggagtctcgctcagtcccccaggctggagtgcagtggcgcgatctcggctcactgcaag62220
ctccacctgccaagttcacaccattctcctacctcagcctctccagtagctgggactaca62280
ggcacccgccaccacgcctggctaatttttttgtatttttagtagagatggggtttcacc62340
atgttagccaggatggtctcgatctcctgacctcgtgatccgcccatctcggcctcccaa62400
agtgctgggattataggtgtgagccaccgtgcccagccgatgactcccatatttctatct62460
cttgctgtgtgggagttctcctcagaactccatactcataaatccaactctcataaatag62520
tatctcaaatgggcaatatgctcaaaagtcaattcctacttttctccctaaacttgcttt62580
cctgcagtctccaccatcttaatgtccaatctaacattaggaggcaaaaactttgaagtc62640
attCttgaCtCttCtCtattaCc'.LC~.CCCtatCCaatCtttCtgCagatCCagtCgaCCCC62700
CaaatCCagttagCtCtCatCatCtCCCCtgttaCCCCCtggtCCaggCCatCttCCtct62760
ctcacctgaatcactgcagcattctcctcactggtctctttggttctgttttcactccac62820
cttagcatagtctccacagagcagtcagagggatccttttaaagtgtaattcccatcctg62880
tccctgctctgctcaaaaccctgtcgtgattcccgttttaatctgtcagattaaaagcca62940
gagtCtttCCagtgaCCtaCatgatctgcctattatCa.CCtCCCICttCtttCCCCttgC63~00
tCd.CtCCa.CtCCagCtCtgCagCtgtCCtttCtgtttCCtgaacagcccagattttgctt63060
ctttagaacctttgtatttgctgtcccctctgtctggaatgtttttccaggaagtcacct63120
ggctctctcctgcacttccttcctgaccaccatgtttaaaaatcactcaaacacacttca63180
ggccggacatggtggctcacgcctgtaatcccagcactttgggaggccaaggtgggtgga63240
tcacctgaggtcaggagttcgagaccagcctggccaacatggtgaaacttcgtctctact63300
acaaatacaaatagtagccaggtgtagtggcacacacctgtaatctcagctactcaggag63360
gctgaggcaggagaatcgcttgaacccagaaggcagaggaggtgcagtgagccaagatca63420
cgccacaacaccccagcctgggtgacagagcaagaccccatctcaaaaaaaaaaaaagaa63480
aaaaaaatcacacaaacacacttctcttcatattccttttccaagttttatttttctcca63540
gaatactttacattgttttaatggaagttctCCgtttCCCCCCaaCtagaatggataCtt636OO
cctgcaggtaggcactctagtcctcccatccaagtactaaccaggctcaaccctgcttag63660
cttctgagagcaggggagatcaggcctgttcagggtggtatggcccaggaattttgattc63720
tgttttattcattgctgttctgttgattctcttttgttcctcctcctagtgctgagaaca63780
ctacttgtacataataagcattcaataaatatttgttgaatgaatgacttgttgaatgaa63840
ttaatctcagaaatgcaggactggttctacattagaaaatttttcaaggtcattctctgt63900
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-28-
tgtcgtaaca cattaagaga ggaaaatttt gtactctaaa tcatttgata aaatacatac 63960
tgatttctgt tttcaaaaac tcttagtggc tgggcgaggt ggctcacatc tataatccca 64020
gcattttggg aggacgaggt gggcggatca cttgaggtca ggagtttgag accagcctgg 64080
ccatcatggt gaaaccctat ctctactgaa aatagaaaaa ttagccgggt gtggtggcgc 64140
atgcctgtag tcccagctac ctgggaggct gaggcaggag aatggcttga acccgggagg 64200
cggaggttgc agtgagccaa gatcatgcca ttgcactcca gcctgggtaa cagagtgaga 64260
ctccatctca aaagaaaact cttagtgagt ttaggaatcc aaggaagacc ctcaaactaa 64320
atagataatc tagctaccag aagccttcag taaaccttaa cactccatgg tgaaacatta 64380
gaaacattcc tactaaaaga caggctaaga atgcctgcaa tcttcacggc tagtccaaga 64440
agtcaaaaag aagaaatgag cgctgattta aaaaaataaa caaacaaaaa actaccgatg 64500
cagaggctgg cagcaaggac tgaaggactg tacagtactt gcctggagca ggcggatggc 64560
cacacccctg cgaagcctgc tcagctggct gggggacgct ccagtgtgtg agtggcagga 64620
tgcagggtac ttcctctgcc agggagttgc actggggaga tCCtCCCCCa CtCa.CaCttt 64680
ggcagctggg gctttggaat gtgacttagc ttctgtcaaa gggtcaatcc accctttgat 64740
atatgatgca aaggcgaaca tatgatgcaa aggtgagaga acagcccaaa ttaggacttt 64800
taccacagct gtggaggtgg acagcgacag tggtgggccc tggccagact tttcatgctc 64860
aaaggtggtg gttgttcttc ctacttcttg tccctccagg gcttcctttg cctgtgtgct 64920
gaacctgctt cttttaattt tttttaactt ttttaaattt ttaattgttt taattaaaac 64980
aaattttgaa aactgtctga acctgctttt gaaccctgct atgatttgaa tgtttgtccc 65040
ctgccaaact gattttgaaa cttaatctcc aaagtggcaa tattgagatg gggctttaag 65100
cagtgactgg atcatgagag ctctgacctc atgagtggat taatggatta atgagttgtc 65160
atgggagtgg catcagtggc tttataagag gaagaattaa gacctgagct agcatggtcg 65220
ccccttcacc atttgatatc ttacactgcc taggggctct gcagagagtc CCCaCCaaCa 65280
agaaggctct caccagatac agctcctcaa ccttgtactt ctcagcctct gtaactgtaa 65340
gaaataaatg ccttttcttt atgaattacc cagtttcaga tattctgtta taaacaatag 65400
aaaacgaact aaggcaaact ctcatgattc tactgccatg ccattccaat aaactccctt 65460
tatgcttaag agagccagag ttggccaggc gtggtgactc acgcctgtaa ttccagcact 65520
ttgggaggcc gaggcaggtg gatcacaagg tcaggagatc gagaccatcc tggctaacac 65580
ggtgaaaccc cgtctctact aaaaatacaa aaaaattagc tgggcgtggt agtgggtgcc 65640
tgtagtccca gctactcggg aggctgaagc aggaggagaa tggcgtggac ccaggaggcg 65700
gagcttgcag tgagtcgaga tcgtgccact gcactccagc ctgggtgaca gaatgagact 65760
ccgtctcaaa aaaaaagaga gccagagttt atttctgttg cttgcaacca agaaatctgg 65820
ctggtgcact gaagtttcca taaataatag caatttaaag actctttcca agccaggcaa 65880
tgcctagcct tgtgtagtcc ttgtggtaat acattcattc attcatttgt tcaaccaact 65940
gtgctccaga gactaagaat acaaaaatgg gggccgggtg tggtggctca cacctataat 66000
cctagcactt tgggaggccg aggcaggtag atcacctgag gtcaggagtt cgagaccaac 66060
ctggccaaaa tggtgaaacc cctactctac taaaaataca aaaaattagc tgggggtggt 66120
ggcggacacc tgtaatccca gctactcgtg agactgaggc aggagaatca cttgaacccg 66180
ggaggcagag gttgcagtga gccgagatcg caccactgca ctccagcctg ggcaacaaga 66240
gcgaaactcc acctcgaaaa aaaaaaaaaa aaaaaaagag ggccgggget gggcgcagtg 66300
gctcacgcct gtaatcccag cactctggga ggccaaggca ggagaattac gaggtcagca 66360
gatcgagacc agcctgacca acatggtgaa accccatctc tactaaaaat acaaaaatta 66420
tccgggcgtg gtggcgcaca cctctagtcc cagctacttg ggaggctgag gcaggagaat 66480
cgcttgaacc cgggaggcag aggttgcagt gagccgaaat catgccactg cactccagcc 66540
tgggtgacag agtgagactc cgtctcaaaa aaaaaataaa aaaaaaaaaa gaattcaaaa 66600
attgtagagt tatagtgtgc ttctagttta gttgagagga catctgtcct tcaaggaagg 66660
ctagaatcta taccctgagt ccttactgaa atcaatccag cagtcaaaac atgggaccaa 66720
cgatcacagc agtaagatag gaagagcacc tttgtacatt tagctcatgt tgagataagc 66780
cactgacaga gctgaaggaa gctcacagtt ctgggttcca tcctttggca tttaaaaaga 66840
aaagtgctaa gaaaattcgg ttggtcacgg tggctcacgc ctgtaatccc aacactttga 66900
gaggccaagg caggcagatc acgaggtcag gagttcgaaa ccagcctggc caacatggtg 66960
aaaccccgtc tctactaaaa acagaaaaat tagccgggca tggtggcgca tgcctataat 67020
cccagctact caggaggctg aggcaggaga attgcttgaa cccgggaggg ggaggttgca 67080
gcgagtgaga gcaggccact gcactccagc ctgggagaca gagcaagact ctgtctcaaa 67140
aaaaaaaaag aaaaaaagaa agaaaggaaa aaaagaaaga aaaaaaaaga aaaaagaaaa 67200
ttcaggccag gccaggcctg gtggctcaca cctgtaatcc caacactttg ggaggctgaa 67260
gcgagacggt gccttagccc aggagtttga gaccagcctg agcaacatag cgagaccctg 67320
tctctataaa aaaaaatttt tttttggcca gacgcagtgg ctcacgcctg taatcccagc 67380
actttgggag gccgaggcag gtggatcacg aggtcaggag atggagacca tcctggctaa 67440
cacggtgaaa ccccatctct actaaaaaat acaaaaaatt aaccgggcgt ggtggcgggc 67500
gcctgtagtc ccagctactc gggaggctga ggcaggagaa tggcgtgaac ccgggaggcg 67560
gagcttgcag tgagccgaga ttgcgccact gcactccaga ctgggagaga gtgagactcc 67620
gtctcaaaaa aaaaaaaaaa aaaaaaaaat taattgtcag gtgtgctggc atgcagctgt 67680
agtcctagct actcgggagg ctgaggtaag aagatcgctt gagcccagga gttcaaggct 67740
gcagtaatag tgcctctcac tctaccctgg gtgacaatga gaccctctct caaaaagaaa 67800
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-29-
gaaaaaagggaaagaagaaaagaaagaaagaaagagaagaaaggaaggaagaaagaaaga67860
aaaagaaaaggaaggaaggaagaagaaaaaaaaagaaagaaagaaaagagagagaagttc67920
aaagaccaaagggtcaggatcccaaaatagtttttatgttttatttatttatttacttat67980
ttatttttgagacagtatggctctgtcgcccaggctggagtgcagtgatgcgattgcggc68040
tcactgcagcctccaaactgggctcaggtggccctcccacctcagcctcccgagtagctg68100
ggaccacaggcgcgtgccaccatgcccagctaattttttaattctttgtagagatgaggt68160
ctctatatgctgcccaggctggtctcgagctcctgggcttaagccatccacccgcctggg68220
cctcccaaagtgctgggattacagaagtgagccaccgcgcctaatcgggtggtttgtttg68280
tttattgacggggtctcgctgctgcccaggctggagtgccagtggctgttcacaggtgca68340
gtcctggagcattgcatcagctcttgggctctagcgatcctccagagtagctgcagctgg68400
gattccaggcgcgccaccgcgcggggctcagaatgggtttttatattgagggttatgctg68460
ccacctagaggatatatgtagtaccgaactgtgtgcgcagggaggctgaggttgcagtga68520
gccaagatgatgccagggcactccagcgtgggtgacagagcaagatttcatctcaaaaaa68580
aaaaaaaaaaaaaaaaaaaaaagaattgaaagtaaggtcttgaagagatatttgtgcctg68640
tatggtcatagcagtattaactttgacccactagctaaaacacaaaagcaacatgtgtct68700
gtcagcaggtgaacggataaacaaaatgtggtatatatgtacaattgaatattattcagc68760
ctttaaaaaggaataaaaggctggatgcgggggctcacgcctgtaatcctaacactttgg68820
gagactgaggtgggtggatcacccgaggttaggagtttgagaacagcctggccaacatgg68880
tgaaacttcatctctactaaaaatactaaaattagccgggcatggtggcacttgtctgta68940
atccaagctactggggaggctaaggcaggagaattgcttgaactcaggagccggaggttg69000
cagtgagctaagatggcaccactgcactccagcctgggcaacagagtgagactccatctc69060
aaaacaaacaaacaaaaaattattatttccaaagaaacaagaccctgggtccatttccca69120
gcccacacctgatgttgactcacaacacacagcctggtttgctatgagcctgcttcattt69180
aattgtcaccttaacttcacatcaccctcaagtcctggaataactctttgctgacctttg69240
tgtgCtgagCCatCtCCatgtCgCtCaaCgtgCagtCCCtctcactgcactgagtcaata69300
gccagacgtggtctgactgc.agggtcatccttggtggcttaggctgactcgggcatagca69360
gggtgctctgagacctcaccgcatataggctttgCCCCCaataaaCtCtatataatattc69420
atattatgtggtctgggtgtgtgtagctttgcactgtcttctcgtgacagtgccctcaac69480
ctctttcccaggatttcctcctctacctcctcaagtcccactgctctgcaaagaccaaaa69540
gCtgCagagtCCCagCtCCCtCCtttaCa.CCCCaCgaCgCagCCtCCCCtCtCagaaCCC69600
tttaaaCagagtCttttaCtgCagatCCCaagaaCagCCaCaCCCCtCtCtCCGaL'CCaC69660
tccagacacacccaggtaattatagcacccagggtaactatgtagatggagtccctggaa69720
catgtggatagtgccccctgggagtatgcaaaagcaacattgctggcacctgcagagaac69780
agggtgacatccaggaatcagagcatgggcctctgggaggtagggatgtggccaggcagg69840
ctgccaaaaattggtagagcaaggccacaggatctttctgaccttccttccaaacagagg69900
CtCCtgtaCtggtgatCCCtgtgttgattgaCCa.CtCCCttCCtgggggtCgtggtCtCt69960
gtcccagttgcccggacttctgtgagtgtcctactgaggtccttttcatgagaagcatgc70020
tgtccttccacctgctgggagcaagagtgacaacttcaatactataatagcagtggcata70080
cagagaagaagaaagatgaagtggcaagaaaaacaggcttccaagcaggagtttttctat70140
aaaaacaaaaacgtttacaagcaaactttttataaagggctagatagtaaatattttagg70200
ctttgagagccacatagacttgtttgcagggactcaatgtcgctattgtagtttgaaagc70260
agccatcagggttatgtaaatgagtgagtctgattttgtttcagcaaaattttatttacc70320
aaaacagacaatgagtgggctggatttggcccatgatccttagtttgccaactcctgctt70380
tgggctcacccagatctgattttgaattctggctctgctactggttagctgcaggagctt70440
ggaaggctctctgagcctgtttcctcatctgtaaaattaaagcaataatttctaacactc70500
aagagtgttacctcacgcctgtaatcccagcactttggaggctgaggcaggcggatcacc70560
tgaggtcagaagttcaagaccagcgtggccaacgtggcaaaaccctgtctetactaaaaa70620
atacaaaaagtagccgggcatggtggcgcgcatctgtaatcccagctacttgggaggctg70680
aggcagggatactgctagaacctgggaggtggagcgtgcagtgagtggagatcacacctc70740
cacactccagcctggccgacagagcgagactccatctcaaaaaaaaaaaaaaaaagagtg70800
ttagaaggttttgagataatgaataaaagatgccttgtgtatactaagtattcaacaact70860
gatagctgcattggtctaattataacagtttagaagcgattgagtcaacaaatgctggat70920
ttgtcagggaggacttcctatcaggaggtagatcttgggctgagtcctgaagcaaagata70980
ggcattggatagaggagttgagagaacaccctaggactgttattattattattcgacacg71040
gagtctcttgctctgtcacccaggctggagtgcagtggcgcgatctcggctcactgcaac71100
CtCtgCCtCCCaggttCaagcgattctcctgcctcctaagtagctgagactacaggtgtg71160
tgccaccacacccggctaatttttatatttttagtagagacagagtttcaccatgttggc71220
catgctggtctcgaactcctgacttcaggtgatccacccgcctcagcctcccaaagtgct71280
ggaataacagatgtgagccaccgcacccagcccagaaccatttttcaatccttggctctg71340
ccttttattagctgcaagatctcaggcaatttatttaacctctccaaagactcattttct71400
cattcacaaaatgaggcaaataataatatctactatcccaggttgtcatgagaattaaat71460
gcaacatgacatttaatgaaatgagaagtcccttggacattaactggctaaagtatgtgc71520
tcgacaaggatatcattttaggtggatacttagcatctcagaactgatgctcacaatgga71580
atatcattgaaacgcattaaaattcattttaaatgattgtaggtagtgaggcaattgaaa71640
gaagaagacaagaggactgattataatgcttcaggctcactagtctccttttaggaggga71700
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-30-
aaaacaatttcaagttaaattttaggctctagatttttacccctgctgctcattagaatc71760
acccagattgatgaaatcagagcccatctgaggctgtgtttttcatctccagaatgagag71820
ctgttgtggggattaagtttttgaaaaagtacatctaacaggtgatcgaaaatgatagtg71880
atattattgcagtgatggtcattattgttgttattattatactgaaagaggcttcagttt71940
tctgatccataaagtgagggaattgcatgagaccattgctaagattccttctagctctgt72000
ttttttgtttttgttttttagacagagtctctgtcgcccaggctggagtgcaatggcatg72060
atcttggctcactgcaacctccgcctcccgggttcaaatgatcctcctgtctcagcctcc72120
gaagtagctgggactacaggcacacaccaccatgcccagctaacttttatatttttaata72180
gaggtggggtttcaccatattggtcaggctggtctcaaactcctgacctcaggtgatcca72240
cccgcctcggcctcccaacatgctgggattacaggcatgagCCa.CtgtgCCCaaCCCCtt72300
ctagctttcttgatcactgattctagggttctctgctgaaatatatttgagacatcctgg72360
ataaaagatcatgcaagagctcccaatatggtattaataattgattctggaggcttagct72420
actcctgatggattagacatgactcaactgcctctcttatgtgtacaacacaacaacaca72480
accaagaaaggttattctggcattccatttattcagtttatttacagcccttacttccag72540
cagcacgttaaagatatggccagggccgggtgcagtggctcaagtctgtaatcccaggac72600
tttgggaggccaaggtgggcggatcacaaggtcaggagtttgagaatctggcaattcttc72660
agacttagaagcaaccagctcgataacacagtcttgtgtgggctctccctctgtccctcc72720
CtCgCttCCCtCatttCtCatCCCtgCCCCtgagactgtgCc~.CCttcacatagccctgcc72780
atgagaccttcatctcaggctttgctttctggggtaactgaggctaaacactgagtggcc72840
ctaaaagaggattgggatttggaagttagattattcaccagagaacagactttgctgatg72900
atcaggcccaggttgtaattgttgaaaaaaagagaggatgcatagtcttatctcatctcc72960
tagtcaaagtcaacaccatgataaataagagtcaaatcctgagatgtgaattggggacat73020
ttgagtggttaaccctgagaagcttgcaccttcagacccctcaatacccctgctccccag73080
agaaggctggacattgacctcagcacaggcaggagccctgcaagatgccatttgtcctac73140
taaagatggacccctccactctgtttctaggtaaataaccaaagtcaagtctccacacag73200
cctgagcaagaaagtcagagcctgctacaggagaaaataccacactggccaaaggattca73260
ctagccctggccactgtgtgtgggaggaaccagggaatcatgtgtgggagtcaatgttga73320
agctgttggactgggggtggggtggaatataagcctggccctggggagtttttcccgttt73380
gagggcctttacccacaactcaagatccagtgctatagcaggagatcccagagctagtcc73440
taacagatggtcaggattgaacttggcctagagtaaaatgaggaggatagtgccagaact73500
ttctcaacatactattgaggaagaggtcagaaggcttaaggaggtagtgtaactggaaag73560
gggtcctgatccagaccccaggagagggttcttggaccttgcataagaaagagttcgaga73620
cgagtccacccagtaaagtgaaagcaattttattaaagaagaaacagaaaaatggctact73680
ccatagagcagcgacatgggctgcttaactgagtgttcttatgattatttcttgattcta73740
tgctaaacaaagggtggattatttgtgaggtttccaggaaaggggcagggatttcccaga73800
actgatggatccccccacttttagaccatatagagtaacttcctgacgttgccatggcgt73860
ttgtaaactgtcatggccctggagggaatgtcttttagcatgttaatgtattataatgtg73920
tataatgagcagtgaggacggccagaggtcgctttCatcaccatcttggttttggtgggt73980
tttggccggcttctttatcacatcctgttttatgageagggtctttatgacctataactt74040
CtCCtgCCgaCCtCCtatCtcctcctgtgactaagaatgcagcctagcaggtctcagcct74100
cattttaccatggagtcgctctgattccaatgcctctgacagcaggaatgttggaattga74160
attactatgcaagacctgagaagccattggaggacacagccttcattaggacactggcat74220
ctgtgacaggctgggtggtggtaattgtctgttggccagtgtggactgtgggagatgcta74280
ctactgtaagatatgacaaggtttctcttcaaacaggctgatccgcttcttattctctaa74340
ttCCaagtaCCa.CCCCCCgCCtttCttCtCCttttCCttCtttCtgattttaCtaCatgC74400
ccaggcatgctaCggCCCCagCtCaCattCCtttCCttatttaaaaatggactggggctg74460
ggcgcggtggctcatgcctgtaatcccagcactttgggaggccgaggcgggcggatcatg74520
aggtcaggagatcgagaccatcctggctaacacggtgaaaceccgtctctactaaaaatg74580
caaaaacattagccaggcgtggttgcaggtgcctgcagtcccagcggctcaggaggctga74640
ggcaggagaatggcgtgaacctgggaggtggaggttgcaatgagccgagattgtgccact74700
gcactccagcctgggtgacagagcgagactccgtctcaaaaaaaaaaaaaaaaaaaaaaa74760
tagctgggcatggtggcgcgtgcctgtaataccagctactctggaggctgaggcaagaga74820
atcgcttgaacccagtaggcggaagttgcagtgagccgagatcttgacactgcactccag74880
cctggtgacagagtgagactctgtctcaaaaaaaaaaaaaagaaaaaaaaagacagaaag74940
aaagagcacagacagagtcacaggtatttgcagtaggaagctgtcaggttagagtgcacg75000
gaaatagaaagtatattttacacttacagcacatcttcgtttgattagccacatttaaaa75060
tactgaatagcaacgtgtggctatttagtattcactaaaatcttggacagtgcaagtcta75120
aagaatccttgatccgtccggcatggtggctcacgcctttaatcccagcactttgggagg75180
ccaaggtggaaggatcacttaaggtcaggagttcgagaccagcctggccaacatggtgaa75240
acctcgtctctactaataatacaaaaaaaattagccgggcatggtggtgcatgcctgtaa75300
tcccaggtacttgggaggctgaggcaggagaatagcttgaatccaggaggcgctgcagtg75360
agccgagatcatgccatgccactactgcactccagcctgggcaacagagtgagactgtct75420
caaaaaaaaaaaaaaaattgttgggcgtggtggctcacgcctgtaatcccagcactttgg75480
gaggctgaggggggtggatcacctgggttctggagttcgagaccagcctggccaacatgg75540
tgaaaccccatctctactaaaaatacaaaaattagctgggcgtggtggtgggcacctgaa75600
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-31-
atctcagcta ctcaggaggc tgaggcagga gaatttcttg aacccaggag gcagaggttg 75660
cagtgagcca agatcgcgcc tctgcactcc atcctgggtg gcagagcaag actatgtctc 75720
aaaaaaaaaa aaaaaaatac ttgattgtct ggacattctg cagaacatca tatggagaca 75780
ctatgttgac gacatcatgc tgattgtaag caagaaatgg caagtgttcc agaaacacag 75840
tcaagacaca tacatgccag aaggtgagat ataaactcta ctaagattca gtggcctgcc 75900
acactggtga catttttaaa cctgctagat gtttgtgtag aaaaggattt aaccttgccc 75960
aaagaggggt ctggcctttg tccccagcta ctggacataa tctctttaaa ctcttgaaat 76020
atcattcctg atagaagtat ttttgttttg actaggggcc ttgggccagc cagatagcaa 76080
caatgtgatc tgggttgggg gctttggatc aggtggcatc agtgtgacct cctgagtggc 76140
tagagactag aatcaaccac atgggcagac aacccagctt acatgatgga attccaataa 76200
agactttgga cacaagggct tgggtaagct ttcctggttg gcaatgctct atactgggaa 76260
acccattctg actccatagg gagaggacaa ctggatattc tcatttggta cctccctggg 76320
ctttgcccta tgcatttttc ccttgtctga ttattattat tattatgaga tggaatctcg 76380
ctctgtcacc caggctggag tgcagtggaa tgatctcaac tcactgcaac ctctgcctcc 76440
ccggttcaag cgattttcct gtctcggcct cccgagtagc tgggactaca gatgcatacc 76500
accacacccg gctaattttt ttgtattttt agtagagacg gggtttcacg ttagccagga 76560
tggtctcgat ctcctgacct catgttccgc ctgcctcggc ctctcaaagt gctaggaata 76620
catgtgtgag ccaccgcgcc cagccccctt ggctgattat taaagtgtat ccttgagctg 76680
tagtaaatta taaccgtgaa tataacagct tttagtgagt tttgtgagca cttctagcaa 76740
attatcaaac ctaaggatag ccttggggac ccctgaactt gcagttggtg tcagaaataa 76800
gggtgctcat gtgtgtacca tgccctctaa ttttgtagtt aattaacttt cacaacttta 76860
ttattaccgc ttacactcaa tgtttattca catttatcca cataccactt attctagtgc 76920
cttgcatcaa agactttcta tctcatgtac tttattctgc ttgaagtaaa tcctttagga 76980
tattcttttt tttttttaaa ctttgcacat acatactttt attttttatt tatttttaat 77040
tttgttattt ttgtgggtac gtagtagata tatgtattta tggagtacat gagatgtttt 77100
gatacaggca tgcaatgtga aataagcaca tcatggagaa tggggtatcc atcctctcaa 77160
gcaatttatc cttcaagtta CaaacaatCC aattacactc tttaagttat tttaaaatgt 77220
acatttaatt ttgtattgac tagagtcact ctgttgtgct atcaaatata attttttttt 77280
tttttgagaC agagtCtCaC tCagtggCCC agaCtgaaag tgcagtggca caagctcggc 77340
tcacttcaat CtCtgCCtCC CtggttCaag cgaatctcct gcctcagcct cccacatagc 77400
tgggattaca ggcacacacc accatgccca gctaattttt atattttttt agtagagacg 77460
ggttttCgCC atgttggCCa ggCtggtCtt gaaCtCCtgg CCtCaaatga tCtgaCCaCC 77520
tcagcctccc aaagtgctag gattacaggc atgagccacc acacctggcc aaaatagaat 77580
attctttagt gaggtctgct ggtgacaatt tttttctttt ttttgagact gagtctcgct 77640
gttgtcagct tgggctggag tgcaatagca cgatctcagc tCaCtgCaaC CtCCaCCtCC 77700
cggattccag caattctcct gcctcagcct cccaagtagc tgagagatta caggcaccca 77760
ccaccacacg cggctaattt ttgtattttt agtagaaatg ggggttcacc gtgttggcca 7a82o
ggctggtctc gaactcctga cctcaggtga tccacccacc ttggcctccc aaagtgetgg 77880
gattacaagc atgagccacc acgcacagcc aattttttcc gtttttgtct gaaatcttat 77940
tttgtgtcat ctttgaaata tatttttgat ggatataaaa ttgttggttg atagttatta 78000
tcattattat tattattttg agacagggtc tcactctgtt gcctatgctg gggtgtagta 78060
atgtgatctc ggttCaCtgC agacttgacc tCCtagggCt CaggtgatCt tCCCaCCtCa 7812~
gCCtCCCtag tagCtgggaC tacagatgca tgCCaCCata CCCaaCtaat ttttCtattt 78180
tttgtagaga tgaggctttg ccacatttcc caggctggtc tctaactcct gagctctagc 78240
aatccaccca ccttggcctt acaaagtgct gggccatgac tagccagcag ttacttttta 78300
tagcatattg aatatttaat atgaatcttc tggcatccac tgtaactgtt taaaaaatca 78360
gCtgtttaCt tggCa.CtCtt tttttttttt ttttttttga gacagagtct tgccctgtcg 78420
cccaggctgg agtgcagtgg cgtgatcttg gctcactgca agctctgcct cccgggttca 78480
CgCCattCtC CtgCCtCagC CtCCggagta gCtgggaCta aaggCgCCCg CCaCCaCgCC 78540
cggctgattt ttttgtattt ttcgtagagt tggggtttca ccgtgttagc caggatggtc 78600
tcgatctcct gacctcgtga tctgtccgcc tcggcctccc aaagtgctgg gattataggc 78660
gtgagccacc gcgcccagcc tctttttttt ttttttttag acggagtctt actctgtcat 78720
ctaggctggt gtacagtggc gtgatctcag CtCagtgCaa CCtCCd.CCtC CtgCCt CagC 78780
ctgccaaata gctgggatta caggtgcgta ccatcacgcc cggctaattt ttgtattttc 78840
agtagagatg gggtttcacc atgttagaca ggctggtctc gaactcctgg cctcaagtga 78900
tctgcctgcc ccagcctccc aaagattaca ggcatgagcc accgcacccg gccaagtagc 78960
actcctttga aggtaatctg cttcccctac ccctagcaat ttttaacaat ttttcttcat 79020
ttttatttcc tgaagttttg ttattaataa tctgtgtgca gatttctttg tatttctttt 79080
gtttgcagtt catagtgatt cttgaattag tgtgttggtt tctgttatca ccacaggaaa 79140
attgtcagcc gttagctttt caaatatttc cttgctaaat tctctcttct cccctttcgg 79200
tacaattgat ttgattaaaa ctaaaaccag ggccgggtgc agtgactcat gcctgtaatc 79260
ccaacacttt gagaggctga ggcaggtgga tcacctaagc tcaggagttc aagaccagcc 79320
tggccaatat ggtgaaaccc cgtctctact aaaaatacaa aaattaccag gcatggtggc 79380
acacatttgt agtcaggagg ctgaggcagg agaattgctt gaatccagga ggtggaggtt 79440
gcagtgagct gagatcccac cactgcagtc tggcctgggc gacagagtga gatgagaatc 79500
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-32-
tgtctcgaaaaaaaaagttatgaatgtttgataaactatatttgttagaatgtttgttgt79560
agaatactattcattgatttttaaacaatgttagattaaaccattcactggatttgtgat79620
aattaacttactgattttacctcactgatttgttgtaattaatacaactggtataaaaag79680
actgtgacgaggccgggcatggtggctcccgcctataatcccagcactttgggaggctga79740
ggcaggcggatcacctgaggtcaggagttcaagaccagcctgaccaacatggtgaaaccc79800
catctttactaaaaatacaaaattagccggtcgtggtggtgcatgcctgtaatcccagct79860
cttcgggaggctgtggcaggagaatcacttgaacccgggaggtggaggttgcagtgagcc79920
gatatcgcgccattgcactccagcctgggcaacaagagcgaaactccgtctaaaaaaaaa79980
aaagaaaaaaaacacataaaacaaaacaacactgtgacggttcccaaaaattaggagcat80040
aattaaaggaactcctgataaaaattaattttatcttacatgtaaactaaaatgacttta80100
tgaagttaattcagaaatacaatgcagggtattagtttgccacagctgcgtattcagcct80160
aatgtaatattcttgttatttttaaattcttcttttaactttactcatatgtggatcatc80220
aaatttcaaaagattaaatgacaatactcttagcagcaagcttccctaagcatataaaca80280
ttttaatgggtgatgattcagaaggtacccgaagaatatgtactgccagatatcattcac80340
ccccatatacctgcccgacagacatcccattttgggaccctggataaatgtgtgggtgga80400
gagaaagataggagaaagtggtataagcaaatggctttggagtctgattgacagcgattg80460
aaatcctgtctctacctcttaacagcctcatgatcctacataagttaccccgatcctcag80520
ggccacatctgtaaattgggggttgcgatggcagccatctcacagggtctcttttcgggg80580
aagggcaggaattatggattaagtgagctagtaattgtaaagcacttaatacaaggaggg80640
cgcataataagtacttcataaataatgacggccattatcatgactgaggtgtatgcagct80700
gtcggggattacggcgacttcagaatttctggtgggcagggctcaaaggcagcaaatcac80760
actggaagtcgaggtgaggcactgcttctgcacagactgcttagctggagagaatgagga80820
aggcttagaggagatttagaggaacttagagtCCtCCgCCtCCaaCtCtgtgggatCtgC80880
tcccgtgccagagacattcaggggatttctcgcactctcccctcccctacgtccctcccg80940
ccccatccaactaaccacacaacacatacaaaatagcccctgcgaggttctgcacgctgg81000
aagggaacaggagaagggcgCtgCgCtttCttgCtgatgCCCtgtaCttgggCCCCtggt81060
agacacagccacttgtcccctcagcctgcagagaaatcccacgtagaccgcgcccgggtc81120
cttggcttcagccaatctccctttggtgggggtgggatgcacgatccaaggttttattgg81180
ctacagacagcggggtgtggtccgccaagaacacagattggctcccgagggcatctcgga81240
tCCCtggtggggCgCCgCtCagCCtCCCggtgCaggCCCggCCgaggCCaggaggaagcg81300
gCCagaCCgCgtCCattCggCgCCagCtCaCtCCggaCgtCCggagCCtCtgccagcgct81360
gcttccgtccagtgcgcctggacgcgctgtccttaactggagaaaggcttcaccttgaaa81420
tccaggcttcatccctagttagcgtgtgaccttgagcagttgactttatttttcagtgcc81480
tagttttccagataccaggactgactccaaggactattactcatctggagggtttagcac81540
agtaccgtcgcatagtaaatttccatgtcagttttggttacctttcatgcacttgcaaac81600
atgccatgctctgaaacgaaataggcacatCttttttttttttttttttaaggagtcttc81660
ctctcgcccaggctggagtgcagtggcgcgatcttggctcactgcaacctccacctcccg81720
tgttcgagattctcctgcctcagcctcctgattagctgggactacaggcatgccacgacg81780
cccagttaatttttgtatttttagtagagacggggtttcgccatcttggccaggctggtc81840
taaCtCCtgaCCtCaggtgatctgactgcctcagcctctcaaagtgttgggattacaggc81900
ataagccactgcatctggccagaaatgaaataagtaaatcttttaacctgctctaacaat81960
atagtgaaaagaccatattattattagagcaggttaagggatttgcctatttcgggttct82020
agttatagtcttaaacttggacattcttgtagaaagtaaaaagtttcctcttcaaagttc82080
cccttcttgttaaagaatacatcataagtgttagaagtaatagtttattttaaagactaa82140
ctttcttcaagCCtCCttgCtttgtgctaataactctttgttaagccctatcctatgtaa82200
ctgttggacatgctcacaggcacgttccagttcacagcctatgccccttccttatttgga82260
aatgttattgcttccttaaacctttcggtaagcaacttcctctccttcttcgttcttcct82320
tgcacttacctatttagaaagttttaggctattagcaaatcggctatcagtttaagagtg82380
tgaggtcccgctccagccaatggatgcaggacatagcagtgaggacgacccaaatgcgta82440
agggataaatatgtttgcttttcctttgttcaggtgtgctC'tCgaCatCgttCCatCtgC82500
gattgagcaccctttctgcagaaagtaaagattgccttgctggagatcttttgtctccgt82560
gctgacttttcttcgtggcaccgattatctatttctaacaattttggtatttctaacatt82620
ctgaacaatcttgggctagttgtctcttctgggcctgtttCCCCatCCgtcacatgataa82680
acttcattggtttaaaaaccccagcgaacatttattgagttactattaccttcctgccct82740
ccccaaccccaaccccagggagcagttacaacctcagccgctgagcgcactcgccgggtg82800
ttaagaagcaccaaagacagggaggcttgattgattttgctttgggagtagagggtcaga82860
agattcacaggaaaatggcatttgagcaaggatgattcactggagctagcttttaaatac82920
tggcgaggcttttatgttgcagtcccttacaaagttgagcattcgcagggactgcactcc82980
gaaataagcccgcttccccttttcattcgctaatgatccagggagctgctggttccgcat83040
gcggcaggttgtgccttttcctaatcagggttctgcatcgcctcgaacccgcaggccgtg83100
gcgggttctcctgaggaagcagggactggggtgcagggtgaagctgctcgtgccggccag83160
cgcctgtgagcaaaactcaaacggaggagcaggaggggtcgagctggagcgtggcagggt83220
tgaccctgccttttagaagggcacaatttgaagggtacccaggggccggaagccggggac83280
ctaaggcccgccccgttccagctgctgggagggctcccgccccagggagttagttttgca83340
gagactgggtCtgCagCgCtccaccgggggccggcgacagacgccacaaaacagctgcag83400
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-33-
gaacggtggctcgctccaggcacccagggcccgggaaagaggcgcgggtagcacgcgcgg 83460
gtcacgtgggcgatgcgggcgtgcgcccctgcacccgcgggagggggatggggaaaaggg 83520
gcggggccggcgcttgacctcccgtgaagcctagcgcggggaaggaccggaactccgggc 83580
gggcgggttgttgataatatggcggctggagctgcctgggcatcccgaggaggcggtggg 83640
gcccactcccggaagaagggtcccttttcgcgctagtgcagcggcccctctggacccgga 83700
agtccgggccggttgctgaatgaggggagccgggccctccccgcgccagtCCCCCCgCaC 83760
cctccgtcccgacccgggccccgccatgtccttcttccggcggaaaggtagctgaggggg 83820
cgccggcggggagtcaggccgggcctcaggggcggcggtggggcaggtgggcctgcgagg 83880
gCtttCCCCaaggCggCagCaaggCCttCagcgagcctcgacctcggcgcagatgccccc 83940
tgagtgccttgctctgctccgggactcttctgggagggagaaggtggccttcttgcgcga 84000
ggtcagaggagtattgtcgcgctggttcagaagcgattgctaaagcccatagaagttcct 84060
gcctgtttggttaagaacagttcttaggtgggggttagtttttttgtgtttctttgagga 84120
ccgtggatcaagatcaaggaaatctctttagaaccttattatggaagtctgaagtttcca 84180
aatgttgagggttttatgtctaaaagcaacacgtgaaaaaattgttttcttcacccagtg 84240
ctgtcttccaatttcctctttggggggaggggtagttactgctgttactaaaataaaatt 84300
acttattgctaaagttccccaacaggaagaccactacttttgatgactttggcaagtttg 84360
ctaactactggaaccctaacttacaaacgaactacttacatttttgatttccagttgtat 84420
tacctgcccaatgtttacgtagaaacagcttaattttgattctgggtaacgttgttgcac 84480
ttcattaaaaatacatatccgaagtgagcaagtatgggtctgtggacagcagtgattttt 84540
cctgtcaattcctgttgcttcagataaaatgtaccagacagaggccgggcgcggtggctc 84600
acgcctgtaatcccagcactttgggaggcttggcgggtggatcacctgagatcgggagtt 84660
caagaccagcctgaccaacatggagaaaccccgtgtctactaaaaatacaaaattagcca 84720
gggtggtggcgcatgcctgtaatgccagctacttgggaggctgaagcaggagaatcgctt 84780
gaacctgggaggcggaggttgcggtgagccgagatagcaccattgcactccagcctgggc 84840
aaaaagagcgaaactccgtctcaaaaaaaaagtaccagacagaaatgggttttgttttct 84900
ttttttgttttgagacggagtttcgctcttgttgcccaggctcgagtgcaatggcgcgat 84960
CtCagtCtCggCtCaCtgCaaCCtCtgtCtCCCaggtttaatCgattCtCCtgCCtCagC 85020
CtCCCaagtagCtgggattaCCCatgCCCCaCCatgCCCggCtaatttttgtatttttag 85080
tagaaacggggcttcaccatgttaggctggtCttgaaCCCCtgaCCtCaagtgggCCtCC 85140
Cc'~.CCtCggCCtCCCaaagtgCCaggattaCaggCatgagCCdCCgCggCCagCCagaaat 85200
gggttttggaaaaagcactaaacaaaatcgaacttggtttcatatgacagctctgctgct 85260
aactgtaacaggggcagaccagttaacctacttttctgtettctgtcagctgagaattag 85320
atgattcccaaaggcccattgaactctgaatgactttaaatacttcttcttaagtgggta 85380
cacggttttggtaactgatgccaggtgatgaatgcatgaaagtgcttaatgaatgaaacc 85440
ggtaaaatagtaggaggaagctttattggtaaggcaggggtatacctaatagctctctaa 85500
tttattggtattgaagtggttaacttttgtttttttaaggggggaaaacattctaagaat 85560
aatgaggcaaactgcatattgeacaagagactgttgtctctattcaacaaataccttttg 85620
agtgtccagagtctgccaggtgctgtgctaggccctcacgattgagtagtgaaccagaga 85680
atgtccctgcacceatggagcttattgtctactggggtagacagataataaataagcaaa 85740
caaatcttctctcttctccctttcgctccatgtaagtgtgtgtgtataggtgtatactta 85800
caagttgagtaaagtgttatgaaagattaagaggagaaatgcattttggttagatgttag 85860
aggactcagcaggtgaccttgaaacttagagctgaaggatcagtaggaggtaactagaga 85920
ggccagggaatcgcatgttcaaaggccaggaggcaagaaagagcatggtgCCCttCaaga 85980
gaggaaagaaggctactgtgactggagcatagatgtaggcaagtgttgggtgattgagag 86040
ctctacgggccatggttaggttttattcctaatgccgagatgccaaacatggtggttcat 86100
atctgtaatcccagtattttaggaggccgaggcaggaatatagcttgaacccaggagttc 86160
aagaccagcctgagcaacatgagacctgtacaaaacatttaaaaaattgctgggtatgat 86220
ggtgcacacctgtggtcccagctactcaggaggctgaggcagaaggatcacttgagccta 86280
ggaggtggaggctacaatgagccatatttgagtcactacactccagcctggatgacaaag 86340
tgagaccatgtgtcaaacaaaatacagaaagaatattaatttaaaattttgaaagaggag 86400
tgatctgaacttatatcttaaaaagatcattctagggcatggtggctcatgcctgtaatc 86460
aagggctttgggaggctgagacaggaggatcacctgaggccagttcgagatcaacctgta 86520
cagcatagagagactccatctctacaaaaagaaaaaataaatagctgggtgttgtgagtt 86580
attcaggaggctgaagcagaaagatcacttgagcccaggagtttgaggctgcagtaagct 86640
atgatcccaccactgcaacacagtgagatcttgtctcaaaaaaaaaaaaaaatcattcta 86700
ggtgctttttggaggctggatgtggtaagagtagaagctggagatggtcctgttagggat 86760
tcgattcagactttaaataccatcaatgcattgagtcccaaatttacatcactacgttgg 86820
atccttgcccctgaatccagactggtatatccaactttaggttcagtttgtatctctacc 86880
tgaccaatatagaggtgtccagtcttttggcttccctaggccacattggaagaagaattg 86940
tcttgagccacacatagagtacactaacgctaacaatagcagatgagctaaaaaaaaatc 87000
gcaaaacttataatgttttaagaaagtttacgaatttgtgttgggcacattcagagccat 87060
cctgggccgcgggatggacaagcttaatccagtagataccttcaacttacaatatctaaa 87120
attttatgccagatttagtcattttaaacctgctcatcagtttttctcaagaagtagtat 87180
tttggctttttttcttttcttttttttgagatggagtttcgctcttatcgttcaagctgg 87240
agtgcagtggcggatcttggctcactgcaacctccgcctcctgggttcaagtgattctcc 87300
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-34-
tgcctcagcctcgcaagtagctggaattacaggcatgcgccaccatgaccagctaatttt87360
tggagacagggtttcaccatgttggtcaggctggttttgtactcctgacctcaggtgatc87420
tgcctgcctcggcctcccaaaggctgggattacaggcatgagccaccgctcccggctgca87480
tttttggatttttagttgctcagcccaaaactttagtacatctttgaacctcttctttcc87540
tcctactctatatctgatccatcagcaaatctgttaggtctacctcacacatatcgaaat87600
cctaccacgtctcaccatctgtgacaattaacaccctggtctaggcagtcatctctgtta87660
agattgagtggttaaggatgtcctctaaggagatgacattcaaatcttagcttaaatgtc87720
aagagggagctggttttataaagattgaggaggcagcattattttgccataggcttccat87780
ttggtttccattccattcttgatacttatggtatatattcaaaacaaatgcacagaaaca87840
gacccaggtatattgggaatttcggatatagagttcctagttgggaaaagatagactgat87900
ctgtaaatgatgctagttatccatcatctggcaaaaaataatttcctgcctcctctcata87960
tatctcagatcaacagactttttctgttaagggccaaatcataaatattttaggctttcc88020
agaccatatggtttctgtcacactctcctttatccttgaagccatagacaatatgtaaac88080
aaatgggcatggctgtgctacgataaaactttacttacaaaaactggtagtgggccagtt88140
taggcatggccagcactttgggaggctaaggcagatggatcacttggggtcaggagtttg88200
agaccagcctggccaacatggtgaaaccctgtctctactaaaaatacaaaaaatagctgg88260
gcatggtggtgggtgtctataattccagctactctggaggctaagacacaagaatcactt88320
gaacccaggaggcagaggttgcagtgagctgagatagcaccactgcactccagccagggt88380
gacggagtcttaaagcaaaacaaaacaaaaggtagtgggttgtatttggcccatgggctg88440
tagtttgccaatccctgatgcagaaacaaattccaggtaaataagagcctggaatgttaa88500
aaaaacaaaacttgaagtcatgtagaagaacaggtagggggaacaatcctgatctcagga88560
taggaagggatattgcttaaaataagacacaggaaaatataatccatgttgtgtaaattt88620
gactacgttaaaacttaaaactttcgccaagcgcggtggctcacgcctgtaataccagta88680
ctttgggaggccgaggtgagcagatcaccaggtcaggagattgagaccatCCtggCtaaC88740
acggtgaaaccccgtctctactaaaaatacaaaacattagccgggcgtggtggcgggcgc88800
ctgtagtcccagctacttgggaggctgaggcaggagaatggcctgaacccgggaggcgaa88860
gcttgcagtgagctgagatcgcgccactgcactccagcctgggcgacagagtgagattcc88920
gtctcaaaaaaacaaaacaaaacaaagcaaaaaacctaaaactttcatacaataaagtat88980
acctaagatacttctagaagagaagatttacatccaggacgtgtatggaatttctgcaag89040
taataagtaaaagacaagggacatgaagaggcagttcacaaaagaggaagccaaaatgac89100
caataaacatgaaaggatgtttaacctcaaaggaaacaaggaaatgaattaaaaacatca89160
aatgccatttcaaaactagtaagttggcaaaattaaaaataccaaggatgagaatatgaa89220
gcatggctatatgagtgcatggaatggtacagtcactttcattaaaaatgcacataattt89280
gttttttatttatttttttgagacagtctatgtcgcccaggctagaatgcagtggcatga89340
tCtCggCtCaCCaCaatCtCtgCCtCCtgggttCaagCaattCtCCtgCCtCagCCtCCt89400
gagtagCtgggattaCaggCaCatgCCaCaaCGgCCCggttaagttttgtatttttagtag89460
agacagggttttgccatgttggccaggctggtctcgaactcctgacctcaggtgagctgc89520
ttcccaaagtgctgggattagaggcgtgagccaatgctcctggctgaaaaaaatgcacat89580
aatttgttacctagcaattccatgtctagaggcttatcctagagaaattcttgcttatat89640
gcataggaagacgtgtactagaatgttcactagttgaatgtttaagtgaaaattaggaaa89700
taaagtaaatgttcattaacaggaaaatgagtaaaggtatatttataaaacaattaagta89760
gctaaaatgaataaactagagctgcgtgaatgaactagaactggttcaatagtcatgtca89820
gattattgaatgaatacaggtcagatatgtatagagtgtcatttgtgtaattaatttttt89880
ttttttttttgagatggagtctcactctgttgcccaggctggagtgcagtggcgtgatct89940
CagCtCaCtgCaaCCtCCaCCtCCtgggttaaagtgattctCCtgCCtCagCCtCCCgag90000
tagttgggattaCaggCatgCaCCa.CCatgCCCagCtCattttCCtatttttagtggCCa90060
cagggtttcaccatgttggccaggctggtcttgaactcctgacctcaagtgttccaccca90120
acttggcctcccaaagtgctaggattacaggcgtgagccaccgtgctcagccatttgcgt90180
gatttttaaagatgtgcagaataatgccattaaaaaaaatacacatacatgtatatatat90240
acacgtttggctgggtgtggtggctcacacctgtaatcccagcactttgggaggctgagg90300
caggaggatcacttgagcccaggtgtacaagactagcctgggcgagatagcaagacccca90360
tctcaacaacagaaaggataattaggtatggtggcatgagaggatcacttgagcccagga90420
gttcgagtgttatcaggccactgcactctagcctggacaacaaagcaagaccgtgtctca90480
aaaaaataaaaataaaaagtatttgtatgtggtcatagtcaaaaaacgtacatggaagga90540
aaatgtctttatttatttatttatttttttttttttaagacagagtcttgCtCtgtC2.CC90600
caggctggggtacagtggtgtaatctcagctcaccgcaatctcggcctcccgggttcaag90660
cgattcttctgcctcagccttctaagtagctgggactacaggtacccgccaccacaccct90720
gctaattcttgtgttttcagtagagacagggtttcaccatgttggcaaggctggtctcga90780
actcctgaccttaagtgagccacccgccttggcctcccaaagtcctgggattacaggtgt90840
gagccactgcgcttggccaggaaatatctaatttagtaagtatttatatctgggaaagga90900
agggtcaggtggtgattcataggaactctaaagtctatgtataatacttagggggacaga90960'
aggaaataaagcaaaatgctgatatttgattgttgagttgtgtatatgttagaagtataa91020
cataggagatctgattgatagtaggagaatgtttttaggtggtaaaagtggaaccgtggt91080
ggtttgttttggcagtagaatcagttggtcatagtttgtatgtggaaggtaataaacaga91140
ccatgttaaggatgacttccggaattttggtctgagtagtgggtggatgacagtgtcatt91200
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-35-
catgagggaagatgaagactgaggtaggaacaggtttgggagaagatgacatgttccctt91260
ttagacaagtggaattatggaagatggcaggtaggtggttagctatatgaatttgagata91320
aaagatttaggatggagatataaatttaggagtaacagcgtatctatggtattgtaagcc91380
ttaagaatgggtaggatcagccaggaaatacagatgtatatgcagaagagaggagtcaag91440
gaagccaagacaagttaatgtttaaagtgagtgatgtagtccatgggcagatgctgctga91500
gagggctgcaaacaccagtgaccctacaacatttttaaatgtcgtcttcctgacagcagt91560
gatcagtacctgcaacgatcttatttatttttttcatgttagtctccacacacttgaatg91620
tagactttttgaaggcaaaatcattgccttttctgagctgggagcatgtctggcacatac91680
caagcactcaacagttgatgtattgacttcatccagatactctgagggcgagttatttcc91740
tgctactagcctttcacctttcaatgtttaagagcacaaatacagagatgggcacgtttt91800
ggcatttcttattttgataaccttttcctggtaagattttttaatgttgaaaaaaaaaaa91860
caagaaaagagggttaaaaatagtcttatgtcagatcctgtgatagaattcacacttggc91920
ttaagctgctgggcaccttcctatcttggatgtcatattagcttatctacagcagaattt91980
ttactgttttatgtagtaaggaagcaattatatgattattttacagacaaattattcttt92040
atcttttatttttttagacggagtctctctttgtctcccaggctggagtacagtgtcgcg92100
atCtCggCtCaCtgCaaCCtCCgCCtCCtgggttcaagcaattctctgcctcagcctccc92160
aagtagctgggcttacaggtgtccgccaccacacccagctcattgttttgtatttttagt92220
agagatggggtttcaccatgttggccaggctggtcttgagctactgacctcaggtgatcc92280
acccgccttggcatcccaaagtgctggaattacaggcgtgagccaccgtgcctggcccag92340
acaaattattatactctgagtgttagaggcttaggatgttttcacttgatgctatgggag92400
gaataagtaataagatatgatacacaaccaaagacctttcttcactatgcttctagtagc92460
tagtactatggatgacacatggtaataatattggttagcatttgtcctcaatttactgtg92520
ctagttactcttctaagccccttacaggtatatattttttttcatcaataatcctctaag92580
gtagtttttattattgacctaattttataaatcaagaaaattaagacccagagaagtaag92640
taacttgtccaagatcacatggcttataagtggtagagccagaatttgaccccagatgtt92700
gtgactacattgtctctccataagcaggttcaactcttttgactggatgctgttccaagg92760
tCaCttCCttagagaagCCtttgCtgaCaaCtaCCCtCCtgtgCCCtCCtCCaaggCtgt92820
ccattgttctagaactttgaatactcatcttagaataaagctggtctaatttttacagtg92880
ttatagaatggatctctgactgcaaaagttggtcataattatctttttatgttctagtga92940
aaggcaaagaacaagagaagacctcagatgtgaagtccattaaaggtaagttctgccctt93000
ggCagtCCa.CtgCattaaaaagtgatgtgCtttgcatttgtgagttctttaatCCtgtta93060
tactctctcttttggcattaatcatttctgccttattttataattacttatgattttgat93120
ttatttccctctttaacctgtataatgctttaacatctagcatataataagtaggctttt93180
tttttttttttttttttggagacggagtcttgctctgttacccaggctggagtgcagtgg93240
cgcgatcttggCtCdCtgCaagCtCtgtCtCCCgggttCaCaCCattCtCCtgCCtCagC93300
CtCCCCagCagCtgggaCtaCaggtgCaCggCgCCa.CgCCtggCtaattttttgtatttt93360
ttagtagagacagagtttcaccatgttagccagtatggtctcgatctcctgaccttgtga93420
tccgcccgcctcggcctcccaaagtgctgggattacaagcgtgagccaccgcacccggcc93480
gtaagtaggctttttttaccttaattttatttttttgagatggagtcttgctcttatccc93540
CaggCtggagtgCagtggtgCCatCtCggCtCaCtgCagCatCCaCCtCCCgggttCaag93600
cgattctcctgcctcagcctCCCgagtagCtgggattacaggtggccgccaccatgccca93660
gctaatttttgtatttttagtagagacagggtttcaccgtgttggccaggCCagtCtCaa93720
aCtCCtgaCCtCaagtgatCCaCtCgCCttggCCtCCCaaagtCCtgggattaCaggCgt93780
gagCCa.CCatgCCtggCCataagtaggCttttactgagccttgtgtgtattggctatcct93840
agtgattaCagtgaaCCagtgCCCttCttattaatCaCaCatttaattgttCCCtaaaag93900
tgattagttcactttatttatttagtaagacaaaaaatgaagaatactcttaactgagca93960
gtctgttaactgtaggaaagcactgacacttataaggcttagttttctgtcatttatcca94020
gaagtatggttgattacagtttttacttttttatttgaatgaacaaccttaatttaaaat94080
atattttgtttattttttgttgggatcgatacattgtccttgtttatagattagagcatg94140
ctttttaaagatgctgtattactcactgattttatttgtccagtgtacagagattgaagt94200
gggaaaattataatggaaattgtttccatagtcattacatattaatttcatcaatttatt94260
tccataaaatctgtagattgctacttatttagatttttccttcaaatgtttttatgttgt94320
attgcttgcactgagtatttattctatatgctcaatttgctggagaagaagactaattat94380
aacttaggcaagttgtaaaattagggaaaaaagtaaggtaccttacagcctagtttactt94440
atttcttatgtaaagccagttagattccacattagttcaaactgccttctttgagcaaaa94500
cttgattggcagtgataaaggcttaaagcccttctcaagcagagacctgtaaagactaga94560
tctgactgtagtagaaggaaggaacttagatgtttcaggcagtgagaacaccagtcttcc94620
actctaaactttgccactaacagtatgaccttgggaagttgtaactttcttcagattctt94680
catttgttgaatggggggattggcctagctaatttctaaatctctactgggctaaaaaat94740
tctgtgcttatactctgattatgaagtacataatctgtgcttaacattcactgacttatc94800
cttaggataatacagaagcagtacaagaaacagcccctcaagatgtttgcagtctggtta94860
gaaagacaaacttatacacagaacagtagcaaatagaccaaaataataatagctgccatt94920
tatagaacacttcttctgttctgggcattagacaaaaactgactataacggtgaacaaaa94980
aagacttaggtcctgccctcattgaacttacagattagtaggggagaggaacattaatca95040
agtaattccacagatggcttagcctagattggtagtgatggaagtaaagagatgtgaacg95100
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-36-
gacttgaaaaaaaattcggaggcaaaatggatagaagtttattattgattaaatatgagg95160
tgtgagagagagggatatttaagattgatacctaccttctggcttgcctaacagaaccaa95220
aacaggaaattatatgttcagttttgttatgttgggtgggaggtgcttttgagtcattca95280
tttatatatgttatatatgttattttatatgcatagtaattttaaggtctgagttttaaa95340
ccaaaggttagagagtgattttttagagtctagcaaacctaagttgaaatcctgcctgtt95400
gaaatggctgtttactagctcattaacctagggcaaagtattcaacttgttttcattttt95460
gtcttcatctctaaaatgaggaaaatatggtcttacaagattgtcctgagagatagatga95520
aataatatccaaaaaaaaaaaaggtacatagagaaactcgtatagtgcctggtatatagt95580
aggtcctccattggtagctatcattatctagttttaacatagccttcagtttgttgaatt95640
agtcaaactgagtgaagcactgcaaggaattcagaggaatttgagatcaacaaatgattt95700
ctgaagtttagggaagacttcatggcaatgacacttaccttgtataaaagttgaagaata95760
agaaagatttgaatgagagattctttctcttctccctaccagcccagcttcttatttgag95820
gatatattgggcaaaggggccttcagacaagtagagggagatttttacagaaagattgag95880
atgaaggtatagaaggctgtaaagaccagaaaagagaattgagacagaggaagcaggaag95940
ccactgtaggtttttgagcaagatattgatgctgtaagtatggtgtttatgaaaggttag96000
tctggaagagatttgcaggatggagaccccggaagtttttttgttataatacagaaagac96060
ttgcactgagggtgaggtgttaaaaataaacaggtaagtaaatgtttaaacatcttgaag96120
gaaaagtcaacaaatcttggcaagtaaacagataacagtgaaaaagaatgggaccaagat96180
tttgagttttggagactggtggattgaacagacagggaaattgagaggagaatcagatga96240
tgatgttttaagttgatatttagacagattgtgcttgagatggtaaagtcaatgtgggtg96300
ggaatgcttagtagcgagtaatcagtgatacaagaccaaagcccaggtcaaagacaagtc96360
acagatacagatcagggctttttcatctgctccacagaggtgtaccctaggagctgttgc96420
aaacagtccatgtggagggtgtgagtaagatgtttcccttgaatttgccagaattacttt96480
tttgttgttgttgttgttttttctgagacagattctcgctctgttgcccaggctggaggg96540
cagtggcgagatcgcgcagctCc'LCtgCaaCCtCtgCCtCtCgggttCgagtgattCtCCt96600
gcctcagcctcccaagtagctgggattacaggcttgtgccaccaagcccagctaatttct96660
tttgtatttttagtagagatggggtttcaccatgttggccagactggtctcgaactcctg96720
gcctcgtgatctgcctgcctcagcctccaaaagttctgggattacaggcgtgaaccactg96780
cacccggtcccttgttaagtttattttggtgggaagcaaaggaggtttcagcttttaaaa96840
agtttgaaaattattgctctggtaataattaaagatttgagagtaaatatgctttctagc96900
agaaagaataaaagaagaacagatagcctcaagaaggggagccaaagaagcaggctatat96960
ctgacacactgggtgttgataaatgggtattaaaagaatgagagcaatgagcagatagaa97020
gaggaaattaggagagtataataccatggagaccaagaaagatagactatcaggaaggag97080
tggtaaaaataagttactagttctaagagagatgttaagagggaccggggaaagccttgt97140
acaaatgagttagtagcattttacattatatacatctaattaagaaacaatgcgagagtc97200
tcaccattcctatagactcttacttgtacttgtctgaacacgaaaactggcttttgttta97260
taaataagctaaaaattattttgctccaatttctcatgaaaataaaaataaaccttcttt97320
taacattgaaaaaatagtttgaagacagtcactcttcattttgtaattcccacaactatt97380
attgaatgactgaaattatctttattctgaagccaaaggggtgatactgatatttcttca97440
gactactaaaaatatattttatgaatttttagtgtgctttatctttttttgttttttttt97500
ttgagatggagtttcactcccgttgctcaggctggagggcagtggtgcaatctcagctca97560
CtgCaaCCttCgCCtCCCagattCaagCaattCtCCtgCCtCggtCtCCCaagtagCtgg97620
gattacaggcacctgcccccacacccagctaattttttgtatttttagtagagacagggt97680
ttCaCCatgttggtcaggCtggtCttgaaCtCCtgaCCtCaggtgatCCaCCCa.CCttgg97740
CCtCCCaaagtaCtgCgattgcaggcatgagccaccatgcctggcctgaggaatattttt97800
ctaggttccccccaccccaagcatttattctgcaattttagttttgttcctaaagcaagc97860
aaggtttaaggatttaaaaataatccgtattttagaatgctttctggctttgttactttt97920
tatccacagtagaagttctcagagaatgatctccctcttttaatttaactttttggcaca97980
gtattttgagaattataaataatattagaatgttttctggctgggtgtggtggctcatgc98040
ctgtaatcctggctacttgggaggctgaggcaggagaatcacttgaacatgggaggcaga98100
ggttgcagtgagccgaggtcatgccactgcactccagcctgggtgacagagcaagactct98160
gtctgggaaaaaaaaaaaaaaaaaaaagagtgttttctttcctattttccaccacttgat98220
taagttacttttcctcttaagtattttttgctgagtatgctgacttaagagtaatgttac98280
aaaatttaatttttaaagttctctgaaagcccctttatgagagttttaggctatcaaatt98340
gtgtttaattcttaacaattttttgaaaaattatagcttcaatatccgtacattccccac98400
aaaaaagcactaaaaatcatgccttgctggaggctgcaggaccaagtcatgttgcaatca98460
atgccatttctgccaacatggactccttttcaagtagcaggacagccacacttaagaagc98520
agccaagccacatggaggccgctcattttggtgacctgggtaagtaactatcatttttta98580
ttaacttgtattagaaggatttgagtacaatatgtgaaacttctgtcataggatacagaa98640
ctatataattggaaagtgctttggaaaaaatgtatttaaaataacagctacaagtataat98700
gggtagctgtgttgtgttcctgtaaatatagaatataaagcatgcccagtagaaaaacaa98760
gcatttccagaagaaatatatctgatcactaaatataaatatatgaaaaagatgtctcac98820
tttattactgagggaagtgcaaattaaaataatcagttaatgttctcctaacacattagc98880
atattttttaaagtttgacaatttgaatgtcagtgaagatgcagggaaatacccctccta98940
tttagtgataatataatctggtgaagactctttggaaagcaatttggaaatcagtataaa99000
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-37-
atatgcatgt catttaggcc actctttcta agacctagcc ctcagatatg ctcattcata 99060
tgtgcaggtg tgtatgtgtg tgtgtgtgtg tgtgtgtgtg tgtatatgta tgtatgtatg 99120
tatgtatgta tgtatgttga aggctattca ttatagtatt gtttgtgata gcaaaaaatt 99180
atggacaaca tataaatatc tgttataggg aaataaccaa attgtggtat acgcatgctc 99240
tggagtataa tatagccatt tgtttctatt tatttatttt cttgagacag ggttttactc 99300
tgttgcccag gctggagtgc agtggtatga tcatggttca ctgcagcctt cacctcctgg 99360
gcacaagcca ttctctcgcc tcagcctcca gagttactag gactgcaggc atgtgtcacc 99420
acacccagat aattttttaa ttttttgtag agacagggtc tcactatgtt gcctaagctg 99480
gtctcaaact cctggcctca agcaattctc ccacacaggc ctcccaaagt gctgggatta 99540
ccaacgtgaa ccaccacacc tggttcagtg tagccattta gaaatctaaa aaagacgtgg 99600
gaaaatgtct aaggcatgtt taaatgtgag aaaagcaagt cacagtatgc atggtaaaat 99660
ccgttatatt aaaataagtt cttccaaaac aaaaacatat gcaggagacc tttattttgt 99720
cagtatttct tacccaaatt tctgcactta gaaaattgca tgtcatgttg tcataagttg 99780
aaaaaaagat ccatgaacca atggacttct aataaaatca gtcctgcttt tgacatctct 99840
ctctactttt gtgtatattc aaaccagagt gtcaatgtgt ttgtggggca cacttagcaa 99900
taatacatag cagacaaaat gcatatagct cagagagtaa aattgtaagt tttgctagat 99960
cactcataaa ttgctgatga gaatttaaaa tggtgcagat gctctggaaa acaggcagtt 100020
tctttctttc tttttttttt tctttttgag acagggtctc actctgttgc gcaggctgga 100080
gtacagtggc gtgattacaa ctcactgcag cctcaccctc ctcaggttca ggtgatcctc 100140
cctcagtctc ctgagtagct gggactatag gcatgcacca ccacgcctgg ctaatttttg 100200
tatttttttt tttttttttt gtagagacgg ggtttcgcca tgtttcccag gctggtctca 100260
aactcctgga atcaagcgat ccacttgcgt aggcctccca aagtgctggg attacgggcg 100320
tgagctactg tgcctggcct aggcagtttg tttgtttgtt tgtttgtttg tttatttatt 100380
tgtagacgga gtctcacagg ctggagtgca gtggcccaat ttttggctca ctgcaacctc 100440
cgcctcccag gttcaagcta ttctcctgcc tcagcctcct gagtagctgg gatgacaggt 100500
gcctgccata atgcctggct gatttttgta tatttagtag atatggggtt tcaccatgtt 100560
ggtcaggctg gttttgaact cctgacctca ggtgatcagc ccgcctcggc ctcccaaagt 100620
gctgggatta caggcatgag ccgtcatccc tggctggtgg tttcttatga cgtgaaacat 100680
gcaattacca tatgacctag cagttgcact Ctgtatttat cccagataaa tgaaaactta 100740
ccttccaata aaaacctgtg cacaaatgtt catagcagct taatattgaa aaactggatg 100800
ttcttcagca ggtgaatgaa ctggttcatt cataccatgg aataccattc agcaataaaa 100860
aggaacaaac tgttgataca tttaaccacc tggatgaata tcaagggaat tatgctgtca 100920
gacaaaaacc agtccctaaa gactacatat agtatgattc cgtttggata atattcttga 100980
aatagagaaa ttaagagaaa tgaaaagatt agtgtttgcc agatgttaga gacagggagg 101040
tgagaggggt aagtgggtgt agttataaaa gtgcaacatg agggatcttt gtgatgttga 101100
agttgtatct tggcagtgga tgcagaaatc tcaatgtgat aaaattacaa agaactaaaa 101160
acaagaatga gtatagataa aactggggaa atctgaacaa gttagagtgt tgtatcactg 101220
tcagtatctt agagtgatat tgtactatag ctttgcaaga tgttaccatg ggagaaacta 101280
aagtgtacaa gggatctcta ggtattatta tttttttaga gatggggttt cactatgttc 101340
cccaggccgg tcttgaactc ctgggctcta gtgatccgcc tgccccagcc tcctaaagta 101400
ctggaattac aggcgtgagc gaccatgcct ggccctttca gtattgtatc ttagaacttc 101460
atgtgaatct agcattatct catagaattt aattaaaaga aattgtaaac ctcacagaag 101520
atcagaattt cctcaagttt gtgatgttga caaagatgaa ctagttgaca ctgacagtaa 101580
gactgaggat gaagacacga cgtgcttcaa aaaaatgatt tgaatatcaa tggattaaga 101640
agaactcttt tgacaaattg atgaaaccct cagtcagttt tataagaatg cccatcttta 101700
tgatcatgct atgaaagcca atttttaaaa aaattttttg tctttcctaa caattagctt 101760
gtggttataa tttaaattta gttaaatata agataaatga ttttttatta agtttagttt 101820
catttttcaa ggtacgatct caaagctact ctttaaccta ctatgaatga ataatgctga 101880
gttcataaca tctttgtaga tatatccaca attttccctc aggataagtg cctacaagtg 101940
gaattactgg actgaaaata atgcagtttg ctaagacttt gctatctgtt cctgaatgct 102000
cctccaaaaa ggttttgcca gtttacatcc tcatgaccag cgaatgagag tgttgcctat 102060
tttcctgtgc ccttgttact gcttaataat ttttgaaaaa aatctaattt gacagacaaa 102120
aatgcatttt atgttaattt gcttttctgg gatttttaat gaggttgagt atagttttta 102180
atatttttat tggccccttt ggaactagta tcataagttt tttttcttaa gaatttatgt 102240
agtctgggct gggcgcagtg gctcacgcct gcaatcccag cactttggga ggccgaggtg 102300
ggtggattgc cgaaggtcag gagtttgaga ccatcctgac caacatggtg aaaccgaatc 102360
tctactaaaa gtacaaaaac tagctcagcg tggtggcggg tgcctgtaat cccagctact 102420
taggaggctg agtcaagaga atcgcttgaa cccgggaggt ggaggttggt tgcattgagc 102480
cgagatcgcg ccattgctct ccagcctagg caacaagagt gaaaagtctc aaaaaaaaaa 102540
aaaaaaaaaa aaaaaagaat ttacatggtc tgaattgcca ttaaaagaga tatgagaatt 102600
attgagtaac aaataacttt ttaataattt aggcaagttt tggacgattg tactttgttt 102660
agaaaccaaa agcatagtat ttgtagtttt tttatttact ttagttgcta ggaagtaaac 102720
tttattcaag gtctctggta ccagttgttg ctaaaagtga ttgactaatc tgtcaatctg 102780
aaattatttg ttgctgaact gctaattctt ttgcttctat cttttaggca gatcttgtct 102840
ggactaccag actcaagaga ccaaatcaag cctttctaag acccttgaac aagtcttgca 102900
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-38-
cgacactatt gtcctccctt acttcattca attcatggaa cttcggcgaa tggagcattt 102960
ggtgaaattt tggttagagg ctgaaagttt tcattcaaca acttggtcgc gaataagagc 103020
acacagtcta aacacagtga agcagagctc actggctgag cctgtctctc catctaaaaa 103080
gcatgaaact acagcgtctt ttttaactga ttctcttgat aagagattgg aggattctgg 103140
ctcagcacag ttgtttatga ctcattcaga aggaattgac ctgaataata gaactaacag 103200
cactcagaat cacttgctgc tttcccagga atgtgacagt gcccattctc tccgtcttga 103260
aatggccaga gcaggaactc accaagtttc catggaaacc caagaatctt cctctacact 103320
tacagtagcc agtagaaata gtcccgcttc tccactaaaa gaattgtcag gaaaactaat 103380
gaaaagtgag tatgtgattt tcttgtgtgt acatatgtgt ctcactttct ttttttaatt 103440
tactaagcag aacttcagat gaggaataaa atgattggaa tatttttttt ctcctctaac 103500
tacttgtaaa tttgggagaa tttggagagt gtagtagagt cagatcagtg tatggaaaag 103560
gagcaggagt gactggacct tctaagaagt gtgttatcag aattagtaaa tgaagggtca 103620
aatgtcctac ttttcccctc cactgatttt gacatcaaac cattatccac atagccttat 103680
ttcctccctc ggtcttaatt ttattaatat tttactgcac tttgcagata aaatttttaa 103740
aaaattttta aaaattgcca ataagtgaca tttattaagt tcagtgctta gtgtatattt 103800
ggattttatt tattagtcac aagacctttg tgcaggtagt aggcatgatt atcttttttt 103860
ttttgagatg gagtcttgct ctgtcgccca ggctggagtg caatggcgcg gtctcggctc 103920
actgcaacct ccgggttcat gccattctcc tgcctcagcc tcccaaatag ctgggactac 103980
aggcgcctgc caccacaccc ggctaatttt tttgtatttt tagtagagac ggggtttcac 104040
catgttcgcc aggatggtct cgatctcctg actttgtgat ccgcctgcct cggcctccca 104100
aagtgctggg attacaggca tgagccaccg cgcccggact gattatctta tttacacatg 104160
agaaaaccag ggcttagaaa ggttaggtaa cttcctctag gttgtacagt aaatgtggac 104220
ctagaagcat tttgacaaga gcacctgttt ttttttcttc tctattagtt tagaaattat 104280
atactcttaa ttatcacctg ggattttgat tagacagcct tcatgttctt tttcatctta 104340
aatgttcttt gtgtcttaaa gggctaagtg atttcttcag atcttttagt tcactcattc 104400
tcagtgaact aaaatgaggt ctaatctgct actgaatcaa gttttcagca tgttatttcc 104460
ttcctccctc cctccctcct tccttccctc aaccaggctc ccgaggagct gggattacag 104520
gcgcccgcca ccactcctgg ctaattttta tattttagta gagacggggt ttcaccatgt 104580
tggtcaggct gatcttgaac tcctgacctc aagtgaccca cctgcctcgg cctcccaaag 104640
tgctgggatt acaggcatga atcaccacac ctgacggcat gttattttca tcgc2aagtt 104700
actgtaagct gggagaagtg gcacacactt gtactcccag ctactcagga agcttaaggt 104760
gagaagattg cttgagccca ggagttttga gaccaacctg ggcaacacag caagacccca 104820
gctcaaacaa agaaaaaaag ttattgaatt ttttatttct atggatcatt ttttgtagtt 104880
tcttattcct ttcacccttc attcccactt ttgatcccat cttttattta tttagtttta 104940
ttaaatgtat atttgtctga taattctgct atctacagtt ttttgtggac ctgactcagc 105000
atttctttgt ttcttcggat tcagactgtt ggtggcttgt gattttagtg atttttggcc 105060
gtgaacatgt ttcttggact tttgtctgtg ggaattctct gtgtactctg tataaattaa 105120
gttacttcag gtgttttgca ttttcttttg ccatgcacct ggggcctggg tcacta_CCCt 105180
tctggtacca cttaaaactg aatttttgtc ttgggtgctc gtactgatcc tgtatgagta 105240
caggtttata cttactgtag aaatatggtg tttgattatg gggtattgtc ccagatggtg 105300
ctggagtatt aatatgctct ctgttaaact taatgtgttg tccctgtaaa actccaaaat 105360
tctgaattcc agaatactac tggccccaaa tgtttaagat aagggcactg cctgtatttg 105420
tttctgcctc ccactatttt ccttagttta acacaaactc ac,ctttttaa aaaacatttt 105480
gagagaattc agtattggga agagtttcta acctgtttct ggaaatggaa gtccaaagtc 105540
tgtttctgta attgtttttt ttttgagatg gagtctcact ctgtcaccca ggctggagtg 105600
caatgacgta CtCtCagCtC aCtgCaaCCt CCa.CCtCCCg ggttcaagcg attctcttgc 105660
CtCagCCCCC tgagtagCtg ggattaCagg tgCCCaCCdC CatgCCtggC tgatttttgt 105720
atttttagaa gagatggggt ttcgccatgt tggccaggct ggtcttgaac tcctgacttt 105780
gtgatctgcc cacctcagcc tcccaaagtg ctaggattat gtttctgtaa ttgtaataca 105840
tttattgttt ttagaaactg tctttgcttt agtggtaatt ttcaataaaa atagaaatag 105900
cagtggagtt attaaaagag cattagttac atttttccct ttttcattat cttcaaatat 105960
tatatatagt aagtttgacc tttttaaaat gtatacttgt atcagtttta acacatacat 106020
agattcctgt aactgtcacc actataaggg taaagaacag ttagttcctt cacctttgaa 106080
gtCaagCCCC aCCtCtatCC CaaCdCttgg CaaCCgCtga tCtttCtCCg tCtCaatagC 106140
tttgcctttt ctcttttttt ttcttatttt tttttttgag acagcgtctt gctctgtcgc 106200
ccgagctgga gtgcagtgag gcaatctcgg CtCaCtgCaa CCtCCgCCtC CtgggttCaa 106260
gcagttctcc tgccttagcc tccctagtag ctgggattat aggcacgcac caccacaccc 106320
ggctgatttt tttgtatttt tagtagaaat ggggtttcac catgttggcc aggctggtct 106380
caaactcttg acctcaagtg atccacctgc ctcggcctcc caaagtgctg ggattacagg 106440
cgtgagccac tgtgcccaat caggactttt tttttttaaa tttacattca acttgtcatt 106500
tttttcttgt atggattgtg ccttcagagt cacacctaag agccctttgc ctaagcaaag 106560
gtcatgaaga ttttctcata tgtttccttt taaaagtatt gtggttggcc aggtgccatg 106620
gcttatgcct gtaatctcag cactttgaga agctgaggtg ggcagattac gaggtcagga 106680
gatcgagacc atcctggcta atgcggtgaa accccatctc tactaaaaat acaaaaaaaa 106740
aaaaaaatta gccgggcgtg gtggcgggca cctgtagtcc cagctacttg agaggttgag 106800
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-39-
gcaggagaat agtgtgaacc cgggaggtgg agcttgcagt gagccgagat cgcgccactg 106860
cactccagcc tgggcaacac agtgagactc catctcaaaa aaaaaaaaaa agtattatgg 106920
ttttacactt tacgtttaga tatatatctt ttttgagtta atgtcgtata agtatgaggg 106980
ttacgtcaga ttttttgttt tttgtttatt tttacatatg gatgtctagt tgttctaata 107040
ccatttgttg aaaagacaac ctttactcca ttgaattgcc tttgtacttt tgccatattt 107100
gtctaggcct gtttttggac tcctttttct gtttcatgat gtgtgtgtct attcctttgt 107160
taataccaca tggtcttaat tactgtatag taagtcttaa aattgggtaa tgctggcctt 107220
ataaaacgaa ttgggaagtt tttattttta ctcttatttc cattttctag aagagattgt 107280
gtagaattgg tgtcatttct tctttagata tttggttgaa ttgggaagtg atgccatctg 107340
ggcctagggt tttgtttttt gtgtgtgaga cagagtctca cttctgtcac ccaggttgga 107400
gtgcagtggt gagatcttgg cttactgcaa cctctgcctc ccaggttcaa gttatcctcc 107460
tgcctcagcc tcccaaatag ctgggattac aagcgtgtgc caccatgccc gactaatttt 107520
tgtattttta atgcagacag ggtttcacca tgttagccaa gctggtctcg aacttgtgac 107580
ctcaagtgat tagcccacct tggcctccca aagtgttagg attatagatg tgagccaccg 107640
tgcctggcag gggcctaggg ttttcttttt cagagtattt taaactatga attcagatta 107700
tttaatagat ataggactat ttaagttatc tgtttcttct tgagtgaatt tttactgtag 107760
tttatggcct ttgagtaatt aattgtattg aattgtcaaa tttatgagcg tgtaattatt 107820
tatagcattt cgggtttgta gtggtatccc tcttttattc ctggtgttgg caattgtgtc 107880
ttgtttttct ttgtcagatt gtatagggat ttattagtct tttcaaagaa ctagcttttg 107940
ttttgatttt tctgttgttt tgttttcaat tttattgatt ttctgctctt tattatttct 108000
tttctattat ttctgcttgc tttgggttta ttttactctt ttttttttct ccaagttgct 108060
taaagtagaa acttagattt ctggtttgag acctttcttt tctaagataa gcatttaata 108120
ctgtaaattt ccttctaacc actgctttag ttacaccccc acaaattctg gtattttgaa 108180
ctgagcacaa atgaaatgtt ctaatttccc ttgaatctta ttcttttacc aatgaattat 108240
ttagaaatat gttatttagt ttgcaagcaa ttggagactt ttttcctgtt atttttctac 108300
catttatttc tcatttcatt atattatggt cagagaatat attttgaatg atttcattta 108360
ttaattttta aaaataacat taaaaaattt tttaaaatgt gaatatacca catacagtat 108420
aaagattgta cattctgttt ttggacagtt ttctataaat gtcaagttga tttagttggt 108480
taatgatggt gttcagtttt tctttattct tgctgatact ttgtatgcag ttatatcact 108540
ttattactca gaagagtgtt gaactttcca actacaattt ttttttccaa ttttactttc 108600
agctctatct ggttttgctt catgtatttt gaggctctgt tgttaggtgt gtacacattc 108660
aggatgatat cttctgggtg aattgcctgt tttatcatta tgtaattccc tctttatggt 108720
aattttcctt gttctaagat cagaaatatc tgttgtccaa tttatataga cactgcagct 108780
ttcatttgat tagtgcttgc atggcatatc tttttccatt tttttacttt tgatctacct 108840
ttataattct atttaaaggg ggcttcttgt aggcagcata tagttgggta gtgttattta 108900
tttatttatt tatttattta tttatttatt tattgagaca gagttttgct cttgttgccc 108960
aagctggagt gcagtggtgc aatcctggct taccacaacc tccacctcct gggttgcagt 109020
gattctcctg cctcagcctc ccaagtagct gggattacag gcacgcgcac catgcctggc 109080
tgattttttg tatttttagt agaaacggat tttcaccatg ttagccaggc tcgtcttgaa 109140
CtCCtgaCCt CaggtgatCC aCCtgCtttg gCCt CCCaaa gtgCtgggat taCaggCgtg 109200
agccactgca cccggctgag tcatgttatt tttaatcttt tctcacaata cagggttttt 109260
gttggtaaat ttaattattt taatataaat tttagtataa ttatttacat taaatgtaac 109320
tgttgCactg gggtatttat aatgtgtaaa tataattatt ggtattaata taattatatt 109380
actcataata atattaatat ctttggattt agattaccag tttagtatat gtttttctgt 109440
ttctccctct ttgatttccc cttttttgct tttttttttt ttttaattct tatttttttt 109500
tagtatttgt tgatcattct tgggtgtttc ttggagaggg ggatttggca gggtcatagg 109560
acaatagttg agggaaggtc agcagataaa catgtgaaca aggtctctgg ttttcctaga 109620
cagaggaccc tgcggccttc tgcagtgttt gtgtccctgg gtacttgaga ttagggagtg 109680
gtgatgaCtC ttaaCgagCa tgCtgCCttC aagCatCtgt ttaaCaaagC aCatCttgCa 109740
ccacccttaa tccatttaac cctgagtggt aatagcacat gtttcagaga gcagggggtt 109800
gggggtaagg ttatagatta acagcatccc aaggcagaag aatttttctt agtacagaac 109860
aaaatggagt ctcccatgtc tacttctttc tacacagaca cagtaacaat ctgatctctc 109920
tttCttttCC CCaCatttCC CCCttttCta ttCgaCaaaa CtgCCatCgt CatCatggCC 109980
cgttctcaat gagctgttgg gtacacctcc cagacggggt ggcagctggg cagaggggct 110040
cctcacttcc cagatggggc agccgggcag aggcgccccc cacctcccag acggggcagt 110100
ggccgggcgg aggCgCCCCC Ca.CCtCCCtC CCggatgggg cggctggccg ggcgggggct 110160
gaccccccac ctccctcccg gacggggcgg ctggccgggc gggggCtgaC CCCCCaCCtC 110220
cctcccagat ggggcggctg gccgggcggg ggctgccccc cacctccctc ccggacgggg 110280
cggctgccgg gctgaggggc tCCtCaCttC gcagaccggg cggctgccgg gcggaggggc 110340
tcctcacttc tcagacgggg cggccgggca gagacgctcc tcacctccca gatggggtgg 110400
cggtcgggca gagacactcc tcagttccca gacggggtcg cggccgggca gaggcgctcc 110460
tcccatccca gacggggcgg cggggcagag gtggtcccca catctcagac gatgggctgc 110520
cgggcagaga cactcctcac ttcctagacg ggatggcagc cgggaagagg tgctcctcac 110580
ttcccagacg gggcggccgg tcagaggggc tcctcacatc ccagacgatg ggcggctagg 110640
cagagacgct cctcacttcc cggacggggt ggcggccggg cagaggctgc aatctcggca 110700
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-40-
ctttgggagg ccaaggcagg cggctgggaa gtggaggttg tagggagctg agatcacgcc 110760
actgcactcc agcctgggca acattgagca ttgagtgagc gagactccgt ctgcaatcct 110820
ggcacctcgg gaggccgagg caggcagatc actcgcggtc aggagctgga gaccagcccg 110880
gccaacacag cgaaaccccg tctccaccaa aaaatgcaaa aaccagtcag gtgtggcggc 110940
gtgcgcctgc aatcccaggc actctgcagg ctgaggcagg agaatcaggc agggaggttg 111000
cagtgagccg agatggcggc agtacagtcc agcctcggct ttcacaactt tggtggcatc 111060
agagggagac cggggagagg gagagggaga cgagggagag CCCCtttttt gctttctttt 111120
ggattatttg aatttttcct taaatttatt tatcttactt atttatttat ttttttgagt 111180
gattctcctg ccacagctcc caagtagctg ggactgcagg catgtgccac tacacccagc 111240
taattttttt gtatttttag tagagacagg gtttcaccat attggccagg ctggtcttga 111300
actcttgacc tcaagtgatc cacctgcctc ggcctcccaa agtgctggga ttacaggcgt 111360
gagccaccat gccctgcctt tttctagaat ttatatattg agttcttgat tgtatctttt 111420
tatgtaggct ttttagtggc ttctctagga attacaatat acatactttt cacagtgtac 111480
tcacatttaa tattttgtaa cttcaagtgg aatgtagaaa acttaaccac cataaaaata 111540
gaactaggga tgaggttaaa aaagagagag aaaagaaatg taataaagat ttaataacac 111600
cgtttttttt tttttttctc tttttttttt gagacagagt ctctctttct gttaccaggc 111660
tggagtgcag tggcgtgatc ttggctcact gcaacctccg cctcctgggt tcaagtgttt 111720
ctcctgcctc agcctactga gtagctggga ttacaggtgc gcgccaccat gcccagctaa 111780
tttttgtatt tttagtagag acggtttcac tgtgttggcc aggatggtct cgatttcttg 111840
accttgtgat tcgctctcct cagcctccca aagtgctggg attacaggcg tgagccaccg 111900
cgcccggcta agtctttaaa tatttttttg acattgcact ttttctcttt tccttctagg 111960
attttagtaa cccaaatgtt agttttgtta ttgtttggca ggttcctgag gctttcctta 112020
cttctttaaa tttttttttc ctgttgttca gcttcgaaaa tttctattca tctgtcttca 112080
aattcactgg ttctttcccg ttatttccat tctgttattg agtctttgta gtgaatttta 112140
aattttgttt attatgtttt ttagttctaa aattttcttt ttttgtgtat gtcttatact 112200
ttgctcctga aactcttatt tgtttcagga gtgatcttat ttcttagagc atggttttag 112260
tagctactta aaatttgttt tatcatccca gcatatgtgt cctcttgatt gtcttttctc 112320
ttgtgagata atgggatttt ctggttcttt atatgacaat taattttgga ttgtatcttg 112380
gacagtttga cttacgttac atgattctga atcttgttta aatcctgtgg aaaatattga 112440
agtttttgct ttaacaagca gttgacctag ttaggttcag tccacaaatt ctaagcagca 112500
ttctgtcggc tctggttcca tcatcagttc agttttgtat cttatctgct tatgtgcctt 112560
tctgtgtcca gtctgggacc tggccaatgg tcaggtccca aagcctttgt acacttttag 112620
aagcagggcc atgcacaccc agctcacgag tggccccggg agtgcacata caactcgacg 112680
ttttcatggg ctccttcttt tctgtgatgt ccctgacacg ttctgccttc taagaacctc 112740
CCtttatCCC tttCCtgttg tCtggCtaga aagtcagggc tttagattCC CtataCttCa 112800
gCaCaCttCC tgtagCtatg tCaaCCtCtg tggCCa.CgaC ttCttCttCt tgggaCtgCa 112860
gtttctcttg tcagaaagta ggattcttgg agctgctgtc attgctgctg tggctgctct 112920
gatgctgcct gggagtcgaa ggagagaaag gaacaaaaca aaacaaccca ggggatttcc 112980
tccactctct ttgatccgtg agagccccct ttcctgttcc tcagaccaga aatagagggc 113040
ctgtcttgga acttcttctt tgtgcatctg gtgtgcagtt tcagcttttg agtccaggcc 113100
aggaggtgct ggacaaactt gtcaggagta cggaggtact gcaagttctg attacttttc 113160
tCagtCCaCC tgcttccaag tccttggatg catttgtcca ttgttttgag ttgcattcca 113220
tgggagagac agaagagtgt gcttatttca tcttgacata cttattagga tttcatatca 113280
aatcaacgga tgatattctc tatattaatt tgctgttttc cctttagcaa gcacattagg 113340
aaaataacac tttaacaccc gcctttggtg gtttctgtca taattattaa tacttgactt 113400
tttttttttt tttgagacgg agtctcactc tgtcctttga ggcattgtcc ccataaactt 113460
ttggtaaagc atcaataatt ttatctttca tccacacaag cttcaccata aatttgatgt 113520
ttattcttcc attttagcag aattcatgtt gctccaatag gggctgtctt caaactgatg 113580
ttttctcctt cttagtgcct cagagtagat cctgttcaga tacgttataa caggttaata 113640
tgagtttatt ttggtgtaaa agtactttga aattcatgca tagttttttc atcatatgca 113700
ttttccatag ctttgaacac ccccatgtaa ctctcctctt ccacaaacca aacaatgaaa 113760
aagcaccttt gtgatggaag tttattttgc aataggaact cacagtgatc taagccctgc 113820
tattcatgaa tataattcat tactggagtc caagttgctt tttggttttt gaagttctct 113880
tcttcccttg caggtataga acaagatgca gtgaatactt ttaccaaata tatatctcca 113940
gatgctgcta aaccaatacc aattacagaa gcaatgagaa atgacatcat aggtaagcag 114000
tgcttgaaac tatggcaaaa aaaaaatgac aaaaaatgca cagaactgac aattttcgtt 114060
attgactaag ataatttttt cttaacatgg aatttagcag ttcccttcct aatttgtttt 114120
ctgagtattt tttatatcgg attatagctc actttaaaag tttctcggct gcattcggtg 114180
cgagggtctt tgcctgggcc agatgggctg cagtgtagcg ggtgctcagg cctgcccgct 114240
gctgagcagc cgggccggcg ggcggctacg ctaaccggca cagaccaccg gatggactgg 114300
ccggcagccc cgcaccagtg cacgaagtgg gcgggacaga aacttctggg gttggaagtc 114360
cagtgaggct aaaagccggt accaaagtct ctaggcatca gggctgcagc ccaagagtct 114420
cacgaccagt gggcaactgg atggccagac aggtgtctca gtggtggcct ctccgtctca 114480
gggcttcatc ccacttctca gtgggcctga cgtccctggg caccctggat gtctacctgc 114540
attagccaga gccatcacat ggcctgtgac ttgccttttt ttgccagttg attgtgccac 114600
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-41-
acacagtgtc atttctgtgt catttggcac agctggaggt gcaaggagga gggcagcctc 114660
atgtccagtc ccagtttcac gtaactttat tcttctgaat aaagacaatt tgctaacctt 114720
aaaaaaaaaa aaaaaaaaaa agtttttctt atatgttgga cccaaattct taggctttaa 114780
cctgaataac aatgacagca agatcaataa atagtacaca tttattaaac actcactgtg 114840
tcccagacaa tattccaagc actttttatg gatagactca ttttaacttc taaagaactt 114900
tgtgggataa atacagttat tttatagatg aagaaactga agcacagaga agttaagtgc 114960
tttgtccagg gtaacagctc agatatggca gagtcaggat ttgaaactag accctcacat 115020
accttaactg ctgtgctgtg gcagtgtttt tcatactgta ggttgggacc agccttctct 115080
tatgccctca ccccctgcca aaaaaaaaaa aaaaaaaaaa aaatatatat atatatatat 115140
atatatatat atatatatat aatatatata tatataaaat atatatatat ataaaatata 115200
tgtattagta tatatgcata tatagtatat attatatatt agtatatata ctaatatata 115260
atatacatat tagtgtgtgt atatatatat atactagaat aaaaaaatca aagtatctca 115320
gagtagtaag gacaaacatt tcagaaaaat gttttcatta tatatacatg tatgtatgtg 115380
tatgctgatt caacaaatat atttcttata ggttatagca aaatagtttg aaagctttta 115440
ctgtgtttta tcaggaagac cttaggtgaa cgtatattca cagataaaag aggttattta 115500
ttcattcaat aaatattaca ttctcataag tcctaatatt atgtattttt attcttcaaa 115560
aaagttagta tttgtgattt atgaaataag acatgttctt gcacttttag cagatctgtc 115620
ccgatgttgg gcttctttaa tccttagtgt gggtgctttg cactcactca ctgctgggga 115680
cagcaagacc cctgttagtc tcagctgtgt ttcttaaatt ggcccactgt accttccagt 115740
tagctattct ggggtccatg tcatgttggc tccattttcc ttttctttct cccacacaga 115800
taCCtataaC ggctataaca taggcctggt ggctgttggt ggcttatccc tatctgcttg 115860
tatttaaggg gtactgtttc actgagtttt gctgacagat gttgtcatga gatttgaggt 115920
tttctgtgtt gttgctctat ttttatgtgg gaatttgcta ctatcatcat ccctagacca 115980
gcttttccta gtaatacaac agggatgttc tgactgatta gagtttgcct gtttgaagaa 116040
ttggttggct agtgattttt ttttgagggg agtctgtacc agttaatagc ctgactggcg 116100
tgtggataaa aaggaagcag tttcaagtca aataaaacac ttaaaatgaa accacactgc 116160
aactctcttt cttttactta agcttaatca aattaatgat gatgtaatcc catgaaggaa 116220
aagtcttctg aaggatcaag ttgataacat tttgtgatca aagaatttga gaaaacctct 116280
atcccagtgt ctatcattat atattttagg atgttaatta cctgtgtggc tttaggcaag 116340
tcatttttcc tccttgagcc ccattcttaa tcctgtccaa attatttgtc tcctcttgca 116400
gttggactat tttaatatag ctgtccttca agtgagtttt gttcaaagga gccttcactt 116460
tagctcttac tgtgtaccca ctttgcatag tcttgtttta aatgtaatcc ttggattttt 116520
ggtgttgcta actaattact gtttttatgt gaggatttag agtgatccag aatctatact 116580
tgcactacct ccttcatctt ccacaaatgt ttgaagtggt agaattttta aaaactttga 116640
aggtacagct gacagaattt gctgatggtt tggaagtgag tggtatgaga gggaaaaaaa 116700
ggaataaagc atgactgcat tttttgtttg tttgtttgtt tgtttttgag acggagtctc 116760
actctcgcca ggctggagtg cagtggcgtg atcttggctc acggcaacct ccgcctcctg 116820
ggttcaagcg attcccctgc ctcagcctcc caagtagctg ggactacagg cgctcgccac 116880
cacgcctggc taattttttt ttttgtattt tagtagaaac ggggtttcaC cgtgttggcC 116940
aggatggtct ccatctcctg acctcatgat ctactcacct tggcctccca aagtgctgag 117000
gttacaggca tatatataag catataaagt gtgttatagc atacaaacag gtatatatat 117060
aaacatgcag tccacacagc tgataggaat gaggcagtag tgaaggagaa gttgatgtag 117120
gagaggggac agttgttaca ggaaagaagt ctggaggcag aagggatgaa ttccagtgct 117180
cacatagaag attgcttaga tgggagcaag gacaatttat ctagagtcac aggaaagaat 117240
gcagtacacg ggtagagatg caggtgagtt gaaagatgtg agagatgatg gaaataattt 117300
tctgattgct tctatattct caaggaagca ggaagcaaag tcctcagcaa agagaataga 117360
agaggtgtta aatatttgag aaaggagatg tactgtagaa aaaaaaaaaa ctcagtttct 117420
ccttctgaac tctcacaaaa cagaaccctt ccatgactct agttgtgtgg ggttttttcc 117480
CtgtCagCta CCaattCtgC agatgattgt tCagtgaaCa CCaaCtgggt gtCCtCtaag 117540
tcagttcagt tctcacactg tttacctgga gatagcatca gatcccacag attgaggact 117600
ctgtcccaca agactgcctc cacttcagat gccagtctca agtacaagtt gtggcctgtg 117660
cttctgactg accttctata aattggagtt cccacagtcc cctccttggg ttcaataaat 117720
ttgctagagc agctctcaga actcagggaa atgctttaca tatatttacc catttattat 117780
aaaggatatt acaaaggata cagattgaac aggcagatgg aagagatgca tgggcaaggt 117840
atgggagagg ggcacagagc ttccatgcac tctccaggtc atgccaccct ccaagaacct 117900
ctacagattt agctattcag aagcccccct ccccattctg tccttttggg ttttttgtgg 117960
agacttcatt atataggcat gattgatcat tggctattgg tgatcagctc aaccttcagc 118020
cccctcatcc cgggaggttg gtgggtaggg ctgaaagtcc caaacgtgta attctgcctt 118080
ggtctttctg gtgattagcc ctcatcctaa agctctttag aggccacagc cacaagtcat 118140
ctcattagcc ttcaaaagaa tccagagatt ccatgaattt taggcgctgt atgctaagaa 118200
actggctaaa ggccagttgc aatgtctcag gcctgtaatc ccagcacttt gggaggctga 118260
ggcaggagga tcgtttcagg ccatgagatc aaaaccagcc tggtcaacat agtgagaccc 118320
ccttacaaaa aatttaaaaa ttggccaggc gtaatagctc ttgtctgtag tctcagctac 118380
tcagaaggct gaggatcact gagccctgga gttgaaggca gcagtgagcc atgatcgtgc 118440
cactgactcc ggcttgggtg acaaagtgag accttgtctc agaagaaaaa ggaaaaaaaa 118500
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-42-
aaaactgggc aaagactaaa taacatattt cacagtatca cagatttgta ttgtctagga 118560
aagtgaatgt aaacagacca ggacactagt atgatccctt ggtttcatga aggtcccact 118620
aaagtcatga acacaaagtg agactaggca tcatgttata tggtttttcc agccatgttt 118680
aacagctagc taaatagcta attgtttcgc tgcagtttat tttagcagtt ccttatttta 118740
gcacatttca tgttttaaaa tttctaccaa taacatttta ataaactttt ttacagataa 118800
cttcacaaat ccataatttt ttaagttaca atcccagaaa tagaattgct cattgaaagg 118860
gtatgttcat ttttaaagtt atgctagaaa ctgccaaatt gccttcagaa aaaggtgttt 118920
gtatccccac taacactagt gttagttttc ttgtgccctt gctcaagtat acatattatt 118980
aaaaacaatg ttgggccagt ttactagata aaaggtgtag tgcctcctta ttctaatcta 119040
tttgattact agtgagtatg tatgtctttt cacgttggtc attttatgtt tgttcctttg 119100
tggattgtca tgtcctttgc tcatttttct tttggaacat ttcttagtag tttataagag 119160
ctcttggtat tttaatgata gtaacctttt aactgtcatg catgctgcaa atcttttttc 119220
tgtttgtttg cctttgtatt ttgtttttgg agggtttcta tgtataggaa ttaaatttta 119280
tgttgttaaa tcttttgatt tctgcttttg catatgtact tcaaaagact ttctatttta 119340
agatcaagtg ttacctgtat tttcttttag ttctatttaa aacctcttaa tttatatgcc 119400
tgtgctgtta actcccaagt tgattcacaa gtgtgtatac atagtttgaa tttagtggca 119460
atttaattat ttacaacttc ttttgcagca aggatttgtg gagaagatgg acaggtggat 119520
cccaactgtt tcgttttggc acagtccata gtctttagtg caatggagca agagtaagtt 119580
agttcatatt ttcacattgt gcatcctagg gaatttgggt tcattgttag gaatgggctt 119640
cactcagcta aaaacaaagt atttttgaga atttaaatat tttggatatt tacaagatca 119700
tataaagcat actctatctt ggttaacagt ttcttttaaa tataaattat gtgaactctt 119760
aaaattttca ttttcatttt caatgttaat atttcctaag ttaaaataat ttgtttttag 119820
ttctgaaata atttggggag tgattgagtc tgtagtgatt atgactatta gaattggttt 119880
atttatttaa ataatgcatg tcttcagatg gctctcctaa tttgttagtt aggctttaag 119940
ctaaatggat gctatataac taaatccaca tagatttgtt gaaatggctc cagaggtttt 120000
ttagatttat tactgctatg tgcccttaaa aaaaatctat tcattctttc acttaacatt 120060
tatcagaaga gtgctctgtg taagacgtgg ttaggcatag tgccagtctt gaaggaagtt 120120
acagcctaat aaaagacata gggcatgttg tttggttact gtaatatgaa gtggcatgtg 120180
ttaaatgtca ggggagaact acaaagtcat aaaaaggtgg gagagattac atacaggtaa 120240
aggaatcagg aatgacacca tggggagtaa ggtagtgttg acctaggcct ttaagataca 120300
atagggacag tatggaaaga gtatattttt cccacttaaa ctctttcctt ggtcgttccc 120360
tcaaattttc ccttttgtcc atgtgcaggc actttagtga gtttctgcga agtcaccatt 120420
tctgtaaata ccagattgaa gtgctgacca gtggaactgt ttacctggct gacattctct 120480
tCtgtgagtC agCCCtCttt tatttCtCtg aggtaaagtc tgcatttctt ttcacactct 120540
attcgagcat tccagcctct aactatcaat gctggggccc tgtctatagg aaataacaca 120600
gaagagccaa gtcatttcca aaaagatgta tcattgtttc aagttgtttc tgatggcaag 120660
agtaatttaa taatatatta gagagaacat gaaaattcaa tgtattaaat aactctaatt 120720
ttgagaaacc taattaaact actgcatgta agagagtgca tgtttttaat tatttggagc 120780
tattttaaaa ccacagaatt tgaaacttgc ttccagtgca taaattgcag accagacttc 120840
agaagagaaa aaaagtagta aattttttct tatgctcatc atttttactt tagtcacttg 120900
ataggattgc ccagtgaaga agcatttgca acagacaatg agtatattaa tctttttgag 120960
gcatacagtt tagtataatg ctctttgtta ggcttcaaca agtgaaatta ttttgttgga 121020
aagcaaatga ctattaagta gaaagaggat tcccagtctc acaaagcagt aatttagaca 121080
ctcgattctg cctctttaca agaatacagg tactcagttg atttgttttc tcactccctt 121140
tctttgctat aagtttaaat caacaatttg tttaggttaa tatgtcctca tggaatggtg 121200
gaaatgatca gatataaaat atttggtttg gttagtttac tctttatatg tttgctggca 121260
aggaaccaca aatccagttt agtataattt ttactctagt tcactaaaag tttgcatcca 121320
gctgtgtagg tagtgtttgt ttcttgttaa cttttttttc gtctaaaaga atactttaaa 121380
acttttcaat ctcaaatgac tgtaacttgc tgacaggtgt taacagaaga agtagatctt 121440
tttgtttttt gcttatgacc tgtattttaa tatttgagct tatagattag agattgtgag 121500
agaaatctgt ttatagtctt attttccctt gtgtattttt tcttcctagt acatggaaaa 121560
agaggatgca gtgaatatct tacaattctg gttggcagca gataacttcc agtctcagct 121620
tgctgccaaa aagggccaat atgatggaca ggaggcacag aatgatgcca tgattttata 121680
tgacaagtga gttatattga tagatggatt cagcagatac ttattgaaca tttgatatgt 121740
tttgtggaaa taaagatgaa taaactcagt ctctgttgtc aaggagctca caggaggcag 121800
cataaaagct gcttttatat ggtgtttgta aagctttggg ggttcttaga acaaaagttt 121860
ctgctgggaa aggggaggtg tatgtggggt aaacaggatg gcaatggtgg tgttcaagga 121920
gtgtttccca gaagagagat tttgtttgga tcccaaagaa agaagggaat tttgctaccc 121980
agagaaggca gaaaacaaca ttctaggcaa aggcattggc ccagaagcca tggaaacgta 122040
ggggaaagtg gcactttcaa gaaacttgag tttagataat caaaggagtg gggaataaat 122'100
atgaggatgc tggtactaat tggaatagat tgtaagggac cttgaatgcc tatttatggg 122160
tatattatac tttctgtata aatctgctca ggcacgttgt taattagttt tttattagtt 122220
ttcactgaaa atgagaggat ggaaacatca tacagtaaac aaaattgaaa atatctggtc 122280
aggcagatga tgagcttgtg gccagctctg taacgtatgg tattcttttc atttaacttt 122340
tcttactctg taaaaaaagt aattcgtggt cgggcacggt ggctcactcc tgtaatcaca 122400
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-43-
acactttgag aggcagaggc aggtgaatcg cttgagccca ggaatttgag accagcctgg 122460
gcaacatggc aaaacccgcc tttactaaaa atacaaaaat tagctgagcg tgatggcgtg 122520
cgcctgttgt cctagctact taggggcctg aggcagaagg atcacctgag ccttgggagg 122580
tcgaggctgc agtgagctgt gatccactgt actccaccct gggcagggca gtagagtgag 122640
accctgtctc caaaaaaaaa aaaaacaaca aaggtaattt gttatttgta tccttaagca 122700
aatgctaaag gggtaacttg gggatagaga aaagtccaca gatgttaggg tttgaagaca 122760
ctaatagtat ctaggccagt ggttcctgaa cattagtctg tgggctcttg ctgggctgtc 122820
tgcataggaa tcacctgaga gcttattaaa aataggtttt caggctggtt gcggtggctc 122880
acgcctataa tcccagcact ttgggaggct gaggcaggcg gattacttga ggtcaggcgt 122940
tcaagaccag cctggccaac atggtaaaac cccgtctcta ctaaaaatac aagaattagc 123000
caggcatgat ggcacacacc tgtaatccca gctactcagg aggctgagga aggagaattg 123060
ctcgagcccg ggaggtggag gttgcagtga gcggagatca tgccactgca ctccaggctg 123120
gctgacagag ggagactctg tctcagaaaa aaaaaaaaaa ataggttttc agtctgggta 123180
ccggtggctc acacctgtaa tcccagcact ttgggaggcc aaggcaggca gatcacttga 123240
ggtcaggagt ttgagaactg cctggccaac atagtgaaac cttgtctcta ctagaaacta 123300
caaaaaatta actgggcatt ttgacgggtg cctataatcc cagctactag ggaggctgag 123360
gcaggagaat tgcttgaacc cgggaggcag aggactgcat ctcaaaaaaa aaaaaaaaaa 123420
aaaggtttcc agtccccctg tctcagaaat tctgattctg caggtttgag gtgtgaccag 123480
gaatctttat ttttagaaga cataccagat aattctgata aatagccagt ttagggatgt 123540
agtctaattt tcctattttg caagtaagga aaataaggcc cagagaggta atgattttct 123600
caaagtcaca gaacaagtta gtggcagaat ttggactgga atgcagttct taatgttctg 123660
tccagtgttt attctggtac agtatgtttg tagaaggtat tacgtaagaa acattgttat 123720
atagatgttg agataggaag agtttacatt tagaaatttg gtctaaaatg cctgaacatt 123780
caagtcgtgg aggagtattg accaacttac tcaatacaac ataggagatt cacattttgt 123840
tacaaaaatg ctgatttaaa aggagagttt tctttttttt cttctttttt attttttgag 123900
atggagtctt getctgteac ccaggctaga gtgcagtgac acgatctcag ctcactgcaa 123960
cctccacctc ctgggttcaa gcggttctcc tgcctcagcc tcctgagtag ctgggattac 124020
aggtgggggc caccacgccc agctaatttt tgtattttta gtagagacag ggtttcacca 124080
tgttggccag gccggtcttg aactcctgac ctcaagtgat ccacccacca ctgcctccca 124140
aagtgctggg attataggcg tgagccactg tgcccagcct gcttgttttt gtatcatata 124200
tatgcatcat cataatcatg cattatcaac ctttgtattt ctgtcaggac atagaaacca 124260
ttagagtgct tggaagagag cctttttttt tttctcgcat ttaatgcttt ttttggtatt 124320
catttcataa tcagcttacc aaaacattac ctgcattata ccccatcaag gtagaaatct 124380
ttgtgttatc aatattggtt actccctttc cacaccgagt catcagtaag tcctgttcta 124440
tccaaatagg tcatatgcat ctagctcacc cctcagtgct gttttgtttt gaatttgtac 124500
atgtttactc ctgatgcctt gtagttatga tgatgtgttc ttattttatt ctgtgcatac 124560
aagttctcag ctcgcttttt agggaaaatg accatgtctt cctttcctat aaattecttt 124620
ctatctatca agtcctcaac agagaatagg tacccataaa tatgtgattg ttagtttctt 124680
tgcctcagtt gtagtctgat ccttacagct tttaaacaac agtagagttc accgtcaaga 124740
actaaggatg gttggcaggc agatagaaag gtagcaagtt gacccaacta tctctgggga 124800
agtgggaaca aagaaaggtt acatcagcac tgtcatcaca tagctctata gttctaggcc 124860
tgcaggctca atcaagtagc cttgtataag attctctgga ggaggtgctg aaagttgctt 124920
atacttgcta tggaatttga ttttacttcg gatatctttt taccataggt acttctccct 124980
ccaagccaca catcctcttg gatttgatga tgttgtacga ttagaaattg aatccaatat 125040
ctgcagggaa ggtgggccac tccccaactg tttCacaact ccattacgtc aggcctggac 125100
aaccatggag aaggtaaccc agaacttcaa acgtatcaaa ctacaagaag ttttattggt 125160
agaactcata aaatataagg tgggaaaacc aagcagaata gcacagtgga aattgaagca 125220
gtccagcaaa gtgattaaga gcagaggcct tgagtctggc ctggtatgta cagtcacgtg 125280
ccacataaca ttttagtcaa cagtggactg cgtgtacgat ggtcctgtac gattataatg 125340
gatcaaagct ggtagtgcaa taataacaaa agttagaaaa aataaatttt aataagtaaa 125400
aaagaaaaaa gaaaaactaa aaagataaaa gaataaccaa gaacaaaaca aaaaaaatta 125460
taatggagct gaaaaatctc tgttgcctca tatttactgt actatacttt taatcattat 125520
tttagagtgc tccttctact tactaagaaa acagttaact gtaaaacagc ttcagacagg 125580
tccttcagga ggtttccaga aggaggcatt gttatcaaag gagatgacgg ctccatgcgt 125640
gttactgccc ctgaagacct tccagtggga caagatgtgg aggtgaaaga aagtgttatt 125700
gatgatcctg accctgtgta ggcttaggct aatgtgggtg tttgtcttag tttttaacaa 125760
acaaatttaa aaagaaaaaa aaaattaaaa atagaaaaaa gcttataaaa taaggatata 125820
atgaaaatat ttttgtacag ctgtatatgt ttgtgtttta agctgttatg acaacagagt 125880
caaaaagcta aaaaaagtaa aacagttaaa aagttacagt aagctaattt attattaaag 125940
aaaaaaattt taaataaatt tagtgtagcc taagtgtaca gtgtaagtct acagtagtgt 126000
acaataatgt gctaggcctt cacattcact taccactcac tcgctgactc acccagagca 126060
acttccagtc ttgcaagctc cattcatggt aagtgcccta tacagatgta ccatttttta 126120
tcttttatac tgtattttta ctgtgccttt tctgtatttg tgtttaaata cacaaattct 126180
taccattgca atagtggcct acgatattca ttatagtaac atgtgataca ggtttgtagc 126240
ccaaaagcaa taggttgtac catatagcca aggggtgtag taggccatac catctaggtt 126300
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-44-
tgtataagta cactctgtga tgttagcaca atggcaagca gcctaacgga aattctgttt 126360
attgattgat tgattgattg attgattgag acagagtttc actccattgt ccaggctgga 126420
gtgcagttgc acagtcttgg cacactgcaa cttctgcctc ccaggttcaa ccaattatcc 126480
tgcctcatcc tcccaagtag ctgggattac aggcaggcac caccatacct ggctaatttt 126540
tgtattttag tagagacagg gtttcaccat tttggccagg ctgttctcga actcctgacc 126600
ttaagtgatc tgcctgcttt ggcctccgaa agtgctggga ttacaggcat gagctaccat 126660
gcctgggcag taactgaaat tctctaatgc cattttcctt atctgtaaag tgacgataat 126720
atgcacgttt acctcaaagt tactttgatg attaaagtaa ggtaatgtat ataaaataca 126780
tattaacata gtacctgaca catggtaagc atcaaaaaat gttaactact tttattacta 126840
ttattattac gtatttttaa ataattagag agcagtatca aaaattagct gggcgtagtg 126900
gcatgcacct atagttccag ctactcagga ggctgaagct ggaggattgc atgagcctgg 126960
gaattaaagg ctgcagtgag CCgtgttCat gCCCCtgCaC tccagccttg gtgacagagc 127020
aagaccctgt cttgaacaat taaagaaggc attatgccgc aacgttagct tagaaatgat 127080
ccacatatat caccagtaac tgtcaacagg attggaaccc tagttttggg tattatgatc 127140
acaaggtatt attaatagct tattaataat aaagcgttgg ctaggcacgg cgactcacat 127200
ctgtaatccc agcactttgg gaggccgagg tgggtggatc acctgaggtc aggagtttga 127260
gaccagcctg accaacatgg agaaacccca tctctactaa aaatacaaaa ttagccgggc 127320
gtggtggtgc atgcctgtaa tcccagctac ttaggaggct gaggcaggaa aatctcttga 127380
acccgggagg cagaggttgc agtgagctga gatcgcacca ttgcactcca gcctgggcaa 127440
caagagcaaa actccgtctc aaaaatataa ttataataaa taaataaaag taaagtattg 127500
atgtttgtga atgatttatt cttctaatga actagaggag atttttccag gaatttcaga 127560
gccagtgagg ttatgttgct tgtatgtgtc atgtgtatcc aggtgaaaaa acttaattaa 127620
acgctattat ataataccat acataaaaac tgaattttag gaatactgaa gaatgacata 127680
tagaagtcaa atcattaaat agctagtagt aaacagaata gagtgtcagc tgttacccaa 127740
tgatgataat attttcacga ttaaaattaa accttttctg attttaaagg aaaagttcag 127800
atctgtatca tataaagaat gtaaattttc agggtaataa aattaaaatg cagagagaaa 127860
aatgcaaaaa tagttcttac tagatgtgtg tatgtaagga acttagacta attttaagaa 127920
cactgtcaag accctggtag ttaggtagga aaaaagacat gaatgattca ttcaacaaaa 127980
actttgagta tttctgtgct agatggtagt gttacagtgg taaacaaaat aaatgtgttt 128040
ctgctatcct ggagcttagt ctacaaaaaa ggtacatatt ggccgggcac ggtggctcac 128100
gcctgtaatc ctagcacttt ggaagatcga ggcgggtgga tcacctgagg tcaggagttc 128160
aagaccagct tggccaacat ggcgaaaccc cgtctctact aaaaatacaa aaattaactg 128220
ggtgtggtgg cggacacctg taatcccagc tactcgggag gctgaggcag gagaatcact 128280
tgaacctggg agacagaggt tccagtgagt cgagatcatg ccactgcatt ccagcccggg 128340
ggacaaaagc gaaaatacgt ctcaaaaaaa caaaaacaaa caacaaaggc acgtattaaa 128400
tacgaacata aatatttaca aattatactg aataagttct catgtttatt atttgcttgt 128460
ccagttacaa acttttcctt cgtagaatta gaaatataaa taataaacat gagaactcat 128520
tcagtataat taataattat taaatgtaaa taaaaacatc tatgtacaat taggcattta 128580
tttaagaatt atttgaaaaa aaaacaatgt ggaaacagat attttgatat attgctagtg 128640
attgaaattg ataatgttct tttgaagagt aaagtgacca tatatattaa agttaaaatt 128700
taactcagca atcacacgcc tggtgagtta tcttaaggaa atcagtttga aagtaaaatc 128760
aatatatgca caaagacttt aacatttatc ataaaccaga aaaatcgagt ttcaaattat 128820
atcctatgga ctattttctg ctaaaaagta ttaatatcaa ctttatgtaa tactttcgtg 128880
acaaatattt tgggggagaa aacccaacaa aattacatgc attgtaattt tttttttttt 128940
ttttttttta gacagtcttg CtCCagCgtC CaggCtggag tgcagtggtg CaatCtCggC 129000
tcactgcaac ctccatctcc caggttcaag caattctcct gcctcaggcc tcccgagtag 129060
ctgggattac aggcgctcac caccatgcct agctaatttt tatagttttt agtagagatg 129120
gggtttcatc atgttggcca ggctggtctt gaactcctgg tctcaagtga tccgtctgcc 129180
tcggcctcct agagtgctga gattacaggt gtaagccact gcacccagcc ttatgcatta 129240
taattttaat ttgtaaactg tacaaaggga taatacttgt agtacaacaa gaagtaaaaa 129300
catttgttat aggtagttaa catttgtaac cagtagaatt ataggtaaaa tttatttatt 129360
taaaacagtt ttagttggat ttgatttcaa ctttaaaata atgcttttca tctctatcag 129420
gtctttttgc ctggcttttt gtccagcaat ctttattata aatatttgaa tgatctcatc 129480
cattcggttc gaggagatga atttctgggc gggaacgtgt cgctgactgc tcctggctct 129540
gttggccctc ctgatgagtc tcacccaggg agttctgaca gctctgcgtc tcaggtattg 129600
actgattgcg tctgccatta gggagaaaag catacacatc ctttccttca catcccagta 129660
acagatccta ttatttgtaa attttaagtt gtggaaaaaa aagataaaag ccaggcacag 129720
tggcctgtgc ctgtaatccc agcactttgg gaggctgcgg tgggcggatc acacgaggtc 129780
aggaattcga gaccagcctg gccgacatgg tgaaacccca tctctactaa aaatacaaaa 129840
attagccggg catggtggca ggcacctgta atcctagcta cttgggaggc tgaggcagga 129900
gaatcgcttg aacccaggag gcagaggttg caatgaacca aaatcacgcc actgcactcc 129960
agcctgggtg acaaagtgag actgtgtctc aaaaaaaaaa aaaaaagaga gaaataaaat 130020
tagcctactt actatcttct aatcaaagca tttgtggtaa cttaaaatat actgtattgt 130080
aaagtatcat gctgtttcat ttaggccatt attctatttg aatctgtggc tgtttctctt 130140
aataaatcaa gtaatatgga atatattcat agcctctgaa gagctcttta tgtaagtatt 130200
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-45-
tatttaggat actttttgta aaataagtga atgaattctt aggtctcctt tttttttctt 130260
ttcttgagac agggtctcct cgctgcaacc tggaaattct gggctcaaat aatccaccca 130320
ccacagcctc ctgaatagct gggactagag gcatgcacca ccacgcctgg ctaatttgaa 130380
attttttttt ggccaggcat gatggttcac gcctgtaatc ccagcacttt gggagaccga 130440
ggcaggcaga tcacgaggtc gggagatgga gaccagcctg gccaacgtgg tgaaaccccg 130500
tctctactaa aaatacaaaa attagctggt tatggtggct catgcctgta atcccagcta 130560
cttgggaggc tgaggcagga gaatggcttc aaccagggag tcggaggttg cagtgagccg 130620
agatcacgcc actgcactcc tgcatggtga cagagtgaga ctccatctca aaaaaaattt 130680
tttttttaaa tgatggagtc ttgctgtgtt gctcaggctg gtcttgaacc cctgacctca 130740
aatgccgcct gcttcagcct aagtttcttt tttttttgta aagagacagg gtcttgctat 130800
gttggccagg gtagtctcaa actcctggct tcaagcagtc ctcccacctt ggcctctcaa 130860
agtgctggga ttacaggcgt gaaccactac ctataatgtt gtgtttcact caaggccttt 130920
tgatttcgtt ttgcattacc gtgccacatt gtgcatttcc ttgacctttt ttgggttttt 130980
tggagtgctt tcatatgtta aaccatacct gattctcctc aaaatcacac aaagtagaat 131040
atcctaagac aagaaatcta aggaggcata aagaagttaa ctggttttat taaactcaca 131100
cagtaaatga tagagccaga aatattcccc ttctagtgtt cttcaccatc agcttaatgt 131160
agcataataa ttttctaatt actgttgaca aataaataac cctttgaatt ttcaatactg 131220
ggccttggat aaattttcct aatttgtaag agagtattat cgtattgcca tttacaaagc 131280
tctcctgagt atctttttct tctgttaagt ttacctagga gataaactgc tgagtatggt 131340
tgccattttg gttttttgat ataggttaga atgtcttggt tttttttttt tttttttttg 131400
gtttttgttg ttgtcattgt ttgagacagc atcttgctct gtcgcccagg etggagtgca 131460
atggcacgat cgtggctcac tgcaacctcc acctcccggg ttcaagcaat tctcctgcct 131520
cagcttcctg agtagctggg attacaggca tgtgcaacca cacctggcta atttttgtgt 131580
ttttagtaga gaaggggttt caccatgttg gtcaggctgg tattgaactg ctgacctcat 131640
gatccacctg cctcggcctc ccaaagtgct gggattgcag gcatgagcca ctgcacctgg 131700
ctgaatgtct tgtttttgat taggcactta agaaaggcct aggtactaac cataaaatat 131760
atttttatac cttttgttga tactatatat atagaaaact gcacttatca taaccttaga 131820
caccttgaag aatgttcaca agcagaacta acccatgtga cccagcatcc agatcaaaaa 131880
CagCattatC agCCCCtCta gaagCCCtCt tgggCCCCtt CCattCaCtg tCCttCttgt 131940
CaCCagggta gCtaCtatCC tgaCttttga tggCatagat tagCattaCC tgttCttgtC 132000
attttataaa taaaaccata ctgtgtattc ttttcttgta cagctttatt gtgctaattc 132060
acatttacat catacaattc agtggttttt atatggtcac agagttaggt aaccattacc 132120
acatcgattt tagaacattt ttttcactcc agatagaaac cccctttact taaactccaa 132180
atcccccact ccaccagccc taggcagcca ctagtctact ttttatctct atagagacaa 132240
tagatttgct tattctggac atttcataaa catggaaccg tatattatgt ggtcttttgt 132300
tgccaactgt ctttcactta gcatcatgtg ttcaaaagag catcatgtta tccatgtttg 132360
gcatgtatca gaattttatt cctcattatg gccaaatatc ccattgcaag gatttatgac 132420
attttatttg aattgtaccc tcctttctgc catttatcaa taatgctact gtgaccattt 132480
gtgtacaagt ttttgtgtgg atacaggttt tctttttgtt tttaaatttg aggtggagtc 132540
ttgctctgtc gcccaggctg gagtgcagtg gcacaatctc ggctcactgc aacctctgtc 132600
tcctgggttc aagcagttct cctgcctcag cctcccgagt atctgggact ataggcacgc 132660
accaccacgc ccagctaatt ttttagtaga gatggggttt caccatgttg gccagtctgg 132720
tctcgaactc ttgacctcaa gtgatccacc catctcggcc tcccaaagtg ctgggattac 132780
aggggtgagc cactatgccc ggctgtggtt ttcatttctt ttgttgtata tacataggag 132840
tagaattgct gagtcaagag gtaactctta aacttattga aaaactgcca gattgttttc 132900
cgaaaaggct gcaccatttt gcaatcccac cagcagtgta tgagttttac agcttctcca 132960
catttcattg gaacttatta tctgtttggc tgtttttaaa aatgatagtc attccaataa 133020
gttctacttc agtgtggttt ttgcacttct ctgatgagta atgatgttga gcatcttttc 133080
atttgcttat tggcctttgt tctagctttg gaaaaatgtt tattcaaatc ctttggccat 133140
ttttattttt atttttattt atttattttt ttttgagacc aagtctcact CtgtCagCCa 133200
ggctggagta caatggtgtg gtctcagctc aCtgCaaCCt CCgCCtCCtg tgttcaagtg 133260
attCtCCtgC CtCagCCtCC CgagtagCtg ggattacatt tcaggcacct gccagcatgc 133320
cgggctgatt tttgtatttt tactagtgac agggtttcac catgttagcc aggctggtca 133380
caaactcctg acctcaggtg atctgcctgc ctaggcttcc caaagtgctg ggattacagg 133440
cgtgagccat tgggcccagc ctagattttc ttttttcttt ttttttttga gaaggagtct 133500
tgctcttgtt gcccaggctg gagtgcaatg gcacaatctt ggctcactgc aacctctgcc 133560
tcctgggttc aagcgatttt cctgcctcag cctccccagt agctgggatt acaggtgcct 133620
accaccacac ccagctaact tttgtatttt ttttagagac agggtttcac catgttggcc 133680
aggctggtct caactcctga cctcaggtga tccacctgcc ttggcctccc gaagtgctgg 133740
gattaccggc atgagctacc aggcccagcc aattttctca ttatattgcc caggctggtc 133800
tcaaactcct gggttcaagt gatcctcctg ccttggcctc ccaaagtgtg gggagtacag 133860
gcgtgagcca ccttgctcag cccctttgcc catttttaaa ttagattgcc tttttatatt 133920
gagtttcagg agtcctttat atattctaga taaatgtccc ttatcaaatt atattatttc 133980
caggtatttt cttcattctg tgagttgtct ttcctctacc ttttaaaaaa ggtgggtttt 134040
tgtttgtttg tttgtttgtt tttttaagat aaggtctcat tctgctgccc aggctggagt 134100
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-46-
gcagtggcac aatcacagct cactgccacc tcaacttcct gggccgaagt gatcctctta 134160
cttcagcctc ctgaatagct agggccatag atacacacta tcacacccag cttttttttt 134220
ctgtttgtag agacagatct tactgtgttg cccaagttgg tctcaaactc taggctcaaa 134280
gtgattCtCC Ca.CCtCtgCC tcccagagtg ctgggattac aggtgtgagc cacacgcaac 134340
ctgtcttttc actattaata gtgtcttcct gcttcagcct cccgagtagc tgggattaca 134400
ggcacccacc accatgcctg gctaattttt ttgcattttt agtagagaca gtgtttcacc 134460
atgttcaccc ggctggtctt gaactcctga cctcaggtga ttcacctgcc atggcctccc 134520
aaagtgctgg gattacaggc gtgagccact gcacccggcc aaaatattgc cttcttaaca 134580
gtattgtctt ctaatttgtg aacatggatg tatcttcatg tatttatgtg ttctttcatt 134640
tcagcagaat tttgtagttt tcagagtaga agcctttcac ctccttgggt catttattcc 134700
tatgttttaa gttcttttcg attccattat aaatagaatt gttttcttaa tttcattttc 134760
agattgtttg atgagagagc atagaaatac aagtgatttt tacatgttga tcttgcaact 134820
tcaactttga taaatctgat tgttagctct aatagttttc ttgtggattc tttaggattt 134880
tcaatatata agatcatgtc atttatggat agagatagtt ttttttctgg ctagaactta 134940
cagagcaatg atgagtagaa gtggcagaag caaaaatctt tgtcttgttt cctatctgac 135000
agggaaagct ttcagtttca tcatttaata tgatgttagg tgtgggtttt caataaatgc 135060
cttttttcag attcaggaat ttccctatca ttcctgattt tttaaggctt tttttttttt 135120
ttaaatcatg aaagggtgtt gaatattgtc atgttctttc tgtatcagta taaatgatcc 135180
tatggatttt gggttttatt ctgttgatgt gaaatattaa ttgattttca gatgttaaac 135240
caaccttgca tacctgagat gaatctcact tggtcatggt gtataatctt ttcaatatgc 135300
tgctggattc catttactgg tattttgttg aagattttgt atctgaacgc ttaagataac 135360
atttacactc tatcagaaat gaattgacca taaatgtgag agtgtatttg tgggttcttg 135420
attctcttcc attccaaaga tagacataca tccgtctgta tgtctgtctt tatgccagta 135480
ccatactctc ttgattacta ttgctttgta ataagttttg aaatcagaaa gtataaatga 135540
gattttggta tctgagtaac agtcctcata gaattagttg ggaaatattc cctctttatt 135600
ctggtccctc tttctttttt gtttaactgt gtatcttgga gattgttcct tctcaacaca 135660
tgagagccgc tttccctacc ctcccacccc tgctatagag aggtctataa gtgtctgttc 135720
aattatttta tttacttaac ctattactta gtcggggaca ttaagcttgt ttatgtcttt 135780
tattttaaac aatgctgcag tgaataatct tgtatataag tcattttcca tcaatataag 135840
tctctctgta actgaatttt tagaagtgga atttctaggt caacctatgg ctctgtattt 135900
cacaaaaata ccaattctgg tttttcttgt ggaggtgggg agtaggaggt agaatgctgg 135960
aggagaactt gctgtactca gctggctagt cattttagaa aggtttcctt agcttctttt 136020
tgtcatatgg cctcaccaag aatcaaaaac attcctattt accctgtaaa catggggctt 136080
tactacccaa gatacatatt tctggatgta tgacagcttt tcatattgaa gaaataatgc 136140
tgtgagtaca gcacatttgt tggaacttag gtcgttaaga atgtcttata aattcataca 136200
ttatacattt tattttattt tattttttag tttttgatac agagtcttcc tctgtcgccc 136260
aggccagcgt gcagtggtac aatcttggct cactgcgacc tccatctcct gggctcaagt 136320
gattctcatg tctcagcctc cagagtagct atggttacag gcatgcacca ccatgcccgg 136380
ctaatttttt tatttttagt agaaactggg tttcaccata ttgaccatgc tggcctcgaa 136440
ctcttggcct caagtgatcg gcctgcctca gcctcccaaa gtgctgggat ccttgtattg 136500
ggtaaaagat gaatattgag ggctgcatgg tggctcatac ctgtaatccc agcactttct 136560
gagactgagg tgggaggagt cctggagccc aggagggtga ggctgcagtg agttgtgatc 136620
gcgccattgc acttcaacct aggaattata ggcttcagtc actgtgcccg gcatgtacat 136680
tttaatattg tgctttcctc ttttagctat agtatgaggt tacatttcag agtcattgtt 136740
gttaagcatc ttaatagtga tgaggttgag tgaaagttac ttctatttca aacactgaag 136800
aaaattttgt acaaatctgt cacattccaa gcccaggact gattgtttca tatacttcta 136860
attttacaat ttctattgta gtccagtgtg aaaaaagcca gtattaaaat actgaaaaat 136920
tttgatgaag cgataattgt ggatgcggca agtctggatc cagaatcttt atatcaacgg 136980
acatatgccg ggtaagctta gctcatgcct agaattttta caagtgtaaa taactttgca 137040
tcttttaaat tttttaatta aattttacat ttttttctaa tctattatta tatgcccaga 137100
actttcactt agagtgtgca gtataatgtg gtggttaagt ataaaggctc tggagtgact 137160
tcctgggttt taatcttggc tctgccattt attggcagcc gctaacctct tggtatctca 137220
gtttcttcat ctgtaaaatg agaataataa agtgaaaaga tgccaacatc atttactctg 137280
ggctgcataa ctgatacttg gaaaaagtat tcctttgagt ttaagaatta agttggttat 137340
tcattttagc ttgtaataaa aagatagtga ttcataggat atgccactta ctgaaattta 137400
ccacagatcc aatcataaaa tcactttctc ttccctaaag atagcttgat taacatgtaa 137460
aggtgtgtaa aggcttgatt acactaccct gatccgtacc ccagttccca gcagcaccat 137520
gaaaaaggga tttcaacata tttaattact ttcagtagaa agtaacagtg gtaggccagg 137580
cgcagtggct cacacctgta atcccagcac tttgggaggc cgaggtgggc ggatcacgag 137640
gtcaggagat tgagaccatc ctggctaaca cgatgaaacc ccgtctctac taaaaataca 137700
aaaaattagc cgggcatggt ggcaggcacc tgtagtccca gctacttggg aggctgagac 137760
aggagaatgg cgtgagcccg ggaggcggag cttgcagtga gcttagattg tgccactgca 137820
CtCCagCCtg cgcagtggag cgagactctt gtctcaaaaa aaaagaaagt aacagtggta 137880
ttgggagact gaggagccta gaaagtactt gaaggaagta aaaggtttgt ttgaccacat 137940
tgtatttgga aagccagctt tttcagctgt gtcagctttg tgtagtgatt tttagttctt 138000
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-47-
cttttagaaa ataacggaca aggccgggca cggtggctca cgcctgtaat cccaccactt 138060
tgggaggccg agacgggcgg attacctgat ctcaggagtt cgagaccagc ctgggcaaca 138120
tggtgaaacc ccgtctctac taaaatacaa aaagttagcc gggcgtggtg gcgtgtgcct 138180
gtagtcccag ctactccgga ggctgaggca ggagaattgc ttgaacccgg gaggcggagg 138240
ttgcagtgag ccaagatcac accattgcac tgcagcctgc gcgacagagt aagactctgt 138300
ctcaaaaaat aataataaaa taaaaaagaa tggacagtaa acctaaatga gttcattccc 138360
aaagatgatg ttattcttaa gggatggttc atttatttaa gaccttacat aaagtctatc 138420
aattgcgtga tttttcactt ctgtaattgt gtgtatgtat aatgtaaata tatatgtttt 138480
tgttttgttt tggttttttg agacggagtc tcgctctgtt gctcaggctg gaatgcagtg 138540
gtgcaatctc agctctctgc aacctctgtc tcccaggttc aagcgtttct tctgcctcat 138600
cctcccaagt agctgggact acaggcacgt gccaccacgc ccggctaatt ttttgtattt 138660
ttagtagaga tggggtttca ccgtgttagc caggatggtc tcaatctcct gacctcgtga 138720
tccacccgcc ttggcttccc aaagtgttgc tattacaggc atgagccacc acacccagca 138780
tgtatttttt aaatgtataa aatgaagcag aaaagagaaa tgataatttt tcttcatctt 138840
gaaagattat cttcaccagg cgcagtggct cacacttgta atcccagcac tttgggaggc 138900
ctcggcaggc ggctcacttg agttcgaaac cagcctggcc gacatggtga aactccgtct 138960
ctactaaaaa taaataaata aagatggttt taatatatgt tttagtttta tgattttagc 139020
atctttctga aatttttctc aaggcaagta aatttgtatc agttggtata ttggtaccca 139080
tctatgaaat aacttattag gaagatatct ctaaaataag atcactttgc ctaaaataaa 139140
ctgatatatt gatgttcaca gaatttttct tttaaccgac ttgataaatg cattattctt 139200
gacgtcaagt gatccacctt cctcagcctc ccaaagtgct gggattacac acatgagcca 139260
ccgcacctgg cattattctt ataaaaggtt aaatttctag ttaagtttaa tgtcctcttt 139320
gttcatgtac cattgcttat tttcttccct tcctactcac agtaatcatt cttatggtat 139380
gcacttttgt ttgcttattt ttatgtaatt gatattacgc tccattctgt acgttgtact 139440
ttcattcaca gtgagttttg gacattccta tgttcatcta tacagactta cttcatttta 139500
actacactgt agtattccgt atgtaatatt tactataact catcactgta gcagagcatc 139560
tcatagtgta tgtattactg ttttgccatt ttggtatcaa tgagtattta agtcatttgc 139620
agtttttccc tcttataccc agtattacag aggatctctt tttatatgct tctttgtacc 139680
aagaggcaga ttaaaaaatt tttttttgaa aaaatttttg aaaaaaaatg aaatgaagtc 139740
tcactatgtt gcccaggctg gtctcaaact cctaggctca agcaatcctt ccatcttggc 139800
ctcccaaagt gctggggtta caggcatgag ccaccatgcc tggcctacat tttaaatttt 139860
gatagctctt acaatttact ttgtaaagta tctgcatcat tttatgttct caccagtctt 139920
taataagaat acttcatact tttggctgga cacagtggct cacgcctgta atcccagcac 139980
tttgggaggc cgaggcgggc agatcaagag atcgagacca ccctggccaa tatggtgaaa 140040
ccetgtctct actaaaaata caaaaattag ctgggcgtgg tggcgcaccc gtagtcccag 140100
ctactcgaga ggctgagaca ggagaatcac ttgaacccgg gaggtggagg ttgcagtgaa 140160
cttagatcac accactgcac tccagcctag caacagagtg agactctgtc tcaaaaaaaa 140220
aaaagaatac ttcagactta attttttttc cagtcttaag tgtttgctaa tgagattgag 14.0280
tttcttttgg tatgtctctt gattgttcag gttttttctt ttatgaattg actgttcatc 140340
tctttttcac attatttctg ttgggtgatt ttattagtga cttgttaaaa ttctgtatat 140400
tttttcagca tgacacttca ttattcaaaa aaaaaaaaag attctctatg tttctcgata 140460
ctaatcattg gttggtaata ccttaaaaat aagaccctta ctgtattttt tgcttttttt 140520
tttttttttt tttttttttt tttgagatag agtcttgctc tgttgcccag gctggagtgc 140580
aatggtatga tctcggctct cagctcactg caactgcaac ctctacctcc ctgtttcaag 140640
caattctcct gccttagcct cccaagtagc tgggattaca ggcatccacc acCacaCCCa 140700
gctaattttt gtatttttag tagagacagg gtttcaccat gttggccagg ctggtctcaa 140760
actactggcc tcaagtgatc cgcctgcctc ggcatcccaa agtactggga ttacaggcat 140820
gagccacagt gcctagccac tttttgcttt ttaactttgt tttatagtac tatagtttta 140880
gtataaacag atgtatgtat acacacaact atggctttat aatatgtttc agtcattgtt 140940
agagcaaggc ctaccttttg ggtgcttctt ttacaaaatt gtcttggcta ttcttgtgcc 141000
ttttttctta tttgtgaatt ttagaattgt gaattacctg ttgactcacc atgttttgta 141060
aactgaggat tttgaatgga attgcactca attaaagatt atcttgcttt ctgtgcagca 141120
atgttttatt tcaaataatc cctactttaa attacttagg atagctataa attgtgtttc 141180
tggctttcta gatttagatg aaacgcttta aattgattgt tttctcctaa atttaaaact 141240
gattgttaga agttaaagtc ttctgttcat tcttatttag gaagatgaca tttggaagag 141300
tcagtgactt ggggcaattc atccgagaat ctgagcctga acctgatgta aggaaatcaa 141360
aaggtttgtg gtgtttttat acttcatatt aagcctttac tcacattagt gattgactgt 141420
aagtcaaaga ccacttaagg tttaaactgt ttattttgta aagtaaccac tgtatctttc 141480
accttgtgtt tatagtcaga agtaagtaca agggcttcct gtagtcacat ctttatgcaa 141540
tctcctctga atcaaaagtt agtgaacttg ctttgccact ccagaaggca catgaatatg 141600
aaaaagcatt gtctattttc ttatttaatg gcaaaatacc cgacctaagt tggacttaat 141660
gtttgagacc gtttatttta ttaaattata ttttttctct tttctttttt ttttttgaga 141720
cagttcttgc tctgtcaccc agaccggagt gcagtggtct gaccgcacct cactgcaacc 141780
tctgcttcct aggttcaagc gattttcctg cctcatcctc ctgagtagct gggactacaa 141840
gtgcgcacca ccacacctgg ctaatttttg tatttttagc agagatgagg tttcaccacg 141900
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-48-
ttggctaggc tggtctcata ctcctgacct caagcaatcc atccgccttg gcttcccaaa 141960
gtgctgggat tacaagtgtg agccaccatg cctggcctta ttaaattatt tttattaaat 142020
ttcctcaaga ttgatgaaag taatgaaata taaaagtaat gaaatatatg tggaaaatag 142080
actggattaa gaaaatgtgg cacatataca ccatggatac tatgcagcca taaaaaagga 142140
tgagttcatg tcctttgtag ggacatggat gaagctggaa accatcattc tgagcaaact 142200
gtctcaagga tagaaaacca aacaccgcat gctctcactc ataggtggga attgaacaat 142260
gagaacactt ggacacaggg tggggaacat cacacgctgg ggcctgtcgt ggggtggggg 142320
gctgggggag gaatagcatt aggagatata cctaatataa atgacgagtt aatgggtgca 142380
gcacaccaac atggtacatg tatacatatg taacaaagct gcacgttgtg cacatgtacc 142440
ctagaactta aagtataata aatttaaaaa aaataaatat atgtggaaaa tattaatagg 142500
tcaaaattca aattgttcat ttaatcagaa gagtagttta gtcaaatcca agggttagac 142560
aacagaaatc ttttttgtca agtgcattct ttgtgactga tttcattttc ttcctggttt 142620
acacaggaag atttcagaaa caaatgtgga tccgtgacag atggtatcta gaagttttta 142680
gtttggttga attgacagta ttttattgag taaaagatac taatttttgt aagaagaaaa 142740
attcaatttt gataagtatg tttaagatta agagctattg gccaggcgct gtggctcatg 142800
cctgtaatcc tagcactttg ggaagctgga gcaggtgggt cacgaggtca agagattgag 142860
accatcctgg ccaacatggt gaaaccctgt ctctactaaa ttagccaggc gtggtggcac 142920
atgcctgtgc acccgcctcc gggtttaagc gatcctactg cctcaggctc ctgagtagct 142980
gggattacag gcgccatggc taatttttgc atttttagta gagacagggt ttcactacat 143040
tggccaggct ggtctggtct caaactcctg acctcaggtg atctgcccgc cttagcctcc 143100
caaagtgctg ggattacagg catgattcac catgtctggc catttatctt attttctttt 143160
tttttttttt ttttgtttga gacggagtct tgctgtgtcg cccagagctg gagtgcaatg 143220
gtgcgatctc agctcactgc aacctctgcc tcctgggttc aagcaattct cctgcctcag 143280
tcttccaagt agctgggatt acaggcgcgt gccaccacat ctagctaatt tttgtatttt 143340
tagtagagac agggtttcac catgttggcc aggctggtct cggaactcct gacctcgtaa 143400
tctgcccacc tcggcctccc aaagtgctga gattacaagt gtgagccact gtgcccagcc 143460
atcttatttt ctttcttttt ttttgtcggg tgggaggggg acagagtcta gctctgtcgc 143520
caggcttggc tcactgcaac ctctgccccc caggttctag caattattct gcctcagcct 143580
cccaagtagc tgggattata ggcacctgcc accacgcctg gctaattttt tgttattttt 143640
agtagagatg gggttttgct atgttgacca tgctggcctc aagtgatccg cccaccttgg 143700
cctcccaaag tactgggctt acaggcgtga gcttgtattg ggtaaaagaa caatattggg 143760
ggctgcatgg tggttcatac ctgtaatctg agcactttgt gagactgaga tggaaggagt 143820
gttggagccc aggagggtga ggctgcggct gcagtgaatt gtgatcacgc cattgcactt 143880
ccacctaggt aatggagcaa gaccatgtct ctaaaaaaca aaacacaatt tttttaagga 143940
atactgggaa gaggtcagtg gtggttttag aacagaggaa gtgccagatg acctttgtga 144000
ggcattggcc aggaagaact ctacagtgtc tttaggtagc ttctgtccat aaggataatg 144060
gggtctcctc cccagtatta atagaaaatc tctgagctgt ttttttttgt ttgtttgttt 144120
tgtttttttt tcctgagatg gagtctctct ctgtcggcca ggctggagtg ctgtggcgcg 144180
atcttggctc actgcaagct ctgcctccca ggttCaCaCC attCtCCtgC CtCagCCtCC 144240
caagtagctg ggactacagg tgtccaccac cacgcccagc taattttttg ttatttttag 144300
tagagatggg gtttcaccat gtcagccagg atggtctcga tctcctgacc tcgtgatccg 144360
CtCgCCtCtg CCttgCaaag tgCtggagtt acaggcgtga gccaccgtgc ctggcctggt 144420
ttttttgttg ttgttattta tttatttatt tatttatttt ttgagacaga ctctcgctct 144480
gtcgcccggg ctggagtgta gtggcacgat gtcggctcac tgcaagctct gcctgccagg 144540
ttCaagCCat tCtCCtgCCt CagCCtCCtg agtagCaggg aCCaCaggCg CtCgCCa.CCa 144600
cgcccggcta attttttgta tttttagaag agacggggtt tcaccgcatt agccaggatg 144660
gtctcgatct cctgatgtcg tgatccgccc acctcggcct cccaaagtgc tgggattaca 144720
ggtgtgagcc accgtgcctg gcctgatttt tttttttttt taatctggtc tcatacctct 144780
gacagctcat gaagaagtge tcctgcttca tatgtatatg tgttagcata gtgttaacat 144840
agcataggtg ttcggtgttt gcagtttctg tttgttttat atgaattaag gtgtattatg 144900
agcagttgaa gatatatagg aaattttttc ccaaaccact atctctgctc gttctattca 144960
ttcagtctgt ttatgttatt ccttcattca ttcattttat agaacagtgg agtgcctact 145020
gtatgcatct attgttctgg gtcctgggga agaaaacaaa gttcctgctt tcatggaact 145080
tacattatat tggcggagac agtaacagac aaacaaatgt agcctgtgta catgtgttac 145140
atgaaaagca gggtaggggg ctgggagaga gtagtaggga gtgctatttt cgaggtggtt 145200
gtcaggaaag gcctcactga ggaggtggca ttttgagtag acctgagcgc agcgggggcg 145260
taagcccagg cagcatgtgg aggaagagtg ttcttggtga aaggaacaag gatagaggcc 145320
cgaagctaga gagctcagca tgatcaagga acagcaagcc ccgtgtggct ggaatggagt 145380
gagcaaagga atgagcagta gaaggtgagt gagttgggag gtcaccagag accatggcaa 145440
ggacttgaaa gtgtcaggga cacattggaa gttggagcag ggaaatgatg ggatttatgt 145500
tttgtttttg ttttatgttt agtgttttta agggattgct ctatcagcta tttggaaaat 145560
ttagtgtagg gcttcaagaa gagaagcaga gaaacaacat tcttgccata gtcatagtct 145620
aagtaaggga tgatggtggt gtggattagg ctggtagtgg aagaccagtc cagttcgggt 145680
tgtatttgaa ggtagaggca aaaagattat atttctacca gcaagcccat ctatgaagtt 145740
acttgtatta ttaatttaat tgagacatgc ccacataaac taataaatag gaatttctgc 145800
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-49-
agtttggtta aacacccctg tatatcctgg ttcttctttt agttgtccag atgtctcttt 145860
aagtcaagta ttttttggtg gtgtaggagc ctagagattg aatttattca cccaaaaggc 145920
atttgagtga ttactatgtg ccaggcacta tgctgaatgc caaggatgta aataagaggg 145980
cgtagtctca gtCtgtttta CtCCagCttg gttCCttttt aatgaccctg acttgttaag 146040
catatcagtt atcctacaga atgtttaatc ttctgtactt tcctggttgt gttatttagc 146100
ttatttctct ttccttgaca tttcttgtaa actggaagtt acacctatag tcttgatgat 146160
tcgtgttaca cattttagat tagaacacat catgtgttgt atatggtgtt tttgaaagcc 146220
tctctgtata ttggtctgta cattaaaatg ttgcctgaat ggatacacat aaaatttaac 146280
agtgattaca ttagagatga gaagaaagag gtgcctttta cttttcaata taccttttcc 146340
tctgcttttt gaactttctt gccctatgca tacgttattg cttaatcatc cacctcatct 146400
cttcccctgt ggctttctgt tgcatttgga atgaaatcta gcctctttgc tgttacctgt 146460
ggatgtccct tgctggcctc tatcacctta ctttgaacca ctcctttcat ggactgagct 146520
ctcattggac tatcttttat tcttttgctg aagtttcttc actttgagtg cctctgcagt 146580
tgctatttca tggctgtggc aagccctgcc atggctttca tgcaaggatg gttcctcctt 146640
ctcatctcaa tattatctct tcagagaggg accttcccaa ctccgatgat ctaaaatcct 146700
ttgtatatac cactcactac cacttctttc ttttcttttc cttttatctt tttttttttt 146760
tttttttttt gagatagggt cttgctctgt tgcccaggct ggaatcacga ctcactgcag 146820
cctcatcttc ttgggctcaa atgatcctct cacctcagcc tctcgagtag ctggaactgc 146880
aggcacacac caccatactt ggcttattat tttacttttt gtagagacag ggtttcacca 146940
aggctggtct caagctcctg ccgcaagcaa tccacatctc tcagcctccc aaagtattgg 147000
gattatagga gtgagccact actcctggcc tattttctta ttcactgtct aaaattatct 147060
tgttcattta tttacatact tgtttatagc ttatttctca gctggacatg gtgcctcaca 147120
cctgtaatct caatactttg ggaggctggg ttggagaatt ggttgagccc aggacttcaa 147180
gaccagcctg ggcaacaaag tgagaccctg tctataaaaa attgtttaaa aattagctgg 147240
gCatggtggC aCatgCCtgt ggtCCCagCt acttgggagg cagaggtggg agaatcgctt 147300
gggcccagga ggttgaggcg acggtgagcc atgattgtgc cactgcactc tagcctagtg 147360
acagagtgag accatgtgtc taaaaagtaa ataaaaatag tttctctttc atgactagaa 147420
tattacctct atgtgggcag ggagtttgtc tatactattt ggcactatat ttectgattc 147480
tgaaattatg cctagcacat ggtaagtact ccttaaatat ttattgactg aattatttaa 147540
tacttaagaa tttcatttgg gattatctga gtggtaagat tacggattat atttatgtaa 147600
gaaaaaatca ttttttaaac ttggttgccc tttgccacac tgacatagac actaagtttt 147660
cttagccaga ttacttccga ggatactcac agaggccatt ctcttctcaa tccccaaata 147720
attgatattt cttagcactt tcaagctaat gcaattctta gatgatgtat ctgtgtatat 147780
catatcctca ttctacaaat gtagaaattg aagtctgggc acagtggctc tcacctgtaa 147840
tctcagcagt ttgggaggcc aaggcgagcg gatcactgag gacaagagtt aagaccagcc 147900
tggccaacat ggtaaagcct tgcctctatt aaaaatacaa caattagggc cgggcgtggt 147960
ggctcacgcc tataatceca gcacgttggg aggccaaggc aggcagatca cgaggtcagg 14.8020
agttcgagac catcctggct aacacagtga aaccccatct ctactaaaaa tacaaaaaat 148080
tagccaggca tggtggcacg cgcttgtagt cccagctatc gggaggctga ggcaggtgaa 148140
tcccttgaac ccgggaggcg gaggttgcaa tgagctgaga ttgcaccgct gaactccagc 148200
ctggtcaaca gagggagact ctgtctcaaa aaaaaaaaaa aaaaacaatt agccaggcgt 148260
ggtggcgggt acgagtacct gtaatcccag ctactaggga ggctgaggga ggagaatcac 148320
ttaaacccag gaggtggagt ttgcagcggg ctgataatgc accactacat tccagcctgg 148380
gcaacagagt gagactctgt cttaaaaaaa aaaaaaagaa agaaagaaat tgaggaatgt 148440
ggagattgtg gtctgtgatt tgttaggaat cacacagcag gttagtagca actacagggc 148500
tttggttCag aatacCacCt tgaCaatggt ttgtttaCag ttCggCtCCC CttCCtCtgC 148560
ctttctctcc ttccttattg agggcagctg gaaagaattt tcatcattta ctagcctata 148620
gctttaattt gagttttgaa accttgataa tagagcacag aggaaaagac tgagttttct 148680
ttttttgaga cagtcttgct ctatggccca ggctggagtg cagtgacacc atctcagctg 148740
gttgcaacct CtgCCtCCCa ggttCaagCa attCtgCCtC agcctctcga gtagctgaga 148800
ttacaggcac gtgtcaccac gcccagctaa ttttctgttt ttgtttcgtt ttgttttttt 148860
ctgagatgga gtcttgctct gtcacccagg ctggagtgca gtggtgcgat gttggctcac 148920
tcaaacctct gtctcctggg ttcaagcaat tcttctgcct cagcctcccc agtagctggg 148980
actacaggta cgtgccacca tccctagttc atttttgtat gtttagtaga gatggggttt 149040
cactatgttg accaggctgg tctcgaactc ctgatctcag gtgatctact cgtctcagtt 149100
tcccaaagtg ctgggattat tggcacacgc ctatttttgt atttttagta gagacggggt 149160
ttcaccatgt tggttagact ggtctcaaac ttctgacctc aagtgatttg cccgccccag 149220
cctcccaaag tgctgggatt acaggcgtga gccaccgtgc ccagccaaga ttgagttttg 149280
aaaagagcct tctgagatta tgagaagggc aagcaagata acttaagaag ttacattaaa 149340
atcatctaag agacagtgta acaagaagga attgtaaaat gatgttatga gcacgtgccc 149400
aatgtagtgg caatcccttg tgcttcgata cattggtggg agacaaaact gtacttaaat 149460
tgataaatcc cttacatgtc attttaagga gcttagactg actcccatca tgtagacatc 149520
agagatttct tttttttttt tttttttttt tttttttttt tttgtgacag agttttgctc 149580
ttgttgccga ggctggagtg caatggcgtg atctcggctc accacaacct ccacctccca 149640
ggttcaagca attctcctgc ctcagcctcc cgagtagctg ggattacagc catgcaccac 149700
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-50-
cacgcctggc taattttgta tttttagtag agacggggtt tctccatgtt gtggctggtc 149760
tcgaactcct gacctcaggt gatcctcccg cctcagccac ccaaagttct gaaattacag 149820
gcgtgagcca ccgcgcccag cccagagatt tctaaacaga gttctaacca gatgcttttc 149880
cctgtcagta gaatgagaat gaattggagg tgggagagac tggcatgagg gacaccagtc 149940
agccagtgga attagctggt aatgttgata ggagaagaaa aagattcaaa gttaggtagt 150000
ggtagcaaga attagaggga aggtcggatt tatgatatgt ccaaggttga attctaaggt 150060
gaaatttggt ggcagatttc atgtgtaaat tgggaaggta gattgagttt ttttaacatg 150120
ggttttctaa catgtcaata gagtgactct gcaggggggc ctgacgagag aacagtgcat 150180
ggggtgattc aacagccagt tgagccttca tgcagagcat ttaacactgt gactctgtag 150240
actctggttg gcagtaaaat ttcattaaac caatatttaa acccttaggt aataataaaa 150300
attgagggaa aaggatccag gttttgtatt ttttatgaat tcagttattg aattaaacag 150360
gaccttgcct caagaaataa tctaccaaca attaacttgt tttaaagcaa agttaggaag 150420
tgagcatgtt caaattatta aataaaaaag taagctgtgt atttcattca tagaaataga 150480
ggctggccta cttcggatga ttctcagcat gtgattacag atgtgggctt atacatccta 150540
gggagttaag gcgtactctg gcttggatag agtagagctc tttgaaactc ttctctcacc 150600
cagctagttt atatagacta gagaactaga atgtagcagc atactctgtc ttagaagccc 150660
ttttatatag gagctggtct ggaaggtttg aaaacataac aaatgtgttg gtgtctccca 150720
atgtattgct agattcttac ccaagagcat tatcctggtt agggtttggt ttggttttgt 150780
tttgtttttt aatgtttgcc acaaactaac actagatgtt agttctttca tcaagtgagg 150840
agagtagaag aaaagtccag aactctgaaa caccttttca aaagtttttc aagccatgat 150900
gtttgcaagt taaatgctct gttatgtaag caatataatc agtttttatt aatgtaacat 150960
tccttagtgt tttggggtat cacacaaaaa agaatatcca tatctggaag caacagcttt 151020
taaataagag cattgtggtg gtggtggtga tagtggtttt tttttttttt tttgagttgg 151080
agtctcgctc tgttgcccag gttggagtgc agtggcacga tctcagctcg cttcaacctc 151140
tgctcccagg ttcaagcaat tcttctgcct cagcctcctg agtagctggg attataggca 151200
cctgctacca tgcctggctg atttttatta ttttagtaga gacaggtttc accatgttgg 151260
ccaggctggt cttgaactct taacctcagg tgaatcaccc acctcggcct cccaaagtgc 151320
tggaattaca ggcatgaacc accatggcca gccaaataag agcattttta atgtaaaatt 151380
atgcatgaaa tgtacattca attttgtctt tgtttactag gatccatgtt ctcacaagct 151440
atgaagaaat gggtgcaagg aaatactgat gaggtaaatc ctacctttag gataaaaaga 151500
tttctgttta taagtgCCa.C CCtCatgtaa gtgaggttta aaattttcct tttctttagg 151560
tcccatgttt aagcagcatg gcacatttat gttctcttac ccagaatgta ccaagaaagg 151620
gtggtccctt cttaacatct aacaattgcc tggtagtagc agtgaaggta tcttcagtca 151680
gaggctagga ccactgaagg atatacatgc attcaagttt ccatcagcca gcaggcatca 151740
gtaatcagtg tgtagatcaa aagctcaaat gtttccttcc ccactggcag ttttacttca 151800
agtagtggag gcttgctttt ttaatagtta attaagtaca ttgagagatg ggaggtgaaa 151860
aaaggaaaat gttttatttt gaccatctaa tatgaaagta gttcggtgtt aggtatccag 151920
tagttgacac tggaagacag ggaatgacat gttaatattc atagccagag ggtggcccag 151980
gttttttcgt acatgggaat gaaattctta tccaaataag tagaaattat gtgcgtaagc 152040
catttgttaa gagcactgag tatgtgcatc tcgatccatc taatgaataa ccattatcac 152100
cagtttaaat tattttcttt aggcccagga agagctagct tggaagattg ctaaaatgat 152160
agtcagtgac attatgcagc aggctcagta tgatcaaccg ttagagaaat ctacaaaggt 152220
aaggatgact tcgttttgtg taaactaaaa agtattattt tccaggtgta aaaataaaaa 152280
agaacataag gggtttcttt gcctttgaag gattaactgc tgtggggatt accttcttat 152340
cataagcaac tagaaaattg acaaactaaa tgaaacaact gtttgcatat attggacaat 152400
gggcaataca gggaaaccat ggaaaccaaa cagagcccag tagtcttgct gaacgaaaga 152460
gttaaatatc aaagttcagg ccaggtgcag tggctcacgc ctgtaatccc agcactttgg 152520
gaggccaagg cgggtgaatc acttgaggtc aggagttcaa gaccagcctg gccaacatgg 152580
tgaaaccctg tcttagccgg gtgtggtggc aggcacctgt aatcccaact atttgggagg 152640
ctgaggcagg agaatcgctt gaaccaggga ggcggaggtt gcagtgagcc gagatcacac 152700
cactgcactc cagcctgggc gacgagcgaa accccatttc aaaaaaaaaa tcaaagttca 152760
gagagctcaa tttgagtaga agttgtagga taaggtagca gaaaagagga agctgcccag 152820
aaagaaagcc gtagagatat ttagagagat tcccatggat ccttggccta ggagtgatct 152880
gtatatgtgt ggggtgaaaa cgcatgtgtc caggtagaga accccccaga aattagtagg 152940
ctgaatgatt gctggaacat agggctaaga aaagttcatg gccagaagga tctggccaga 153000
gtagagagac ttagtaatac acaaggcatt gggtagtgtc ttcacagagg ttatgcctta 153060
ctactgaaga taaattagtc ctagagtaca agcacctgaa ccaagtttca aagcaaattt 153120
ttaaagggtc aaattaccta acaactgcat gccaaaacaa aggcctaacc ctctttacag 153180
taacacaaca aaattcagca cttcacagtg taaagttaga atgtctgacg tccaggctgg 153240
gcgcagtggc tcatgcctgt aatcccagca ctttgggagg ccgaggcagg tagatgacct 153300
gaggtcagga gttcaagacc agcctggcta acatggtgca accccgtctc tattaaaaat 153360
acaaaaactt agccaggcat ggtggccggc acctgtgatc ccggctactt gggaggctga 153420
ggcaggagaa ttgcctgaac ccaggaggtg aaggttgcag tgagccgaga tcgcaccact 153480
gcactctggt ctgggcaaaa agagcaaaac tcaggctcaa aaaaaaaaaa gaatgtctga 153540
cgtcaatcac aaattaccaa gcatgacatg aagttgacct ataaccagga gaaaactcaa 153600
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-51-
tctatagaaa cagacccaga tgtgagaaag atgatgaatt tagcagacaa agaccatcaa 153660
gtggctattt taaatattaa aaatatgttc aagtggccag gtgcagtggc tcatgcctgt 153720
aatcccagca ctttgggagg ccaaggtggg taggagttca agaccagctt ggccaatatg 153780
gtgaaacccc ttctctacta aaaatacaaa aaaattagct gggcatggtg gcaggtgcct 153840
atagtcccag ctatatggga ggctgaggca caagaatcac ttgaacccgg gaggtggagg 153900
ttgaggttgc agtaagccga gattgtgcca cttgtactcc agcctggaca acagagtgag 153960
actctgtctc aaaaaaaaaa aaaaaaaagt taaagaaaac aagagtataa tgagaaaaat 154020
gcaaaatagt tttaaaagaa ccaaatggaa tttcttaaaa taaaaaatac cagaaatggg 154080
ggccgggcgt ggtagctcac gtctataatc ccagcacttt gtgggggctg aggcaggcag 154140
atcacctgag atcggtagtt caaggccagc ctgaccaaca tggagaaacc tcatctctac 154200
taaaaataca aaattagctg ggcgtggtgg cgcattgcct gtaatcccag ctacttggga 154260
ggctgaggca ggagaattgc ttgaacccgg gaggcagagg ttgcggtgag ctgagattgc 154320
accagtgcac tccagcttgg gccacaagag tgaaactccg tctcaaaaaa aaaacaaaaa 154380
aaaacagtag actcgaagaa ctagctgagt ttttctttac tttaggcagt aagtgtgacc 154440
ttttgcaggt gactacttta gttcctcatg tcctcattag tagatcagag aaattcgaca 154500
ccaaaacccc aaaagaaaaa ccccttctaa tcctcattcc atgattttat gaatgcatga 154560
agtcctaggc ctgcgaagga atactcattc tctttatcct gtgttgatac ctctctgctt 154620
caacctccaa ctcgacattt gcctatagga tgtacttgga cattcagcat aaactacctc 154680
acaccattac tgaattgctt catgtgcaca tgtcccatgc cacaataccg gggaccttgt 154740
cttccgtgat atttgtccgc agtgctgtga ctacaggagg gagtcagtga atgtctgcat 154800
gtgtgtcttt aCCatCCCtC ttgaatatgc tctagggtta attcctagaa gtagaattac 154860
tctattgaaa attggcaata tttttcattc taatatctat tgccaacatg ggaaagcaag 154920
tctggatgcc agtccttgtt atatgcccct tgggtaagtt acgtaacctc tttaagcttc 154980
tgttcactca tattttaaca aggaaaatta caatatttta cctcacaaaa ttgtagtcag 155040
cttctggctg tcttaaactc tggtatatag taaacactaa gtgttggtgt ccatccttaa 155100
tttgtaataa taggtcactt gttagagaaa tgcaccttac CattttCttt tCttttcttt 155160
tttcagttat gactcaaaac ttgagataaa ggaaatctgc ttgtgaaaaa taagagaact 155220
tttttccctt ggttggattc ttcaacacag ccaatgaaaa cagcactata tttctgatct 155280
gtcactgttg tttccaggag agaatgggag acaatcctag acttccacca taatgcagtt 155340
acctgtaggc ataattgatg cacatgatgt tcacacagtg agagtcttaa agatacaaaa 155400
tggtattgtt tacattacta gaaaattatt agttttccaa tggcaataac ccatttatga 155460
gagtgtttta gcctactgga atagacaggg accacatcct ctgggaagca gataagcata 155520
gaactgatac ttgatgcaca ctcgtagtgg taactcatcc ctaatcagca ttgtaaagca 155580
ggtgccagag gtggtttgct ttgtccttcc aaagcaggtg agtcagcccc accgagagcc 155640
aggcagcttt gagtggcagc gtggtgctag cagcttcagc ggaacagggt gagagttaat 155700
tatgcagtct tcttgacagc ggcattaatt tggaaggaaa ctgacaagtc atgggtcaag 155760
tttcagtgac ttcctccttc ctctgatggc agtatatagt tttcacattt taattcctcc 155820
tcctgagatg cactatactt aaaaccattc tctcccctgc taacagaagg gtgtgaatct 155880
ggtttacttt gagcattagg atttgcccct ttggaattct gcactccagt tacttaactt 155940
tcccttcaga atacatgtgg aaagaaagaa agaaatagcg atgactccac ttttgcccct 156000
gtggcacctt gaacaaagca gttcttccca aattatactt tttttttttt taaataaggt 156060
gagcaggatg actggggaga gagaaacatt tgactttgac tgcctccccc attctttgct 156120
gtgagctgga aagtgtgcag ttggtcgtct ttcttctcct ttctttagga tagtaagaga 156180
ctcactcact gcacttctgc tcagttggct tctgcatcgg gatcacacag ccatcagcag 156240
gactgcccag ttggtgagca cactccattg accacgtggc gccagcgctt cctcaatgca 156300
catgattgag aggaaagaaa gttctcttag atgttactgc ttttgctcag actttgcaaa 156360
aaaaaaaata tatatatata tgtataaata tataattatt aatcactttt gtccttgaga 156420
aagtcttgaa tgaacagaga atttattcca ttgcaatatt tgattgtata gaggcacact 156480
gtttcatcga cagaagaagc aaaaaggctt tgtgtaagtt tttggtacta tgtaccacct 156540
ctgttattct tttaaagctg aagtattcat gtacttaaac catattatat ttaattgtgt 156600
ttgattttaa aatatatata tatgaattct atttaaaatt gtgtcaactt tctgctttca 156660
gggcatttat ggctcttctg ttgaaatata ttgatctttc caaatatttt catttgcttt 156720
ctaaaaaccc agaacatgag ccactactgg actttgcctt gtgtttgaag tgtatggcat 156780
aaacccaagg tttttattag tcatctatgc tgtgattaat tcattttgtt cttttaacaa 156840
aatatttcca tccacttcac attgcttcaa tctttaacag aaaagcaata taaaggttat 156900
agaataaaat gtggttttgg gcaactcttg ctgcctctgc atgttttgga ataacaattt 156960
ctacaagact ctaggctgtt taaactagtg ctttcagtta agataaattc taatcatttc 157020
tttgtatata cattttgtgc ttctgagcta gagatgccaa gtagttgtaa actgcttata 157080
aagagaatag cagcaaattt gagactcggc tacttttttc tgccccacct gctttgagac 157140
acagaagcgg agtgtggccc gaaattatta gccagattta atatttgatc taaagtaggt 157200
ccttgtactc attttaaagt tggaatttga ttcctccaac attgagcacc caccatgttc 157260
caggctctgt gcattgtgcc cacaaaataa gattccctgg tggagttttt atgggttcaa 157320
ataatcagtt gaacaccctt catctttatc atgttgttga cattgacaca aattgtttaa 157380
aaagaaaaga tattagagag aaagtggtac ctttgtaact tgatgtgtct tcatcattcg 157440
gtaagatttg atgaaagtaa aaagcaaatg tcagccaaat ccagtgaaca gcaataaaac 157500
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-52-
agggagtaac tttttataac tttttctact tggatttcaa cattcagtag agcttttcga 157560
aatgtaagta gtttacagta ctggaggttt gactagttca gtaggaattt ggaggggaag 157620
gtcattctga attgtaacaa agtacaaact tctttgctgt tttatttaag tactgagagc 157680
taagcacctg atgaagtgac tgacctctct ccagtgacag tgtttgggta cctgcctgac 157740
ttcaggagtg gggtttatgt ttctacacag tgaccttttc tctcgccctc tcctccctct 157800
tgcccacaca ccagttgatt ggacctgggt tgaactcctg atccagacag gcccaagaca 157860
gttcttaatg ttaagaattt tggggccggg cacggtggct catgcctgta attgcaacac 157920
tttgggaggc cgagacaggc ggatcacttg aggtcagggg ttcgaggcca gcctggccaa 157980
catggtgaaa ccctgtcttt actaaaaata caaaaattag ctgggcatgg tggcgcacgc 158040
ctgtaatccc agctacgtgg gtggctgaga caggggaatc gcttgaacct ggaggcggag 158100
gttgtgcaat gagccgagac cgtgtcactg cattccagcc tgggtgacag agggagactc 158160
tgtctccaaa aataaaaata agaaaaagaa ttttgggcta ggtgcagtgg ctcacgcctg 158220
taattacagc attttggaag gcccaagatg ggcagatcac ttgaggacag gagttcgaga 158280
ccagcctgga caacatggtg aaactccatc tctactaaaa agacaaaagt tagccagatg 158340
tggtgatggg cacctataat cctagctcct cgggaggctg gggcaggaga atcacttgaa 158400
cccaggaagc agagattgca gtgagccaag atcacatctc tgcactccag cctgggcaac 158460
agagcaagac tctgtctcaa aaaaaaaaga atttggccag gcgcagtggt tcacgcctgt 158520
aatcccagca ctttgggagg ccaaggcagg cagatcacga ggtcaggaga tcgagattgt 158580
cctggctaac atggtgaaac cctgtctcta ctaaaaatac aaaacattag ccgggtgtgg 158640
tggtgggcac ctgtagtccc agctactagg gaggctgagg cagaggaagg atgtgaaccc 158700
aggaggcgga gcttgcagta agccaagatc gtgccactgc actacagtct gggcgacaga 158760
gtgagactcc gtctcaaaaa aaaaaagaat tttggccggg tgcggtggca catgcctgta 158820
gtcccagcac tttgggagac caaagtgggc ggattacctg aggtcaggag ttcaagacca 158880
gtccggccaa tatggcgaaa ccctgtctct tactaaaaaa aatacaaaaa ttagccaggt 158940
gtggtggcgg gcacctgggg aggctgaggc agggagaaat gcttgaaccg gggaggcaga 159000
ggttgcagta agccaagatc gtgccactgc actccagagc aagactcttt ctcaaaaaaa 159060
aaaaaaaaag aattttgcat ggggaaggag agatactgtt caccatctgg aatggtgctt 159120
ggatgtggca cttacaaaat caggagccag cactgcatgg acaaacagaa gcatgtgggc 159180
CtgagatagC aggtaCCttg ataaCCCtga agaCatCCtt ggtttCtgCa tCtattCCtg 159240
catccttgca ttggactaca ttaatctgtc agttatcctt ataatgattt ttgatttttt 159300
ttttttgaga tggagtttcg ctcttgttgc ccaggctgga gtgcaatggc acgatctcgg 159360
ctcaccacaa cctccacctc ccaggttcaa gtgattctgc tgcctcagcc tcctgagtaa 159420
ctgggattac aggcatgcgc caccacacct ggctaatttt gtatttttag tagagacggg 159480
gtttctccat gttggtcagg ctggtctcga actcccaacc tcaggtgatc accctgtctc 159540
ggcctcccaa agtgctggga ttacaggcgt aagccatggt acccggtctg ttttttgatt 159600
ttttgaaacc agtctgaagt gagttttttt aattacgtga aaggagtttg gctaaaatac 159660
tgccatactg ccctaatgcc taatgattat gtattctcag catgtctgca aagtactgct 159720
gatttctgga gaataatttt tctttagtaa acttcactta agtcgtcatg tgtattctct 159780
caaaatggta tcctaaccta atggagctaa aagacacccc ttgtttttat aacaagcagt 159840
tactgaggcc caggaagggg agaagtccct ggcttgtgag atgatcacca ttagaactca 159900
ggcctgggcc agtgcctttt catgcttctc agatccttcc aaagaataat gaagattata 159960
accgctttta gcaattgtaa taaacccaga aatagaaagc tttttggtta gagtactggt 160020
agaagtttgg cgggagagat aatttttaca aaatttgtaa atacctgcca attctatata 160080
ctaggcaagg tctctggcct tgtaaaaccc ctcaaggtta caactttggt ggcccacact 160140
aatagttacc cactgaggcc ctctccgggt gaacattgag cactagagga agcccctctg 160200
cttgggcagg actgggcgtg gtgcagagta ggagcggtga tactgtggat tctgggcagg 160260
tggagatggc cagtgatgtc caataaagga cactggaggg agcagtgtga gtaaaggccc 160320
tgagggcatt catgttcagg gagggttgct gcccactggc ttgcttggca cacaggagag 160380
tgggtattcc tgccttagta actttatgta aacaagtatt tcctcagtct gttcctctca 160440
aactgcctgc tctggcacat tcagaatgtc acagaactca cctggatgca ttcagcccct 160500
tgcctaaagg tgacagtgca tCtCCttCCC CaCCCCaCCC CtCataCCaC tgaagcacct 160560
gtcagactgg cccagtctgt gggcaaggag cctagagagg gcttagtttc agcttgaaag 160620
gagctgggat ttaccaagaa gcaaatgaga gacgaggatt gcaacaactg tgccatttcc 160680
ccagcttcag ctgactcctg tatattgact gtgccttcag actcatccgt aagtgacccc 160740
aggctggcct ctcccacatc acagtaagaa ttccacacac catacaactt ggaaagaggc 160800
tccagctgaa ggaagcccca cacttctttc aagtttttct tagtcttctc ttcttggcaa 160860
agagtacctt ttgtttettc taattatgta actattggtt tagtaaatat tcacccattc 160920
agtcaccctg taagtggcag gcactgttta cagggacaca ggaaggaata aaaacttgca 160980
ggcaccttgg agcttgcatt ctattgaaga ggtaatggaa gttgggatag cagctaaact 161040
atgctggtat tggccaggcg cagtggctca cacctgtaat cccagcactt tggaggccaa 161100
ggtgggcaga tcatgaagtc aggagatcga gaccatcctg gctaacatgg tgaaaccccg 161160
tctctactaa aagtaaaaaa aaaaattagc caggtgtggt ggcgggcgcc tgtagtccca 161220
gctacttggg aggctgaggc aggagaatgg tgtgaaccca ggaggcgaag attgcagtga 161280
gccgagatgg caccactgca ctccagcctg ggtgacagag cgagactctg tctcagaaaa 161340
aaaaaatatg ctggtagttt tgattcaaga tggectttgg agcccatgat ttaggtctcg 161400
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-53-
tacccaccaa ggtctactgg aaaacatcag gctctcctgc tatagaccca tagggagagc '161460
tgcagccgag agggggagct gaagagaagt gccccttctg tgtcctgtca gcctcatcct 161520
tccgcaagga ccagttgctg tgccactcca ttcacttgct gcaagactgg aggtttttcc 161580
tcaggtgttg agcacctggt ttacaagatg tcagcatctt gatgcctgag accatcaagg 161640
caagtctctg aacagggctt accttagagt aaggcttaga agaggccgta aagtcagtct, 161700
cagctccgtg gctctgcaga gctttgggac atgtgaattc ttaaaaacaa gactattgta 161760
cagttactat atgcatgcag tataaaatta taaccttgga aaatcctagc tagctgttga 161820
gctaattcca taaagtaatc agctcctgag ttctgcagtg gtaataataa tcagcataat 161880
gagtaaacac tgtgtgtgcc aggcagcgtc tcatttgatc cttgtgataa tcttgtaagt 161940
actgattttc tcccttcttt aaacaaagtt tttttttttt ttttagagag ggtctcacta 162000
tgttgcccag gctagtcttg aattc 162025
<210> 14
<211> 162025
<212> DNA
<213> Homo Sapien
<220>
<221> allele
<222> 129600
<223> Nucleotide sequence of chromosome 17 containing
the genomic sequence of the allelic variant
AKAP10-7
<400>
14
gaattcctatttcaaaagaaacaaatgggccaagtatggtggctcatacctgtaatccca60
gcactttgggaggccgaggtgagtgggtcacttgaggtcaggagttccaggccagtctgg120
ccaacatggtgaaacactgtctctactaaaaatacaaaaattagccgggcgtggtggcgg180
gcacctgtaatcccagctactcaggaggctgaggcaggagaattgcttgaacctgggaga240
tggaggttgCagtgagCCgagatCgCgCCaCtgCtCtCCagCCtgggtggcagagtgaga300
ctctgtctcaaaaagaaacaaagaaataaatgaaacaattttgttcacatatatttcaca360
aatttgaaatgttaaaggtattatggtcactgatatcctgtttcattctttatataatca420
ttaagtttgaaatgtatacttgcactactaacacagtagttaatcttagtcctacaagtt480
actgcttttacacaatatattttcgtaatatgtatgcactggtgtttatgtacgtgttta540
tgtttatatctgttaaaattagcagtttccatctttttctattttgtaccatcacatcag600
ttcagaaggattgaeagagcaaaatgatttgatgaagtataaaagtcacatggtgagtgg660
cataaatacaactctgaacaattaggaggctcactattgactggaactaaactgcaagcC720
agaaagacacatatcctatatgtcaagagatgtaccacccaggcagttaaagaagggaag780
tacacatagaaagcacaatggtgaataattaaaaaattggaatttatcagacactggatt840
catttgctcctaaagtcagagtcctctattgtttttttgtttttgtgggtttctttttaa900
atttttttattttttgtagagtcggagtctcactgtgttacccgggctggtctagaactc960
ctggcctcaaacaaacctcctgcctcagcttcccaaagcattgggattacagacatgagc1020
cactgagcccagcccagacgctttagcatttatgaagcttctgaaatagttgtagaaacc1080
gcataagctttccatgtcactttcaaagtttgatggtctctttagtaaaccaaccaagtt1140
attcctcaagggcaaaataacatttctcagtgcaaaactgatgcacttcattaccaaaag1200
gaaaagaccacaactatagaggcgtcattgaaagctgcactcttcagaggccaaaaaaaa1260
aggtacaaacacatactaatggaacattctttagaagagccccaaagttaatgataaaca1320
ttttcatcaaagagaaaagagaacaaggtgttagcaaattcctctatcaaataacactaa1380
acatcaaggaacatcaatggcatgccatgtggaagaggaagtgctagctcatgtacaaac1440
cagtagataatttcaacttgctgccgaatgaaacctctttgcaaggtatgaatcagcact1500
tctcatgtttgttttgctttgttttgttttgtttttagagaCaggCCCttgCt CtgtCa.C1560
acaggctggagtgcagtggcacgatcagagctcactgcaacctgaaactcctgggctcaa1620
gggatcctcctgccttagcctcccaagtagctgggactacaggcccaccatgcccagcta1680
attttttaaattttctatagagatgggatctcactagcacctttcatgtttgatgttcat1740
atacaacgaccaaggtacaatgtggaaaagggtctcagggatctaaagtgaaggaggacc1800
agaaagaaaaggggttgctacatagagtagaagaagttgcacttcatgccagtctacaac1860
actgctgttttcctcagagcagagttgatgatctaaatcaggggtccccaacccccagtt1920
catagcctgttaggaaccgggccacacagcaggaggtgagcaataggcaagcgagcatta1980
ccacctgggcttcacctcccgtcagatcagtgatgtcattagattctcataggaccatga2040
accctattgtgaactgagcatgcaagggatgtaggttttccgctctttatgagactctaa2100
tgccggaagatctgtcactgtcttccatcaccctgagatgggaacatctagttgcaggaa2160
aacaacctcagggctcccattgattctatattacagtgagttgtatcattatttcattct2220
atattacaatgtaataataatagaaataaaggcacaataggccaggcgtggtggctcaca2280
cctgtaatcccagcacttcgggaggccaaggcaggcggatcacgaggtcaggagatcgag2340
accatcctggctaaaacggtgaaaccccgtctactaaaaattcaaaaaaaaattagccgg2400
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-54-
gtgtggtggtgggcacctgtagtcccagctactcgagaggctgaggcaggagaatggtgt2460
gaacctgggaggcagagcttgaggtaagccgagatcacgccactgcactccagcctgggc2520
gacagagcgatactctgtctcaaaaaaaaaaaaaaaaaaaaaagaaataaagtgaacaat2580
aaatgtaatgtggctgaatcattccaaaacaatccccccaccccagttcacggaaaaatt2640
ctcccacaaaaccagtccctggtgccaaaaaggttggggaccgctaatctaaataatcta2700
atcttcattcaatgctaaaaaatgaataaacttttttttaaatacacggtctcactttgt2760
tgcccaggctggagtacggtggcatgatcacagctcactgtagcctcaatcacccaggcc2820
ccagcgatcctcccacctaaacttcctgagtagctgggactacaggcacgcaccaccatg2880
cccagctaatttttaaattttttatagagatgggggtctcaccatgttgcccagactggt2940
ctcaaaccctgggctcaagtgatcctccctcaaactcctggactcaagtgatcctccttc3000
cttggcctcccaaagtgctgggattacaagcatgagccactgtacccagctggataaaca3060
ttttaagtcgcactacagtcatggacaatcaggcttttcaacatgcagtatggacagtga3120
gtcccagggtctgcttttccatactgaaatacatgtgatactaaggagaaaggtgctcgc3180
aaggatatttaaaatgaagaatatttaaaatgaggaaaaaactgtttcttcatgactttg3240
ataaggctgataaagaccatttctgtgatctcaggtgattcactcaagtagtatatttca3300
gtaatcattatctggaacagcctgaatcttaaccaaaataccatgattttttaatgctgt3360
tatgataccttgatgatatgaccaaactgcaatgtaggcagctaaatctccacgagtttg3420
acttccccgagagttgacagttttcttcacaaattaaagaaatatattttttgatacatg3480
attggcatatttaaaaactacactgaaatgctgcaaaatgatataaagaaacattttcca3540
gaatcaaatgcaatcaaagagtggattaggaatctactcaccattatcaactaaatagaa3600
acacttggactgggtgtggtggctcacatctgtaatctcagcactttgggaggccaaggc3660
aggtggattgcttgaggccaggagctcaagaccagcctgagcaacatagcaaaactctgt3720
ctctacaaaaaaaaaaaaaaattaaccaggcatggtggcagatgcttgtaatcccagcta3780
ctctggaagctgaagtaggaggactgcttgagcccaggagatcaagactgcagtgagccg3840
tggtcatgctgcgccacagcctgagtgacagagagagaccctgtctcaaaaacaaaaaca3900
aacaaaaaacacttaaccttcctgttttttgctgttgttgttgttgtttgtttgttttga3960
gatggagtctcactctgttgcccaggctggagtgcagtggcgtgatcttggctcactgca4020
agCtCtgCCtCCCgggttCaCgCCattCtCCtgCCtCagCCtCCCgagtagCtgggaCta4080
taggcgcccgCCaCCaCgCCCggCtaCttttttgCatttttagtagagatggggtttcac4140
CgtgttagCCaggatggtCttgatCtCCtgaCCtCgtgatCCaCCtgCCtCggCCtCCCa4200
aagtgCtgggattaCaggcatgagCCaCCgCaCCCggCCaaCCtttCtgttttttagttt4260
gatatgcttgttaactcagcagctgaaagaatgctgaaagtggccttcagtaaaaaaatt4320
tcactagaatctctacatccatatttaatctgaatgcatatccagattgatcagttagag4380
caaaaacactcatcatcattcctgatgacctctaattctggtttcggctttctatttcaa4440
tggaaacagaataaggaaagaaatggaagggctctggaaatttgtcctgggctatagata4500
ctatcaaagatCaCCaacaataagatctctcctataaatataaaacaagtataattaatt4560
ttttaattatttttttctcttcagaggattttatttcaagataaaacataacttctaccc4620
ata~ctattgattccaaaggttagaaaaagtgtttttcctcatcttatccttcaaagaggt4680
cacagcaatgcaaacatctataaaatgcctctgcataattgtcagaagctatagtccaga4740
aatcattgaaaatgcttttccattttaagcttaggtgaggtgtcttaggaaacctctatg4800
acaacttactctatttattgggaggtaaactcccagactctCCCagggtCtCCtgtattg4860
atCtCattttttaggCttCCtaatCCCttgaagcacaatcgaaaaagccctggatctctt4920
ttctgcacatatcatcgcggaattcattcggcttccagcaagctgacactccatgataca4980
agcggcctcgCCCttCtCCggacgccagtcCttgCtgCggttagCtaggatgaggggttt5040
gctgggcttcagtgcaggcttctgcgggttcccaagccgcaccaggtggcctcacaggct5100
ggatgtcaccattgcacactgagctcctggcaggctgtaccaattttttaattatttaat5160
atttatttttaaaattatggtgaatattttggtattctgctctaaaataggcccataaat5220
gcacagcagatatctcttggaacccacagctttccactggaagaactaagtatttttctt5280
ttaaagatgctactaagtctctgaaaagtccagatcctctaCCtCtttCCatCCCaaaCt5340
aagacttggaatttatgagagatctagctaacagaaatcccagacacatcattggttctt5400
cccagagtgcagtcctcctaaagaggctcagccctaagcaggcccctgcaccaggagggt5460
gggtctgagacccacatagcacttcccaaggtgcatgctccagagaggcactgaaacagc5520
tgagcacaagcctgcaagcctggagaactctcacagtcagaacggagggggcccagtggg5580
actaacataaagagaaaagggaacacagagaaatggatggcaccaacaaccagcaaagcc5640
ttcatggccaatgaaagcatcagtgacggggccagaaccctcatccccaaagactcttca5700
ctgcctttagtgaaaaacaatggctagagagtgaagttatgatcatgtatagagaggtaa5760
agttacatttttatattctgactctgctaatgtgaaattccctatctgctagactaaaag5820
tttcagacaccctgttcaaatatcccattagttgctagagacttaaaatgaacagaacgc5880
acattgtcaggatgactattaccaaaaaatcaaaagacagcaagtattggtgaggatgta5940
gagaaactggaacttttgtgcactgtttatgagaatgtaaaatggagcagctgctgtgga6000
aaagagtatgcaggttcctcaaagagtaaaaccaagatgtggaaacaactaaatgcccat6060
cagtggatgaaggggtagacaatatgtggtatatacataccatggagtactattcagcct6120
ctaaaaaaaaaaaaggaaattctataacatgcaacagcatggatgaatcttgaggacatt6180
ttgctaatgaaataaggcagtcatagaaagacaaatactgcacgactccacttatatgag6240
ataccaaaaatagacaaattcatagaatcaaagagtacaatggaggttacctggagctgc6300
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-55-
agggcgggaaacgaggagttactaatcaacgaacataacgttgcagttaagtaagatgaa6360
taagctctcaagatcagctgtacaacactgtacctagagtcaacaataatgtattgtaca6420
cttaaaaatttgttaagggtagattaacaaatgtagtagatccacaaatgtggttaagtg6480
ttcttaccacagtaaaataaaaaaagaatatcaagcccaggagttcgagactagcctggg6540
taacatggtgaaaccctgtctctacagaaaatacaaaaattagccagctgtggaggtgca6600
ctcctagggaggctgaggtgggaggcttgcttgagcccaggaggtcaaggctgcagtgag6660
ccatgattgcaccactgtactccagcccagatgacagagcaagacaccaccccccccaaa6720
aaaagaaaaagaatatcaaacattttaaaagatcagatacgcaagaacaacaacaaaaaa6780
gagatgaacagagcatcgaccctcatctagtgggattcttggtctaactgaaaaacagac6840
attgagagacaaacaatgacagtgatgtgatcacagcaattacacaggtatcccctgggg6900
actgcagaagaaaggaggaatgcctaactttcagaaaatagagaaagcgtcaaacagttg6960
gtgaaagccttccaaaactagagagaactgcacacaccaaatcacagaaagaagaaaagc7020
cgtgggagattctgggacccaccggctatttttgatggctgaacaccctgctgcaggaga7080
gacaggagctggaaagcatggtgggatgaaacctcaaacagctttgcctgcattgcttaa7140
gatgactgggcttgattaactctagtcaatggggacaattcaatcaaagaagaaagatgc7200
tcaaattcacattttagaatgattttttatggcagtatggggaatagattaaaagagagt7260
gaagctggaggcaagaaacttgttaagaggcaactgaaacagtctagatgataaataata7320
aactgacagagtgactagaaaaatcagaacaggctgaatcaacagatacctagatgaaaa7380
taacaggacttgatcaccagttgtatcttggagaggaaggagttgtttccttgctttccc7440
tacgactgggaatacggaaggtttgccgtgtgtattggttatatactggtgtgtagccaa7500
tcactgacaaccatttagcagcttaaaacacaaaggcttatctcccagtttctgtgggcc7560
aggaatctaagataggcttagctggctggttctggctcagagtttctcaagaggttgcaa7620
tcaagatgtcagctggggttgcatcatctgaaggctcaactggggccggagggtccactt7680
ccaaggagttCaCt CaCCtgcctgacaaggcagtgctggttgttggcaggagatctcaat7740
tcattgccaagtgagcctctctatagcattgctggaacatCCtCCCCatCtggcagttgg7800
CttCtCtCagCatgagtgatctgagagagagagcaaggaggaagccacagtgttcttcct7860
actcctactcctaacactatggacctactcctaacactctcacttctgccttattccatt7920
agttagaaagggaactaagctccacctcttgaaataagaagtgtcaaagaatttgtggat7980
atatttaaaaatcatcacaCtgtggaagtggatagggggttcaattaatgctgaacttga8040
aatgcctgagacattcaaatgtccaacaggcaatgaacatacccatagatggtcatgact8100
ttagcaagaatagaggaagatcacagaattaaggaggaattgaaaggtaaaagaagtgga8160
gtcagattccccctgaaaagtgagccatgaaaggaactttaactattgagttagaggtca8220
gagtaggaaatttcggtggaattcttttttaaagaaaggaaccatataagcatgttttga8280
ggtagagggagaataaatcagtagacagggagaggtaaaaaacataaatgataggggata8340
gttgacaaaggtcttggcagaatcccttacccattgacttggggccaagagagggacact8400
tctttgtttgagggataaggaaaataagaaagaatgggtgCtatttagtgtggtcctgtc8460
tctagggcaaacgcataggtaacaaactgtgtgtgttaggaatatagatgtgacctcaca8520
ttgagattctcacctcaaatccattttgttgttacctgtaccttcctaccttctcttttt8580
gctacatgcagactgctgttttgtcttcctggcctgttccaggtttcagcattctggcat8640
atCtgCtaCCCtgttCCCaaaCCtCtCtagagtCCatgCtCCttCCttggatagtgtttg8700
attgggCCaCgtatCtaagaagtgatgCCttCagttaggCCtgagaaCCtcctctatgga8760
aatCtCCatCagtgaccctgacagacttggtatcttggagatgtcactgctcccagcctg8820
tggtctaggagaatctcagcctgggcctctagtagtatggataaggcgttaaggtatctt8880
tgaaccagagtctgtcatattcctcaatgtgggacagataaaacagtggtagtgctggtg8940
tttctgagctagaactctggtttttggtctagattctttgatgtatgacctttcagaggt9000
attaaaatttgttctaatacaatgttcaatacaaatgtagttccttttctgttaggacct9060
caacaaaacatgaccaactgtagatgaacattaaactatgacaattcatggaaatgaata9120
cagtaatacctgcggttcccccattttagcagtcactatggtgacatttggcacaaatgg9180
CtatttaagggtgcttttgttaaaaCCtaCCatCttaCtaggcacatgatattgaaacta9240
atgaaataatggagaaacttcttaaaaacttttaatgaataaagtgatgaagtgataata9300
ttttagctgctatttataaagtgactattacaggtcaaacattcttctagggtttttttg9360
ttgaagttgtcacatttaatccttaataacccactatgagtcaggtattcttctctcccc9420
tttggacagttggggaaatgggggtcagagaggttaggtaatttgctcagggccacacaa9480
cctgcatgtagaaaatctgagatttgtacaggaacgtatcaaactctgaagtccatgctt9540
ctattttcccatgctgcctttctaataaaaggtaactaatgctactggatgctgccccca9600
aagtgagtcactttcaccccaccctacttgattttctccataaaactaatcacatcctga9660
caacttatttattgctgatctcccccactagattataaactcaataaaagcaagatcctt9720
gtctgctgaatatcagtacctaaaacgctgtctagcacagagcaagtaattaatatttgt9780
tgaatgaacaaataaaggaaaaaaattcaaaggaagaaaaagccctaaaacagatgttta9840
cctaaacatacattttaaaagaaagcatataacaaattcaggacagaatttaaatttgat9900
tttttaaagaaataaccaagtgctagctgggcacagtggctcacacctgtaatcctagca9960
ctctgggaggccgaggcaggcagatcacttgaggtcaagagttcaagaccagcctggcca10020
acatggtgaaacctgtctctactaaaaatacagaaattatccaggcatggtggcaggtcc10080
ctgtaaccccagctactcaggaggctgagtcaggagaattgcttgaacccaggaggcaga10140
ggttgcagtgggccaagattgcaccactgcactccagcctgagtaacaaagcaagactct10200
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-56-
gtctgaaggagaaggaaagaaagaaggaaagaaggaaagaaggaaagaaggaaagaagga10260
aagaaagaaagaaagaaagaaagaaagaaagaaagaaagaaagaaagaaagaaagaaaga10320
aagaaagaaaaagaaagaaagaaagaaagaaccaagtgcttatttgggacctactatgct10380
atgtttttccatgcacgctattttcagtaaagcagttagcaaacttgcaagatcataaca10440
acaaatatatgcttctataactctaaaattgtgctttaagaagttcctctttaccagctc10500
atgtatgcattagttttctaagagttactagtaactttttccctggagaatatccacagc10560
cagtttatttaaccaaaggaggatgcttactaacatgaagttatcaaatgtgagcctaag10620
ttgggccagttcatgttaatatactccagaacaaaaaccatcctactgtcctctgacaat10680
tttacctgaaaattcattttccacattaccaaggagccagggtaggagaatatagaaaga10740
ccacccaagaatccttacttctttcagcaaaatcaattcaaagtaggtaactaaacacat10800
gccctaacaatgaatagcagattgtgctcagaagaatgatctacaacatcttactgtgaa10860
ggaactactgaaatattccaataagacttctctccaaaatgattttattgaatttgcatt10920
ttaaaaaatattttaagcctaaattttaaaaggtttgatattggtacatgaatagacaaa10980
cagacatggactagaccaagaattaggttcaaacatatacaggaatttaatatacgataa11040
atctagtattccaaaggaaccaacaaatggtgttcagacagcaggataggcatcaggaaa11100
aacacagttgggcaccctaccttactcctaacaccaggagtaactgaaggagcaccaaat11160
atttatttattttaattatagttttaagttctagggtacgtgtgcacaacatgcaggttt11220
attacataggtatacatgtgccatgttggtgaggagcaccaaatatttaaaagaaaaaaa11280
ttggccaggggcggtggctcaCaCCtgtaatcccagcactttgggaggccaaggtgggca11340
gatcacctgaggtcgggagttcgagaccagCCtgagCaaCatggagaaaccccatCtCta11400
ctaaaaatacaaaattagccaggcatggtggcacatgcctgtaatcccagctacttggga11460
ggctgaggcaggagaatagctttaatctgggaggcacaggttgcggtgagctgagatatt11520
gcactccagcctgggcaacaagagcaaaacttcaactcaaaaaaattaataaataaataa11580
aaataaagaaagaaaagaaaaaaatgaaaatagtataattagcagaagaaaacaccgtag11640
aatcctcggactcttaggatggggaatgcctataatataaaaaccctgaagttataaaag11700
agaaaatcacctacatacaaaccaaatctttctacatgcctaaaacatagcacaaacaca11760
gctaaataatcatagctgaatgaactgggaaaacaaaacttgactcatatCcagacagag11820
ttaattttcCtacacataaagagtacctatataaacccaacaaaaaaaccaccactaacc11880
caaaataaaaatgtgacaggtaatgaacaggtagttcacagagaatacaaatggctcttc11940
ggcacataagatgctcagactgacttttacttatttattttttgagagacagggtctcac12000
gatgttgcccaggttaggctcaaactcctgggctcaaatgatagtaccaggactacaggt12060
gtgccccaccgcacctggctcctcaaccacctgtattaacaggaaatgcaaaataaaact12120
ttcaaatctattttacctattagaatggcaaaaatttgaaaaacttcaaacatcatcatg12180
ttggtgagaatgtgaggagactggcactctcattttttgctgatagcatatatatactga12240
tggcttctatggaaagcaatctggcagcgtctatcaaatgtacaagtgcatatatccttt12300
gacaaagcaattccactctaggaatgtgttctatatggttgtgcttcctggggctgggaa12360
ctgggagctaagggacaggggcagaagataatcttcttttccctccttccccgttddaca12420
tgttgaattttatatactgtaatatattatttttcacaaaagataatttttaagcgatat12480
gtctgggaatttttttttttCttttctgagacagggtctcactctgtcatccaggctgga12540
atgCCatggtatgatCtCagCtgaCtgCagCCtCgaCCtCCtgggttCaagCaatCCtCC12600
cacctcagcctcctgagtagctgggactacaggcacgtgccatcatgctaatttttgtat12660
ataCagggtCtCa.CtatgttgcccaggctaatgtcaaactCCtaggCtCaagCaatCCaC12720
ccacctcaggctccaaagtgctgggattacaggcgtgagccaccgcgcctggccctggga12780
attcttacaaaagaaaaaatatctactctccccttctattaaagtcaaaacagagaagga12840
aattcaacctataatgaaagtagagaagggcctcaaccctgagcaacaaacacaaaggct12900
atttctgagacaggaatttgctgaacaaaatcgagggaagatgacaagaatcaagactca12960
cttctcggctgggcgcagtggctcacacctgtaatcccagcactttgggaggccgaggcg13020
gacagatcacgaggtcaggagattgagaccatactggctaacacagtgaaacccagtctc13080
tactaaaaatacaaaaaattagccgggcgtggtggcaggtgcctgtagtcccagctactt13140
gggaagctgaggcaggagaatggcgtgaacccaggaagcggagcttgcagtgagccgaga13200
tCaCgCCaCtgCaCtCCagCCtgggtgaCagagcaagactctgtctcaaaaaaaaaaaaa13260
aagactcatttctctagatcttgagccgtattcaaatttatctcagcttagtgagaggtt13320
aaagcaaggaatatccttccctgtgggccctgctccttactgaaggaaggtaacggatga13380
gtcaaggacaccaatggagaaaagcactaacaccattatctgatgaacattacgtgaaga13440
agggtaagaagtgaagtggaattgctgaagaagtcagtgaaagcggacattcatttgggg13500
aaatggaatataggaaatccataaaagtgattaaaaagatgttagaggctgaggcggggg13560
gaccacagggtcaggagatcgagaccatcctggctaacacggtgaaaccccatctctact13620
aaaaatacaaaaaattagccaggcgtggtggcaggcacctgtagtcccaactactcggga13680
gactgaggcaggagaatggcatgaacctgggagacggagcttgcagtgagccgagatcac13740
gccactgcactccagcctgggtgacagagtgagactccatctcaaaaaaaaaagttagat13800
acgagagataaagatccaacagacacacaactgctaattctgaacagaacaaaacaaatg13860
gcacaggaaaagaaaatttaagatataacaccggaaaactttcctgaaattgagtaactg13920
aatctatagcttgaaagggtttagcatatgccaagaaaaatcagtagagtccaaccagca13980
caagacacatctagcaaggctggtgattctaccaacacagagaaagaagtgggtgaccca14040
taatgcggaaaaaggcagaccatctgcagtcttctccagaacactggagtctgaagacaa14100
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
_57_
aagaatgctgcctactgagccagaagggagagaaagtgacccaacacatctttaccaagt14160
tagaatgtcacgcattatttaaaggctgcaaaagccatgaaagacatgaaagaacacaag14220
catttacaacatgaaagaacacaagcattctcatactcaagaatccttaagaaaaatgta14280
gtcctaatccagcccactgaaagttaaatgtacttaatgtgctcattaatgggaacttca14340
tagcttcaaatcagtctggtcccatctaccaacatctctcgcccggctttcctgcaatag14400
tcagcacctttccctcctcccagtcttgtcCCCtggagtCtgCtCtCagCatagcagagt14460
gaccacatcaacacccaagtCagagCCCtCCagtgCgCa.Ctggtctacaaagcccttccc14520
aCCCCCC3CCCCaCgtgCCCtccggatccttgtgacgtgtctcctgcataccctagcagc14580
cctggcctcctCaCtgCCCCtcctgtacatcaggaaggcgactccttgagtcttggctct14640
ggccgcctcctccacctgcagtgagttaactcccttacctactctaggtcattgctcaaa14700
tgtcagcatctcaatggggccctccctgactaccctatttaaattctacataCtcCCCtt14760
gaccccatggaCCtCaCtCaCCCtattCCaCttttattCttaCaatttagCacttgttct14820
CttCtaaCgtattCtaagaCttaCtCatttattaCattgtttgCCaCCCCCtCtagtaCa14880
taaactccagaggggcagggatttctgtctatttattcatttctttatccctaggacata14940
gaacagggcatagttcagagtattcaatgttatcaatgaatgaactagcagtagtaccag15000
ttccagttaggcacagaattaaatctaaatagaattaaatctcatggtctgggttaacta15060
tggatagaaaattagatataattttaagaagcctagaaagaaaaaattaataatgtaaaa15120
ataatattaatttgataataataacaaaaactctgccaggcactgtggctcaaatctgca15180
atcccagctactcaggaggctgaggtggaaggatcacttgagaccagagttcaagactca15240
gcctaggcaacacggcaagaaactgtctctaaaaaaattaaaacttaaatttttaaaaaa15300
gaattctcaaagcgtcacaaaaactggagattaaggtacaggaagtgtgaagtaatatta15360
ctatgctaatggttttttttttttttagaaaggtataaccaaaagatttctttctcaagt15420
cgataaactgagaaagataagcatatcttccaattaacagagggggaggaaaagccagat15480
acaacaaaataagatataaattagtttccagttgaaaacaagagtaggagttattttgca15540
tcaCCtCaCCtgtgaCCtCCCCCagCCCaaaaaaCaCtaCtgataaaCagggtagaaaag15600
catcatctcagataaagcaggaaaaactgccacagtctcaaaccacaaactataagcaca15660
CaCCtggCCaaCCCtgCCaagtCtgggCtCagtaggaggaaCgtgCtgagagctaggatg15720
taccaacttagacattctgtgggatacagatgtccctggaagggtcacaccatctcaaag15780
gcacctgtaatgcccactgattacagccaccatatgtgagagagaaactcagggcactta15840
gagagtataacaagaaccttatgtcatctgagatgaggaatcctcagccctgcaaattaa15900
ccaactctttagaacaactggcaaaacataaatatccacaacttttgtttcagtaattcc15960
actcttagatatcaatccaaagtacatgagacagcagatacacacacaaaatggtattta16020
ctgcagcattgtttataatagcaaaaaacaagaaataatccatatgtctcaataggatac16080
tgggtacatgagggtatgtacccatcattcaaccatcaaaaagagtgatatggatgtcca16140
cagatggacataaaaagctgtgtgttacgtgaaaacaaactcaagcagcagcaggatggg16200
cttatgatagtcagtatgagctaatttctggaaaaaaaaatctagtgtgtgcacagaaaa16260
catctgaaagaacagaaacaaaactatcagcagaatattgagatgttttactaagttgta16320
tatctatactgcttgtaatttttaccccaagcaagaattactttttggaaaaagaaaatt16380
caggaaataaagcatttctttaaacttcatgtttaaacaaatggtgatggaataaaagag16440
ttcttattcatcataaacacacacagcacacatgcacgcatgtgcgtgagcacacccttt16500
acttgataaataceatgttgaatattttagtctttccttttaggttctatcccttcactc16560
aaaatgcggttataaataaatgtacttttcatgtgccttctgcctaaacccactttaata16620
taactttacagtcccattatcattatagtctcaaagctagactcagcctgaaactaccct16680
ttcatttggaacccttattaaaatgccacatacagctccttcaaataaaaacaaacccta16740
ggacctgacactaggcttcctttgttgctactcataatggccaagttctgtgcttataat16800
acatcttctttcattttattgctacatatccaagggttttatatgtttttCttattatat16860
cttaattcaaaacaccatcacgctcttttccagatgaaaataaggaaaagaaattgagca16920
actgactgacttaaaggtcataaaactatatagtagcagagtcagcaaaagaagaaacac16980
acatctcccaagtagaggctgaaaaccagtaccattcacctccagggtgagctatataca17040
gattacaaagtcaccttctctaaatgttcaaactgaatcccatacccatactttaccact17100
acctcgtaagaacagcctcagatcttgttatagccttttttttagcatgctgaagccaat17160
aaaatgcttcccattcagcaagagaaacaagttctgaaacactgaataatctgcccaggg17220
cctatgaacatttccactgtgagaaatgttctccactgtgtggagaagatCCttactctt172$0
ctccacacaggcagaacattagaaaaattcttggattctatgatgcacagcttaggagtc17340
tgtttagcacaatttaagtccaaatagttattaaatcctcctctgttccagaaacagtgc17400
taaatactgtgaatataaaaattgaaaagatactctcctggctcccaagaaagtcagcca17460
gatagaggagacacaggcacacaaatcactgtcacatgaagctctacctccctaacttca17520
aacgagggcctaagtcaccaagaatacagtagcagttgtgactacgagtaactactataa17580
ttcaatactttatcttcccttagaaaactcttctcccttggaaatttatttgcatttcta17640
aataccattccttactaaaaggaagcagggctccttggggaaatagctgattctaggtgt17700
ggactatgaaatgaaaatggtgagtctgggacatcccatgttgcccagaaatcaaggaac17760
tgcccaaagattaacagagtcatgttaaatggacctaagagtgaaccagaaggagctcac17820
tttgccccgcgtggaacaatttcaagaaaaacatgacagtaatgaattataaaacatgaa17880
ttaaaatacatattggtactaaaaagagaacaaaaggatgtggctttggataaagctctt17940
cttcatggaagaataccagctaataaatgtaaaggaaatgagagaattagaaaaattatc18000
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-58-
attttgtaaaccttaatatattcacctagacatgctaaaaccactgagtaaaaggctgct18060
tgggaagaggatgctcacatgatctcagagtttcacaccacagataatttattagataca18120
ggaaggaagatgtgatcaagCttCCtgtgaCCCCCagCCaggccccacaacactatgtgc18180
ctccttgtgatgtgggagctacacagcatcgcccacacagcttctcgccaaaactgtttg18240
aagctaatcacaagggaagaactggacagcttctgaccatgagacgctccaccagacaac18300
ttgcttggcctctccaaagaaacttgcttggcctctccaaagaaaactcagtttcattta18360
aaaacaaaactaattatttaaaaacaaacgaaaagcaagttgtggacttgagctccaggg18420
acagagcagacatacttttccctgttcttcccagtaagtggtaataaaaaccctcaacac18480
tagatataaaacaaatataagaaggttctggaaggggaagaggaggcagactatccaggt18540
gccttgaggcccacagaacaacccagtgatgggttcactgggtcttctttttgcttcatt18600
atctcagacttggagctgaagcagcaggcaacttcaaaacaccaaggggcacagattgaa18660
aagccccaagaaaagcctgccctctctagccaaaggaccaggaaggagacagtctaatga18720
gatggaacacatttagacagtaactgcccatttaccagcaataactgagcagggagccta18780
gacttccagtcttgtgaggacgtaccaaggtacccaacacccccaccaaggctgagtaag18840
gactgcgacttttatccctgcatggcagtagtaaggagcccatccctcacccgccagcag18900
tgtcaggggaacctggacttccactcccacccaggagtgatgaggccctccctgctgggg18960
tcatgtcagaggaggcctagtggagattcagtgacttaaccttttcccagagataatgag19020
gCCa.CCtttCCtCCCtCttCCCCCatggtgacagtgaaagcactgtggcaagcagtaggc19080
actcctacccctcctagccagggaggtatcagggaggccaagtagggaaccagaataccc19140
acaaccacccagcagcaacaggggtcccccaccccattgggtgtcaatggaagcagagcg19200
gaaagcctggatatttacccccatctagaagtaacaagctgatgtcccccttcttctact19260
acaatggtgttcaaaacaggtttaaataaggtctagagtctgataacgtaatacccaaat19320
cgttgaagttttcattgaggatcatttataccaagagtcaggaagatcccaaactgaaag19380
agagaaaagacaattgacagacactagcactaagagagcacagatattagaactacctga19440
aaggatgttaaagcacatatcataagcctcaacaggctgggcgcggtggctcacgcctgt19500
aaccccagcactttgggaggccgaggcaggtggatcacaagatcaggagatcgagaccat19560
cctggctaacacggtgaaaccccgtctctactaaaaatacaaaaaaaaatagcaaggcat19620
ggtggtgggcacctgtagtcccagctactcgggagcctgaggcaggagaatggcatgaac19680
ctgggaagaggagcagtgagccgagatcgcaccaccgcactccagcctgggcaacagagc19740
aagacttcgtcccaaaaaaaaaaaaaaaaaaaaaaaaagcctcaacaaacaactacaaac19800
gtgcttgaaacaaatgaaaaaaaaatcttggcaaagaaataaaagatatatattttggcc19860
aggtgcagtggctcacagcctgtaatccctgcactttgggaggctgaggcaggcggatca19920
cctgaggtcaggagtttgagaccagcctgaccaacatggagaaaccccgtctctactaaa19980
aatacaaaattagccagtcatggtggcacatgcctgtaatcctagctactcaggaggccg20040
aggcaggagaatcgcttgaactcaggaggtggaggttgcggtgagccgagatcccgccat20100
tgcacattgcactccagcctgggcaacaagagcaaaactccatctcaaaaaaatagatac20160
atattttaatggaaattttagaattgaaaaatacagtaaccaaattgaatggaaagacaa20220
catagaatggagggggcagacaaaataatcagtgaacttcaacagaaaataatagaaatt20280
acccaatatgaagaacagaaagaaaatagactggccaaaaaataaagaagaaaaaagagg20340
agcagcaggaggaatgatggaaaaagagaaaggaaggaaggaagggaaggagggagggaa20400
ggagtgagggagaaagtctcaaagacctctgagactaaaataaaagatctaacacttgtc20460
atcagggtccaggaaagagacaaagatggcacagctggaaacgtattcaaaaaataatag20520
ctgaaaacttcccaaatttggcaagagacataaacctatagattcgaaatgctgaacccc20580
aaataaaaagcccaataaaatccacaccaaaatacatcatagtcaaacttctgaaaagac20640
gaaaagagaaaacgtcttgaaagcagtgagtgaaacaacacttcatgtataagggaaaaa20700
caattcaagtaacagatttcttacagaaattaaggaagccagaaggaaatgacacaatgg20760
ttttcaagtgctgaaagaaaagaagtgtcaacacaaaattctagattcagtaaaaatatc20820
cttcaagaatcaatgggaaatcaagacagtctcagataaagcaaaataagagaatatgtt20880
gccagcagatctcccctaaaggaatggcaaaaggaagatcatgcaacagaccaaaaaatg20940
atgaaagaaggaatccagaaacatcaagaagaaagaaataacatagtaagcaaaaataca21000
tgtaattacaataaaatttctatctcctcttaagacttctaaattatattgatggttgaa21060
gcaaaaattataaccctgtctgaagtgcttctactaaatgtatgcagagaattataaatg21120
gggaaagtataggtttctatacctcattgaagtggtaaaatgacaacactgtgaaaagtt21180
acatacacacacacacgtaagtatatataaatatatgtgtgtatatgtgtgtgtatatat21240
atatatacatataatgtaatacagcaaccactaacaacactatacaaagagataataacc21300
aaaaacaatttagataaattgaaatggaattctaaaaaatattcaaatactctacaggaa21360
gacaagacaaaaagagaaaaaaagaggaggacaaactaaattttttaaaaacataaataa21420
aatggtagacttaagccctaacttatcaataattacataaatgtaaatgatctaattata21480
tcaattaaaagacagagatagcagagttaatttaaaaacatagctataagaaacctgctt21540
tgggctgagtgcagtgactcacacttgtaatcccagcacttcgggaggccaaggcgggtg21600
gatcacctgaggtcaggagttccagaccagcctggacaacatggtaataccccatctcta21660
ctaaaaatacaaaaaaattagccaggcatggtggcacacgcctgtagtcccaactactca21720
ggaggctgcgacacaagaactgcttgaacccgggcagcagaggtagcagtgggccaagat21780
tgcgccactccagcctgaacgacagagtgagactccacctcagttgaaaaacaaaaaaga21840
aacctgctttaaatataccaacatatgttggttgaaattaaaagaataaaatatatcatg21900
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-59-
aaaacattaatcaaaagaaaggagtggctatattaataacataaaatagacttcagagaa21960
aagaaaatttcaagagacaggaataaaaggatcaagaaaagatcctgaaagaaaagcagg22020
caaatcaatcattctgcttggagattcaacaccctctcttaacaactgatagaacaacta22080
gacaaaaaaatcagcatggagttgagaagaacttaacaccactgaacaacaggatctaat22140
agacatttacggaacactctacccaacaatagcaaaataaacattcttttcaagtattca22200
ctgaacatatccttagaccctaccctgggccataaaacaaagctcactagtgattgccga22260
aggcttggatggacagtggaagagctgcatggggagggagaaggtgacagttaaagagtg22320
taggatttctttttgggataatgaaaatgttccaaaattgattgtggtgatgttggcgca22380
actctacaaatataaaaaaggccattgaattgtacgttttaagtgggtgaaacatatggt22440
atgtggattatatctaacgctttttaaaaacttaacacatttcaaagaatagaagtcata22500
cagagtgtgctctactggaatcaaactagaaagaggtaactggaggataacgagaaaagc22560
ctccaaatacttgaaaactggacagcacatttctaaaatcatccgtgggtcaaagatatt22620
catttctgatattcatttttattgtttaatgtatttttaaaaatttcttaagggaaataa22680
actgactaaaaatgaatatggctgggtgcggtggctcacgcctgtgatcccagcactttg22740
ggaggccgaggctggtggatcacaagatcaggagttcgagaccagcctggccaagatggt,22800
gaaaccccgtctcaactaaaaaactacaaaaagtagccaagcgcagtggcgggagcctgt22860
ggtcccagctacttgggaggctgaggtaggagaatcgcttgaacacaggcagcagaggtt22920
gcagtgagccaagattgtgccactgcacgccagcctgggcgacagagactgcctcaaaaa22980
aaaaaaaaaaaaaaagaatatcaaaatttgtgggacatagttaaagcaatgctgagaggg23040
aaatttataacactaaatgtttacattagaaaagagaaaaagtttcaaatcaatagtctc23100
cactcccatctcaagaacacagaagatgaagagcaaaataaacccaaagcaagcaaaaga23160
aagaaaatataaaaataaatcagtaaaattgaaaacagaaacacaataaagaaaatcagt23220
gaaacaaagtactgattcttcgaaagattaataaaattgacaaacctctagcaaggctaa23280
caaacaaaaaagaaagaagacacggattaccagttattagaatgaaagcataattagaaa23340
caactctacacattataaatttgacaatgtagatgaaatggactaattactgaaaaaaca23400
caaattaccacaactcacccaatatgaaatagataattgggatagcctgataactactga23460
gaaaattgaatttgtaattttaacactcttaaaacagaaacattaaacttaatattttat23520
aaatattagataaggtaattatacccttccttaacaaataaaaacgacaaattattttgc23580
agctaaagagatgtatgtactgtgaaaaatatcttcagaaaaatagaactttgtttgaag23640
aataaggatttaaaaaatgtttttaactctcaagaagcaaatatctgggcccagatggtt23700
tcactgaagaattctaccaaatgtttaatgaagaattaccaccaactctacatagcatct23760
ttgagaaaactgaagagaagggaacatctcccagttcattttatgaagtgggtgttactc23820
tgatactagaactgtataaggacagctactcttgacacactgcctatgggtagctctgct23880
ctgcaggaacagtcagaaaaaaaaaaaaaagaagcactggacaagggcagtataaaaaaa23940
gaaaactgggccaggtgcagtggctcacacctgtaatctcagcactttgggaggctgacg24000
ctggtggatcacctgaggtcaggagtttgagactagcctggccaacatggtaaaaccctg24060
tctctactaaaatacaaaaattagccaggcagggtggtggggaaaataaaaaggaaaaaa24120
aaacaaaaataaactgcagaccaatatccttcatgagtatagacacaaaactccttaaac24180
tccttaacaaaatattagcaagtagaagcaatatataaaaataattatacaccatgatca24240
agtgggacttattccagaaacgcaagtctggttcaacatttgaaaacaaggtaacccact24300
atatgaacgtactaaagaggaaaactacataatcacatcaatcaatgcagaaaaaagcat24360
ttgccaaaatCCaatatCCattcatgatactctaataagaaaaataagaataaaggggaa24420
attccttgacttgataaagcttacaaaagactacaaaagcttacagctaacctatactta24480
atggtgaaaaactaaatgctttcccctacgatcaggaacaaagcaaggatgttcactctc24540
attgctcttatttaacatagccctgaagttctaacttgtgcaaaacgataagaaagggaa24600
atgaaagacctgcagattggcaaagaagaaataaaactgttcctgtttgcagatgacatg24660
attgtctcatagaaaatgtaaagcaactaggggtaggggggcagtggagacacgctggtc24720
aaaggataccaaatttcagttaggaggagtaagttcaagatacctattgcacaacatggt24780
aactatacttaatatattgtattcttgaaaatactaaaagagtgggtgttaagcgttctc24840
accacaaaaatgataactatgtgaagtaatgcatacgttaattagcacaacgtatattac24900
tccaaaacatcatgttgtacatgataaatacacacaattttatctgtcagtttaaaaaca24960
catgattttggccaggcacagtggctcatacctgtaatcccagcattttaggaggctgag25020
gcgagcagaaaacttgaggtcgggagtttgagaccagaatggtcaacatagtgaaatccc25080
gtctccactaataatacaaaaattagcaggatgtggtggcgtgcacctgtagacccagct25140
acttgggaggctgaggcacgagaattgcttgaacaagggaggcagaggttgcagtgagct25200
gggtgccactgcattccagcctggtgacagagtgagactccatctcaaaaaaaataaaat25260
aaagcatgacttttcttaaatgcaaagcagccaagcgcagtggctcatgcctgtaatccc25320
accactttgggaggccgaggcaggcagatcacaaggtcaggagtttgagaccagcctgac25380
caacatggtgaaaccccatctctactaaaaaatatataaattagccaggcatgtgtagtc25440
tcagctactcaggaggctgaggcaggagaatcacttgaacccggaggcagaggttgcagt25500
gttgagccaccgcactccagcctgggtgagagaacgagactccgtctcaaaaaaaaaaag25560
caaaataacctaattttaaaaacactaaaactactaagtgaattcagtaagtctttagga25620
ttcaggatatatgatgaacatacaaaaatcaattgagctggacaaaggaggattgtttta25680
ggtcagtagtttgaggctgtaatgcacaatgattgtgcctgtgaatagctgctgtgctcc25740
agcctgagcagcataatgagaccacatctctatttaaaaaaaaaaaaattgtatctctat25800
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-60-
gtactagcaataagcacatgggtactaaaattaaaaacataataaatactgtttttaatt25860
gcctgaaaaaaatgaaatacttacatataaatctaacaaaatgtgcaggacttgtgtgct25920
gaaaactacaaaacgctgataaaagaaatcaaagaagacttaaatagcgtgaaatatacc25980
atgcttataggttggaaaacttaatatagtaaagatgccaattttatccaaattattaca26040
caggataacattattactaccaaaatcccagaaaaattttacatagatatagacaagatc26100
atacaaaaatgtatacggaaatatgcaaaggaactagagtagctaaaacaaatttgaaaa26160
agaaaaataaagtgggaagaatcagtctatccagtttcaagacttacatagctacagtaa26220
tcaagactgtgatattgacagagggacagctatagatcaatgcaaccaaatagagaacta26280
agaaagaagcacacacaaatatgcccaaatgatttctgacaaaggtgttaaaacacttca26340
acgggggaagatatgtctctcattaaagggtgtagagtcattgcacatctataggcaaaa26400
agatgaacctgaacctcacaccctacagaaaaattaactcaaaatgactcaaggactaaa26460
cataagatatacatctataaaacatttagaaaaaggccacgcacggtggctcacgctcgt26520
aatcccagcactttgggaggccaaggcaggtggatcacctaaggtcaggagtttgagacc26580
agccggatcaacatggagaagccccatctctactaaaaatacaaaattagctggacgtgg26640
tggcacatgcctgtaatcccagctacttgggaggctgaggcatgagaatcgcttgaaccc26700
ggggggcagaggttgcggtgagccaagatcacaccattgcactccagcctgggcaacaag26760
agcaaaactccaactcaaaaaaaaaaaaaaaaaggaaaaatagaaaatctttgggatgta26820
aggcgaggtaaagaattcttacacttgatgccaaactaagatctataaggccagtcgtgg26880
tggetcatgcctgtaattccagcactttggtcaactagatgaaaggtatatgggaattca26940
ctgtattattctttcaacttttctgtaggtttgacatttttttagtaaaaaattggggga27000
aagacctgacgcagtggctcacacctgtaatCCCagCdCtttgggaggCCggggcaggtg27060
gatcacacggtcaggagttcgagaccagcctggccaacatggtgaaaccccgtctctacc27120
aaaaatataaaaaattagccgggtgtcatggtgcatgcctgtaatcccagctactgagga27180
ggctgaggcaggagaatcacttgaacctgggaggtggaagttgcagtgagccgagattgt27240
gccactgcactccagccttgggtgacagagcgagactccgtctcaaaagaaaaaaaaaaa27300
aaagaatatcaaacgcttactttagaaactatttaaaggagccagaatttaattgtatta27360
gtatttagagcaatttttatgctccatggcattgttaaatagagcaaccagctaacaatt27420
agtggagttcaacagctgttaaatttgctaactgtttaggaagagagccctatcaatatc27480
actgtcatttgaggctgacaataagcacacccaaagctgtacctccttgaggagcaacat27540
aaggggtttaaccctgttagggtgttaatggtttggatatggtttgtttggccccaccga27600
gtctcatgttgaaatttgttccccagtactggaggtggggccttattggaaggtgtctga27660
gtcatgggggtggcatatccctcctgaatggtttggtgccattcttgcaggaatgagtga27720
gttcttactcttagttcccacaacaactggttattaaaaacagcctggcactttccccca27780
tCtCtCgCttCCtCtCtCaCcatgtgatctCa.CtggttCCCCttCCCtttatgcaatgag27840
tggaagcagcctgaagccctcgccagaagcagatagtgatgccatgcttcttgtacagcc27900
tacaaaaccatgagcccaataaaccttttttctttataaattatccagcctcaggtattc27960
ctttatagcaagacaaatgaaccaagacagggggaaatcaacttcattaaaataa_tetat28020
gcagtcactaaacaaataagaacaagaggctccagaagtgggaagccaatacccagagtt28080
cctacaatacagtatctgaaaagtccagtttCCaaCCaaaaaatatatatatacaggccg28140
gacatggtagcttatgtctgtaatcccagcactttgggatgctgaggcgggcagatcacc28200
ctaggtcaggagttcgagaccagcctggccaatatggcaaaaccccgtctctactaaaaa28260
taeaaaaattagccaggcatggtggtggatgcctgtaatcccagctactcgggaggctga28320
ggcagggaatcacttgaacccaggaggcagaggttgcagtgagccgagatcacgccactg28380
aactccagcctgggcaacaaagtgagactccacctcaaaaaaaaaaaaaatatacatata28440
tatatgtgtgtgtgtgtgtgtgcgcgcgtgtgtgtatatacacatacacatatatacata28500
tatacagacacacatatatatatgaagcatgaaaagaaacaaggaagtatgaaccatact28560
ttctgtggttatgataggatggggtatcacgggggaagtagacaagggaaactgcaagtg28620
agagcaaacagttatcagatttaacagaaaaagactttggagtaaccattataaatatgt28680
ccacagaattaaagaaaagcgtgattaaaaaaggaaaggaaagtatcataacaatattac28740
tccaaatagagaatatcaataaaggcatagaaattataaaatataatacaatggaaattc28800
cggagttgaaaggtagaataactaaaatttaaaattcactagagaaggttcaacactata28860
tttgaactggcagaagaaaaatttagtgagacaaatatacttcaatagacattattcaaa28920
tgaaaaataaaaagaaaaaagaatgaagaaaaataaacagaatctcagcaaaatgtggca28980
caccattaatcacattaacatatgcatactgagagtaccggaagcagatgagaaagagga29040
agaaaaaatattcaaatgatggccagtaacttcctagatttttgttttaaagcaataacc29100
tatacaatcaagaaactcaatgaattccaagtaggataaatacaaaaagaaccacaaaca29160
gatacaccatggtaaaaatgctgtaagtcaaaaacagagaaaatattgaaagcagctaga29220
ggaaaacttataagagaacctcacttacaaaagaacatcacttataaaagaaccacaata29280
atagaaacagttgacctctcatcagaaacaatgaatgataacatatttgaagtgctcaaa29340
gaaaaaaaataaagattcctatatacgacaaagctgtctttcaaaaatatacatccaaaa29400
ggattgaaaccagggtcttgaagagttatttgtacatccatgttcatagcagcattattc29460
acaatagccaaaaggtagaagcaacccaagggtccatcgacaaataaataaaatgtggta29520
tatgtatacacaatggaatttattcagtattaaaaaggaatgaaattctgacacatgcta29580
caacatggctaaaccttgagaacactatgctaagtgaaataagccagccacaaaaggaca29640
aataccatattacttcacttgtatgaaatacctagggtagtcaaattcagagatagaaag29700
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-61-
taaaacagtggttgccaagggctgagggagggagtaacgtggagttattgttgaatgggt29760
acagaatttcagttttgcaagataaaaagagttctggagacagatggtggtgagggtggt29820
acaacaatacaaatatactttatactactgaacagtatacttaaaaatgattaacatggt29880
gaaaccccgtctctactaaaaatacaaaaaaattagctgggtgtggtggcgggcacctgt29940
aatcccagctacttgggaggctgaggcagcagaattgcttgaaaccagaaggcggaggtt30000
gcagtgagctgagattgcgccaccgcactctagcctgggcaataagagcaaaactccgtc30060
tcaaaaaataaaaaataaaaaaaatttaaaaatgattaagcaggaggccaggcacggtgg30120
ctcacacctataatgccagcactttgggaggccgaggcaggcgatcacttgagacca'gga30180
gtttgagaccagcctggccaacatggcaaaaccctgtctctgctaaaaatacaaaaatta30240
gccaggcatggtggcatatacttataatcccagctactggtgagactgagacacgagaat30300
tgcttgaacccaggaggcagagattgcagtgagtcgagatcgcgccactgaattccagcc30360
tgggcgacagagcaagattctgtctcgaaaaaacaaaaacaaaaacaaaaagcaaaacca30420
aaaaataattaagcaggaaacgagattgctgctgaggaggagaaagatgtgcaggaccaa30480
ggctcatgagagcacaaaacttttcaaaaaatgtttaatgattaaaatggtaaattttat30540
atgtatcttaccacaaaaaaaagggctggggggcaggaaatgaaggtgaaataaagacat30600
cccagagaaacaaaagtagagaatttgttgccttagaagaaacaccacaggaagttcttc30660
aggctgaaaacaagtgaccccagagggtaatctgaattctcacagaaaattgaagcatag30720
cagtaaaggttattctgtaactatgacactaacaatgcatattttttcctttcttctctg30780
aaatgatttaaaaagcaattgcataaaatattatatataaagcctattgttgaacctata30840
acatatatagaaatatacttgtaatatatttgcaaataactgcacaaaagagagttggaa30900
caaagctgttactaggctaaagaaattactacagatagtaaagtaatataacagggaact30960
taaaaataaaattttaaaaaatttaaaaataataattacaacaataatatggttgggttt31020
gtaatattaatagacataatacaaaaataccacaaaaagggaagaagacaatagaactac31080
ataggaataacattttggtatctaactagaattaaattataaatatgaagtatattctgg31140
taagttaagacacacatgttaaaccctagatactaaaaagtaactcacataaatacagta31200
aaaaaataaataaaataattaaaatgtttgtattagtttcctcagggtacagtaacaaac31260
taccacaaattgagtggcttaacacaacttaaatgtattttctcccagttctggaggcta31320
aacacctgcaatcaaggtgagta_cagggccatgctccctgtgaaggctctaggaaagaat31380
ccteccttgtctcttccagcttccagtggttctcagtaaccctaagtgctccttggcttg31440
tagctatatcattcctagcaaccagaaagaagaaaataataaagattatggcaaaaaata31500
atgaaatcaaaaggagaaaaatggaaaaaaataaataaaaccaaaagctagttctttgaa31560
aagatcaaccaagttaacaaaccttttaactagactgacaaaaaggaggtaagactcaaa31620
ttactagaatcagaaataaaagaggggacattactaatgagggattagaaaagaatacta31680
cgaacaaatgtgtgccaacaaattagaaaacttagatgaaatggacaggttcctaggaca31740
acatcaactaccaaaatttactcaagaagaaagagacaatttgaatgagctataacaagg31800
gaagagactgaattgacaaccaagaaactatccacaaagaaaatcccaggcecagaagat31860
ttcactgtgaaattctttcaaacttataaatataaattaacatcagttcttcaeaaactc31920
ctccaaaaaaaagaacagatctctatttacaggcgatacgatctttagaaaatcctaagg31980
gaactactaagaCactatgataactgataaacaagttcagcaaggctgcaggatagaaaa32040
ccaatatacaaaaatctattatatttctatacacttgcagtgaacaacccaaaaatgaga32100
ttaagaaaataattcaatttacaataacatcaaaaagaataaaaacactcaaaaataaat32160
ttattcaagtaagtgcaaaacttatactctagaagctacaaaacactgttaaaagaaatt32220
aaaggtttacataaatgaaaaactatcccatgttcatggatcaaaagacttattactggc32280
aatgctctccaaattgatctataaattcaacaaaatccttatcaaaatcccagatgaggc32340
tgggggtggcggttcatgcctgtaatcccagcactttgggaggctgaggcacgcagatta32400
cctgaggtcgggagctcgagatcagcctgaccaacatggagaaaccctatctcttctaaa32460
aatacaaaattagtcaggcgtggtggcacatgcctataatcccagctactcgggaagctg32520
aggcaggagaatcgcttgaacccaggaggcagaggttgcagtgagccaagatcgtgccat32580
tgcactccagcctgggcaacaagagcaaaattccatctcaaaaaaaaaaaaaaaaaaatc32640
ccagatgacttcactgttgaaattgaaaagattattctaaaattcacatggaattgcaag32700
accttgagaatagccaaaacaaacttgaaaaacacgaacaaaatataggatgactcactt32760
gccaattgcaaatgttacgacacagcaacagtaatcaagactgtgtggtactggcaaaag32820
acacatacatacatacatatcaatggaatataattgagagtacagaaacaagcctaaaca32880
tctatggtaagtgcttttctatttttttctttttttttttcttttttgtagagatagaat32940
ctcaccatgttgcccaggctggtcttcaacttctgggctcaagcaatcctcccactgtgg33000
cctcccaaagtgctgggataactggcatgagccaccacatccagcccagatgattttcaa33060
aaaagtcaacaagaccattcttttcaacaaataggtctgggatgatcagatagtcacatg33120
aaaaaaaaaatgaagttggaccctccatcacactaaagtgctgcgattataggcatcagc33180
caccacatccagcccaaatgattttcaaaaaggtcaacaagaccattcttttcaacaaat33240
aggtctgggataatcagatagtcacatgaaaaaaaaaatgaagttggaccctccatcaca33300
ccatatgcaaaaattaattcaaaaatgaattgatgacttaaacgtaagagttacgactgt33360
aaaactcttagaaggaaacatacgggtaaatcttaaagacgttaggtttgacaaagaatt33420
cttagacatgacaccaaaagcatgaccaactaaggtaaaatagggtaaattgtacctacc33480
aaaatgaaaaacctttgtgctggaaaggacaccatcaagaaatggaaagccaaaatagcc33540
aaggcaatattaagcaaaaagaacaaagctggaggcatcatactacctgacttcaaagca33600
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-62-
acagtaacca aaacagcatg gtactagtag aaaaacagac acatagacca atggaacaga 33660
ataaagaacc caaaaataaa tccacatatt tatagtcaac tgatttttga caatgacacc 33720
ccttcaataa atgatactag gaaaactgga tatcgatatg cagaagaata aaactagacc 33780
cctatctctc accatataga aaaatcaact cagactgaat taaagacttg aatgtaagac 33840
ccaaaactat aaaactactg gtagaaaaca taaggaaaaa cgcttcagga cattggtcca 33900
ggcaaagatc ttatggctaa aacctcaaaa acacaggcaa caaaaacaaa aatggaaaaa 33960
tagcacttta ttaaactaaa aagctcctgc acagcaaagg aaacaacaga atgaaaagac 34020
aacctgtaga atgggagaaa atatttgcaa actatccatc catcaaggga ctagtatcca 34080
gaacacacaa gtgactaaaa caactcaaca gcaaaaaagc aaataatctg gtttttatat 34140
gggcaaaaga tctgaataaa cattctcaaa ggaagacata caaatgtcac tatcattctg 34200
ccagtaccac actgtcttga ttacttgtta gtgtataaat ttttaaattg ggaagtgtga 34260
gtcatcctac actttgttct tgtttttcaa gtttgttttg gctattctgg gagccttgca 34320
agtataaaat agccaacaag tatgaaaaaa tgctcaccat cactaatcat cagagaaata 34380
aaaatcaaga ccactatgag atatcctctc actccagtta gaatggctac tatcaaaaag 34440
acaaaatata atggatgctg gcaaagattt ggagaaaggg gaactcctat acactgtggg 34500
tagggatgca aattggtaat ggccattatg gaaaataata ctgaggtttt tcaaaaaact 34560
gaaaatagaa ctaccatatg atccagcaac cctactactg ggtatttatc caaaggaaag 34620
aagtcagtat actgaagaaa tatatgcact ctcatgttaa ttgcaacact gttcacaaca 34680
gccaagacag ggaataaatc taaatgtgca tcaacagatg aatggataaa gaaaatgtgg 34740
catatacact caatagaata ctattcagcc attaaagaag aatgaaatcc tgtcatccca 34800
gcaacatgga tgaacctgga ggacattata tttaatgaaa taagtaaagc acaaaaagat 34860
aaacagtaca tgttctcact cagacatggg tgctaaaaag aaaatggggt cacagaatta 34920
gaaggggagg cttgggaaaa gttaatggat aaaaatttac agctatgtaa gaagaataag 34980
ttttagtgtt ctatagaact gtagggcgag tatagttacc aataacttat tgtacatgtt 35040
caaaaagcta gaagagattt tggatgttcc cagcacaaag gaatgataaa tgtttgtgat 35100
gatggatatc ctaattaccc tgattcaatc attacacatt gcatacatgt atcaaattat 35160
cactctgtac ctcataaata tgtataatta ttacgtcaac aaaaaaagga aaaaaaagaa 35220
aattaagaca acccacataa tggaagaaat aaaatatctg caaattatat atatctgata 35280
aatatttaat atttataata tataaagaac tcctacaact caagaacaac aacaaaacaa 35340
cccaattcaa aaatgggtaa aagccttgaa tatacactta tctaaagact atatacaatt 35400
ggccaataaa gacacgaaaa gatgctcaac atcactagtc atcagggaaa tataaatcaa 35460
aaccacaatg tagaatgtag acaccacttc atatgcacta ggatggctag aataaaaagg 35520
taataacaaa tgttggtaag gatgtgaaaa aatcagaaac ctcattcgct gctgttggga 35580
atgtaaagtg atgcagccac tttggaaaac agtctggcag ctcctcaaat tattaaatac 35640
agagttaccg tatgacccag gaatattcct cctgggtcta taaccaaaaa aatgaaaaca 35700
tatatccaca taaaaacttg tacatgggca tttatagcaa cattattcat aacagcaaag 35760
gtggtaagaa cccatatgcc catcatctga tgaacaggta aataacatgc ggtattatcc 35820
atacactaga atattatctg cccatacaag gagtgacatc Cagctacatg ctacaaggat 35880
gaatctcgga aaccttatgc taagtgaaag aagccagtca caaatgacca cagattatga 35940
ttccatgcat cggaaatgac cagaataggg aaatctatag agacagaaag tagattagtg 36000
gttgggtggg gctgggagga caggtagtac actactttcc cagaactact ggaacaaagt 36060
accacaaact ggggagctta aacatagaaa ttgatttcct cacagttctg gagactagga 36120
ctctgagatc aaggtgtcag cagagctggt tctttctgag ggccctgagg caaggctctg 36180
tCCCaggCCt CtCtCCttgg ctggcaggtg gccatcttct CCCtgCgtCt tCaCatCatC 36240
ttttCtCtgt gtgtgCCCat gtCCaaattt tgattggCtC attCtgggtC atggccaatt 36300
gctatgcaca aagtgaagtc tacttccaaa agaagggaag agggaacact gactaggcta 36360
aacttatagt cattttaatg tccgcttttc ctatgagatt gtgaacacac agaagtaggg 36420
tttttatcta cattgtgcaa agtttaataa gaaaaataga attcaagaga agcagttcaa 36480
tagcaggaat ttaatatggg aactaattac aaggtttagg gcaggactaa aaagccagtt 36540
gggatggtga gccaacccag agattagcaa cagtgggacc ccatCtaCCt accacccatg 366OO
aagctggaag gataaaggag gggctattat cagagtccac aagccagtgt cagagtcctt 36660
ggctggagct gggaccaccc tagagacact gtgcaaagca gaaaacaagg gggaaaaacc 36720
ctgacttctc CCttCCtCCC aCCtttcaat CtCCCaCtag tgcttcctac tagccatact 36780
tggccagaga cagtgacaag gaacactgca aaatgaagtt tgtaggaatc atctccctct 36840
gagacagaga aatatggaag ggtagaaaat gaatcagagg ataaagagaa aaaaccctga 36900
gtactatctt atttatcttt gtatctccag tgcctaatct gtctctcaaa aaaggaaagc 36960
aattgagaga aactgaaaac tccaattgaa atgaaagaat ggagaattac tggactagaa 37020
gagaagagaa aaatttattc cgcatagagt aaacaagaat ggattcacaa aggacgtgat 37080
gaatgaaaag ctataatcag caaagatttg ccagagaaat taaaaagtgg taaactcagc 37140
cacgctgtac aacctgaagg cacaatgcat gaaaacgttt caagaaatga caagatttga 37200
agtcaaattc taagtgcttt tccagaatct ctcaagacga ttatatagct accccatttt 37260
attaaataaa atggaaactt actaaacttt ccccttgtat taaactaaca tatgtcctaa 37320
tagcaaacga ttctggaatt cctagagtaa aatatatttc gtcaaagtgt attgctcttt 37380
taatattctg ctgacctcct tttgctattt aggatatttg tatacacatc acacgtaaat 37440
ttggtctata gtttacatet acgggcttat actgttcttt ttttcatttt tttaaaattt 37500
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-63-
ccaacccccagtatccatatactgctctctatcagggttattttaactttgtaaaatcag37560
ctgagatgctttccatgttttttttttttattttctgccacatttgaatagcataggagt37620
taccaccatcaaccttggattatttaagcattcacgattccacgtgtggattttttattc37680
agagtctttcttgtcattcctgctatcagcacagaacccaatctcagctttccagctata37740
ctctcaccccatggaatttgcagatgaagttcaaaaggacctttgcattatcctgcctcg37800
ccctcttcccccttcatttagacatcaccttcttctagaacgtcttacctgacatgccct37860
gctcccaacccctgctgcccaattgtgtgctctcccgtgtCCtggCCtgCCatCCt 37920
Cttt
agtaattgcctgctccctcatctgtctccccacccagacattaagctgaatagactggat37980
ttgtgtcttgtccatcactataatctcagcacctagtacctagtaggtacttaccatgta38040
ttcattagcaaaatgttatgtataaccttgcaccttaaaaacaagagaaggaagacaaaa38100
ttaagtcttaagactatggtttagaacatggatcagaaactacagtctgcagcccaaatc38160
cagaccaaatgaagagaccatgttcatttacatacaacctatagcagctttcacactaca38220
ggagcagagctaagtagttccaagggaacacacggccctgcaaagcctaaaatatttact38280
ctatagctcttcacagaaaaagttttcagatccctcgtttagaactcttgttcatatgca38340
atttcactaaaccatagttttttgggtttgtttggttttttttggcaaaaaggaatgagc38400
cgatccagaaaaggttgaaaagaatgaatcattactgctgaaagaatgtgcacacagtcc38460
gtcagtattctgctgccatgctgacacccatccaatagtgtcatgagatgcagcagctac38520
tactgtgttctcaatgccgagtccacccactccataaccatgtccaagcaatcttgggaa38580
catcatcaccatgcttgtttatccttaaggtattgcctcacatacagcagtggctggtca38640
taaagtcaaatgacactagtggccaggaggtcaagagaatgagtgaggacaggtgggtag38700
gcagcccaggccctagcaacagcaggagctcacccctcagtcactctagccaggactgaa38760
atacttttcaccctttcaagagagactaggaatctggatttttatgtgaaatatcttgat38820
tactaaatgttgtcaacagacatgtcaaaaggtaaaactaagtaagttcatggggcagat38880
tgactattcaggttatagaattaaggattcttatccaacacagataccaaccaaaaagct38940
gacgtataacatattaggagaaactatgtgcactgtcgaaacatcaacaaggggctaatg39000
tctaaaatagtctatattggattccagttgaaacatggggaaaggacatgaacaggcaac39060
ttatgtcaatggaaactcaaaaagataacaagcatatataaaagcattctcaaattcagt39120
agtaaacagacagatgcaaataaaaagagggaaactgctgccgggcacagtggctcacac39180
ctgtaatcccagcactttgggaggccgaggcgggcggatcatgaagtcaggagatcgaga39240
ccatcctggctaacatggtgaaaccccgtctctaetgaaaacacaaaaaattagccaggc39300
gtagtggtgggcaccagtagtcccagctactcaggaggttgaggcaggagaatggcatga39360
acccaggaggcggagattgcagtgagccgagaccatgccactgcactccagcctgggcga39420
ctgagtgaaactccatctcaaaaaatataataataattataattataataataataaata39480
gtaaataaataaaaagagagagactgctaaagtctagaaagttgaatgatgccaagcgca39540
tgcaaagatcagggccttgggatggccgggtgcagtggctcacgcctgtaatcccaccac39600
tttgggaggCCaaggCgggCggatCatgaggtCaagagatCaagaCCatCCtggCCgaCa39660
cagtgaaacccggtctctactaaaagtacaaaaaaatatatatatatatatatattatta39720
tattatatatatatatatcagagccttgggaC.tccttgtgtgctgctggggaaggtagtg39780
gtgcagccacccttgacagcaatctggcagtacttggttatattaagtataggcacacac39840
cacgaccaggcagtcctactcctgggtctaaatcccaaagaattctcacacaagtccata39900
aggagacatgtacgaggctcattcagcattactgggagtgggaatcaacctgggtgtcca39960
tctacaggagacgagatggacaaaatgtggtggatattaagaccagaatcaccaagtaac40020
agagatgggtggtgagtgacaatcctaagatacagaataaaggctagaacatgatgccat40080
tcatgtaaattaaaaatagatgCaCaCaaagcagtatacgcgtgacccttgaatagcaca40140
ggtttgaactgCCtgtgtCCaCttaCatgtggattttcttCCaCttCtgCtaCCCCCaag40200
aCagCaagaCCaaCCCCtCttCttCCt CCCCCtCagCCtaCtCaaCatgaagatgac40260
CCt
aaggatgaagacttttatgataatccaattccaaggaactaatgaaaagtatattttctc40320
ttccttatgattttctttatctctagcttacattattctaagaatatggtacataataca40380
catcacacgcaaaataaatgttaattgactgtttatattatgggtaaggcttccactcaa40440
cagtaggctgtcagtagttaagttttgggagtcaaaagttatacacagattttcaactgt40500
gcaggcaatcagttcccctgaccccctcattgttcacgggtcaactgtatatacacaaaa40560
gtattatatgaacctcattagaatagctgtctatagggagaagagaatgagagtgggata40620
aaacggaatgaacaaataaaccaacaaatgcattaacaagcaaaacaacagaggggcttg40680
catgggccagtgatgataaagggctaagaatgagaatataattaattcaattcctcacac40740
ctgaggtctaaaaccaaggaaagggagggccaggcgtggaggctcacgcctgtaatccca40800
gcactttgggaggctgaggcgggcggatcacaagattaggagtttgagatcagcctggcc40860
aacacagtgaaagcccatctctacaaaaaatacaagaattacccaggtgtggtggcacat40920
gcctgtagttagctactctggaggctgaggcaggagaatcacttgaacccaggaggcgga40980
ggttgcagggagccgagatcacaccattgcactccagcctgggtgacagagtaagactct41040
gtctcaaaaaaataaaaaaaataaaaaaacagagaaagggaggaaactagatccaggctg41100
actagatacagcctttagagttagaaaagatgatttgacaatctaagcccacactcagat41160
tgaatgaaattgaaaagcctttcaaactaaaacatttaattacaccatctgctgcagaca41220
gaactcagacaactcaaacaggtaatgtcagcgtggtgttttatatcaccaccctcaaca41280
cagaataaaaatcagctgcatgtgaagcagtgactagaatgaagaaaaggctgcttctta41340
cttccttctagtggttctttccgaaaacattaataggcaccagctctatgcatgtcaccc41400
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-64-
tgcagggaga catggggtat ataactatga cttactgttc attcctcaag gaattcccaa 41460
tcttgtggaa gattatacac aatgaggcaa caaaaactat ccaataaaac cacggaaaag 41520
aagccagtga caaagaagcc agtgatgaaa ggccctgtga gcagagctga tggccatttg 41580
gggaagaaag accaacatgg atgggggtga tcagggtggc tccgtgggaa agctggaaga 41640
gaagtggcag atctctgagc tggatgatgg gccactacca tctgtatatg gctaattaaa 41700
gaccatgtgt ggatttttta ttcagctctt tcgtgtcatt cctgctatca gcacagaacc 41760
caatctcaac tttccagcta tattgagcta aacttctcac ctcatggaat ttgcagataa 41820
agttcaaaag gatccttgcc ttttcaaaat aattttgaat ggttgagtag tccctctgtg 41880
ctctctcact gacaccctct caaggctgct gagcacgtgc catgctatgg ctttctccaa 41940
catcaggaaa tgttctccac tcagtttcac cttaatacaa atgtgttctc tcttcagaga 42000
aggcaaaaaa attcatgacc atctgactgg gagaagtcat ttctaggtaa agtgtccatc 42060
tttttctgag gaacacagga ggaaaatctt acagaaaaga gttaacacag caggcctaag 42120
actgcttttt aaaataaata aataaataaa taaataaata aataaataaa taaataaata 42180
aataaatgaa tgatagggtc ttctgtattg gccaggctag tctcaaattc ctggcttcaa 42240
gagatcctcc caccttggtc tcccacagtg ttgggattat agacatgagc cattgtgctt 42300
ggcccaagac tgttattctt aaaaagtctc ataaaaagca tggttaatcc ttggctggca 42360
cctgggaact tagatttcag aagggttccc accatccaac ctggaaagag ggactcactg 42420
tgcctaaatt attgtgtggt ttatgctgaa ctcctgcttt tcttcaggta gcgtggaatg 42480
tggtatgtgc tgggcaaagg gggcctgcat gaccagcccc caataaaaac cctgggtgtt 42540
gggtctctag tgagtttccc tggtagacag CatttcaCat gcgttgtcac agctccttcc 42600
tcggggagtt aagcacatac atcctgtgtg actgcactgg gagaggatgc ttggaagctt 42660
gtgcctggct tcctttggac ttggccccat gcacctttcc ctttgctgat tgtgctttgt 42720
atcctttcac tgtaataaat tacagccgtg agtacaccac atgctgagtc ttccaagtga 42780
accaccagat ctgagcatgg tcctgggggc ccccaacaca gaaataaatt ataaaagacc 42840
aaggactggg catggtggcc catgccggta atctcagcgc tttgggaggc cgaggcagga 42900
ggaCCagtta agCCCaaaag ttCaaagtta CagtgaCCta tgaCtgCgCC aatgCaCtCt 42960
aacctgggag acagagcaag accctgtccc caaaacaata aactaaacac atacttctgc 43020
cttccaagtg tcttaaaatt caatggaatg gtagaaacat ttttaaaaca ctaaatcaaa 43080
agaaacctgg aaaacaagag tgccgatggc caactaaaat gtctaggaaa tttctgaaaa 43140
gtaaaaagta ctcagaacca gattacctga gcaaaccata gcccaataca agcttgggag 43200
gaggctgtta tgcagaagga aatggtaaca ggtttccagg aacagacttg taacagcaga 43260
tagaacagca gaggtagaac ctgacaaggt gattacctgg ggaactgcag tctgaatgac 43320
caggactgtt ggacccttcc cctcacatgg aatacacacg ccactcagca gcacaccaca 43380
gctcttcaac aatcacagga ggcacgctac gcctagtaag acaggaaaaa aggaattctc 43440
aaacttcgaa gatgaacaca taaagaatca ccaagttttt attcagtatg atgaaacagg 43500
gacactgaat caacagaaca caaacccaag caaagataat tactagagca catagaagaa 43560
attattagat attcttggga agacctaagg ggacattata aagagcaagc agttggtatg 43620
tgacgatctt tgtgatatac caagaaataa aaacacagga tgaagaccag atagagaata 43680
atgctactat ttgtgcaaaa aaggagaaat ggagaatctg attcatattt gcttgtattt 43740
gcatgaagaa actttggaag gtacataagt aactaacaac aatggttacc tacttgtaag 43800
gcgagagaag taagaggaca ggaatggtgg gaacaccttt tgtgtccgga attggtgggt 43860
tcttggtctg acttggagaa tgaagccgtg gaccctcgcg gtgagcgtaa cagttcttaa 43920
aggcggtgtg tctggagttt gttccttctg atgtttggat gtgttcggag tttcttcctt 43980
ctggtgggtt cgtagtctcg ctgactcagg agtgaagctg cagaccttcg cggcgagtgt 44040
tacagctctt aagggggcgc atctagagtt gttcgttcct cctggtgagt tcgtggtctc 44100
gctagcttca ggagtgaagc tgcagacctt cgaggtgtgt gttgcagctc atatagacag 44160
tgcagaccca aagagtgagc agtaataaga acgcattcca aacatcaaaa ggacaaacct 44220
tcagcagcgc ggaatgcgac cgcagcacgt taCCaCtctt ggctcgggca gcctgctttt 44280
attctcttat ctggccacac ccatatcctg ctgattggtc cattttacag agagccgact 44340
gctccatttt acagagaacc gattggtcca tttttcagag agctgattgg tccattttga 44400
cagagtgctg attggtgcgt ttacaatccc tgagctagac acagggtgct gactggtgta 44460
tttacaatcc cttagctaga cataaaggtt ctcaagtccc caccagactc aggagcccag 44520
ctggcttcac ccagtggatc cggcatcagt gccacaggtg gagctgcctg ccagtcccgc 44580
gccctgcgcc cgcactcctc agccctctgg tggtcgatgg gactgggcgc cgtggagcag 44640
ggggtggtgc tgtcagggag gctcgggccg cacaggagcc caggaggtgg gggtggctca 44700
ggcatggcgg gccgcaggtc atgagcgctg ccccgcaggg aggcagctaa ggcccagcga 44760
gaaatcgggc acagcagctg ctggcccagg tgctaagccc ctcactgcct ggggccgttg 44820
gggccggctg gccggccgct cccagtgcgg ggcccgccaa gCCCaCgCCC aCCgggaaCt 44880
cacgctggcc cgcaagcacc gcgtacagcc ccggttcccg CCCgCgCCtC tCCCtCCaCa 44940
cctccctgca aagctgaggg agctggctcc agccttggcc agcccagaaa ggggctccca 45000
cagtgcagcg gtgggctgaa gggctcctca agcgcggcca gagtgggcac taaggctgag 45060
gaggcaccga gagcgagcga ggactgccag cacgctgtca cctctcactt tcatttatgc 45120
ctttttaata cagtctggtt ttgaacactg attatcttac ctattttttt tttttttttt 45180
tgagatggag tcgctctctg tcgcccagac tggagtgcag tggtgccatc ctggctcact 45240
gcaagctccg cctcccgggt tcacaccatt ctcctgcctc aacctcctga gtagctggga 45300
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-65-
ctacaggcaatcgccaccacgcccagctaattttttattttattttttttttagtagaag45360
cggagtttcaccatgttagccagatggtctcaatctcctgacctcgtgatccatccgcct45420
cggcctcccaaagtgctgggattacagacgtgagccactgcgccctgcctatcttaccta45480
tttcaaaagttaaactttaagaagtagaaacccgtggccaggcgtggtggctcacgcctg45540
taaccccagcactttgggaggccgaggcgggcggatcacgaggtcaggagatcgagatca45600
tcctggttaacacagtgaaaccccgtcgctactaaaaatacaaaaaattagccgggcgtg45660
gtggtgggcaccggcagtcctcgctactggggaggctgaggcaggagaatggcgtgaacc45720
tgggaggcagagcttgcagtgagccgagatagtgccattgccttccagcctgggcgacag45780
agcgagactccacctcaaaaaaaaaaaaaaaaaatagagacccggaaagttaaaaatatg45840
ataatcaatatttaaaaacactcaagagatgggctaaagagttgacggaacaaatctaaa45900
tattagattggtgacctgcaaaaccagcccaaggaacatcccagaatgcagcccataaag45960
ataaagagagcatttccgctgggcacagtggtatggcaggggaattgcctgagtccaaga46020
gttgcaggtcacattgaaccacaccattgcactccaggcctgggcaacacagcaatactc46080
tgtctcaaaaaaaaaaaaaattaaattaaaaaagacagaatatttgagagaaaaaaatgc46140
ttatttcaagaaacatgaaagataaatcaagatattctaattcccaagtaagaataattc46200
cagaagcagaaaatagaatagaggcaaggaaacactcaaaacttctccagtgccatagaa46260
atgtgtattaatctttagaatgaaacggactaccaaatgctgagcaggaagaacaaaaga46320
gatccactcttaagccagtgtggtgcccaagcgcagtggctcatgcctgtaatcccagca46380
ctttgggaggccgaggcaggtggatcacctgaggtcaggagtttgagatcagtcaggcca46440
acatggtgaaaccctgtctgtactaaaaatacaaacattagctgggtatggtggtgcaca46500
tctgtaatcccaactacttgggaggctaaggcaggagaatcacttgaaaccaggaggtgg46560
aggttgtagtgagccgagatcatgccacactcccagcctgggtgacagagcaagattcca46620
tctcaaaaaaaaaatccactcctagacaaataatagttaaattttagaacaccaaggaga46680
aagaaaaaaaattgtaaagcttcagagaaaataaacattaactacaaagaaacgagagtc46740
agacgcgtgcacttcttcctagataccagcagataaagcaatatctccaaaattcagaag46800
gttttaacgtagaatcctatacccagtcaagaatattcacatggaaaagtgaaataaaaa46860
acattgtttaaacatgcaagggttcagaaagtttaccattcacagaatccctgaaaacaa46920
aaccaaataatcacttaaggactcattaagaaaacaaatgaaataaaagcaccaatgatg46980
agtaaataatcagaaaaatttacagtttacctaaataactgtttatgcataatgtatgaa47040
aacccaaaaatttaatatgggacagaattaaaatcatgataagattcttttttgctttac47100
tcatggagagttcacataaacagattatcttttaatagcaagagaaaaaaatgtttagat47160
atgtgtgaaaaactaagggtaccaaaacagtgcaaattcatttatcatcaggaaaatcca47220
aattaaaaccacagtatccaccagaataactaaaaggtaaaagacagaaattaccaagag47280
ttggcaagaatgtggagcaaccacatatacttctggggtaaataagttggtgcaaccggt47340
actgaaaactgtttgctagtatctactaaaaccgagcacatgcacagactacaaccaagc47400
agttccactcccagatacacactcaacagaaatgcacacactcactcaacaaaagacgtg47460
tactagagtgttcatgtacttactattcataatagtccaaaaatgcaaacaaccaactgc47520
caatcaaagtcaaatgtatatctatattagggatatatacaatggcatatacacagcaat4.7580
gagaatgaaatgaaccagctcggcacagtggttcatgcctgtaatctcagcactttgggc47640
gggtaaggcaggcagatcacttgaggtcagaaatttgagactagcctggccaacacggtt47700
aaaacctgtccccactaaaaacacaaaaattagccgggcatagtggttgcaggcctgtaa47760
ttccagctactcgggaggctgggttgggagaatcgtttgaacccgaaagccggaggtcgc47820
agtgagcggagatcgtgccactgcactccagcctggacgatagagcaagactccgtctca47880
aaaaaggaaatcaaaaatataaaataagatgacaggaataatccgcaaaagatcagtaat47940
caaaataaatataaatgggctaaagctacctattaaaagacaaagatttcacacccataa48000
ggatagctactatcaaaaaaagagagagaataacagatgttagcaaggatgtatggaaac48060
tgaaattctcacgcattgctggtgagaatataaaatggttcagcctctgcggaaaacact48120
atgctgggtcatcaaaaaattaaaaatagaagtactacttgatccaacaattctacttct48180
gggtatatacccaaataactgaaagcagggtcttgaagagatatttgtacacccatgatc48240
atggcagcattattcataatagctatgatgtggaaccaacataaatatcctttgataaat48300
atatggataagcaaaatgtggtgtatacattcaatggaatattaattagcaataaaaatg48360
aagaaaattctgacacatgctacaacatggatgaaccttgagggcattacattaaatgaa48420
ataagccagttataaaaagacaaatactatatgaggtactatattagatactcatgcaag48480
gtacctaaaataggcaaattcatagagacaaaaagcagaatggtggttgccaggggctgc48540
ggtaatggatacagagcttcaattttgtaagatgaaaaaattctggagattggttgcata48600
acaatgtgcacacacttaacactggggaactgtaaacttaaaagtagtaaatggtaaaaa48660
taaaaataataaataataaattttatgttattttaccacaatatttattaaaagacaaag48720
attaactaattaaacaaaatccagccataagctaatggtaagagtaacaattaaagaaga48780
cacagaaaattgaaaatcagtgactagaaaaagatattccatataaatgctaacaaaaag48840
caagtacagcaatataaagagaatgaacaaaaaaaaaattaaataagatggctcgtttat48900
tcccaaaaggtacaattcaccaagaagatacaagaattgtgaacctttaagcacataaaa48960
cagcttcaaaaatacaacatttaaagaaaaatatatattaaacatagaaatagtacaaaa49020
acccctacaagaatcataatgggagtcttcaatacaactctccatatcaacaggtcaaac49080
agagaaaaaaaataagttaaggatgcagaaaacctgaattaccatcaataaacttgagat49140
taatatagaactgtatacccaatatactaagagttcagggaacagtcgtgactgacagtg49200
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-66-
gactgcaaat taatctgttc ttaatctttg tttttctttc agcactgtgg cagaatagag 49260
atcctaaaaa ccttccagct acaaaacatc tttttaaaaa tataaaaaaa tacaaaaata 49320
actctgaaat caatagaaga cacatggtga aaccaaaatt ctagaataca gggagaataa 49380
aggcattttc agatattaca aaaacagaaa attgatcatt gctgaagtaa tttctaaaga 49440
atgtacttga gggagaagaa aaatgttcca aagaaaagta tctgtgatac aagaaggaat 49500
ggaaagtgaa gaaatggtaa acaggtagat aaagctaata aatgttgacc tagaaaataa 49560
caaaaacaat agcaataatg tctcgttgga agggttgaag taaaaataca attaaggcca 49620
aatgtgaggt aagtggaatg aaagaattag aagtccttgc cttgttcaca ggactgatta 49680
aataaatgag ccaggttttc cattcaaaca gttaaaactt gaacaaaata aactcaaatt 49740
aagtagaaag ataaaaaaca gaaattaatg tcatagaaaa ataaaaaatc aatagaatta 49800
atcaataaat cctggttaat aaaagctggt tctttgaaag gattaataaa ataatcatta 49860
agcaagtctg atcaaaaaaa aagagaaaag gtaccaaaaa aagtactgta tcagaaagag 49920
aacatacaga tacatacaga tatgtaagag tctgttttct tacaccagaa tactatatac 49980
aacattatgc tagcatatat taaatttcaa taatgttaat gattttctag gaaaacagaa 50040
aatattaaat ttactttgaa gaaacagaaa aactgagaaa aataaatgat catgaaaaaa 50100
atgaaaaggt aattaaatac tgatattaac tgcctaaaca acaccagcag cagcccaggc 50160
agtctgcagt caagttctgc caaacttgag ggaacagata attcttctat tccagagcat 50220
agaaaatgat ggaaagtttc ccaatttaat cagagaggac agcctgatcc ttgttatgaa 50280
cacagataaa aatggggtaa actatatgcc aaactcagat accaaaaccc taaataagat 50340
gctagcttat tgatgtgaac aatccaaaag tgcattttaa attagcccag ggttttagag 50400
aaagaaaatc tagcaatgtg accaccactt atgttaacaa ttttaagacg aaaatctaca 50460
tgatcatatc aatgcatgct acacaaaagc atttgggcaa aaaacccaac acccaccctt 50520
gactttttaa actcttagta attaggcata aacagaaatg tacttaatgt gatagaatac 50580
actcggtgaa gatacagagg gaatgctccc taaaaccaag cccaagacaa agattcctat 50640
ttaacctcaa tagtcaacac tgcagcgaga gtaatctatg gaagacaagg aaaaaagtaa 50700
aaacatgaga gacatctgtt gtttaacaga caataagatc acctacttgg aagaggcaaa 50760
cgaatcaagc gaaaaactat taaaactgag acaggcttta gtatggaggc tcagcttcag 50820
ctgtagtttg ggctaccaaa ttcaactcgc ttgcttggag agttaatcct gcaaagctaa 50880
tttctgttga ggtattagga ttgacaagcc tgtgCtCCtC CCtCCtCCCC CatCttCaaC 50940
actgaaataa cacggtgttt ggaactggat aacagaatct tccaaaaaca aaaattgtcc 51000
tgaagggctg acttgtgccc ttactcaaaa aacactttat ctgctgcctg cagctcctac 51060
agttgctggt ggataagcct gccaaccagc tcggcgtaat tcttcctgca gagggcaagg 51120
aagagcactt tcacaggaaa atttttttcc gaactgtatg ccgcttatta cataaactta 51180
cgtgctggca aatggagctc cagcaaaata agatattcag agtcaaactt ccttaggaaa 51240
aaaaaaaaaa aaaagcaagc acataacact aatttccttg catgggcact ggggaaggag 51300
gtcgttactt CCgCaCgCCC gcaggtccgc accaccggga aacccacggg caccgcgcgc 51360
tgcccccggg ccttccaggt gcactgcgcc gcggcgcccc agctgacccg ggatgcgcag 51420
ccctagccct tcccctgtca ccccggccag gaaggggcgg gagcgcggcg gacgccgagg 51480
gcgaagggct tctcggtcct ctgcaccacg cagcaccccc aaggcacaac agggagggtg 51540
cgggaggctc ccgagaccca ggagccgggg ccgggcgtgc ccgcgcacct gtcccactgc 51600
ggcgagggct ggggtcgcct ccagggccgc agctgtcggg agccacctgg ctctcagtcc 51660
cgggtccctg cgacaaccct cgggcccgga ggggaggagg cggccacctg ccgctgccac 51720
CtgCggCa.CC ggtCCC2CCg CtCCgggCCg ggCaggaCag gCCaggaCgt CCCt.CCtggg 51780
ctggggacag gacacgcgac gaggggaccg gggcccccgc ggcgaagacg cagcacgcct 51840
tCCCagaaag gCagtCCCgt gCCCCCaCga CggaCtgCCg gaCCCCCgCg CtCgCCCgCC 51900
catcccttca gaccacgcgg ctgaggcgca aagagccggc cggcgggcgg gctggcggcg 51960
cggctagtac tC2CCggCCC CgCtggCtCa gCgCCgCCgC aaCCCCCagC ggccacggct 52020
ccgggcgctc actgatgctc aggagaggga cccgcgctcc gCCggCgCCt CCagCCatCg 52080
ccgccagggg gcgagcgcga gccgcgcggg gctcgctggg agatgtagta cccggaccgc 52140
CgCCtgCgCC gtCCtCCttC agCCggCggC CgggggCCCC CtCCCtCCCa gCtCtCagtg 52200
tCtCatCtCC CtatCtgCtC atCCtCtggt cgcacataat cgatgtttgg gcgtcccaag 52260
ccagatgtgg accccatttc cgcactctac actggaggtt ttctaagggt ggtgcccgga 52320
ccagcagctt cagcctcatc tgggaacttg agaaaatgca gattctccgt cccacccagc 52380
ctattcggtt tttcctgcac taaaaccatg aaggtggggc ccagcagtcc acattctcgc 52440
aagcccgtca agtgattctg aggcgccctc cagtttgaga gctatgctca cggcctcacc 52500
tccgccccgc aaggagcccg gtcttgcctg tggcgctagc cgcacacgga cacctcatcc 52560
tgcggggccc gcccccccgc tgcaccctca ccgcccaacg cctcctccgg gatgcagcgg 52620
aggcgcctgg aagtcggcaa ggtcaacatc cccctcagca tcttccctac cctcacggct 52680
cctcctccag gggtgcctca tggccagggg ttagaaagag ccactgtgtt tcttgacatg 52740
gaagtggcct aagaccttaa tgaaaactgc aggagtggaa tgacagaacc tttggtcata 52800
cttgagggcg tgaagctcaa atgaggagga aggaaaggat ccagggagaa taaccaaccc 52860
tggcaagttg tggcgcccag gtagaggggc gagcctaggc tagcggttct cgaccagggc 52920
cggtgttgcc cctcctcgcc gccccgcgta catttgggga ggtctggaga catttttggt 52980
tgtcatgatg cgggagttgc tactgttgcc taagtgggta gacacgaggg tgctcctcaa 53040
catcctacct gaaggacagg actgccccac aaggaagaat gatccggccc caaataagaa 53100
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-67-
accctgggct ggtcagcaac aacccctttg ttctgagaag agaggaggaa agaataaaag 53160
aagtggggtg aagttttggt ttggtagagg aaacttgaag acattttcac tggaaaggaa 53220
gagaggaaga ggagggagat gtctgtaagg acgagcaaac cgggtgacag ctgatttcct 53280
catattgaag taatgagtcc tagttataat aaattcctaa taaaaaccca gtttatccct 53340
gcaataaact tgtctttttt ttttaaatat actgcttgat tctgtttgct aatattttat 53400
ttacaggctt tgcattgata tgcaaaaatg agatgggcaa taattttctt tttgaatgtc 53460
taatgttgtt tggtttcaga atcaatgtta tgctcacatc ataaaaaatt tggaaccgag 53520
gcaggaggag tgcttgaggc cagaagttcg agaccagtct aggaaacaca gtgagacccc 53580
cccatctcta caaaaaaaaa aaaagaaaaa aaaatgggca tgtttgcttt ttccttttac 53640
tctgaacaat ttaaggagca ttaaaattat ctattctttg aggtttgatc atttcccagt 53700
taaaaatgtt cctcccagcc tgatgctttc tttggggagg gtaaatcttt taaggctaga 53760
aaagtttctt ctgtggcaat tttattattt acattttaaa aattattcta gagttaattt 53820
tgataaagca tgtatttctt aaaacaaatt atcctttttt tccagatgtt caagtgtatt 53880
tgcataaagt tgaggaaagt agtcttttgt gaatctttta acttctccca aatatcttat 53940
tttgtgtatt tttgcttctt tattttgtta acttttaaaa gtgtattttt ttttcaaaga 54000
atcagctctt aggtttatgt ttttggttat actggagctt ttttcttctt ctttttaaaa 54060
tattttttct cctttatttt ttagacgtat tttgatctaa cgtaatcgga agaaggtaaa 54120
ttagaatctt ttgttactat tgtgttttta tttctcctta tttctctgaa gtcctgcttt 54180
ataaatagta ccatgttatt tgtgcataaa tattcatttg tcttatattc ttgggaattt 54240
tcccacttca tcataaaatg accttccttg tctcatttaa tgtgttcaaa ctttgccctg 54300
aatttaactt tgtctgatat tttaccatcc tgctgaattt tgtttgttac cccaaacaac 54360
ctttgctgtt ttcgtctttt ctgaaccctt tattttaggt aatcccttga attagagcac 54420
taagttttgc tttgtgatta aatctgaaaa tctttatctt gccatagatg agttgagccc 54480
tattcatgtg acagctatat tatgctgttt CatagCCCtt ttggtCCttt tttCaCtCtt 54540
gcattgcata ttttgtgttt attgtgtttt gtgtttcttc tgataatttg gaaggtttgt 54600
atttttattc agggagttgc cttataatca tactccgcaa tacacatcgt cctcagtttc 54660
ttcagactgt ctgttaactc cctattctga ataaaaatga cattgtaatt tccctctttt 54720
ttctttaccc cttttcttct cctcacctaa tgtaaatgat tttatccttc tttagtattt 54780
gcttttttaa ttaactacat ttataaatat ctttatcact tgatttttaa atcagctttg 54840
aatgagatat ttggattcct agatataaaa gatgttaatt ataccatttc cacgttagta 54900
ggtttataaa atcatacatt ctgctgtgta accataatcc cacgtttgtt ttagttccac 54960
tcctacagtt aaaagattca gaagtattat taacagttat tttgccatag ttttttcccc 55020
aacccatttt gtggtaagtt atgatcctgc tttagtttct taagaataat ttatagagca 55080
gagtgtggtg gctcacgttt gtaatcccag cactttggga gacaagaggt agaaggatcg 55140
cttgaagcca gcagttcaag accaccctga gcaacatagt gagaccttgt ctctacaaaa 55200
aattttaaaa tttagccaga cgtagtggcg tgtgcctata gtcccagcta ctcaggaggc 55260
tgaggcaaga ggattgctag agcccagaag tttgaggctg cagtgacctc tgattgtgcc 55320
actgcacccc agtctgggca agaaagtgag aacctatctc tttaaaataa caataataac 55380
ttatgaaaat tatattccct gagtttttca tgtttaaaaa tatttgttgc ctttatcctg 55440
taaaagtttg agtataaatt cttgggttat actttattta ttgaagaatg tataagtatt 55500
gtcttctaga attgagtgtt gctgtaatga aaccagaagt cagcctggtt tatttttcct 55560
Cagaaatgag gtaattgCCg gCCggaCaCC gtggctcatg cctgtaatcc caacactttg 55620
ggaggccgag acaggtggat cacgaggtca ggagattgag accatcctgg ctaacatggt 55680
gaaaccccgg ctctactaaa agtacaaaaa gttagctggg catggtggtg gacgcctgta 55740
atCCCagCta CCCgggaggC tgaggCagga gaatggcgtg aacctgggag gaggagcttg 55800
CagagagCtg agatCgCgCC aCtgCaCtCC agCCtgggCg acagagtgag actccgtctc 55860
aaaaaaacaa aaaaaaaaca aagaagtgaa gtaattgcca tgatgctcca agaattatct 55920
ctttgtctat gaaatccaga aatctcactg ttatacattt tggaattatt attctgggcc 55980
aatatttcct gggacacaat agattgactc tatagattta attttttttt tttttttgag 56040
acagagtctc actgcaatct cagcttactg caacctctgc ctcacgggtt caagcaattc 56100
tcctgcctca gcctcccaag tagctgggac tacaggcgcg tggcaccatg cctggctaat 56160
ttttgtcttt ttagtagaga cagggtttca ccatgttggc caggctggtc ttgaacgcct 56220
aacctcaagt gatccacctg cctcagcctc ccaaagtgct gggattacag gcgtgagcca 56280
ccatgcccag cctcaattcc tctttctatc tggtaatttt tctgaagttg aaaacatttg 56340
ttctaatacg ttatttcagt gttcttctaa gatgtgtaaa gcaccctatt cccaggtcag 56400
cccccatctt gctagtgagc tcggctggtt cttcacaaga gctctggttt tctcctgctt 56460
aatctcaagt acctctgtca gcctccacct ggtttatgat ttggagtttt ttggtttttg 56520
ttttttgttt ttgacagagt cttactctgt cacccaggct ggagagcagt ggcataatct 56580
cagctcactg caacctctgt ctcccaggtt tgagcgattc tcctgcctca gcctactgag 56640
tagctgggat tacaggcgcg tgccaccaca cccggctaat ttttgtattt ttagtagaga 56700
tggggtttca ccatgttggc cagggtggtc ttgaactcct gacctcaggt aatccacctg 56760
cctcagcctc ccaaagtgct gagattacag gcgtgagcca ccgcgcctgg catggtttgg 56820
agttttaatc tgtagtttta ataaagatag tgcttatgtt tgtgtttctt atatttcttg 56880
gtactcttgg gtaatttgta agatccccat atctacacaa gaagtccatt ttcaattctt 56940
ttcttcagac tgtttatttt attttatttt attttatttt tatgtttgag atggagtctc 57000
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-68-
gctgtgtcacttctggaggctggagtgcagtggcgcgatctcaggtcactgcaacctccg57060
tctcccgggttcaagcaattctcctgcctcagcctcccgagtagctgggattacaggcac57120
ctgccactttttaatttttttagagacagagtctcgctttgttgaccaggctggagtgcg57180
gtggtgcaatcatggctgactataacctccaaatcctgggctcaagtgatcctcctgcct57240
cagcctcctgagtagctgggactacaggcacatgccaccatgcccagttaattttaattt57300
ttttgtagagaCagggtCtCCatatgttgCCCaggCtggCCtCCtaCtCCtggcctcaag57360
taatcctcctacctcagcctcccaaattactaggattataagcatgagccaccatgccca57420
gccttgttctactactttaatttcatatgttaggtgaccatgtaattgatcatccaaacc57480
aggatactgtaagaatgaaagaggctgacagtagtatgatgctgggactagcattgtgca57540
ctgagattatttctgggaaagcaggagatacggtcaccctacttatagtgtgcttgtctt57600
tggattgttgaatttggagtttctatttgcaggcttatttcaactgggcagccttgatcc57660
gccctgcccagcaatgctaccgttctctccaccgggtctctgggaccccttcagtcacta57720
tacttagctcagttccccaccctcccactccctaaaagcgtaaccaggaatcctgcctca57780
ggtctactgccgtcttccgtgggctgtttcagttcctattacccagagtcaaactcccag57840
cattccctacctgattccagacttggagtccagagctttaacctcttcaggccaactccc57900
cactttgcatttctgtccctatatcttagtccatggagatacatttcatgtctttgagtc57960
tacttacaaagtaaattttgctgttttttaattttttttttgagatggagtcttgccctg58020
tcacccaggctgtggtgcaatgacgccatctcggctcactgcaacctccgcctcctgggt58080
tcaagcgattCatCtgCCtCagCCtCCCaagtagctgtgattacagacaggcaccaccac58140
gcccagctaattttttttatcttttagtagagacagggtttcaccatgttggccaggctg58200
gtcttgaattCCtgaCCtCgtgatCtgCCCatCtCggCCtcccaaagtgctgagattaca58260
ggcgtgagccactgtgcccagccaattttgctttttttatatttcattgctatatgttta58320
gaggataagtttacagtgctatatgcattcccaaatattagaccaaaaaaatctccaaaa58380
aattagaaagaaaatccaaaaaatctcaaaaaataccaaaaagcaacaatctcacagacc58440
atactcactgacccccaataaaataaaattagaaattaaccacaacttaacaaaataaag58500
tactcaagtcagagaggaaagaggaaataaacatcaaaattacaaagtctaggcggtggc58560
tcacgcctgtaatcccagcactttgggaggccaaggcgggcagatcacaaggtcaggaat58620
tCgagaCCagCCtggCCaatatggtgaaaccccgtttccactaaaaatacaaaaattagc58680
caggcatagtgatgtgtgcctgtaatccagccacttgggaggctgaggcaggagaatcac58740
tgaacccagggagacgaagattgcagtgagccaaaatcgtgccactgcacttcggcctgg58800
gtgacaaagcgagactccatctcaaaaaaaaaaaaattacaaactctttagatagaaatt58860
ttggtgtttttttttgagaCggagtCtCaCtCtgtCC~CagaggCtggagtgCagtgggaC5892~
tatgtcagctcaccgcaacctCCatCtCCtggattcaagcaattctcctgtCtCagCCtC58980
ccaagtagctaggattacaggcgcccaccaccagacccagctagtttttatatttttagt59040
agagatggtgtttcaccatgttggccaggctggtctcaaactcctgacctcaagtgatcc59100
aCCtgCttCagCCtCCCaaagtgCtCagattaCaggCgtgagCCaCCgCaCCCCaCCtag59160
atagaaatttcaacatgaggccgggcacaatggctcacgcctgtaatctcagcacttcag59220
gaggctgaggcgtgggaggatcacttgggcccaggagttcagg2ccagcatgggtgacag59280
agacagaccctgtctctatttatttgaaaaaaaaaaaaaaaaagagagagagaaagaaat59340
ttcaacatgaaaagtatctctcaaacccttcgagatgttggcaaaaagcgactcaaagga59400
aaatgtattactgtgtgtgaatttgcttgaaaataagaaagaggccgggtgtggtggcta59460
acacctgtaatcccaacactctgggagtccgaatcaagtggatcatgaggtcaggagatc59520
gagaccatcctggctaacatggtgaaaccctgtctctactaaaaatacaaaaaattagct59580
aggCgCggtggCtCatgCCtgtaatCCCagcactttgggaggctgaggcaggtggatcac59640
ctgaggtcaggggtttgagaccagcctggcctacatggtgaaacctcgtctcttctacaa59700
ataCaaaaattagCtgggCgtggtggtgggtgCCtgtaatCCCagCtaCtcagaggctga59760
ggcaggagaatcgcttgaacccgggaggcggaggttgcggtgagccgagatcgcaccact59820
acactccagcctgggcaacagcctgggtgacacagtgagactccatctcaaaaaatacaa59880
aaaattagctgggtgtggtggcctgcgcctgtagtcccagctacccgggaggctgaggca59940
ggagaatggagtgaacctgggaggaggagcttgcagtgagccgagatcccaccactgcac60000
tccagcctgggcgacagagcaagactcttgtctcaaaaaaaagaaaaaaaaaggaaaaaa60060
gaaccctgataataaagaaaccaaatgttcaactctcaaagctcggacactttaaagaaa60120
taattaataaaggcagaagttaaagggaggatgataaagcaattttttttgttggttttt60180
ttgagatggagtcttgctctgtcacccaggctggagtgcagtgatgcgatcttggctcac60240
tgcaacctctgcctcccgggttcaagcaattctcctgcctcagcctcctgagtagctggt60300
actacaggtgcgcgccacctggcccagctaatttttgtatttttattagagacggggttt60360
caccatatttgttaggctggtctcaaactcctgatctcaggtaatctgcccacctcggcc60420
tctcaaagtgctgggattacaggcaggcgccaccgcgcctggcctaaagcaaaatattgg60480
ttctgtgcaaaaggtcaataaaaagagcaaacgtttacaaactggagccagcacccattc60540
agctcagtgtgtctggagaaaaaacaatctcgcttcagaattcatgattacgcagccctt60600
tttgcttcctaaaaatcctactatgttgctgttgaccattctctctctttctctctctct60660
tgctttctctccagaaaagctattcagacattctcctctttcctcaaacctccaacactt60720
CCtCCtCCatccttagcctcagctgctgacctcacttctaatcattgagaaaccaggaga60780
agcatttaagagtgaacctccgcctccccgcacgggcaaaaccacccacccacagaattg60840
tgccccaattctgcgtcctctcctctcaccatggatggacggtccaggctccgagccaaa60900
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-69-
gccaggcctcccctggagctctggatccaccacctgcagcttctcaggcagggccccagc60960
agctcccctgCtCCCttgtaCCatCaatCCCtCCCCtCaCtgggtcactcccaacaatat61020
atatatttagtgatgtttctcccatgtggtaaaatcacttagCCtCtCtCCtCCCCCagC61080
tactatcctatttgtttctttccattctctgcaaaacttctcaaagcattgtgtctatgt61140
gctgactccatttatcttctcccgttctctgctgagtccttcccacagactctcacccca61200
gttactccatgaaatgacctctgcactgccacatccaatggtgaatgttcagttcttaat61260
tttattcagtctttcagcagcatttgacctggccgatcactccctcttcttaaaaatact61320
tttctcagccaggcgtgatggctcacacctgtaatcccaacactttgggaggccaaggcg61380
ggaggatcatgagagcccaggagttcaagatcagcctgggcaacatggcaaga~cctatc61440
tctacaaaaactaaaaagtagccagtgtgatggcatgcacctgtagtcccatctacttag61500
gaggctgaggcagtaggatgacttgagcctgggaaatcaaggctgcagtgagccatgatt61560
gcaccactgcactccagcctgagtgacagcgagaccctgtctcaaaaagacaaaatagga61620
aacttttctcagcatattcctctgattctcctgctgcttctgtctgcacagattcagtct61680
CCtttgCCggttCttCCtCatcctcctgatctcttgaccttgaagtgccccagagtacag61740
tctttttttttttttttgagacgcagtctcgtctgtcacccaagctggagtgcaatggcg61800
aggtctcagctcatgcaacctctgcctcctgggttcaagcgattctcctgcctcagcctc61860
ccaagtagccaggactacaggcacatgccaccatgcccagcaaattgttgtatttttagt61920
agagacagggttttactatattggccacgctggtctcaaactcctgaactcgtgaaccac61980
ccgcctcggcctcccaaagtgctgagattacaggcatgagccaccacacccggcccagag62040
taCagtCtttagaCggCCtCtCtaCCtataCttgCtCCCCtCataaaCtCCtCCtgCCtC62100
atggctttaaataccatcggtagactgatgactcccatatttctcttttttttttggaga62160
cggagtctcgctcagtcccccaggctggagtgcagtggcgcgatctcggctcactgcaag62220
ctccacctgccaagttcacaccattctcctacctcagcctctccagtagctgggactaca62280
ggcacccgccaccacgcctggctaatttttttgtatttttagtagagatggggtttcacc62340
atgttagccaggatggtctcgatctcctgacctcgtgatccgcccatctcggcctcccaa62400
agtgctgggattataggtgtgagccaccgtgcccagccgatgactcccatatttctatct62460
cttgctgtgtgggagttctcctcagaactccatactcataaatccaactctcataaatag62520
tatctcaaatgggcaatatgctcaaaagtcaattcctacttttctccctaaacttgcttt62580
cctgcagtctccaccatcttaatgtccaatctaacattaggaggcaaaaactttgaagtc62640
attcttgactcttctctattacacaccctatccaatctttctgcagatccagtcgacccc62700
CaaatCCagttagCtCtCatCatCtCCCCtgttaCCCCCtggtCCaggCCatCttCCtCt6276~
CtCaCCtgaatCaCtgCagCattCtCCtCaCtggtCtCtttggttCtgttttCaCtCCaC62820
cttagcatagtctccacagagcagtcagagggatccttttaaagtgtaattcccatcctg62880
tccctgctctgctcaaaaccctgtcgtgattcccgttttaatctgtcagattaaaagcca62940
gagtctttccagtgacctacatgatctgcctattatcacctcccacttctttccccttgc63000
tCaCtCCaCtCCagCCCtgCagCtgtCCtttCtgtttCCtgaaCagCCCagattttgCtt63060
ctttagaacctttgtatttgctgtcccctctgtctggaatgtttttccaggaagtcacct63120
ggctctctcctgcacttccttcctgaccaccatgtttaaaaatcactcaaacacacttca63180
ggccggacatggtggctcacgcctgtaatcccagcactttgggaggccaaggtgggtgga63240
tcacctgaggtcaggagttcgagaccagcctggccaacatggtgaaacttcgtctctact63300
acaaatacaaatagtagccaggtgtagtggcacacacctgtaatctcagctactcaggag63360
gctgaggcaggagaatcgcttgaacccagaaggcagaggaggtgcagtgagccaagatca63420
cgccacaacaccccagcctgggtgacagageaagaccccatctcaaaaaaaaaaaaagaa63480
aaaaaaatcacacaaacacacttctcttcatattccttttccaagttttatttttctcca63540
gaatactttacattgttttaatggaagttctccgtttccccccaactagaatggatactt63600
cctgcaggtaggCaCtCtagtCCtCCCatCCaagtaCtaaCCaggCtCaaCCCtgCttag63660
cttctgagagcaggggagatcaggcctgttcagggtggtatggcccaggaattttgattc63720
tgttttattcattgctgttctgttgattctcttttgttcctcctcctagtgctgagaaca63780
CtaCttgtaCataataagCattcaataaatatttgttgaatgaatgacttgttgaatgaa63840
ttaatctcagaaatgcaggactggttctacattagaaaatttttcaaggtcattctctgt63900
tgtcgtaacacattaagagaggaaaattttgtactctaaatcatttgataaaatacatac63960
tgatttctgttttcaaaaactcttagtggctgggcgaggtggctcacatctataatccca64020
gcattttgggaggacgaggtgggcggatcacttgaggtcaggagtttgagaccagcctgg64080
ccatcatggtgaaaccctatctctactgaaaatagaaaaattagccgggtgtggtggcgc64140
atgcctgtagtcccagctacctgggaggctgaggcaggagaatggcttgaacccgggagg64200
cggaggttgcagtgagccaagatcatgccattgcactccagcctgggtaacagagtgaga64260
ctccatctcaaaagaaaactcttagtgagtttaggaatccaaggaagaccctcaaactaa64320
atagataatctagctaccagaagccttcagtaaaccttaacactccatggtgaaacatta64380
gaaacattcctactaaaagacaggctaagaatgcctgcaatcttcacggctagtccaaga64440
agtcaaaaagaagaaatgagcgctgatttaaaaaaataaacaaacaaaaaactaccgatg64500
cagaggctggcagcaaggactgaaggactgtacagtacttgcctggagcaggcggatggc64560
cacacccctgcgaagcctgctcagctggctgggggacgctccagtgtgtgagtggcagga64620
tgcagggtacttcctctgccagggagttgcactggggagatCCtCCCCCaCtCa.CaCttt64680
ggcagctggggctttggaatgtgacttagcttctgtcaaagggtcaatccaccctttgat64740
atatgatgcaaaggcgaacatatgatgcaaaggtgagagaacagcccaaattaggacttt64800
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-70-
taccacagctgtggaggtggacagcgacagtggtgggccctggccagacttttcatgctc64860
aaaggtggtggttgttcttcctacttcttgtccctccagggcttcctttgcctgtgtgct64920
gaacctgcttcttttaattttttttaacttttttaaatttttaattgttttaattaaaac64980
aaattttgaaaactgtctgaacctgcttttgaaccctgctatgatttgaatgtttgtccc65040
ctgccaaactgattttgaaacttaatctccaaagtggcaatattgagatggggctttaag65100
cagtgactggatcatgagagctctgacctcatgagtggattaatggattaatgagttgtc65160
atgggagtggcatcagtggctttataagaggaagaattaagacctgagctagcatggtcg65220
ccccttcaccatttgatatcttacactgcctaggggctctgcagagagtccccaccaaca65280
agaaggctctcaccagatacagctcctcaaccttgtacttctcagcctctgtaactgtaa65340
gaaataaatgccttttctttatgaattacccagtttcagatattctgttataaacaatag65400
aaaacgaactaaggcaaactctcatgattctactgccatgccattccaataaactccctt65460
tatgcttaagagagccagagttggccaggcgtggtgactcacgcctgtaattccagcact65520
ttgggaggccgaggcaggtggatcacaaggtcaggagatcgagaccatcctggctaacac65580
ggtgaaaccccgtctctactaaaaatacaaaaaaattagctgggcgtggtagtgggtgcc65640
tgtagtcccagctactcgggaggctgaagcaggaggagaatggcgtggacccaggaggcg65700
gagcttgcagtgagtcgagatcgtgccactgcactccagcctgggtgacagaatgagact65760
ccgtctcaaaaaaaaagagagccagagtttatttctgttgcttgcaaccaagaaatctgg65820
ctggtgcactgaagtttccataaataatagcaatttaaagactctttccaagccaggcaa65880
tgcctagccttgtgtagtccttgtggtaatacattcattcattcatttgttcaaccaact65940
gtgctccagagactaagaatacaaaaatgggggccgggtgtggtggctcacacctataat66000
CCtagCaCtttgggaggCCgaggcaggtagatcacctgaggtcaggagttcgagaccaac66060
ctggccaaaatggtgaaacccctactctactaaaaatacaaaaaattagctgggggtggt66120
ggcggacacctgtaatcccagctactcgtgagactgaggcaggagaatcacttgaacccg66180
ggaggcagaggttgcagtgagccgagatcgcaccactgcactccagcctgggcaacaaga66240
gcgaaactccacctcgaaaaaaaaaaaaaaaaaaaaagagggccggggctgggcgcagtg66300
gctcacgcctgtaatcccagcactctgggaggccaaggcaggagaattacgaggtcagca66360
gatcgagaccagcctgaccaacatggtgaaaccccatctctactaaaaatacaaaaatta66420
tccgggcgtggtggcgCacacctctagtcccagctacttgggaggctgaggcaggagaat66480
cgcttgaacccgggaggcagaggttgcagtgagccgaaatcatgccactgcactccagcc66540
tgggtgacagagtgagactccgtctcaaaaaaaaaataaaaaaaaaaaaagaattcaaaa66600
attgtagagttatagtgtgcttctagtttagttgagaggacatctgtccttcaaggaagg66660
ctagaatctataccctgagtccttactgaaatcaatccagcagtcaaaacatgggaccaa66720
cgatcacagcagtaagataggaagagcacctttgtacatttagctcatgttgagataagc66780
cactgacagagctgaaggaagctcacagttctgggttccatcctttggcatttaaaaaga66840
aaagtgctaagaaaattcggttggtcacggtggctcacgcctgtaatcccaacactttga66900
gaggccaaggcaggcagatcacgaggtcaggagttcgaaaccagcctggccaacatggtg66960
aaaccccgtctctactaaaaacagaaaaattagccgggcatggtggcgcatgcctataat67020
cccagctactcaggaggctgaggcaggagaattgcttgaacccgggagggggaggttgca67080
gcgagtgagagcaggccactgcactccagcctgggagacagagcaagactctgtctcaaa67140
aaaaaaaaagaaaaaaagaaagaaaggaaaaaaagaaagaaaaaaaaagaaaaaagaaaa67200
ttcaggccaggccaggcctggtggctcacacctgtaatcccaacactttgggaggctgaa67260
gcgagacggtgccttagcccaggagtttgagaccagcctgagcaacatagcgagaccctg67320
tctctataaaaaaaaatttttttttggccagacgcagtggctcacgcctgtaatcccagc67380
actttgggaggccgaggcaggtggatcacgaggtcaggagatggagaccatcctggctaa67440
cacggtgaaaccccatctctactaaaaaatacaaaaaattaaccgggcgtggtggcgggc67500
gcctgtagtcccagctactcgggaggctgaggcaggagaatggcgtgaacccgggaggcg67560
gagCttgCagtgagCCgagattgCgCCaCtgCc3.CtCCagaCtgggagagagtgagactcc67620
gtctcaaaaaaaaaaaaaaaaaaaaaaaattaattgtcaggtgtgctggcatgcagctgt67680
agtcctagctactcgggaggctgaggtaagaagatcgcttgagcccaggagttcaaggct67740
gcagtaatagtgcctctcactctaccctgggtgacaatgagaccctctctcaaaaagaaa67800
gaaaaaagggaaagaagaaaagaaagaaagaaagagaagaaaggaaggaagaaagaaaga67860
aaaagaaaaggaaggaaggaagaagaaaaaaaaagaaagaaagaaaagagagagaagttc67920
aaagaccaaagggtcaggatcccaaaatagtttttatgttttatttatttatttacttat67980
ttatttttgagacagtatggctctgtcgcccaggctggagtgcagtgatgcgattgcggc68040
tcactgcagcctccaaactgggctcaggtggccctcccacctcagcctcccgagtagctg68100
ggaccacaggcgcgtgccaccatgcccagctaattttttaattctttgtagagatgaggt68160
ctctatatgctgcccaggctggtctcgagctcctgggcttaagccatccacccgcctggg68220
cctcccaaagtgctgggattacagaagtgagccaccgcgcctaatcgggtggtttgtttg68280
tttattgacggggtctcgctgctgcccaggctggagtgccagtggctgttcacaggtgca68340
gtcctggagcattgcatcagctcttgggctctagcgatcctccagagtagctgcagctgg68400
gattccaggcgcgccaccgcgcggggctcagaatgggtttttatattgagggttatgctg68460
ccacctagaggatatatgtagtaccgaactgtgtgcgcagggaggctgaggttgcagtga68520
gccaagatgatgccagggcactccagcgtgggtgacagagcaagatttcatctcaaaaaa68580
aaaaaaaaaaaaaaaaaaaaaagaattgaaagtaaggtcttgaagagatatttgtgcctg68640
tatggtcatagcagtattaactttgacccactagctaaaacacaaaagcaacatgtgtct68700
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-71-
gtcagcaggt gaacggataa acaaaatgtg gtatatatgt acaattgaat attattcagc 68760
ctttaaaaag gaataaaagg ctggatgcgg gggctcacgc ctgtaatcct aacactttgg 68820
gagactgagg tgggtggatc acccgaggtt aggagtttga gaacagcctg gccaacatgg 68880
tgaaacttca tctctactaa aaatactaaa attagccggg catggtggca cttgtctgta 68940
atccaagcta ctggggaggc taaggcagga gaattgcttg aactcaggag ccggaggttg 69000
cagtgagcta agatggcacc actgcactcc agcctgggca acagagtgag actccatctc 69060
aaaacaaaca aacaaaaaat tattatttcc aaagaaacaa gaccctgggt ccatttccca 69120
gcccacacct gatgttgact cacaacacac agcctggttt gctatgagcc tgcttcattt 69180
aattgtcacc ttaacttcac atcaccctca agtcctggaa taactctttg ctgacctttg 69240
tgtgctgagc catctccatg tcgctcaacg tgcagtccct ctcactgcac tgagtcaata 69300
gccagacgtg gtctgactgc agggtcatcc ttggtggctt aggctgactc gggcatagca 69360
gggtgctctg agacctcacc gcatataggc tttgccccca ataaactcta tataatattc 69420
atattatgtg gtctgggtgt gtgtagcttt gcactgtctt ctcgtgacag tgccctcaac 69480
ctctttccca ggatttcctc ctctacctcc tcaagtccca ctgctctgca aagaccaaaa 69540
gctgcagagt CCCagCtCCC tCCtttacaC cccacgacgc agcctcctct ctcagaaccc 69600
tttaaacaga gtcttttact gcagatccca agaacagcca cacccctctc tcccacccac 69660
tccagacaca cccaggtaat tatagcaccc agggtaacta tgtagatgga gtccctggaa 69720
catgtggata gtgccccctg ggagtatgca aaagcaacat tgctggcacc tgcagagaac 69780
agggtgacat ccaggaatca gagcatgggc ctctgggagg tagggatgtg gccaggcagg 69840
ctgccaaaaa ttggtagagc aaggccacag gatctttctg accttccttc caaacagagg 69900
ctcctgtact ggtgatccct gtgttgattg accactccct tcctgggggt cgtggtctct 69960
gtcccagttg cccggacttc tgtgagtgtc ctactgaggt ccttttcatg agaagcatgc 70020
tgtccttcca cctgctggga gcaagagtga caacttcaat actataatag cagtggcata 70080
cagagaagaa gaaagatgaa gtggcaagaa aaacaggctt ccaagcagga gtttttctat 70140
aaaaacaaaa acgtttacaa gcaaactttt tataaagggc tagatagtaa atattttagg 70200
ctttgagagc cacatagact tgtttgcagg gactcaatgt cgctattgta gtttgaaagc 70260
agccatcagg gttatgtaaa tgagtgagtc tgattttgtt tcagcaaaat tttatttacc 70320
aaaacagaca atgagtgggc tggatttggc ccatgatcct tagtttgcca actcctgctt 70380
tgggctcacc cagatctgat tttgaattCt ggCtCtgCta CtggttagCt gCaggagCtt 70440
ggaaggctct ctgagcctgt ttcctcatct gtaaaattaa agcaataatt tctaacactc 70500
aagagtgtta cctcacgcct gtaatcccag cactttggag gctgaggcag gcggatcacc 70560
tgaggtcaga agttcaagac cagcgtggcc aacgtggcaa aaccctgtct ctactaaaaa 70620
atacaaaaag tagccgggca tggtggcgcg catctgtaat cccagctact tgggaggctg 70680
aggcagggat actgctagaa cctgggaggt ggagcgtgca gtgagtggag atcacacctc 70740
cacactccag cctggccgac agagcgagac tccatctcaa aaaaaaaaaa aaaaagagtg 70800
ttagaaggtt ttgagataat gaataaaaga tgccttgtgt atactaagta ttcaacaact 70860
gatagctgca ttggtctaat tataacagtt tagaagcgat tgagtcaaca aatgctggat 70920
ttgtcaggga ggacttccta tcaggaggta gatcttgggc tgagtcctga agcaaagata 70980
ggcattggat agaggagttg agagaacacc ctaggactgt tattattatt attcgacacg 71040
gagtctcttg ctctgtcacc caggctggag tgcagtggcg cgatctcggc tcactgcaac 71100
ctctgcctcc caggttcaag cgattctcet gcctcctaag tagctgagac tacaggtgtg 71160
tgccaccaca cccggctaat ttttatattt ttagtagaga cagagtttca ccatgttggc 71220
catgctggtc tcgaactcct gacttcaggt gatccacccg cctcagcctc ccaaagtgct 71280
ggaataacag atgtgagcca ccgcacccag cccagaacca tttttcaatc cttggctctg 71340
CCttttatta gCtgCaagat CtCaggCaat ttatttaaCC tCtCCaaaga CtCattttCt 71400
cattcacaaa atgaggcaaa taataatatc tactatccca ggttgtcatg agaattaaat 71460
gcaacatgac atttaatgaa atgagaagtc ccttggacat taactggcta aagtatgtgc 71520
tcgacaagga tatcatttta ggtggatact tagcatctca gaactgatgc tcacaatgga 71580
atatcattga aacgcattaa aattcatttt aaatgattgt aggtagtgag gcaattgaaa 71640
gaagaagaca agaggactga ttataatgct tcaggctcac tagtctcctt ttaggaggga 71700
aaaacaattt caagttaaat tttaggctct agatttttac ccctgctgct cattagaatc 71760
acccagattg atgaaatcag agcccatctg aggctgtgtt tttcatctcc agaatgagag 71820
ctgttgtggg gattaagttt ttgaaaaagt acatctaaca ggtgatcgaa aatgatagtg 71880
atattattgc agtgatggtc attattgttg ttattattat actgaaagag gcttcagttt 71940
tctgatccat aaagtgaggg aattgcatga gaccattgct aagattcctt ctagctctgt 72000
ttttttgttt ttgtttttta gacagagtct ctgtcgccca ggctggagtg caatggcatg 72060
atcttggctc actgcaacct ccgcctcccg ggttcaaatg atCCtCCtgt CtCagCCtCC 72120
gaagtagctg ggactacagg cacacaccac catgcccagc taacttttat atttttaata 72180
gaggtggggt ttcaccatat tggtcaggct ggtctcaaac tcctgacctc aggtgatcca 72240
cccgcctcgg cctcccaaca tgctgggatt acaggcatga gccactgtgc ccaacccctt 72300
ctagctttct tgatcactga ttctagggtt ctctgctgaa atatatttga gacatcctgg 72360
ataaaagatc atgcaagagc tcccaatatg gtattaataa ttgattctgg aggcttagct 72420
actcctgatg gattagacat gactcaactg cctctcttat gtgtacaaca caacaacaca 72480
accaagaaag gttattctgg cattccattt attcagttta tttacagccc ttacttccag 72540
cagcacgtta aagatatggc cagggccggg tgcagtggct caagtctgta atcccaggac 72600
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
_72_
tttgggaggccaaggtgggcggatcacaaggtcaggagtttgagaatctggcaattcttc72660
agacttagaagcaaccagctcgataacacagtcttgtgtgggctctccctCtgtCCCtCC72720
CtCgCttCCCtCatttCtCatCCCtgCCCCtgagactgtgCaCCttCacatagccctgcc72780
atgagaccttcatctcaggctttgctttctggggtaactgaggctaaacactgagtggcc72840
ctaaaagaggattgggatttggaagttagattattcaccagagaacagactttgctgatg72900
atcaggcccaggttgtaattgttgaaaaaaagagaggatgcatagtcttatctcatctcc72960
tagtcaaagtcaacaccatgataaataagagtcaaatcctgagatgtgaattggggacat73020
ttgagtggttaaccctgagaagcttgcaccttcagacccctcaatacccctgctccccag73080
agaaggctggacattgacctcagcacaggcaggagccctgcaagatgccatttgtcctac73140
taaagatggacccctccactctgtttctaggtaaataaccaaagtcaagtctccacacag73200
cctgagcaagaaagtcagagcctgctacaggagaaaataccacactggccaaaggattca73260
ctagccctggccactgtgtgtgggaggaaccagggaatcatgtgtgggagtcaatgttga73320
agctgttggactgggggtggggtggaatataagcctggccctggggagtttttcccgttt73380
gagggcctttacccacaactcaagatccagtgctatagcaggagatcccagagctagtcc73440
taacagatggtcaggattgaacttggcctagagtaaaatgaggaggatagtgccagaact73500
ttctcaacatactattgaggaagaggtcagaaggcttaaggaggtagtgtaactggaaag73560
gggtcctgatccagaccccaggagagggttcttggaccttgcataagaaagagttcgaga73620
cgagtccacccagtaaagtgaaagcaattttattaaagaagaaacagaaaaatggctact73680
ccatagagcagcgacatgggctgcttaactgagtgttcttatgattatttcttgattcta73740
tgctaaacaaagggtggattatttgtgaggtttccaggaaaggggcagggatttcccaga73800
aCtgatggatCCCCCCa.CttttagaCCatatagagtaaCttCCtgaCgttgccatggcgt73860
ttgtaaactgtcatggccctggagggaatgtcttttagcatgttaatgtattataatgtg73920
tataatgagcagtgaggacggccagaggtcgctttcatcaccatcttggttttggtgggt73980
tttggccggcttctttatcacatcctgttttatgagcagggtctttatgacctataactt74040
ctcctgccgacctcctatctcctcctgtgactaagaatgcagcctagcaggtctcagcct74100
cattttaccatggagtcgctctgattccaatgcctctgacagcaggaatgttggaattga74160
attactatgcaagacctgagaagccattggaggacacagccttcattaggacactggcat74220
ctgtgacaggctgggtggtggtaattgtctgttggccagtgtggactgtgggagatgcta74280
ctactgtaagatatgacaaggtttctcttcaaacaggctgatccgcttcttattctctaa74340
ttCCaagtaCCaCCCCCCgCCtttCttCtCttttCCttCtttCtgattttaCtaCatgC74400
C
CCaggCatgCtaCggCCCCagCtCaCattCCtttCCttatttaaaaatggaCtggggCtg74460
ggcgcggtggctcatgcctgtaatcccagcactttgggaggccgaggcgggcggatcatg74520
aggtcaggagatcgagaccatcctggctaacacggtgaaaccccgtctctactaaaaatg74580
caaaaacattagccaggcgtggttgcaggtgcctgcagtcccagcggctcaggaggctga74640
ggcaggagaatggcgtgaacctgggaggtggaggttgcaatgagccgagattgtgccact74700
gCaCtCCagCCtgggtgaCagagcgagactccgtctcaaaaaaaaaaaaaaaaaaaaaaa74760
tagctgggcatggtggcgcgtgcctgtaataccagctactctggaggctgaggcaagaga74820
atcgcttgaacccagtaggcggaagttgcagtgagccgagatcttgacactgcactccag74880
cctggtgacagagtgagactctgtctcaaaaaaaaaaaaaagaaaaaaaaagacagaaag74940
aaagagcacagacagagtcacaggtatttgcagtaggaagctgtcaggttagagtgcacg75000
gaaatagaaagtatattttacacttacagcacatcttcgtttgattagecacatttaaaa75060
tactgaatagcaacgtgtggctatttagtattcactaaaatcttggacagtgcaagtcta75120
aagaatccttgatccgtccggcatggtggctCa.CgCCtttaatcccagcactttgggagg75180
ccaaggtggaaggatcacttaaggtcaggagttcgagaccagcctggccaacatggtgaa75240
acctcgtctctactaataatacaaaaaaaattagccgggcatggtggtgcatgcctgtaa75300
tcccaggtacttgggaggctgaggcaggagaatagcttgaatccaggaggcgctgcagtg75360
agccgagatcatgccatgccactactgcactccagcctgggcaacagagtgagactgtct75420
caaaaaaaaaaaaaaaattgttgggcgtggtggctcacgcctgtaatcccagcactttgg75480
gaggctgaggggggtggatcacctgggttctggagttcgagaccagcctggccaacatgg75540
tgaaaccccatctctactaaaaatacaaaaattagctgggcgtggtggtgggcacctgaa75600
atctcagctactcaggaggctgaggcaggagaatttcttgaacccaggaggcagaggttg75660
cagtgagccaagatcgcgcctctgcactccatcctgggtggcagagcaagactatgtctc75720
aaaaaaaaaaaaaaaaatacttgattgtctggacattctgcagaacatcatatggagaca75780
ctatgttgacgacatcatgctgattgtaagcaagaaatggcaagtgttccagaaacacag75840
tcaagacacatacatgccagaaggtgagatataaactctactaagattc-agtggcctgcc75900
acactggtgacatttttaaacctgctagatgtttgtgtagaaaaggatttaaCCttgCCC75960
aaagaggggtctggcctttgtccccagctactggacataatctctttaaactcttgaaat76020
atcattcctgatagaagtatttttgttttgactaggggccttgggccagccagatagcaa76080
caatgtgatctgggttgggggctttggatcaggtggcatcagtgtgacctcctgagtggc76140
tagagactagaatcaaccacatgggcagacaacccagcttacatgatggaattccaataa76200
agactttggacacaagggcttgggtaagctttcctggttggcaatgctctatactgggaa76260
acccattctgactccatagggagaggacaactggatattctcatttggtacctccctggg76320
ctttgccctatgCatttttCCCttgtCtgattattattattattatgagatggaatctcg76380
ctctgtcacccaggctggagtgcagtggaatgatctcaactcactgcaacctctgcctcc76440
ccggttcaagcgattttcctgtctcggcctcccgagtagctgggactacagatgcatacc76500
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-73-
accacacccg gctaattttt ttgtattttt agtagagacg gggtttcacg ttagccagga 76560
tggtctcgat ctcctgacct catgttccgc ctgcctcggc ctctcaaagt gctaggaata 76620
catgtgtgag ccaccgcgcc cagccccctt ggctgattat taaagtgtat ccttgagctg 76680
tagtaaatta taaccgtgaa tataacagct tttagtgagt tttgtgagca cttctagcaa 76740
attatcaaac ctaaggatag ccttggggac ccctgaactt gcagttggtg tcagaaataa 76800
gggtgctcat gtgtgtacca tgccctctaa ttttgtagtt aattaacttt cacaacttta 76860
ttattaccgc ttacactcaa tgtttattca catttatcca cataccactt attctagtgc 76920
cttgcatcaa agactttcta tctcatgtac tttattctgc ttgaagtaaa tcctttagga 76980
tattcttttt tttttttaaa ctttgcacat acatactttt attttttatt tatttttaat 77040
tttgttattt ttgtgggtac gtagtagata tatgtattta tggagtacat gagatgtttt 77100
gatacaggca tgcaatgtga aataagcaca tcatggagaa tggggtatcc atcctctcaa 77160
gcaatttatc cttcaagtta caaacaatcc aattacactc tttaagttat tttaaaatgt 77220
acatttaatt ttgtattgac tagagtcact ctgttgtgct atcaaatata attttttttt 77280
tttttgagac agagtctcac tcagtggccc agactgaaag tgcagtggca caagctcggc 77340
tCaCttcaat CtCtgCCtCC CtggttCaag CgaatCtCCt gCCtCagCCt CCCa.CatagC 77400
tgggattaca ggcacacacc accatgccca gctaattttt atattttttt agtagagacg 77460
ggttttcgcc atgttggcca ggctggtctt gaactcctgg cctcaaatga tctgaccacc 77520
tcagcctccc aaagtgctag gattacaggc atgagccacc acacctggcc aaaatagaat 77580
attctttagt gaggtctgct ggtgacaatt tttttctttt ttttgagact gagtctcgct. 77640
gttgtcagct tgggctggag tgcaatagca cgatctcagc tcactgcaac ctccacctcc 77700
cggattccag caattctcct gcctcagcct cccaagtagc tgagagatta caggcaccca 77760
ccaccacacg cggctaattt ttgtattttt agtagaaatg ggggttcacc gtgttggcca 77820
ggctggtctc gaactcctga cctcaggtga tccacccacc ttggcctccc aaagtgctgg 77880
gattacaagc atgagccacc acgcacagcc aattttttcc gtttttgtct gaaatcttat 77940
tttgtgtcat ctttgaaata tatttttgat ggatataaaa ttgttggttg atagttatta 78000
tcattattat tattattttg agacagggtc tcactctgtt gcctatgctg gggtgtagta 78060
atgtgatCtC ggttCa.CtgC agaCttgaCC tCCtagggCt CaggtgatCt tCCCaCCtCa 78120
gcctccctag tagctgggac tacagatgca tgccaccata cccaactaat ttttctattt 78180
tttgtagaga tgaggctttg ccacatttcc caggctggtc tctaactcct gagctctagc 78240
aatCCaCCCa CCttggCCtt aCaaagtgCt gggCCatgaC tagCCagCag ttaCttttta 78300
tagcatattg aatatttaat atgaatcttc tggcatccac tgtaactgtt taaaaaatCa 78360
gctgtttact tggcactctt tttttttttt ttttttttga gacagagtct tgccctgtcg 78420
cccaggctgg agtgcagtgg cgtgatcttg gctcactgca agctctgcct cccgggttca 78480
CgCCattCtC CtgCCtCagC Ct CCggagta gctgggacta aaggcgcccg ccaccacgcc 78540
cggctgattt ttttgtattt ttcgtagagt tggggtttca ccgtgttagc caggatggtc 78600
tcgatctcct gacctcgtga tctgtccgcc tcggcctccc aaagtgctgg gattataggc 78660
gtgagccacc gcgcccagcc tctttttttt ttttttttag acggagtctt actctgtcat 78720
ctaggctggt gtacagtggc gtgatctcag ctcagtgcaa cctccacctc ctgcctcagc 78780
ctgccaaata gctgggatta caggtgcgta ccatcacgcc cggctaattt ttgtattttc 78840
agtagagatg gggtttcacc atgttagaca ggctggtctc gaactcctgg cctcaagtga 78900
tCtgCCtgCC CCagCCtCCC aaagattaCa ggCatgagCC aCCgCaCCCg gCCaagtagC 78960
actcctttga aggtaatctg CttCCCCtaC ccctagcaat ttttaacaat ttttCttCat 79020
ttttatttcc tgaagttttg ttattaataa tctgtgtgca gatttctttg tatttctttt 79080
gtttgcagtt catagtgatt cttgaattag tgtgttggtt tctgttatca ccacaggaaa 79140
attgtcagcc gttagCtttt CaaatatttC CttgCtaaat tCtCtCttCt CCCCtttCgg 79200
tacaattgat ttgattaaaa ctaaaaccag ggccgggtgc agtgactcat gcctgtaatc 79260
ccaacacttt gagaggctga ggcaggtgga tcacctaagc tcaggagttc aagaccagcc 79320
tggccaatat ggtgaaaccc cgtctctact aaaaatacaa aaattaccag gcatggtggc 79380
acacatttgt agtcaggagg ctgaggcagg agaattgctt gaatccagga ggtggaggtt 79440
gcagtgagct gagatcccac cactgcagtc tggcctgggc gacagagtga gatgagaatc 79500
tgtctcgaaa aaaaaagtta tgaatgtttg ataaactata tttgttagaa tgtttgttgt 79560
agaatactat tcattgattt ttaaacaatg ttagattaaa ccattcactg gatttgtgat 79620
aattaactta ctgattttac ctcactgatt tgttgtaatt aatacaactg gtataaaaag 79680
actgtgacga ggccgggcat ggtggctccc gcctataatc ccagcacttt gggaggctga 79740
ggcaggcgga tcacctgagg tcaggagttc aagaccagcc tgaccaacat ggtgaaaccc 79800
catctttact aaaaatacaa aattagccgg tcgtggtggt gcatgcctgt aatcccagct 79860
cttcgggagg ctgtggcagg agaatcactt gaacccggga ggtggaggtt gcagtgagcc 79920
gatatcgcgc cattgcactc cagcctgggc aacaagagcg aaactccgtc taaaaaaaaa 79980
aaagaaaaaa aacacataaa acaaaacaac actgtgacgg ttcccaaaaa ttaggagcat 80040
aattaaagga actcctgata aaaattaatt ttatcttaca tgtaaactaa aatgacttta 80100
tgaagttaat tcagaaatac aatgcagggt attagtttgc cacagctgcg tattcagcct 80160
aatgtaatat tcttgttatt tttaaattct tcttttaact ttactcatat gtggatcatc 80220
aaatttcaaa agattaaatg acaatactct tagcagcaag cttccctaag catataaaca 80280
ttttaatggg tgatgattca gaaggtaccc gaagaatatg tactgccaga tatcattcac 80340
ccccatatac ctgcccgaca gacatcccat tttgggaccc tggataaatg tgtgggtgga 80400
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-74-
gagaaagataggagaaagtggtataagcaaatggctttggagtctgattgacagcgattg80460
aaatcctgtctctacctcttaacagcctcatgatcctacataagttaccccgatcctcag80520
ggccacatctgtaaattgggggttgcgatggcagccatctcacagggtctcttttcgggg80580
aagggcaggaattatggattaagtgagctagtaattgtaaagcacttaatacaaggaggg80640
cgcataataagtacttcataaataatgacggccattatcatgactgaggtgtatgcagct80700
gtcggggattacggcgacttcagaatttctggtgggcagggctcaaaggcagcaaatcac80760
actggaagtcgaggtgaggcactgcttctgcacagactgcttagctggagagaatgagga80820
aggcttagaggagatttagaggaacttagagtcctccgcctccaactctgtgggatctgc80880
tcccgtgccagagacattcaggggatttctCgCaCtCtCCCCtCCCCtaCgtccctcccg80940
ccccatccaactaaccacacaacacatacaaaatagcccctgcgaggttctgcacgctgg81000
aagggaacaggagaagggcgctgcgctttcttgctgatgccctgtacttgggcccctggt81060
agacacagccacttgtcccctcagcctgcagagaaatcccacgtagaccgcgcccgggtc81120
cttggcttcagccaatctccctttggtgggggtgggatgcacgatccaaggttttattgg81180
ctacagacagcggggtgtggtccgccaagaacacagattggctcccgagggcatctcgga81240
tccctggtggggcgccgctcagcctcccggtgcaggcccggccgaggccaggaggaagcg81300
gccagaccgcgtccattcggcgccagctcactccggacgtccggagcctctgccagcgct81360
gcttccgtccagtgcgcctggacgcgctgtccttaactggagaaaggcttcaccttgaaa81420
tccaggcttcatccctagttagcgtgtgaccttgagcagttgactttatttttcagtgcc81480
tagttttccagataccaggactgactccaaggactattactcatctggagggtttagcac81540
agtaccgtcgcatagtaaatttccatgtcagttttggttacctttcatgcacttgcaaac81600
atgccatgctctgaaacgaaataggcacatcttttttttttttttttttaaggagtcttc81660
ctctcgcccaggctggagtgcagtggcgcgatcttggctcactgcaacctccacctcccg81720
tgttcgagattctcctgcctcagcctcctgattagctgggactacaggcatgccacgacg81780
cccagttaatttttgtatttttagtagagacggggtttcgccatcttggccaggctggtc81840
taactcctgacctcaggtgatctgactgcctcagcctctcaaagtgttgggattacaggc81900
ataagccactgcatctggccagaaatgaaataagtaaatcttttaacctgctctaacaat81960
atagtgaaaagaccatattattattagagcaggttaagggatttgcctatttcgggttct82020
agttatagtcttaaacttggacattcttgtagaaagtaaaaagtttcctcttcaaagttc82080
cccttcttgttaaagaatacatcataagtgttagaagtaatagtttattttaaagactaa82140
ctttcttcaagcctccttgctttgtgctaataactctttgttaagccctatcctatgtaa82200
ctgttggacatgctcacaggcacgttccagttcacagcctatgccccttccttatttgga82260
aatgttattgcttccttaaacctttcggtaagcaacttcctctccttcttcgttcttcct82320
tgcacttacctatttagaaagttttaggctattagcaaatcggctatcagtttaagagtg82380
tgaggtcccgctccagccaatggatgcaggacatagcagtgaggacgacccaaatgcgta82440
agggataaatatgtttgcttttcctttgttcaggtgtgctCtCgaCatCgttCCatCtgC82500
gattgagCaCCCtttCtgCagaaagtaaagattgccttgctggagatcttttgtctccgt82560
gctgacttttcttcgtggcaccgattatctatttctaacaattttggtatttctaacatt82620
ctgaacaatcttgggctagttgtctcttctgggcctgtttccccatccgtcacatgataa82680
acttcattggtttaaaaaccccagcgaacatttattgagttactattaccttcctgccct82740
ccccaaccccaaccccagggagcagttacaacctcagccgctgagcgcactcgccgggtg828OO
ttaagaagcaccaaagacagggaggcttgattgattttgctttgggagtagagggtcaga82860
agattcacaggaaaatggcatttgagcaaggatgattcactggagctagcttttaaatac82920
tggCgaggCttttatgttgCagtCCCttaCaaagttgagCattCgCagggaetgcactcc82980
gaaataagCCCgCttCCCCttttCattCgCtaatgatCCagggagCtgCtggttCCgCat83040
gcggcaggttgtgccttttcctaatcagggttctgcatcgcctcgaacccgcaggccgtg83100
gcgggttctcctgaggaagcagggactggggtgcagggtgaagctgctcgtgccggccag83160
cgcctgtgagcaaaactcaaacggaggagcaggaggggtcgagctggagcgtggcagggt83220
tgaccctgccttttagaagggcacaatttgaagggtaCCCaggggCCggaagCCggggaC83280
CtaaggCCCgCCCCgttCCagCtgCtgggagggCtCCCgCCCCagggagttagttttgCa83340
gagactgggtCtgCagCgCtccaccgggggCCggCgaCagaCgCCa.CaaaaCagCtgCag83400
gaacggtggctcgctccaggcacccagggcccgggaaagaggcgcgggtagcacgcgcgg83460
gtcacgtgggcgatgcgggcgtgcgcccctgcacccgcgggagggggatggggaaaaggg83520
gcggggccggcgcttgacctcccgtgaagcctagcgcggggaaggaccggaactccgggc83580
gggcggcttgttgataatatggcggctggagctgcctgggcatcccgaggaggcggtggg83640
gcccactcccggaagaagggtcccttttcgcgctagtgcagcggcccctctggacccgga83700
agtccgggccggttgctgaatgaggggagccgggccctccccgcgccagtCCCCCCgCaC83760
cctccgtcccgacccgggccccgccatgtccttcttccggcggaaaggtagctgaggggg83820
cgccggcggggagtcaggccgggcctcaggggcggcggtggggcaggtgggcctgcgagg83880
gctttccccaaggcggcagcaaggccttcagcgagcctcgacctcggcgcagatgccccc83940
tgagtgccttgctctgctccgggactcttctgggagggagaaggtggccttcttgcgcga84000
ggtcagaggagtattgtcgcgctggttcagaagcgattgctaaagcccatagaagttcct84060
gcctgtttggttaagaacagttcttaggtgggggttagtttttttgtgtttctttgagga84120
ccgtggatcaagatcaaggaaatctctttagaaccttattatggaagtctgaagtttcca84180
aatgttgagggttttatgtctaaaagcaacacgtgaaaaaattgttttcttcacccagtg84240
ctgtcttccaatttcctctttggggggaggggtagttactgctgttactaaaataaaatt84300
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
_75_
acttattgctaaagttccccaacaggaagaccactacttttgatgactttggcaagtttg84360
ctaactactggaaccctaacttacaaacgaactacttacatttttgatttccagttgtat84420
tacctgcccaatgtttacgtagaaacagcttaattttgattctgggtaacgttgttgcac84480
ttcattaaaaatacatatccgaagtgagcaagtatgggtctgtggacagcagtgattttt84540
cctgtcaattcctgttgcttcagataaaatgtaccagacagaggccgggcgcggtggctc84600
acgcctgtaatcccagcactttgggaggcttggcgggtggatcacctgagatcgggagtt84660
caagaccagcctgaccaacatggagaaaccccgtgtctactaaaaatacaaaattagcca84720
gggtggtggcgcatgcctgtaatgccagctacttgggaggctgaagcaggagaatcgctt84780
gaacctgggaggcggaggttgcggtgagccgagatagcaccattgcactccagcctgggc84840
aaaaagagcgaaactccgtctcaaaaaaaaagtaccagacagaaatgggttttgttttct84900
ttttttgttttgagacggagtttcgctcttgttgcccaggctcgagtgcaatggcgcgat84960
CtCagtCtCggCtCaCtgCaaCCtCtgtCtcccaggtttaatcgattctcctgcctcagc85020
ctcccaagtagctgggattacccatgccccaccatgcccggctaatttttgtatttttag85080
tagaaacggggcttcaccatgttaggctggtcttgaacccctgacctcaagtgggcctcc85140
cacctcggcctcccaaagtgccaggattacaggcatgagccaccgcggccagccagaaat85200
gggttttggaaaaagcactaaacaaaatcgaacttggtttcatatgacagctctgctgct85260
aactgtaacaggggcagaccagttaacctacttttctgtcttctgtcagctgagaattag85320
atgattcccaaaggcccattgaactctgaatgactttaaatacttcttcttaagtgggta85380
cacggttttggtaactgatgccaggtgatgaatgcatgaaagtgcttaatgaatgaaacc85440
ggtaaaatagtaggaggaagctttattggtaaggcaggggtatacctaatagctctctaa85500
tttattggtattgaagtggttaacttttgtttttttaaggggggaaaacattctaagaat85560
aatgaggcaaactgcatattgcacaagagactgttgtctctattcaacaaataccttttg85620
agtgtccagagtctgccaggtgctgtgctaggCCCtCdCgattgagtagtgaaccagaga85680
atgtccctgcacccatggagcttattgtctactggggtagacagataataaataagcaaa85740
caaatcttctCt CttCtCCCtttcgctccatgtaagtgtgtgtgtataggtgtatactta85800
caagttgagtaaagtgttatgaaagattaagaggagaaatgcattttggttagatgttag85860
aggactcagcaggtgaccttgaaacttagagctgaaggatcagtaggaggtaactagaga85920
ggccagggaatcgcatgttcaaaggccaggaggcaagaaagagcatggtgcccttcaaga85980
gaggaaagaaggctactgtgactggagcatagatgtaggcaagtgttgggtgattgagag86040
ctctacgggccatggttaggttttattcctaatgccgagatgccaaacatggtggttcat86100
atctgtaatcccagtattttaggaggccgaggcaggaatatagcttgaacccaggagttc86160
aagaccagectgagcaacatgagacctgtacaaaacatttaaaaaattgctgggtatgat86220
ggtgcacacctgtggtcccagctactcaggaggctgaggcagaaggatcacttgagccta86280
ggaggtggaggctacaatgagccatatttgagtcactacactccagcctggatgacaaag86340
tgagaccatgtgtcaaacaaaatacagaaagaatattaatttaaaattttgaaagaggag86400
tgatctgaacttatatcttaaaaagatcattctagggcatggtggctcatgcctgtaatc86460
aagggctttgggaggctgagacaggaggatcacctgaggccagttcgagatcaacctgta86520
cagcatagagagactccatctctacaaaaagaaaaaataaatagctgggtgttgtgagtt86580
attcaggaggctgaagcagaaagatcacttgagcccaggagtttgaggctgcagtaagct86640
atgatcccaccactgcaacacagtgagatcttgtctcaaaaaaaaaaaaaaatcattcta86700
ggtgctttttggaggctggatgtggtaagagtagaagctggagatggtcctgttagggat86760
tcgattcagactttaaataccatcaatgcattgagtcccaaatttacatcactacgttgg86820
atccttgcccctgaatccagactggtatatccaactttaggttcagtttgtatctctacc86880
tgaccaatatagaggtgtccagtcttttggcttccctaggccacattggaagaagaattg86940
tcttgagccacacatagagtacactaacgctaacaatagcagatgagctaaaaaaaaatc87000
gcaaaacttataatgttttaagaaagtttacgaatttgtgttgggcacattcagagccat87060
cctgggccgcgggatggacaagcttaatccagtagataccttcaacttacaatatctaaa87120
attttatgccagatttagtcattttaaacctgctcatcagtttttctcaagaagtagtat87180
tttggctttttttcttttcttttttttgagatggagtttcgctcttatcgttcaagctgg87240
agtgcagtggCggatCttggCtCaCtgCaaCCtCCgCCCCCtgggttCaagtgattCt 87300
CC
tgcctcagcctcgcaagtagctggaattacaggcatgcgccaccatgaccagctaatttt87360
tggagacagggtttcaccatgttggtcaggctggttttgtactcctgacctcaggtgatc87420
tgcctgcctcggcctcccaaaggctgggattacaggcatgagccaccgctCCCggCtgCa87480
tttttggatttttagttgctcagcccaaaactttagtacatctttgaacctcttctttcc87540
tcctactctatatctgatccatcagcaaatctgttaggtctacctcacacatatcgaaat87600
cctaccacgtctcaccatctgtgacaattaacaccctggtctaggcagtcatctctgtta87660
agattgagtggttaaggatgtcctctaaggagatgacattcaaatcttagcttaaatgtc87720
aagagggagctggttttataaagattgaggaggcagcattattttgccataggcttccat87780
ttggtttccattccattcttgatacttatggtatatattcaaaacaaatgcacagaaaca87840
gacccaggtatattgggaatttcggatatagagttcctagttgggaaaagatagactgat87900
ctgtaaatgatgctagttatccatcatctggcaaaaaataatttcctgcctcctctcata87960
tatctcagatcaacagactttttctgttaagggccaaatcataaatattttaggctttcc88020
agaccatatggtttctgtcacactctcctttatccttgaagccatagacaatatgtaaac88080
aaatgggcatggctgtgctacgataaaactttacttacaaaaactggtagtgggccagtt88140
taggcatggccagcactttgggaggctaaggcagatggatcacttggggtcaggagtttg88200
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
-76-
agaccagcct ggccaacatg gtgaaaccct gtctctacta aaaatacaaa aaatagctgg 88260
gcatggtggt gggtgtctat aattccagct actctggagg ctaagacaca agaatcactt 88320
gaacccagga ggcagaggtt gcagtgagct gagatagcac cactgcactc cagccagggt 88380
gacggagtct taaagcaaaa caaaacaaaa ggtagtgggt tgtatttggc ccatgggctg 88440
tagtttgcca atccctgatg cagaaacaaa ttccaggtaa ataagagcct ggaatgttaa 88500
aaaaacaaaa cttgaagtca tgtagaagaa caggtagggg gaacaatect gatctcagga 88560
taggaaggga tattgcttaa aataagacac aggaaaatat aatccatgtt gtgtaaattt 88620
gactacgtta aaacttaaaa ctttcgccaa gcgcggtggc tcacgcctgt aataccagta 88680
ctttgggagg ccgaggtgag cagatcacca ggtcaggaga ttgagaccat cctggctaac 88740
acggtgaaac cccgtctcta ctaaaaatac aaaacattag ccgggcgtgg tggcgggcgc 88800
ctgtagtccc agctacttgg gaggctgagg caggagaatg gcctgaaccc gggaggcgaa 88860
gcttgcagtg agctgagatc gcgccactgc actccagcct gggcgacaga gtgagattcc 88920
gtctcaaaaa aacaaaacaa aacaaagcaa aaaacctaaa actttcatac aataaagtat 88980
acctaagata cttctagaag agaagattta catccaggac gtgtatggaa tttctgcaag 89040
taataagtaa aagacaaggg acatgaagag gcagttcaca aaagaggaag ccaaaatgac 89100
caataaacat gaaaggatgt ttaacctcaa aggaaacaag gaaatgaatt aaaaacatca 89160
aatgccattt caaaactagt aagttggcaa aattaaaaat accaaggatg agaatatgaa 89220
gcatggctat atgagtgcat ggaatggtac agtcactttc attaaaaatg cacataattt 89280
gttttttatt tatttttttg agacagtcta tgtcgcccag gctagaatgc agtggcatga 89340
tCtCggCtCa CCaCaatCtC tgcctcctgg gttcaagcaa ttctcctgcc tcagcctcct 89400
gagtagctgg gattacaggc acatgccaca acgcccggtt aagttttgta tttttagtag 89460
agacagggtt ttgccatgtt ggccaggctg gtctcgaact cctgacctca ggtgagctgc 89520
ttcccaaagt gctgggatta gaggcgtgag ccaatgctcc tggctgaaaa aaatgcacat 89580
aatttgttac ctagcaattc catgtctaga ggcttatcct agagaaattc ttgcttatat 89640
gcataggaag acgtgtacta gaatgttcac tagttgaatg tttaagtgaa aattaggaaa 89700
taaagtaaat gttcattaac aggaaaatga gtaaaggtat atttataaaa caattaagta 89760
gctaaaatga ataaactaga gctgcgtgaa tgaactagaa ctggttcaat agtcatgtca 89820
gattattgaa tgaatacagg tcagatatgt atagagtgtc atttgtgtaa ttaatttttt 89880
tttttttttt gagatggagt ctcactctgt tgcccaggct ggagtgcagt ggcgtgatct 89940
cagctcactg caacctcCac ctcctgggtt aaagtgattc tcctgcctca gcctcccgag 90000
tagttgggat tacaggcatg caccaccatg cccagctcat tttcCtattt ttagtggcca 90060
CagggtttCa CCatgttggC CaggCtggtC ttgaaCtCCt gaCCtCaagt gttCCaCCCa 90120
acttggcctc ccaaagtgct aggattacag gcgtgagcca ccgtgctcag ccatttgcgt 90180
gatttttaaa gatgtgcaga ataatgccat taaaaaaaat acacatacat gtatatatat 90240
acacgtttgg ctgggtgtgg tggctcacac ctgtaatccc agcactttgg gaggctgagg 90300
caggaggatc acttgagccc aggtgtacaa gactagcctg ggcgagatag caagacccca 90360
tcteaacaac agaaaggata attaggtatg gtggcatgag aggatcactt gagcceagga 90420
gttcgagtgt tatcaggcca etgcaetcta gcctggacaa caaagcaaga ccgtgtctca 90480
aaaaaataaa aataaaaagt atttgtatgt ggtcatagtc aaaaaacgta catggaagga 90540
aaatgtcttt atttatttat ttattttttt ttttttaaga cagagtcttg ctctgtcacc 90600
caggctgggg tacac~tggtg taatctcagc tcaccgcaat ctcggcctcc cgggttcaag 90660
cgattcttct gcctcagcct tctaagtagc tgggactaca g~taCCCt~CC aCCaCa.CCCt 9O72O
gctaattctt gtgttttcag tagagacagg gtttcaccat gttggcaagg ctggtctcga 90780
aCtCCtgaCC ttaagtgagC CaCCCgCCtt ggCCtCCCaa agtCCtggga ttacaggtgt 90840
gagccactgc gcttggccag gaaatatcta atttagtaag tatttatatc tgggaaagga 90900
agggtcaggt ggtgattcat aggaactcta aagtctatgt ataatactta gggggacaga 90960
aggaaataaa gcaaaatgct gatatttgat tgttgagttg tgtatatgtt agaagtataa 91020
cataggagat ctgattgata gtaggagaat gtttttaggt ggtaaaagtg gaaccgtggt 91080
ggtttgtttt ggcagtagaa tcagttggtc atagtttgta tgtggaaggt aataaacaga 91140
ccatgttaag gatgacttcc ggaattttgg tctgagtagt gggtggatga cagtgtcatt 91200
catgagggaa gatgaagact gaggtaggaa caggtttggg agaagatgac atgttccctt 91260
ttagacaagt ggaattatgg aagatggcag gtaggtggtt agctatatga atttgagata 91320
aaagatttag gatggagata taaatttagg agtaacagcg tatctatggt attgtaagcc 91380
ttaagaatgg gtaggatcag ccaggaaata cagatgtata tgcagaagag aggagtcaag 91440
gaagccaaga caagttaatg tttaaagtga gtgatgtagt ccatgggcag atgctgctga 91500
gagggctgca aacaccagtg accctacaac atttttaaat gtcgtcttec tgacagcagt 91560
gatcagtacc tgcaacgatc ttatttattt ttttcatgtt agtctccaca cacttgaatg 91620
tagacttttt gaaggcaaaa tcattgcctt ttctgagctg ggagcatgtc tggcacatac 91680
caagcactca acagttgatg tattgacttc atccagatac tctgagggcg agttatttcc 91740
tgctactagc ctttcacctt tcaatgttta agagcacaaa tacagagatg ggcacgtttt 91800
ggcatttctt attttgataa ccttttcctg gtaagatttt ttaatgttga aaaaaaaaaa 91860
caagaaaaga gggttaaaaa tagtcttatg tcagatcctg tgatagaatt cacacttggc 91920
ttaagctgct gggcaccttc ctatcttgga tgtcatatta gcttatctac agcagaattt 91980
ttactgtttt atgtagtaag gaagcaatta tatgattatt ttacagacaa attattcttt 92040
atcttttatt tttttagacg gagtctctct ttgtctccca ggctggagta cagtgtcgcg 92100
CA 02518238 2005-09-06
WO 2004/081576 PCT/US2004/006740
_77_
atctcggctc actgcaacct ccgcctcctg ggttcaagca attctctgcc tcagcctccc 92160
aagtagctgg gcttacaggt gtccgccacc acacccagct cattgttttg tatttttagt 92220
agagatgggg tttcaccatg ttggccaggc tggtcttgag ctactgacct caggtgatcc 92280
acccgccttg gcatcccaaa gtgctggaat tacaggcgtg agccaccgtg cctggcccag 92340
acaaattatt atactctgag tgttagaggc ttaggatgtt ttcacttgat gctatgggag 92400
gaataagtaa taagatatga tacacaacca aagacctttc ttcactatgc ttctagtagc 92460
tagtactatg gatgacacat ggtaataata ttggttagca tttgtcctca atttactgtg 92520
ctagttactc ttctaagccc cttacaggta tatatttttt ttcatcaata atcctctaag 92580
gtagttttta ttattgacct aattttataa atcaagaaaa ttaagaccca gagaagtaag 92640
taacttgtcc aagatcacat ggcttataag tggtagagcc agaatttgac cccagatgtt 92700
gtgactacat tgtctctcca taagcaggtt caactctttt gactggatgc tgttccaagg 92760
tcacttcctt agagaagcct ttgctgacaa ctaccctcct gtgccctcct ccaaggctgt 92820
ccattgttct agaactttga atactcatct tagaataaag ctggtctaat ttttacagtg 92880
ttatagaatg gatctctgac tgcaaaagtt ggtcataatt atctttttat gttctagtga 92940
aaggcaaaga acaagagaag acctcagatg tgaagtccat taaaggtaag ttctgccctt 93000
ggcagtccac tgcattaaaa agtgatgtgc tttgcatttg tgagttcttt aatcctgtta 93060
tactctctct tttggcatta atcatttctg ccttatttta taattactta tgattttgat 93120
ttatttccct ctttaacctg tataatgctt taacatctag catataataa gtaggctttt 93180
tttttttttt tttttttgga gacggagtct tgctctgtta cccaggctgg agtgcagtgg 93240
cgcgatcttg gctcactgca agctctgtct cccgggttca caccattctc ctgcctcagc 93300
ctccccagca gctgggacta caggtgcacg gcgccacgcc tggctaattt tttgtatttt 93360
ttagtagaga cagagtttca ccatgttagc cagtatggtc tcgatctcct gaccttgtga 93420
tCCgCCCgCC tCggCCtCCC aaagtgCtgg gattacaagc gtgagccacc gcacccggcc 93480
gtaagtaggc tttttttacc ttaattttat ttttttgaga tggagtcttg CtCttatCCC 93540
caggctggag tgcagtggtg ccatctcggc tcactgcagc atccacctcc cgggttcaag 93600
cgattctcct gcctcagcct cccgagtagc tgggattaca ggtggccgcc accatgccca 93660
gctaattttt gtatttttag tagagacagg gtttcaccgt gttggccagg ccagtctcaa 93720
actcctgacc tCaagtgatC Ca.CtCgCCtt ggCCtCCCaa agtCCtggga ttacaggcgt 93780
gagccaccat gcctggccat aagtaggctt ttactgagcc ttgtgtgtat tggctatcct 93840
agtgattaca gtgaaccagt gcccttctta ttaatcacac atttaattgt tccctaaaag 93900
tgattagttc actttattta tttagtaaga eaaaaaatga agaatactct taactgagca 93960
gtctgttaac tgtaggaaag cactgacact tataaggctt agttttctgt catttatcca 94020
gaagtatggt tgattacagt ttttactttt ttatttgaat gaacaacctt aatttaaaat 94080
atattttgtt tattttttgt tgggatcgat acattgtcct tgtttataga ttagagcatg 94140
ctttttaaag atgctgtatt actcactgat tttatttgtc cagtgtacag agattgaagt 94200
gggaaaatta taatggaaat tgtttccata gtCattaCat attaatttca tcaatttatt 94260
tccataaaat ctgtagattg ctacttattt agatttttcc ttcaaatgtt tttatgttgt 94320
attgcttgca ctgagtattt attctatatg ctcaatttgc tggagaagaa gactaattat 94380
aacttaggca agttgtaaaa ttagggaaaa aagtaaggta ccttacagcc tagtttactt 94440
atttcttatg taaagccagt tagattccac attagttcaa actgccttct ttgagcaaaa 94500
cttgattggc agtgataaag gcttaaagcc cttctcaagc agagacctgt aaagactaga 94560
tctgactgta gtagaaggaa ggaacttaga tgtttcaggc agtgagaaca ccagtcttcc 94620
actctaaact ttgccactaa cagtatgacc ttgggaagtt gtaactttct tcagattctt 94680
catttgttga atggggggat tggcctagct aatttctaaa tctctactgg gctaaaaaat 94740
tctgtgctta tactctgatt atgaagtaca taatctgtgc ttaacattca ctgacttatc 94800
cttaggataa tacagaagca gtacaagaaa cagcccctca agatgtttgc agtctggtta 94860
gaaagacaaa cttatacaca gaacagtagc aaatagacca aaataataat agctgccatt 94920
tatagaacac ttcttctgtt ctgggcatta gacaaaaact gactataacg gtgaacaaaa 94980
aagacttagg tcctgccctc attgaactta cagattagta ggggagagga acattaatca 95040
agtaattcca cagatggctt agcctagatt ggtagtgatg gaagtaaaga gatgtgaacg 95100
gacttgaaaa aaaattcgga ggcaaaatgg atagaagttt attattgatt aaatatgagg 95160
tgtgagagag agggatattt aagattgata cctaccttct ggcttgccta acagaaccaa 95220
aacaggaaat tatatgttca gttttgttat gttgggtggg aggtgctttt gagtcattca 95280
tttatatatg ttatatatgt tattttatat gcatagtaat tttaaggtct gagttttaaa 95340
ccaaaggtta gagagtgatt ttttagagtc tagcaaacct aagttgaaat cctgcctgtt 95400
gaaatggctg tttactagct cattaaccta gggcaaagta ttcaacttgt tttcattttt 95460
gtcttcatct ctaaaatgag gaaaatatgg tcttacaaga ttgtcctgag agatagatga 95520
aataatatcc aaaaaaaaaa aaggtacata gagaaactcg tatagtgcct ggtatatagt 95580
aggtcctcca ttggtagcta tcattatcta gttttaacat agccttcagt ttgttgaatt 95640
agtcaaactg agtgaagcac tgcaaggaat tcagaggaat ttgagatcaa caaatgattt 95700
ctgaagttta gggaagactt catggcaatg acacttacct tgtataaaag ttgaagaata 95760
agaaagattt gaatgagaga ttctttctct tctccctacc agcccagctt cttatttgag 95820
gatatattgg gcaaaggggc cttcagacaa gtagagggag atttttacag aaagattgag 95880
atgaaggtat agaaggctgt aaagaccaga aaagagaatt gagacagagg aagcaggaag 95940
ccactgtagg tttttgagca agatattgat gctgtaagta tggtgtttat gaaaggttag 96000
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