Note: Descriptions are shown in the official language in which they were submitted.
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TITLE: Lipase Genes and Proteins
FIELD OF THE INVENTION
The invention relates to the field of lipase structure and function,
triglyceride
metabolism, lipoprotein metabolism and energy homeostasis, and is concerned
with novel
lipase proteins, I,PDL and LPDLR, nucleic acids encoding the proteins, nucleic
acids
controlling their gene expression and methods and agents for their
manipulation for the
modulation of cellular processes, and their use in basic research, industry,
disease prevention,
diagnosis and therapy.
BACKGROUND OF THE INVENTION
(i) Tri~lyceride metabolism
Plasma triglyceride (TG) is associated with increased atherosclerosis risk and
TG
metabolism is crucial for whole body and local energy homeostasis. A number of
thorough
reviews of TG metabolism have been published (Zilversmit 1995; Adeli et al.
2001; Hegele
2001; Moghadasian et al. 2001; Jin et al. 2002). Briefly, in the intestine,
pancreatic lipase
(PNLIP) hydrolyses dietary TG to liberate free fatty acid (FFA), that is
absorbed both
passively and actively. Several different classes of membrane proteins have
been proposed as
FA acceptors or transporters (Glatz and Storch 2001). FA trafficking by
soluble intracellular
FA binding proteins may involve interaction with specific membrane or protein
targets, such
as FABP2. Within enterocytes, processing by partially characterized
biosynthetic pathways
prepare TG for assembly together with cholesterol esters [CE], apolipoprotein
(apo) B-48 and
apo E, which is mediated by microsomal TG transfer protein (MTP). The assembly
process
creates chylomicrons (CM) for secretion into lymph and plasma. In the liver,
fat or
carbohydrate that is not required for energy is converted to TG through
several partially
characterized biosynthetic pathways (Hegele 2001 ). Acyl-CoA: diacylglycerol
acyltransferase
(DGAT) is a microsomal enzyme that catalyzes the terminal and only committed
step in TG
synthesis. DGAT had been considered necessary for adipose tissue formation and
essential for
survival. Two groups (Oelkers et al. 1998; Cases et al. 1998) independently
cloned the DGAT
gene. While there are no human DGAT mutations, targeted disruption produces
lean mice that
resisted diet-induced obesity, but still synthesized TG (Smith et al. 2000),
suggesting the
existence of other DGAT-like enzymes. Indeed a new diacylglycerol
acyltransferase gene
DGAT2 was recently identified which conferred high levels of DGAT activity
(Lardizabal et
al. 2001), The systhesized TG as the major form of energy storage within the
adipocytes
increases body fat and weight. However, another important control of adipose
triglycerides is
hormone sensitive lipase which hydrolyzes adipocyte TG and provide the body
with energy
(Kahn 2000).
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Within hepatocytes, MTP directs the assembly of TG and CE together with apo B-
100 and
apo E to produce very low-density lipoproteins (VLDL) for secretion into
plasma. In the
capillaries of adipose tissue and muscle, CM and VLDL core TG are hydrolyzed
to FFA by
endothelial-bound lipoprotein lipase (LPL), using apo CII as a co-factor. FFA
are re-esterified
and stored as TG within adipocytes, or oxidized to provide energy in muscle.
CM and VLDL
are remodeled into smaller, denser, more CE-rich CM remnants (CMR) and
intermediate
density lipoprotein (IDL), respectively. CMR and some IDL are cleared by apo E-
mediated
endocytosis through hepatic remnant receptors, contributing to the hepatic
lipid pool. >DL that
is not cleared is then hydrolyzed by hepatic lipase (HL or LIPC) making
smaller, CE-rich LDL
particles.
While all plasma lipoprotein metabolic pathways are complex and
interconnected, TG
and HDL metabolism are especially closed linked. Clinically, elevated TG
occurs almost
always together with depressed HDL cholesterol. Liver and small intestine
produce nascent
HDL particles, which attract excess FC from both extra-hepatic cells and other
circulating
lipoproteins. Phospholipids (PL) and FC that accumulate in the intimal layer
of the arteries are
transferred to apo AI of nascent HDL, a process mediated by the ATP-binding
cassette A-1
transporter (ABCA1). Using apo AI as a cofactor, plasma lecithin: cholesterol
acyltransferase
(LCAT) converts FC to CE, providing a source of core lipid for HDL. Plasma
cholesteryl
ester transfer protein (CETP), and PL transfer protein (PLTP), modify HDL by
shuttling CE
and PL between HDL and TG-rich lipoproteins (VLDL and CM). HL hydrolyzes HDL
TG,
thus reducing HDL size. HDL delivers cholesterol to the liver, and scavenger
receptor BI
(SRBI) mediates selective uptake of lipids. Macrophages depend on cholesterol
efflux through
transfer to HDL to prevent lipid accumulation (Nicholson et al. 2000; van
Berkel et al. 2000).
It is clear that enzymes and transfer proteins within the plasma compartment,
such as
LPL and CETP, have pleiotropic effects on many classes of lipoproteins (Hegele
2001). Thus,
newly identified gene products, such as LPDL, may be reasonably expected to
have pleiotropic
effects on plasma lipids and lipoproteins, and likely on tissue lipids.
(ii) Plasma TG and coronary heart disease (CHD) risk
The complexity of mechanisms that underlie the association between
hypertriglyceridemia and atherosclerosis obscures ascertainment of a direct
causal relationship
(Forrester 2001). Pro-atherogenic metabolic and biochemical abnormalities,
such as obesity,
diabetes, decreased HDL cholesterol, increased small-dense LDL, increased FFA,
dysglycemia, hyperinsulinemia, increased plasma viscosity, increased
inflammatory
molecules, impaired fibrinolysis and pro-thrombosis, are often associated with
elevated TG.
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Any of these associated factors will increase atherosclerosis risk. Recent
epidemiologic
consensus opinion is that moderately elevated plasma TG (between 2.3 and 9.3
mmol/L),
usually due to excess VLDL and/or remnant particles, appears to be
independently associated
with increased CHD risk (Austin 1999; Yarnell et al. 2001), especially in
familial
hypertriglyceridemia (Austin et al. 2000). In contrast, grossly elevated
plasma TG (> 12
mmol/L), usually due to excess CM, is associated with increased risk of
pancreatitis, but not
necessarily CHD (Santamarina-Fojo 1998). Studies in which treatment was given
to lower TG
have shown improved CHD outcomes, although this is often hard to attribute to
an affect on
TG specifically (Hodis et al. 1994; Frick et al. 1997; Rubins et al. 1999).
Mechanistically, CM,
VLDL or their remnants may act directly in atherogenesis, contributing to
arterial wall foam
cell formation (Gianturco et al. 1982; Evans et al. 1993). Because post-
prandial FFA released
from lipolysis impair physiological endothelial response, a newer concept is
that post-prandial
lipemia may independently predict CHD. While factors such as diet, alcohol,
obesity and
diabetes contribute to moderate hypertriglyceridemia, the primary molecular
mechanisms
underlying inter-individual variation in response to such secondary factors
remain
incompletely characterized in most hypertriglyceridemic patients (Hegele 2001
).
(iii) Lipase and lipase mutations
Lipases hydrolyze a wide range of esterified FA species within triglyceride
(TG), and
are often active against other substrates, such as phospholipids (PLs). At
least 20 lipases or
lipase-like molecules have been given names and accession numbers in OMIM
(http://www.ncbi.nlm.nih.gov/entrez/quervl. These have been characterized
based upon factors
such as their anatomical distribution, localization intra- or extra-
cellularly, substrate.
specificity, and or homology with other lipases (Hide et al. 1992). For
instance, lipases that
function within the plasma compartment, anchored to endothelium by heparan
sulfate
proteoglycans, include, in order from most-to-least-potent TG lipase activity,
and least-to-
most-potent PL lipase activity, LPL, HL, and EL (Jin et al. 2002). Other
lipases are non-
secreted and have predominantly intracellular hydrolytic activity, such as
hormone sensitive
lipase (HSL) and lysosomal acid lipase (LAL) (Holm et al. 1988). The activity
of some other
lipases is extra-corporeal, such as that of pancreatic lipase (PNLIP) within
the intestine.
Lipases are evolutionarily conserved within superfamily members and between
species. One highly conserved motif includes the lipase consensus sequences G-
X-S-X-G that
contains the active site serine which forms a catalytic triad with His and Asp
that minics the
catalytic triad of trysin (Emmerich et al. 1992; Lowe 1997). The crystal
structure analysis of
pancreatic lipase revealed Ser153, His264 and Asp177 as its triad (Lowe 1997).
Other
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structural features include conserved cystine residues in disulfide bridge
formation for tertiary
structure and the lid motif which determines the substrate specificity (van
Tibeurgh et al. 1994,
Dugi et al. 1995). For most triglyceride lipases, the lid loop is composed of
1923 amino acids
between two conserved systine residue (Dugi et al. 1995). Lipase genes usually
contain
910 exons. Human LPL gene contains 10 exons that spans a genomic region of 30
kb on
chromosome 8 with mutations mostly detected in exon 5 and 6 (Deeb et al, 1989;
Ishimura-
Oka, et al., 1992). Human hepatic lipase on chromosome 15 contains 9 exons and
was reported
to 35 kb in size (Ameis et al., 1990).
More than ten phospholipases (PLases) and related genes are cited in OMIM.
These
include PLases Al, A2, C, D and lysophospholipase. Almost all of these have no
disease
causing mutation and no disease association (Jin et al. 2002). A new member of
the PLase
family, phosphatidylserine phospholipase A1 (PS-PLA1), has recently been
described.
Surprisingly PS-PLA1 does not show any homology to PLase members, but shows
about 30%
homology to mammalian TG lipases HL, LPL and PL (Sato et al. 1997), a finding
that in
consistent with the observed reactivity of other members of the lipase
superfamily against
various substrates.
Naturally occurring loss-of function mutations in LPL cause chylomicronemia
(Santamarina-Fojo 1998; Hegele 2001), some LPL SNPs are fairly consistently
associated with
metabolic and cardiovascular phenotypes (Busch and Hegele 2000), and LPL knock-
out and
transgenic mice have instructive phenotypes involving the expected alterations
in plasma TG
and HDL (Goldberg and Merkel 2001). Similarly, naturally occurring loss-of-
function
mutations in HL cause a complex hyperlipidemia with early atherosclerosis
(Hegele et al.
1993), some HL SNPs, especially -514C>T, are fairly consistently associated
with metabolic
and cardiovascular disease phenotypes (Cohen et al. 1999; Hegele 2001 ), and
HL knock-out
and transgenic mice have instructive phenotypes that reflect the human
phenotypes (Kawano
et al. 2002). In contrast, neither naturally occurnng human mutations, nor
induced murine
mutations in EL have yet been reported, although several common SNPs have been
associated
with variation in plasma concentrations of HDL cholesterol (deLemos et al.
2002). Mild to
moderate hypertriglyceridemia (Fredrickson Type IV) with low HDL cholesterol
is among the
most common hyperlipidemia seen in many lipid clinics, but most patients have
no mutation in
either LPL or HL. Thus, it remains important to identify new candidate genes
for TG
metabolism using a variety of experimental approaches as required.
(iv) lnd insertional mutation
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Microinjection of cloned DNA directly into the pronucleus of a fertilized egg
is the
most widely used method for generating transgenic mice. The injection of
foreign DNA into
the zygote normally results in chromosomal integration. The transgene probably
integrates
randomly into the mouse genome. Occasionally this integration will disrupt an
endogenous
gene and results in a mutation, which is designated an "insertional mutation".
The overall
frequency of generating insertional mutations by microinjection is estimated
to be 5-10%
(Palmiter et al. 1986).
We previously identified a mouse transgenic insertional mutation, lpd (for
"lipid
defect"), characterized by perinatal accumulation of TG in both plasma and
liver (Wen et al.
_ 10 1998). The lpd mutation was recessive and induced by LacZ transgene.
Apart from elevated
plasma triglyceride level, the lpd homozygotes are runts and develop fatty
livers. Molecular
cloning of the transgene-flanking sequences led to mapping of the lpd locus to
the distal part
of murine chromosome 16 (Wen et al. 1998). Further mapping studies ruled out
the identity of
lpd with a recently identified phospholipase gene psplal in its vicinity (Wen
et al. 2001).
The locus in a mouse insertional mutation is genetically tagged by the
transgene,
which provides a unique marker to clone the genetic locus and to identify the
affected gene.
The fortuitous observation of plasma and tissue TG disturbances in the
insertional mutation
from the yF-crystallin promoter/LacZ Z 14 transgenic mouse experiments
provided an
important clue - actually a positional clue - that some gene within the
disrupted lpd locus on
murine chromosome 16 was a key determinant of TG metabolism.
SUMMARY OF THE INVENTION
The present invention relates to two novel lipase proteins, LPDL and LPDLR and
to
the nucleic acid molecules encoding them as well as to expression vectors
comprising the
nucleic acid molecules of the invention and host cells transformed with the
expression vectors.
The protein sequences of LPDL and LPDLR are described in Figure 1 B
(SEQ.)D.N0:2), Figure 2B (SEQ.ID.N0:4), Figure 2D (SEQ.ID.N0:6), Figure 3B
(SEQ.m.N0:8), Figure 4B (SEQ.ID.NO:10) or, Figure 4D (SEQ.>D.N0:12).
The invention also relates to fragments, analogs, homologs, derivatives or
mimetics of
the LPDL or LPDLR proteins and to antibodies that can bind the LPDL or LPDLR
proteins or
fragments, analogs, homologs, derivatives or mimetics thereof.
The nucleic acid sequences of LPDL and LPDLR include the cDNA sequences of the
genes including a nucleic acid sequence as shown in Figure lA (SEQ.)D.NO:1),
Figure 2A
(SEQ.>D.N0:3), Figure 2C (SEQ.>J7.N0:5), Figure 3A (SEQ.>D.N0:7), Figure 4A
(SEQ.1D.N0:9), Figure 4C (SEQ.ID.NO:11); including promoter and regulatory
sequences
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for controlling gene expression as shown in Figure 21-A for mouse Ipdl
(SEQ.117.N0:77), 21-
B for human LPDL gene (SEQ.ID.N0:78), Figure 22-A for mouse lpdlr
(SEQ.ID.N0:79), 22-
B for human LPDLR gene (SEQ.ID.N0:80); and including exon/intron sequences of
the
genes as shown in Figure I S of human LPDL gene: Exon/Intron 1 (SEQ.1D.N0.17),
Exon/Intron 2 (SEQ.1D.N0.18), Exon/Intron 3 (SEQ.ID.N0.19), Exon/Intron 4
(SEQ.ID.N0.20), Exon/Intron 5 (SEQ.1D.N0.21 ), Exon/Intron 6 (SEQ.1D.N0.22),
Exon/Intron 7 (SEQ.ID.N0.23), Exon/Intron 8 (SEQ.1D.N0.24), Exon/Intron 9
(SEQ.1D.N0.25), and Exon/Intron 10 (SEQ.1D.N0.26); and in Figure 16-A as exon
sequences
and adjacent intron sequences of human LPDLR gene: Exon/Intron 1
(SEQ.1D.N0.27),
Exon/Intron 2 (SEQ.1D.N0.28), Exon/Intron 3 (SEQ.ID.N0.29), Exon/Intron 4
(SEQ.1D.N0.30), Exon/Intron 5 (SEQ.117.N0.31), Exon/Intron 6 (SEQ.ID.N0.32),
Exon/Intron 7 (SEQ.1D.N0.33), Exon/Intron 8 (SEQ.ID.N0.34), Exon/Intron 9
(SEQ.ID.N0.35) and Exon/Intron 10 (SEQ.1D.N0.36).
The invention also relates to fragments, analogs, homologs, derivatives of the
nucleic
acids of LPDL and LPDLR including both cDNA, exon and intron seuqences and the
promter
and regulatory elements for gene expression.
The invention also relates to methods for identifying substances which can
bind with
LPDL or LPDLR protein, to methods for identifying compounds that affect LPDL
or LPDLR
protein activity or expression and to methods for identifying compounds that
affect the binding
of LPDL or LPDLR with an LPDL or LPDLR binding protein.
The invention also relates to the use of agents capable of modulating the
expression of
a nucleic acid molecule of the invention to modulate tissue or plasma lipid
and lipoprotein
metabolism. It further relates to the use of an agent that can stimulate the
activity or expression
of an LPDL or LPDLR protein to treat conditions selected from the group
consisting of lipase
deficiency, lipoprotein defects, hypertriglyceridemia (primary genetic defect
or secondary
from other diseases), fatty liver diseases, cardiovascular disorders
(including but not limited to
hypertriglyceridemia, dyslipidemia, atherosclerosis, coronary artery disease,
cerebrovascular
disease hypertension, and peripheral vascular disease), body weight disorders
(including but
not limited to obesity, cachexia and anorexia), inflammation (including but
not limited to
sinusitis, asthma, pancreatitis, osteoarthritis, rheumatoid arthritis and
acne), eczema, Sjogren's
syndrome, gastrointestinal disorders, viral diseases and postviral fatigue,
psychiatric disorders,
cancer, cystic fibrosis, endometriosis, pre-menstrual syndrome, alcoholism,
congenital liver
disease, Alzheimer's syndrome, hypercholesterolemia, autoimmune disorders,
atopic
disorders, acute respiratory distress syndrome, articular cartilage
degradation, diabetes and
diabetic complications. In preferred embodiments, the conditions are selected
from the group
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consisting of lipase deficiency, atherosclerosis, fatty liver disease and
dyslipidemias, such as
hypercholesterolemia, hypertriglyceridemia, mixed (combined) dyslipidemia,
lipid or
lipoprotein deficient states, and/or any other tissue or plasma disorders of
lipid or lipoprotein
metabolism.
Additionally, the invention relates to methods for detecting conditions
associated with
increased or decreased LPDL or LPDLR expression. Such conditions include
disorders
selected from the group consisting of lipase deficiency, lipoprotein defects,
hypertriglyceridemia (primary genetic defect or secondary from other
diseases), fatty liver
diseases, cardiovascular disorders (including but not limited to
hypertriglyceridemia,
dyslipidemia, atherosclerosis, coronary artery disease, cerebrovascular
disease hypertension,
and peripheral vascular disease), body weight disorders (including but not
limited to obesity,
cachexia and anorexia), inflammation (including but not limited to sinusitis,
asthma,
pancreatitis, osteoarthritis, rheumatoid arthritis and acne), eczema,
Sjogren's syndrome,
gastrointestinal disorders, viral diseases and postviral fatigue, psychiatric
disorders, cancer,
cystic fibrosis, endometriosis, pre-menstrual syndrome, alcoholism, congenital
liver disease,
Alzheimer's syndrome, hypercholesterolemia, autoimmune disorders, atopic
disorders, acute
respiratory distress syndrome, articular cartilage degradation, diabetes and
diabetic
complications. In preferred embodiments, the conditions are selected from the
group
consisting of lipase deficiency, atherosclerosis, fatty liver disease and
dyslipidemias, such as
hypercholesterolemia, hypertriglyceridemia, mixed (combined) dyslipidemia,
lipid or
lipoprotein deficient states, and/or any other tissue or plasma disorders of
lipid or lipoprotein
metabolism.
The invention also relates to pharmaceutical compositions comprising a nucleic
acid
molecule of the invention, an antisense oligonucleotide complimentary to a
nucleic acid
molecule of the invention, a LPDL or LPDLR protein or gene, a compound
identified by the
methods of the invention, or a substance capable of modulating the expression
or activity of an
LPDL or LPDLR protein in admixture with a suitable diluent or carrier
including an antisense
oligonucleotide complimentary to a nucleic acid molecule of the invention.
The invention also relates to methods for screening a subject for a mutation
in a LPDL
or LPDLR protein which comprises obtaining a sample from the subject,
comparing the
sequence of the LPDL or LPDLR gene from the sample with the corresponding wild
type gene
sequence, wherein a difference indicates a mutation in the LPDL or LPDLR gene
in the
sample.
The invention also teaches a nucleic acid molecule comprising: (a) a nucleic
acid
sequence as shown in Figure lA (SEQ.>D.NO:1), Figure 2A (SEQ.)D.N0:3), Figure
2C
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(SEQ.1D.N0:5), Figure 3A (SEQ.1D.N0:7), Figure 4A (SEQ.ID.N0:9), Figure 4C
(SEQ.ID.NO:11),Figure 15 (SEQ.ID.N0.17),(SEQ.ID.N0.18),(SEQ.>D.N0.19),
(SEQ.ID.N0.20),(SEQ.ID.N0.21), (SEQ.ID.N0.22),(SEQ.ID.N0.23),(SEQ.ID.N0.24),
(SEQ.>D.N0.25),(SEQ.ID.N0.26); Figure (SEQ.117.N0.27),(SEQ.ID.N0.28),
16-A
(SEQ.ID.N0.29), (SEQ.ID.N0.30), (SEQ.ID.N0.31), (SEQ.ID.N0.32),
(SEQ.ID.N0.33),
(SEQ.ID.N0.34), (SEQ.ID.N0.35), (SEQ.ID.N0.36); Figure 21 (SEQ.>D.N0:77),
(SEQ.>D.N0:78); or Figure 22 (SEQ.ID.N0:79), (SEQ.ID.N0:80); (b) nucleic acid
sequences
that have substantial sequence homology to a nucleic acid sequences of (a) or
(b); (c)
sequences which are at least 90% homologous with a sequence of any of (a) to
(b); (d)
sequences which are at least 95% homologous with a sequence of any of (a) to
(b); (e)
sequences which are at least 98% homologous with a sequence of any of (a) to
(d); and (f) a
sequences which are at least 99% homologous with a sequence of any of (a) to
(b).
The invention teaches a method for identifying a compound which inhibits or
promotes
the activity of a polynucleotide sequence of the invention, comprising the
steps of: (a)
selecting a control animal having the sequence and a test animal having the
sequence; (b)
treating the test animal using a compound; and, (c) determining the relative
quantity of an
expression product of the sequence, as between the control animal and the test
animal.
The invention further teaches a method for identifying a compound which
inhibits or
promotes the activity of a polynucleotide sequence of the invention,
comprising the steps of: (a)
selecting a host cell of the invention; (b) cloning the host cell and
separating the clones into a
test group and a control group; (c) treating the test group using a compound;
and (d)
determining the relative quantity of an expression product of the sequence, as
between the test
group and the control group.
The invention also teaches a process for producing a polypeptide sequence of
the
invention comprising the step of culturing the host cell of the invention
under conditions
sufficient for the production of the polypeptide.
The invention teaches a method for identifying a compound which inhibits or
promotes
the activity of a polypeptide sequence of the invention, comprising the steps
of: (a) selecting a
control animal having the sequence and a test animal having the sequence; (b)
treating the test
animal using a compound; (c) determining the relative quantity or relative
activity of an
expression product of the sequence or of the the sequence, as between the
control animal and
the test animal.
The invention teaches a composition for treating a disorder of tissue or
plasma lipid
and lipoprotein metabolism comprising a compound which modulates a
polynucleotide
sequence of the invention and a pharmaceutically acceptable carrier. The
disorder may be
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selected from the group consisting of lipase deficiency, lipoprotein defects,
hypertriglyceridemia (primary genetic defect or secondary from other
diseases), fatty liver
diseases, cardiovascular disorders (including but not limited to
hypertriglyceridemia,
dyslipidemia, atherosclerosis, coronary artery disease, cerebrovascular
disease hypertension,
and peripheral vascular disease), body weight disorders (including but not
limited to obesity,
cachexia and anorexia), inflammation (including but not limited to sinusitis,
asthma,
pancreatitis, osteoarthritis, rheumatoid arthritis and acne), eczema,
Sjogren's syndrome,
gastrointestinal disorders, viral diseases and postviral fatigue, psychiatric
disorders, cancer,
cystic fibrosis, endometriosis, pre-menstrual syndrome, alcoholism, congenital
liver disease,
Alzheimer's syndrome, hypercholesterolemia, autoimmune disorders, atopic
disorders, acute
respiratory distress syndrome, articular cartilage degradation, diabetes and
diabetic
complications. In preferred embodiments, the conditions are selected from the
group
consisting of lipase deficiency, atherosclerosis, fatty liver disease and
dyslipidemias, such as
hypercholesterolemia, hypertriglyceridemia, mixed (combined) dyslipidemia,
lipid or
lipoprotein deficient states, and/or any other tissue or plasma disorders of
lipid or lipoprotein
metabolism. The compound may be selected from the group consisting of small
organic
molecules, peptides, polypeptides, antisense molecules, oligonucleotides,
polynucleotides,
triglycerides and derivatives thereof.
The invention teaches a method for diagnosing the presence of or a
predisposition for a lipase disorder or lipid metabolism disorder in a subject
by detecting a
germline alteration in a sequence of the invention in the subject, comprising
comparing the
germline sequence of a sequence of the invention from a tissue sample from the
subject with
the germline sequence of a wild-type of the sequence, wherein an alteration in
the germline
sequence of the subject indicates the presence of or a predisposition to the
triglyceride disorder.
The disorder may be selected from the group consisting of lipase deficiency,
lipoprotein
defects, hypertriglyceridemia, fatty liver diseases, cardiovascular disorders
(including but not
limited to hypertriglyceridemia, dyslipidemia, atherosclerosis, coronary
artery disease,
cerebrovascular disease hypertension, and peripheral vascular disease), body
weight disorders
(including but not limited to obesity, cachexia and anorexia), inflammation
(including but not
limited to sinusitis, asthma, pancreatitis, osteoarthritis, rheumatoid
arthritis and acne), eczema,
Sjogren's syndrome, gastrointestinal disorders, viral diseases and postviral
fatigue, psychiatric
disorders, cancer, cystic fibrosis, endometriosis, pre-menstrual syndrome,
alcoholism,
congenital liver disease, Alzheimer's syndrome, hypercholesterolemia,
autoimmune disorders,
atopic disorders, acute respiratory distress syndrome, articular cartilage
degradation, diabetes
and diabetic complications. In preferred embodiments, the conditions are
selected from the
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group consisting of lipase deficiency, atherosclerosis, fatty liver disease
and dyslipidemias,
such as hypercholesterolemia, hypertriglyceridemia, mixed (combined)
dyslipidemia, lipid or
lipoprotein deficient states, and/or any other tissue or plasma disorders of
lipid or lipoprotein
metabolism. The comparing may be performed by a method selected from the group
consisting
of immunoblotting, immunocytochemistry, enzyme-linked immunosorbent assay, DNA
fingerprinting, in situ hybridization, polymerase chain reaction, reverse
transcription
polymerase chain reaction, radioimmunoassay, immunoradiometric assay and
immunoenzymatic assay. The alteration may occur at a SNP selected from the
group
consisting of but not limiting to. CSSY, G364E, E431K and D444E of LPDL gene
and SNPs
within LPDLR gene.
The invention teaches a method for identifying a compound which modulates a
triglyceride disorder, comprising identifying a compound which modulates the
activity of a
polynucleotide, wherein the polynucleotide is a coding sequence selected from
the group
consisting of LPDL, LPDLR, and the control regions thereof, comprising the
steps of (a)
selecting a control animal having the polynucleotide and a test animal having
the
polynucleotide; (b) treating the test animal using a compound; and, (c)
determining the relative
quantity of an expression product of the polynucleotide, as between the
control animal and the
test animal.
The invention teaches a method for identifying a compound which modulates a
triglyceride disorder, comprising identifying a compound which modulates the
activity of a
polynucleotide, wherein the polynucleotide is a coding sequence selected from
the group
consisting of LPDL, LPDLR, and the control regions thereof comprising the
steps of: (a)
selecting a host cell having the polynucleotide, wherein the host cell is
heterologous to the
polynucleotide; (b) cloning the host cell and separating the clones into a
test group and a
control group; (c) treating the test group using a compound; and (d)
determining the relative
quantity of an expression product of the polynucleotide, as between the test
group and the
control group.
The invention teaches a method for identifying a compound modulates a
triglyceride
disorder, comprising identifying a compound which modulates the activity of a
polypeptide
selected from the group consisting of LPDL and LPDLR, comprising the steps o~
(a) selecting
a control animal having the polypeptide and a test animal having the
polypeptide; (b) treating
the test animal using a compound; (c) determining the relative quantity or
relative activity of
an expression product of the polypeptide or of the the polypeptide, as between
the control
animal and the test animal.
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The invention teaches a method for identifying a compound which modulates a
triglyceride disorder, comprising identifying a compound which modulates the
activity of a
polypeptide selected from the group consisting of LPDL and LPDLR, comprising
the steps of:
(a) selecting a host cell comprising the polypeptide, wherein the host cell is
heterologous to the
polypeptide; (b) cloning the host cell and separating the clones into a test
group and a control
group; (c) treating the test group using a compound; and (d) determining the
relative quantity
or relative activity of an expression product of the polypeptide or of the the
polypeptide, as
between the test group and the control group.
The invention teaches a method for identifying a compound which modulates the
activity of a polynucleotide, wherein the polynucleotide is a control region
of a gene selected
from the group consisting of LPDL and LPDLR, comprising the steps of: (a)
selecting a
control animal having the polynucleotide and a test animal having the
polynucleotide; (b)
treating the test animal using a compound; and, (c) determining the relative
quantity of an
expression product of an operably linked polynucleotide to the polynucleotide,
as between the
control animal and the test animal.
The invention teaches a method for identifying a compound which modulates the
activity of a polynucleotide, wherein the polynucleotide is a control region
of a gene selected
from the group consisting of LPDL and LPDLR, comprising the steps of: (a)
selecting a host
cell comprising the polynucleotide, wherein the host cell is heterologous to
the polynucleotide;
(b) cloning the host cell and separating the clones into a test group and a
control group; (c)
treating the test group using a compound; and (d) determining the relative
quantity of an
expression product of an operably linked polynucleotide to the polynucleotide,
as between the
test group and the control group.
The invention teaches a composition for treating a lipase or lipid disorder
comprising a
compound which modulates a polynucleotide from the coding sequence selected
from the
group consisting of LPDL and LPDLR, and a pharmaceutically acceptable carrier.
The
disorder may be selected from the group consisting of lipase deficiency,
lipoprotein defects,
hypertriglyceridemia (primary genetic defect or secondary from other
diseases), fatty liver
diseases, cardiovascular disorders (including but not limited to
hypertriglyceridemia,
dyslipidemia, atherosclerosis, coronary artery disease, cerebrovascular
disease hypertension,
and peripheral vascular disease), body weight disorders (including but not
limited to obesity,
cachexia and anorexia), inflammation (including but not limited to sinusitis,
asthma,
pancreatitis, osteoarthritis, rheumatoid arthritis and acne), eczema,
Sjogren's syndrome,
gastrointestinal disorders, viral diseases and postviral fatigue, psychiatric
disorders, cancer,
cystic fibrosis, endometriosis, pre-menstrual syndrome, alcoholism, congenital
liver disease,
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Alzheimer's syndrome, hypercholesterolemia, autoimmune disorders, atopic
disorders, acute
respiratory distress syndrome, articular cartilage degradation, diabetes and
diabetic
complications. In preferred embodiments, the conditions are selected from the
group
consisting of lipase deficiency, atherosclerosis, fatty liver disease and
dyslipidemias, such as
hypercholesterolemia, hypertriglyceridemia, mixed (combined) dyslipidemia,
lipid or
lipoprotein deficient states, and/or any other tissue or plasma disorders of
lipid or lipoprotein
metabolism. The invention further teaches a method for identifying a compound
which
modulates a biological activity of a polypeptide selected from the group
consisting of LPDL
and LPDLR, comprising the steps of: (a) providing an assay which measures a
biological
activity of the selected polypeptide; (b) treating the assay with a compound;
and (c)
identifying a change in the biological activity of the selected polypeptide,
wherein a difference
between the treated assay and a control assay identifies the compound as
modulator of the
polypeptide.
The invention further teaches the use of a cell containing a transgene
comprising a
polypeptide of the invention for cell therapy by administration to a patient
in need thereof.
The invention further teaches a process for expression of a protein product of
a
polypeptide selected from the group consisting of LPDL and LPDLR comprising
the steps of:
(a) providing a recombinant DNA cloning vector system which integrates into
the genome of
an host single cell organism, a vector system comprising: DNA-sequences
encoding functions
facilitating gene expression comprising a promoter, transcription initiation
sites, and
transcription terminator and a polypeptide selected from the group consisting
of LPDL and
LPDLR; (b) transforming the host with the recombinant DNA cloning vector
system from
step (a); and (c) culturing the transformed host in a culture medium.
Other features and advantages of the present invention will become apparent
from the
following detailed description. It should be understood, however, that the
detailed description
and the specific examples while indicating preferred embodiments of the
invention are given
by way of illustration only, since various changes and modifications within
the spirit and scope .
of the invention will become apparent to those skilled in the art from this
detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in which:
Figures 1 A (SEQ.ID.NO:1 ) and B (SEQ.>D.N0:2) show the nucleic acid and amino
acid sequences, respectively for human LPDL.
Figures 2A (SEQ.)D.N0.3), B (SEQ.ID.N0.4), C (SEQ.ID.NO:S) and D
(SEQ.)D.N0:6) show the nucleic acid or amino acid sequences for mouse lpdl.
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Figures 3A (SEQ.ID.N0.7) and B (SEQ.>D.N0.8) show the nucleic acid and amino
acid sequences, respectively, for mouse lpdlr.
Figures 4A (SEQ.ID.N0.9), B (SEQ.m.N0.10), C (SEQ.ID.NO:11) and D
(SEQ.ID.NO:12) show the nucleic acid or amino acid sequences for human LPDLR.
Figure 5 shows the shotgun sequencing of BAC clone for identification of LPDL
gene
exons and lpd lipase sequences.
Figure 6 shows the nucleic acid sequence of contig #6 from the BAC clones
(SEQ.ID.N0.13).
Figure 7 shows the nucleic acid sequence of contig #28 from the BAC clones
(SEQ.m.N0.14).
Figure 8 shows the nucleic acid sequence of contig #86 from the BAC clones
(SEQ.ID.NO.15).
Figure 9 shows the nucleic acid sequence of contig #98 from the BAC clones
(SEQ.m.N0.16).
Figures 10 A and B show gene expression of LPDL and LPDLR
Figure 11 shows the cDNA and amino acid sequences of human LPDL highlighting
the ORF, lipase consensus sequence, conserved cystine residues and catalytic
triads.
Figure 12 shows the cDNA and amino acid sequences of mouse lpdlr highlighting
the
ORF, lipase consensus sequence, conserved cystine residues and catalytic
triads.
Figure 13 shows a protein sequence comparison of LPDL and LPDLR with other
lipases and PS-PLA1.
Figure 14 shows the phylogenetic relationship of the lipase family and PS-
PLA1.
Figure 15 shows exon sequences and adjacent intron sequences of human LPDL
gene:
Exon/Intron 1 (SEQ.>D.N0.17); Exon/Intron 2 (SEQ.ID.N0.18); Exon/Intron 3
(SEQ.)D.N0.19); Exon/Intron 4 (SEQ.>D.N0.20); Exon/Intron 5 (SEQ.ID.N0.21);
Exon/Intron 6 (SEQ.)D.N0.22); Exon/Intron 7 (SEQ.ID.N0.23); Exon/Intron 8
(SEQ.ID.N0.24); Exon/Intron 9 (SEQ.>D.N0.25); Exon/Intron 10 (SEQ.>D.N0.26);
Figure 16-A shows exon sequences and adjacent intron sequences of human LPDLR
gene: Exon/Intron 1 (SEQ.ID.N0.27); Exon/Intron 2 (SEQ.ID.N0.28); Exon/Intron
3
(SEQ.ID.N0.29); Exon/Intron 4 (SEQ.B7.N0.30); Exon/Intron 5 (SEQ.>D.N0.31);
Exon/Intron 6 (SEQ.ID.N0.32); Exon/Intron 7 (SEQ.)D.N0.33); Exon/Intron 8
(SEQ.m.N0.34); Exon/Intron 9 (SEQ.)D.N0.35); Exon/Intron 10 (SEQ.)D.N0.36);
Figure 16-B shows the assembled LPDLR gene sequences from exons together with
encoded protein sequences.
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Figure 17 shows schematic map of genetic disruption of exon 10 of lpdl in the
Ipd
locus.
Figure 18-A s hows primer s equences for a mplifying a nd d etecting a xons o
f h uman .
LPDL: Primer 1F (SEQ.)D.N0.37); Primer 1R (SEQ.ID.N0.38); Primer 2F
(SEQ.ID.N0.39);
Primer 2R (SEQ.>D.N0.40); Primer 3F (SEQ.ID.N0.41); Primer 3R (SEQ.ID.N0.42);
Primer
4F (SEQ.ID.N0.43); Primer 4R (SEQ.)D.N0.44); Primer SF (SEQ.ID.N0.45); Primer
SR
(SEQ.)D.N0.4G); Primer 6F (SEQ.ID.N0.47); Primer 6R (SEQ.)17.N0.48); Primer 7F
(SEQ.D.~.N0.49); Primer 7R (SEQ.ID.NO.50); Primer 8F (SEQ.ID.NO.51); Primer 8R
(SEQ.117.N0.52); Primer 9F (SEQ.ID.N0.53); Primer 9R (SEQ.ID.N0.54); Primer
lOF
(SEQ.T~.NO.55); Primer lOR (SEQ.)I?.hrO.56);
Figure 18-B s hows p Timer s equences for amplifying a nd d etecting a xons o
f human
LPDLR: Primer 1F (SEQ.1D.NO.57); Primer 1R (SEQ.m.N0.58); Primer 2F
(SEQ.ID.N0.59); Primer 2R (SEQ.ID.N0.60); Primer 3F (SEQ.ID.N0.61); Primer 3R
(SEQ.ID.N0.62); Primer 4F (SEQ.ID.N0.63); Primer 4R (SEQ.)D.N0.64); Primer SF
(SEQ.ID.N0.65); Primer SR (SEQ.ID.N0.66); Primer 6F (SEQ.1D.N0.67); Primer 6R
(SEQ.1D.N0.68); Primer 7F (SEQ.1D.N0.69); Primer 7R (SEQ.)D.N0.70); Primer 8F
(SEQ.ID.N0.71); Primer 8R (SEQ.ID.N0.72); Primer 9F (SEQ.ID.N0.73); Primer 9R
(SEQ.)I3.NO.74); Primer lOF (SEQ.ID.N0.75); Primer lOR (SEQ.)D.N0.76).
Figure 19 shows SNPs identified for human LPDL gene.
. Figure 20 shows significant (P<0.05) quantitative lipoprotein associations
with LPDL
SNPs.
Figure 21-A shows promoter and regulatory sequences of murine lpdl
(SEQ.ID.N0.77), the primer sequences used to clone the promoter fragments and
the sizes of
cloned fragments; Figure 21-B shows promoter and regulatory sequences of human
LPDL
(SEQ.>D.N0.78).
Figure 22 shows computing analysis of transcription factor binding sites in
200 by of
murine lpdl promoter.
Figure 23-A shows promoter and regulatory sequences of murine LPDL
(SEQ.1D.N0.79). Figure 23-B shows .promoter and regulatory sequences of human
lpdl
(SEQ.m.N0.80).
DETAILED DESCRIPTION OF THE INVENTION
The inventors previously identified a mouse perinatal, transgenic insertional
mutation,
Ipd (lipid defect), which is characterized by accumulation of triglycerides in
the liver and in
the plasma. It was hypothesized that this triglyceride accumulation resulted
from the
transgenic disruption of a putative gene involved in triglyceride metabolism.
Molecular
RECTIFIED SHEET (RULE 91)
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cloning of the transgene-flanking sequences led to mapping of the lpd locus to
the distal part
of murine chromosome 16 (Wen et. el. 1998). Since the identified human PS-PLA1
demonstrates significant homology to mammalian triglyceride lipases, the
inventors first
characterized the murine ps plal to investigate whether it is encoded from lpd
locus. Using
mouse whole-genome radiation hybrid (WG-RH) mapping, the ps-plal gene was
mapped to
the same chromosome as the lpd locus but with a genetic distance of 17 cM
which suggest that
ps plal and lpd are different genes (Wen et. al. 2001 ). To further identify
the putative lipase
gene in the lpd locus, the inventors used a positional cloning strategy.
Since the insertional locus of lpd mutation involves gene rearrangement,
identifying
the disrupted gene directly from the mutant locus is not straight forward. The
inventors cloned
the entire wild-type region of lpd locus with bacteria artificial chromosome
(BAC). By
sequencing one BAC clone 0500 kb sequenced) and in connection with and
bioinformatic
studies, the inventors have identified lipase-related sequences and discovered
a new mouse
gene lpdl (lpd lipase) that belongs to the triglyceride lipase gene family.
Using mouse lpdl
gene fragments as probes, the inventors cloned the human LPDL cDNA and
identified its
nucleic acid and amino acid sequences. Based on the LPDL sequences and
bioinformatic
studies, the inventor further identified a second novel lipase related to but
distinct from LPDL
which is designated LPDL-related lipase (LPDLR).
LPDL and LPDLR demonstrates extensive homology to other members in the lipase
gene family with about 3040% identity at protein level. But interestingly, the
lid sequence
(12 amino acids) of both LPDL and LPDLR are much shorter than that of the
other lipases
(1923). In contrast, it demonstrates similarity with the PS-PLA1 lid sequences
that is also
composed of 12 amino acids. Previous studies in HL and LPL demonstrate the 22-
amino acid
loops ("lids") are critical for the interaction with lipid substrate (Dugi et
al., 1992). Using the
GrowTree program of the web-base SeqWeb Wisconsin GCG package, the inventors
have
demonstrated that LPDL, LPDLR and PS-PLAT are very closely related in
evolution and they
form a subfamily in the lipase family.
By Northern blot and/or RNA in situ hybridization, LPDL is expressed strongly
in the
testis and weakly in the liver while LPDLR is expressed in colon prostate and
testis. The
genomic structure and exon/intron boundaries of both LPDL and LPDLR genes has
been
characterized. Ten exons were discovered for both genes. Human LPDL locates on
chromosome 21 and spans a huge genetic region of over 100 kb while LPDLR
locates on
human chromosome 3. The comparison of mouse genomic sequences adjacent to
transgene
insertion with the gene structure of mouse lpdl gene revealed that exon 10 was
deleted in the
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mutant lpd insertional locus, suggesting disruption of lpdl lipase gene
resulted in the observed
phenotype in the lpd mutant mice.
Following the characterization of human LPDL and LPDLR genomic structure, the
inventors performed mutation screening of both genes in hypertriglyceridemic
subjects and
normal populations. For LPDL gene, seven transcribed and six non-transcribed
SNPs were
discovered. Their allele frequencies were determined from.genotyping of normal
populations.
One coding SNP, CSSY, was only detected in hypertriglyceridemic subjects but
not in the
normal population, suggesting the discovery of a potential human disease
mutation. By
sequence comparison with other lipases whose tertiary structure were well
characterized (van
Tilbeurgh et al. 1994), C55 of LPDL belongs to the conserved cysteine residues
required for
disulfide bridge formation and is therefore structurally important.
Interestingly, further studies
of SNP association with lipid traits demonstrated association of a few LPDL
SNPs to be
associated with HDL cholesterol in a few independently sampled populations.
Five SNPs for
LPDLR gene have been identified including two transcribed SNPs. However, these
two
LPDLR coding SNPs appeared to be silent without amino acid substitution.
Since the LPDL gene is highly expressed in testis and weakly expressed in the
liver but
not in any other tissues examined, LPDL promoter activity is very tissue
specific. By
comparing the mouse lpdl and human LPDL gene sequences, the promoter region
demonstrates significant sequence homology indicating structural conservation
of LPDL
promoter across the species. From the BAC#16 DNA of mouse lpdl gene, the
inventors have
cloned the promoter region up to ~6 kb and generated differently-sized
promoter/reporter gene
constructs. With computing analysis of the promoter sequences, the inventors
also identified
potential binding sites for variety of transcription factors. Two recombinant
adenoviruses
carrying human LPDL and marine lpdlr lipases have been generated and their
function in
regulating lipid metabolism is being investigated in animal models. The
inventors also
expressed the recombinant proteins of human LPDL and mouse lpdlr in
Baculovirus system.
I. Nucleic Acid Molecules of the Invention
The present invention provides an isolated nucleic acid molecule comprising a
sequence encoding a LPDL or LPDLR protein. In one embodiment, the LPDL nucleic
acid
molecule is preferentially but expressed in but not limited to testis and
liver, and the LPDLR
nucleic acid molecule is preferentially expressed in but not limited to
testis, prostate, colon,
mammary and salivary gland.
The term "isolated" refers to a nucleic acid substantially free of cellular
material or
culture medium when produced by recombinant DNA techniques, or chemical
precursors, or
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other chemicals when chemically synthesized. The term "nucleic acid" is
intended to include
DNA and RNA and can be either double stranded or single stranded.
In one embodiment of the invention, the LPDL and LPDLR nucleic acid includes a
nucleic acid sequence as shown in Figure lA (SEQ.ID.NO:1), Figure 2A
(SEQ.)D.N0:3),
Figure 2C (SEQ.ID.NO:S), Figure 3A (SEQ.ID.N0:7), Figure 4A (SEQ.ID.N0:9),
Figure 4C
(SEQ.ID.NO:11).
In another embodiment of the invention, an isolated nucleic acid molecule is
provided
having a sequence which encodes a LPDL protein or LPDLR protein having the
amino acid
sequence as shown in Figure 1B (SEQ.ID.N0:2), Figure 2B (SEQ.ID.N0:4), Figure
2D
(SEQ.>D.N0:6), Figure 3B (SEQ.ID.N0:8), Figure 4B (SEQ.)D.NO:10) or, Figure 4D
(SEQ.)D.N0:12).
In another embodiment of the invention, an isolated nucleic acid molecule is
provided
having a sequence as promoter and/or regulatory elements which control the
gene expression
of LPDL protein or LPDLR protein as shown in Figure 21-A for mouse lpdl
(SEQ.ID.N0:77);
21-B for human LPDL gene (SEQ.ID.N0:78); Figure 22-A for mouse lpdlr
(SEQ.ID.N0:79);
22-B for human LPDLR gene (SEQ.ID.N0:80);
In one more embodiment of the invention, an isolated nucleic acid molecule is
provided having a exon/intron sequence of LPDL or LPDLR gene having the
nucleotide acid
sequence as shown in Figure 15 of human LPDL gene: Exon/Intron 1
(SEQ.)D.N0.17),
Exon/Intron 2 (SEQ.ID.N0.18), Exon/Intron 3 (SEQ.ID.N0.19), Exon/Intron 4
(SEQ.ID.N0.20), Exon/Intron 5 (SEQ.ID.N0.21), Exon/Intron 6 (SEQ.ID.N0.22),
Exon/Intron 7 (SEQ.ID.N0.23), Exon/Intron 8 (SEQ.ID.N0.24), Exon/Intron 9
(SEQ.ID.N0.25), and Exon/Intron 10 (SEQ.)D.N0.26); and in Figure 16-A as exon
sequences
and adjacent intron sequences of human LPDLR gene: Exon/Intron 1
(SEQ.ID.N0.27),
Exon/Intron 2 (SEQ.ID.N0.28), Exon/Intron 3 (SEQ.ID.N0.29), Exon/Intron 4
(SEQ.ID.N0.30), Exon/Intron 5 (SEQ.ID.N0.31), Exon/Intron 6 (SEQ.ID.N0.32),
Exon/Intron 7 (SEQ.ID.N0.33), Exon/Intron 8 (SEQ.ID.N0.34), Exon/Intron 9
(SEQ.ID.N0.35) and Exon/Intron 10 (SEQ.>D.N0.36).
In a preferred embodiment, the invention provides an isolated nucleic acid
sequence
comprising:
(a) a nucleic acid sequence as shown in Figure lA (SEQ.ID.NO:1), Figure 2A
(SEQ.)D.N0:3), Figure 2C (SEQ.ID.NO:S), Figure 3A (SEQ.ID.N0:7), Figure 4A
(SEQ.1D.N0:9), Figure 4C (SEQ.>D.NO:11), Figure 15 (SEQ.ID.N0.17),
(SEQ.ID.N0.18),
(SEQ.ll~.N0.19), (SEQ.117.N0.20), (SEQ.117.N0.21), (SEQ.ID.N0.22),
(SEQ.)D.N0.23),
(SEQ.m.N0.24), (SEQ.ID.N0.25), (SEQ.1D.N0.26); Figure 16-A (SEQ.ID.N0.27),
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(SEQ.)D.N0.28), (SEQ.ID.N0.29), (SEQ.m.N0.30), (SEQ.m.N0.31), (SEQ.ID.N0.32),
(SEQ.ID.N0.33), (SEQ.ID.N0.34), (SEQ.m.N0.35), (SEQ.)D.N0.36); Figure 21
(SEQ.ID.N0:77), (SEQ.ID.N0:78); or Figure 22 (SEQ.m.N0:79), (SEQ.ID.N0:80),
wherein
T can also be U;
(b) a nucleic acid sequence that is complimentary to a nucleic acid sequence
of
(a);
(c) a nucleic acid sequence that has substantial sequence homology to a
nucleic
acid sequence of (a) or (b);
(d) a nucleic acid sequence that is an analog of a nucleic acid sequence of
(a), (b)
or (c); or
(e) a nucleic acid sequence that hybridizes to a nucleic acid sequence of (a),
(b),
(c) or (d) under stringent hybridization conditions.
The term "sequence that has substantial sequence homology" means those nucleic
acid
sequences which have slight or inconsequential sequence variations from the
sequences in (a)
or (b), i.e., the sequences function in substantially the same manner and can
be used to
modulate triglyceride levels. The variations may be attributable to local
mutations or structural
modifications. Nucleic acid sequences having substantial homology include
nucleic acid
sequences having at least 65%, more preferably at least 85%, and most
preferably 90-95%
identity with the nucleic acid sequence as shown in Figure 1 A (SEQ.ID.NO:1 ),
Figure 2A
(SEQ.ID.N0:3), Figure 2C (SEQ.117.N0:5), Figure 3A (SEQ.ID.N0:7), Figure 4A
(SEQ.ID.N0:9), Figure 4C (SEQ.ID.NO:11), Figure 15 (SEQ.ID.N0.17),
(SEQ.1D.N0.18),
(SEQ.1D.N0.19), (SEQ.ID.N0.20), (SEQ.ID.N0.21), (SEQ.ID.N0.22),
(SEQ.117.N0.23),
(SEQ.1D.N0.24), (SEQ.lD.N0.25), (SEQ.ID.N0.26); Figure 16-A (SEQ.117.N0.27),
(SEQ.117.N0.28), (SEQ.ID.N0.29), (SEQ.)D.N0.30), (SEQ.m.N0.31), (SEQ.m.N0.32),
(SEQ.~.N0.33), (SEQ.ID.N0.34), (SEQ.ID.N0.35), (SEQ.ID.N0.36); Figure 21
(SEQ.ID.N0:77), (SEQ.m.N0:78) or Figure 22 (SEQ.ID.N0:79), (SEQ.ll~.N0:80).
The term "sequence that hybridizes" means a nucleic acid sequence that can
hybridize
to a sequence of (a), (b), (c) or (d) under stringent hybridization
conditions. Appropriate
"stringent hybridization conditions" which promote DNA hybridization are known
to those
skilled in the art, or may be found in Current Protocols in Molecular Biology,
John Wiley &
Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the following may be employed:
6.0 x sodium
chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0
x SSC at 50°C; 0.2 x
SSC at 50°C to 65°C; or 2.0 x SSC at 44°C to 50°C.
The stringency may be selected based on
the conditions used in the wash step. For example, the salt concentration in
the wash step can
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be selected from a high stringency of about 0.2 x SSC at 50°C. In
addition, the temperature in
the wash step can be at high stringency conditions, at about 65°C.
The term "a nucleic acid sequence which is an analog" means a nucleic acid
sequence
which has been modified as compared to the sequence of (a), (b) or (c) wherein
the
modification does not alter the utility of the sequence as described herein.
The modified
sequence or analog may have improved properties over the sequence shown in
(a), (b) or (c).
One example of a modification to prepare an analog is to replace one of the
naturally occurnng
bases (i.e. adenine, guanine, cytosine or thymidine) of the sequences shown in
Figure lA
(SEQ.ID.NO:1), Figure 2A (SEQ.ID.N0:3), Figure 2C (SEQ.m.N0:5), Figure 3A
(SEQ.ID.N0:7), Figure 4A (SEQ.ID.N0:9), Figure 4C (SEQ.m.N0:11), Figure 15
(SEQ.ID.N0.17), (SEQ.ID.N0.18), (SEQ.ID.N0.19), (SEQ.ID.N0.20),
(SEQ.ID.N0.21),
(SEQ.ID.N0.22), (SEQ.ID.N0.23), (SEQ.ID.N0.24), (SEQ.ID.N0.25),
(SEQ.ID.N0.26);
Figure 16-A (SEQ.m.N0.27), (SEQ.ID.N0.28), (SEQ.ID.N0.29), (SEQ.ID.N0.30),
(SEQ.ID.N0.31), (SEQ.ID.N0.32), (SEQ.ID.N0.33), (SEQ.ID.N0.34),
(SEQ.B7.N0.35),
(SEQ.ID.N0.36); Figure 21 (SEQ.m.N0:77), (SEQ.ID.N0:78) or Figure 22
(SEQ.ID.N0:79), (SEQ.B7.N0:80), with a modified base such as xanthine,
hypoxanthine, 2-
aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-
halo cytosine, 6-
aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-
halo adenine, 8-
aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and
other 8-
substituted adenines, 8-halo guanines, 8 amino guanine, 8-thiol guanine, 8-
thiolalkyl guanines,
8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza
uracils, thymidines,
cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro
cytosine.
Another example of a modification is to include modified phosphorous or oxygen
heteroatoms in the phosphate backbone, short chain. alkyl or cycloalkyl
intersugar linkages or
short chain heteroatomic or heterocyclic intersugar linkages in the nucleic
acid molecules
shown in Figure lA (SEQ.ID.NO:l), Figure 2A (SEQ.ID.N0:3), Figure 2C
(SEQ.ID.NO:S),
Figure 3A (SEQ.ID.N0:7), Figure 4A (SEQ.ID.N0:9), Figure 4C (SEQ.ID.NO:11)
Figure 15
(SEQ.117.N0.17), (SEQ.ID.N0.18),(SEQ.m.N0.19),(SEQ.ID.N0.20),(SEQ.m.N0.21),
(SEQ.ID.N0.22), (SEQ.ID.N0.23),(SEQ.117.N0.24),(SEQ.ID.N0.25),(SEQ.ID.N0.26);
Figure 16-A (SEQ.m.N0.27),(SEQ.ID.N0.28),(SEQ.ID.N0.29),(SEQ.ID.N0.30),
(SEQ.ID.N0.31), (SEQ.ID.N0.32),(SEQ.m.N0.33),(SEQ.ID.N0.34),(SEQ.m.N0.35),
(SEQ.ID.N0.36); Figure 21 (SEQ.ID.N0:77), (SEQ.m.N0:78) or Figure 22
(SEQ.ID.N0:79), (SEQ.ID.N0:80). For example, the nucleic acid sequences may
contain
phosphorothioates, phosphotriesters, methyl phosphonates, and
phosphorodithioates.
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A further example of an analog of a nucleic acid molecule of the invention is
a peptide
nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in
the DNA (or
RNA), is replaced with a polyamide backbone which is similar to that found in
peptides (P.E.
Nielsen, et al Science 1991, 254, 1497). PNA analogs have been shown to be
resistant to
degradation by enzymes and to have extended lives in vivo and in vitro. PNAs
also bind
stronger to a complimentary DNA sequence due to the lack of charge repulsion
between the
PNA strand and the DNA strand. Other nucleic acid analogs may contain
nucleotides
containing polymer backbones, cyclic backbones, or acyclic backbones. For
example, the
nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506).
The analogs
may also contain groups such as reporter groups, a group for improving the
pharmacokinetic
or pharmacodynamic properties of nucleic acid sequence.
It will be appreciated that the invention includes nucleic acid molecules
encoding
truncations of proteins of the invention, and analogs and homologs of proteins
of the invention
and truncations thereof, as described below. It will further be appreciated
that variant forms of
nucleic acid molecules of the invention which arise by alternative splicing of
an mRNA
corresponding to a cDNA of the invention are encompassed by the invention.
Isolated and purified nucleic acid molecules having sequences which differ
from the
nucleic acid sequence of the invention due to degeneracy in the genetic code
are also within
the scope of the invention. Such nucleic acids encode functionally equivalent
proteins but
differ in sequence from the above mentioned sequences due to degeneracy in the
genetic code.
An isolated nucleic acid molecule of the invention which comprises DNA can be
isolated by preparing a labelled nucleic acid probe based on all or part of
the nucleic acid
sequences of the invention and using this labelled nucleic acid probe to
screen an appropriate
DNA library (e.g. a cDNA or genomic DNA library). For example, a genomic
library isolated
can be used to isolate a DNA encoding a novel protein of the invention by
screening the
library with the labelled probe using standard techniques. Nucleic acids
isolated by screening
of a cDNA or genomic DNA library can be sequenced by standard techniques.
An isolated nucleic acid molecule of the invention which is DNA can also be
isolated
by selectively amplifying a nucleic acid encoding a novel protein of the
invention using the
polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is
possible to
design synthetic oligonucleotide primers from the nucleic acid sequence of the
invention for
use in PCR. A nucleic acid can be amplified from cDNA or genomic DNA using
these
oligonucleotide primers and standard PCR amplification techniques. The nucleic
acid so
amplified can be cloned into an appropriate vector and characterized by DNA
sequence
analysis. It will be appreciated that cDNA may be prepared from mRNA, by
isolating total
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cellular mRNA by a variety of techniques, for example, by using the
guanidinium-thiocyanate
extraction procedure of Chirgwin et al., Biochemistry, 18, 5294-5299 (1979).
cDNA is then
synthesized from the mRNA using reverse transcriptase (for example, Moloney
MLV reverse
transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse
transcriptase
available from Seikagaku America, Inc., St. Petersburg, FL).
An isolated nucleic acid molecule of the invention which is RNA can be
isolated by
cloning a cDNA encoding a novel protein of the invention into an appropriate
vector which
allows for transcription of the cDNA to produce an RNA molecule which encodes
a protein of
the invention. For example, a cDNA can be cloned downstream of a bacteriophage
promoter,
(e.g., a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7
polymerase, and
the resultant RNA can be isolated by standard techniques.
A nucleic acid molecule of the invention may also be chemically synthesized
using
standard techniques. Various methods of chemically synthesizing
polydeoxynucleotides are
known, including solid-phase synthesis which, like peptide synthesis, has been
fully automated
in commercially available DNA synthesizers (See e.g., Itakura et al. U.S.
Patent No.
4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent
Nos. 4,401,796
and 4,373,071).
Determination of whether a particular nucleic acid molecule encodes a novel
protein
of the invention may be accomplished by expressing the cDNA in an appropriate
host cell by
standard techniques, and testing the activity of the protein using the methods
as described
herein. A cDNA having the activity of a novel protein of the invention so
isolated can be
sequenced by standard techniques, such as dideoxynucleotide chain termination
or Maxam-
Gilbert chemical sequencing, to determine the nucleic acid sequence and the
predicted amino
acid sequence of the encoded protein.
The initiation codon and untranslated sequences of nucleic acid molecules of
the
invention may be determined using currently available computer software
designed for the
purpose, such as PC/Gene (IntelliGenetics Inc., Cali~). Regulatory elements
can be identified
using conventional techniques. The function of the elements can be confirmed
by using these
elements to express a reporter gene which is operatively linked to the
elements. These
constructs may be introduced into cultured cells using standard procedures. In
addition to
identifying regulatory elements in DNA, such constructs may also be used to
identify proteins
interacting with the elements, using techniques known in the art.
The sequence of a nucleic acid molecule of the invention may be inverted
relative to
its normal presentation for transcription to produce an antisense nucleic acid
molecule which
are more fully described herein. Preferably, an antisense sequence is
constructed by inverting
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a region preceding the initiation codon or an unconserved region. In
particular, the nucleic
acid sequences contained in the nucleic acid molecules of the invention or a
fragment thereof,
may be inverted relative to its normal presentation for transcription to
produce antisense
nucleic acid molecules.
The invention also provides nucleic acids encoding fusion proteins comprising
a novel
protein of the invention and a selected protein, or a selectable marker
protein (see below).
Also provided are portions of the nucleic acid sequence encoding fragments,
functional
domains or antigenic determinants of the LPDL or LPDLR protein. The present
invention also
provides for the use of portions of the sequence as probes and PCR primers for
LPDL or
LPDLR and related proteins and well as for determining functional aspects of
the sequence.
One of ordinary skill in the art is now enabled to identify and isolate LPDL
or LPDLR
genes or cDNAs which are allelic variants or spliced isoforms of the disclosed
LPDL and
LPDLR sequences, using standard hybridization screening or PCR techniques.
II. Novel Proteins of the Invention
The invention further broadly contemplates an isolated LPDL or LPDLR protein.
The
terms "LPDL protein" or "LPDLR protein" as used herein include all homologs,
analogs,
fragments or derivatives of the LPDL or LPDLR which can modulate triglyceride
and lipase
related function.
In one embodiment, the isolated LPDL or LPDLR protein has an amino acid
sequence
as shown in Figure 1B (SEQ.ID.N0:2), Figure 2B (SEQ.1D.N0:4), Figure 2D
(SEQ.lD.N0:6), Figure 3B (SEQ.ID.N0:8), Figure 4B (SEQ.>T7.N0:10) or, Figure
4D
(SEQ.117.N0:12).
Within the context of the present invention, a protein of the invention may
include
various structural forms of the primary proteins which retain biological
activity. For example,
a protein of the invention may be in the form of acidic or basic salts or in
neutral form. In
addition, individual amino acid residues may be modified by oxidation or
reduction.
In addition to the full length amino acid sequences, the protein of the
present invention
may also include truncations of the protein, and analogs, and homologs of the
protein and
truncations thereof as described herein.
The invention further provides polypeptides comprising at least one functional
domain
or at least one antigenic determinant of a LPDL or LPDLR protein.
Analogs of the protein of the invention and/or truncations thereof as
described herein,
may include, but are not limited to an amino acid sequence containing one or
more amino acid
substitutions, insertions, and/or deletions. Amino acid substitutions may be
of a conserved or
non-conserved nature. Conserved amino acid substitutions involve replacing one
or more
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amino acids of the proteins of the invention with amino acids of similar
charge, size, and/or
hydrophobicity characteristics. When only conserved substitutions are made the
resulting
analog should be functionally equivalent. Non-conserved substitutions involve
replacing one
or more amino acids of the amino acid sequence with one or more amino acids
which possess
dissimilar charge, size, and/or hydrophobicity characteristics.
One or more amino acid insertions may be introduced into the amino acid
sequences
of the invention. Amino acid insertions may consist of single amino acid
residues or
sequential amino acids ranging from 2 to 15 amino acids in length. For
example, amino acid
insertions may be used to destroy target sequences so that the protein is no
longer active. This
procedure may be used in vivo to inhibit the activity of a protein of the
invention.
Deletions may consist of the removal of one or more amino acids, or discrete
portions
from the amino acid sequence of the LPDL or LPDLR. The deleted amino acids may
or may
not be contiguous. The lower limit length of the resulting analog with a
deletion mutation is
about 10 amino acids, preferably 100 amino acids.
Analogs of a protein of the invention may be prepared by introducing mutations
in the
nucleotide sequence encoding the protein. Mutations in nucleotide sequences
constructed for
expression of analogs of a protein of the invention must preserve the reading
frame of the
coding sequences. Furthermore, the mutations will preferably not create
complementary
regions that could hybridize to produce secondary mRNA structures, such as
loops or hairpins,
which could adversely affect translation of the receptor mRNA.
Mutations may be introduced at particular loci by synthesizing
oligonucleotides
containing a mutant sequence, flanked by restriction sites enabling ligation
to fragments of the
native sequence. Following ligation, the resulting reconstructed sequence
encodes an analog
having the desired amino acid insertion, substitution, or deletion.
Alternatively, oligonucleotide-directed site specific mutagenesis procedures
may be
employed to provide an altered gene having particular codons altered according
to the
substitution, deletion, or insertion required. Deletion or truncation of a
protein of the invention
may also be constructed by utilizing convenient restriction endonuclease sites
adjacent to the
desired deletion. Subsequent to restriction, overhangs may be filled in, and
the DNA religated.
Exemplary methods of making the alterations set forth above are disclosed by
Sambrook et al
(Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory Press,
1989).
The proteins of the invention also include homologs of the amino acid sequence
of the
LPDL or LPDLR protein and/or truncations thereof as described herein. Such
homologs are
proteins whose amino acid sequences are comprised of amino acid sequences that
hybridize
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under stringent hybridization conditions (see discussion of stringent
hybridization conditions
herein) with a probe used to obtain a protein of the invention. Homologs of a
protein of the
invention will have the same regions which are characteristic of the protein.
A homologous protein includes a protein with an amino acid sequence having at
least
70%, preferably 80-95% identity with the amino acid sequence of the LPDL or
LPDLR
protein.
The invention also contemplates isoforms of the proteins of the invention. An
isoform
contains the same number and kinds of amino acids as a protein of the
invention, but the
isoform has a different molecular structure. The isoforms contemplated by the
present
invention are those having the same properties as a protein of the invention
as described
herein.
The present invention also includes a protein of the invention conjugated with
a
selected protein, or a selectable marker protein to produce fusion proteins.
For example, the
LPDL or LPDLR cDNA sequence is inserted into a vector that contains a
nucleotide sequence
encoding another peptide (e.g. GST-glutathione succinyl transferase). The
fusion protein is
expressed and recovered from prokaryotic (e.g. bacterial or baculovirus) or
eukaryotic cells.
The fusion protein can then be purified by affinity chromatography based upon
the fusion
vector sequence and the LPDL or LPDLR protein obtained by enzymatic cleavage
of the fusion
protein.
The proteins of the invention (including truncations, analogs, etc.) may be
prepared
using recombinant DNA methods. Accordingly, nucleic acid molecules of the
present
invention having a sequence which encodes a protein of the invention may be
incorporated
according to procedures known in the art into an appropriate expression vector
which ensures
good expression of the protein. Possible expression vectors include but are
not limited to
cosmids, plasmids, or modified viruses (e.g., replication defective
retroviruses, adenoviruses
and adeno-associated viruses), so long as the vector is compatible with the
host cell used. The
expression "vectors suitable for transformation of a host cell", means that
the expression
vectors contain a nucleic acid molecule of the invention and regulatory
sequences, selected on
the basis of the host cells to be used for expression, which are operatively
linked to the nucleic
acid molecule. "Operatively linked" is intended to mean that the nucleic acid
is linked to
regulatory sequences in a manner which allows expression of the nucleic acid.
The invention therefore contemplates a recombinant expression vector of the
invention
containing a nucleic acid molecule of the invention, or a fragment thereof,
and the necessary
regulatory sequences for the transcription and translation of the inserted
protein-sequence.
Suitable regulatory sequences may be derived from a variety of sources,
including bacterial,
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fungal, or viral genes (For example, see the regulatory sequences described in
Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San Diego,
CA
(1990). Selection of appropriate regulatory sequences is dependent on the host
cell chosen,
and may be readily accomplished by one of ordinary skill in the art. Examples
of such
regulatory sequences include: a transcriptional promoter and enhancer or RNA
polymerase
binding sequence, a ribosomal binding sequence, including a translation
initiation signal.
Additionally, depending on the host cell chosen and the vector employed, other
sequences,
such as an origin of replication, additional DNA restriction sites, enhancers,
and sequences
conferring inducibility of transcription may be incorporated into the
expression vector. It will
also be appreciated that the necessary regulatory sequences may be supplied by
the native
protein and/or its flanking regions.
The invention further provides a recombinant expression vector comprising a
DNA
nucleic acid molecule of the invention cloned into the expression vector in an
antisense
orientation. That is, the DNA molecule is operatively linked to a regulatory
sequence in a
manner which allows for expression, by transcription of the DNA molecule, of
an RNA
molecule which is antisense to a nucleotide sequence of the invention.
Regulatory sequences
operatively linked to the antisense nucleic acid can be chosen which direct
the continuous
expression of the antisense RNA molecule.
The recombinant expression vectors of the invention may also contain a
selectable
marker gene which facilitates the selection of host cells transformed or
transfected with a
recombinant molecule of the invention. Examples of selectable marker genes are
genes
encoding a protein such as 6418 and hygromycin which confer resistance to
certain drugs, 13-
galactosidase, chloramphenicol acetyltransferase, or firefly luciferase.
Transcription of the
selectable marker gene is monitored by changes in the concentration of the
selectable marker
protein such as 13-galactosidase, chloramphenicol acetyltransferase, or
firefly luciferase. If the
selectable marker gene encodes a protein conferring antibiotic resistance such
as neomycin
resistance transformant cells can be selected with 6418. Cells that have
incorporated the
selectable marker gene will survive, while the other cells die. This makes it
possible to
visualize and assay for expression of recombinant expression vectors of the
invention and in
particular to determine the effect of a mutation on expression and phenotype.
It will be
appreciated that selectable markers can be introduced on a separate vector
from the nucleic
acid of interest.
The recombinant expression vectors may also contain genes which encode a
fusion
moiety which provides increased expression of the recombinant protein;
increased solubility of
the recombinant protein; and aId in the purification of a target recombinant
protein by acting as
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a ligand in affinity purification. For example, a proteolytic cleavage site
may be added to the
target recombinant protein to allow separation of the recombinant protein from
the fusion
moiety subsequent to purification of the fusion protein.
Recombinant expression vectors can be introduced into host cells to produce a
transformed host cell. Accordingly, the invention includes a host cell
comprising a
recombinant expression vector of the invention. The term "transformed host
cell" is intended
to include prokaryotic and eukaryotic cells which have been transformed or
transfected with a
recombinant expression vector of the invention. The terms "transformed with",
"transfected
with", "transformation" and "transfection" are intended to encompass
introduction of nucleic
acid (e.g. a vector) into a cell by one of many possible techniques known in
the art.
Prokaryotic cells can be transformed with nucleic acid by, for example,
electroporation or
calcium-chloride mediated transformation. Nucleic acid can be introduced into
mammalian
cells via conventional techniques such as calcium phosphate or calcium
chloride co-
precipitation, DEAF-dextran-mediated transfection, lipofectin, electroporation
or
microinjection. Suitable methods for transforming and transfecting host cells
can be found in
Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbor
Laboratory press (1989)), and other such laboratory textbooks.
Suitable host cells include a wide variety of prokaryotic and eukaryotic host
cells. For
example, the proteins of the invention may be expressed in bacterial cells
such as E. coli,
Pseudomonas, Bacillus subtillus, insect cells (using baculovirus), yeast cells
or mammalian
cells. Other suitable host cells can be found in Goeddel, Gene Expression
Technology:
Methods in Enzymology 185, Academic Press, San Diego, CA (1991).
As an example, to produce LPDL or LPDLR proteins recombinantly, for example,
E.
coli can be used using the T7 RNA polymerase/promoter system using two
plasmids or by
labeling of plasmid-encoded proteins, or by expression by infection with M13
Phage mGPI-2.
E. coli vectors can also be used with Phage lamba regulatory sequences, by
fusion protein
vectors (e.g. lacZ and trpE), by maltose-binding protein fusions, and by
glutathione-S-
transferase fusion proteins.
Alternatively, the LPDL or LPDLR proteins can be expressed in insect cells
using
baculoviral vectors, or in mammalian cells using vaccinia virus. For
expression in mammalian
cells, the cDNA sequence may be ligated to heterologous promoters, such as the
simian virus
(SV40) promoter in the pSV2 vector and introduced into cells, such as testis
cells to achieve
transient or long-term expression. The stable integration of the chimeric gene
construct may be
maintained in mammalian cells by biochemical selection, such as neomycin and
mycophoenolic acid.
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The LPDL or LPDLR DNA sequences can be altered using procedures such as
restriction enzyme digestion, fill-in with DNA polymerase, deletion by
exonuclease, extension
by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA
sequences, site-
directed sequence alteration with the use of specific oligonucleotides
together with PCR.
The cDNA sequence or portions thereof, or a mini gene consisting of a cDNA
with an
intron and its own promoter, is introduced into eukaryotic expression vectors
by conventional
techniques. These vectors permit the transcription of the cDNA in eukaryotic
cells by
providing regulatory sequences that initiate and enhance the transcription of
the cDNA and
ensure its proper splicing and polyadenylation. The endogenous SAP gene
promoter can also
be used. Different promoters within vectors have different activities which
alters the level of
expression of the cDNA. In addition, certain promoters can also modulate
function such as the
glucocorticoid-responsive promoter from the mouse mammary tumor virus.
Some of the vectors listed contain selectable markers or neo bacterial genes
that permit
isolation of cells by chemical selection. Stable long-term vectors can be
maintained in cells as
episomal, freely replicating entities by using regulatory elements of viruses.
Cell lines can also
be produced which have integrated the vector into the genomic DNA. In this
manner, the gene
product is produced on a continuous basis.
Vectors are introduced into recipient cells by various methods including
calcium
phosphate, strontium phosphate, electroporation, lipofection, DEAF dextran,
microinjection,
or by protoplast fusion. Alternatively, the cDNA can be introduced by
infection using viral
vectors.
LPDL or LPDLR proteins may also be isolated from cells or tissues, including
mammalian cells or tissues, in which the protein is normally expressed.
The protein may be purified by conventional purification methods known to
those in
the art, such as chromatography methods, high performance liquid
chromatography methods or
precipitation.
For example, an anti-LPDL or anti-LPDLR antibody (as described below) may be
used
to isolate a LPDL or LPDLR protein, which is then purified by standard
methods.
The proteins of the invention may also be prepared by chemical synthesis using
techniques well known in the chemistry of proteins such as solid phase
synthesis (Merrifield,
1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution
(Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and
II, Thieme,
Stuttgart).
III. Uses
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The present invention includes all uses of the nucleic acid molecules and LPDL
or
LPDLR proteins of the invention including, but not limited to, the preparation
of antibodies and
antisense oligonucleotides, the preparation of experimental systems to study
LPDL or LPDLR,
the isolation of substances that modulate LPDL or LPDLR expression and/or
activity as well as
the use of the LPDL or LPDLR nucleic acid sequences and proteins and
modulators thereof in
diagnostic and therapeutic applications. Some of the uses are further
described below.
(i) Experimental S std
Eukaryotic expression systems can be used for many studies of the LPDL or
LPDLR
genes and gene products) including determination of proper expression and post-
translational
modifications for full biological activity, identifying regulatory elements
located in the 5'
region of the LPDL or LPDLR gene and their role in tissue regulation of
protein expression,
production of large amounts of the normal and mutant protein for isolation and
purification, to
use cells expressing the LPDL or LPDLR protein as a functional assay system
for antibodies
generated against the protein or to test effectiveness of pharmacological
agents, or as a
component of a signal transduction system, to study the function of the normal
complete
protein, specific portions of the protein, or of naturally occurring and
artificially produced
mutant proteins.
Using the techniques mentioned, the expression vectors containing the LPDL or
LPDLR cDNA sequences or portions thereof can be introduced into a variety of
mammalian
cells from other species or into non-mammalian cells.
The recombinant cloning vector, according to this invention, comprises the
selected
DNA of the DNA sequences of this invention for expression in a suitable host.
The DNA is
operatively linked in the vector to an expression control sequence in the
recombinant DNA
molecule so that LPDL or LPDLR protein can be expressed. The expression
control sequence
may be selected from the group consisting of sequences that control the
expression of genes of
prokaryotic or eukaryotic cells and their viruses and combinations thereof.
The expression
control sequence may be selected from the group consisting of the lac system,
the trp system,
the tac system, the trc system, major operator and promoter regions of phage
lambda, the
control region of the fd coat protein, early and late promoters of SV40,
promoters derived from
polyoma, adenovirus, retrovirus, baculovirus, simian virus, 3-phosphoglycerate
kinase
promoter, yeast acid phosphatase promoters, yeast alpha-mating factors and
combinations
thereof.
Expression of the LPDL or LPDLR gene in heterologous cell systems may also be
used
to demonstrate structure-function relationships as well as to provide cell
lines for the purposes
of drug screening. LPDL or LPDLR DNA sequence into a plasmid expression vector
to
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transfect cells is a useful method to test the proteins influence on various
cellular biochemical
parameters including the identification of substrates as well as activators
and inhibitors of the
phosphatase. Plasmid expression vectors containing either the entire coding
sequence for LPDL
or LPDLR, or for portions thereof, can be used in in vitro mutagenesis
experiments that will
identify portions of the protein crucial for regulatory function.The DNA
sequence can be
manipulated in studies to understand the expression of the gene and its
product. The changes in
the sequence may or may not alter the expression pattern in terms of relative
quantities, tissue-
specificity and functional properties.
The invention also provides methods for examining the function of the LPDL or
LPDLR protein encoded by the nucleic acid molecules of the invention. Cells,
tissues, and
non-human animals lacking in expression or partially lacking in expression of
the proteins may
be developed using recombinant molecules of the invention having specific
deletion or
insertion mutations in the nucleic acid molecule of the invention. A
recombinant molecule
may be used to inactivate or alter the endogenous gene by homologous
recombination, and
thereby create a deficient cell, tissue or animal. Such a mutant cell, tissue
or animal may be
used to define specific cell populations, developmental patterns and in vivo
processes,
normally dependent on the protein encoded by the nucleic acid molecule of the
invention. To
confirm the importance of the LPDL or LPDLR proteins in lipid metabolism, a
LPDL or
LPDLR knockout mouse can be prepared. By way of example, a targeted
recombination
strategy may be used to inactivate the endogenous LPDL gene. A gene which
introduces stop
codons in all reading frames and abolishes the biological activity of the
protein may be
inserted into a genomic copy of the protein. The mutated fragment may be
introduced into
embryonic stem cells and colonies may be selected for homologous recombination
with
positive (neomycin)/negative (gancyclovir, thymidine kinase) resistance genes.
To establish
germ line transmission, two clones carrying the disrupted gene on one allele
may be injected
into blastocyts of C57/B 16 mice and transferred into B6/SJL foster mothers.
Chimeras may be
mated to C7B1/6 mice and progeny analysed to detect animals homozygous for the
mutation
(LPDL -/-). The effects of the mutation on the triglyceride metabolism in
comparison to non
mutated controls may be determined, and the survival and histologic pattern of
disease may be
analyzed.
(ii) Antibodies
The isolation of the LPDL and LPDLR proteins enables the preparation of
antibodies
specific for the proteins. Accordingly, the present invention provides an
antibody that binds to
a LPDL or a LPDLR protein. Antibodies may be used advantageously to monitor
the
expression of either protein. Antibodies can be prepared which bind a distinct
epitope in an
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unconserved region of the protein. An unconserved region of the protein is one
that does not
have substantial sequence homology to other proteins.
Conventional methods can be used to prepare the antibodies. For example, by
using a
peptide of LPDL or LPDLR, polyclonal antisera or monoclonal antibodies can be
made using
standard methods. A mammal, (e.g., a mouse, hamster, or rabbit) can be
immunized with an
immunogenic form of the peptide which elicits an antibody response in the
mammal.
Techniques for confernng immunogenicity on a peptide include conjugation to
carriers or other
techniques well known in the art. For example, the protein or peptide can be
administered in
the presence of adjuvant. The progress of immunization can be monitored by
detection of
antibody titers in plasma or serum. Standard ELISA or other immunoassay
procedures can be
used with the immunogen as antigen to assess the levels of antibodies.
Following
immunization, antisera can be obtained and, if desired, polyclonal antibodies
isolated from the
sera.
To produce monoclonal antibodies, antibody producing cells (lymphocytes) can
be
harvested from an immunized animal and fused with myeloma cells by standard
somatic cell
fusion procedures thus immortalizing these cells and yielding hybridoma cells.
Such
techniques are well known in the art, (e.g., the hybridoma technique
originally developed by
Kohler and Milstein (Nature 256, 495-497 (1975)) as well as other techniques
such as the
human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4, 72 (1983)),
the EBV-
hybridoma technique to produce human monoclonal antibodies (Cole et al.
Monoclonal
Antibodies in Cancer Therapy (1985) Allen R. Bliss, Inc., pages 77-96), and
screening of
combinatorial antibody libraries (Huse et al., Science 246, 1275 (1989)).
Hybridoma cells can
be screened immunochemically for production of antibodies specifically
reactive with the
peptide and the monoclonal antibodies can be isolated. Therefore, the
invention also
contemplates hybridoma cells secreting monoclonal antibodies with specificity
for LPDL or
LPDLR as described herein.
The term "antibody" as used herein is intended to include fragments thereof
which also
specifically react with LPDL or LPDLR, or peptides thereof, having the
activity of the LPDL
or LPDLR. Antibodies can be fragmented using conventional techniques and the
fragments
screened for utility in the same manner as described above. For example,
F(ab')2 fragments
can be generated by treating antibody with pepsin. The resulting F(ab')2
fragment can be
treated to reduce disulfide bridges to produce Fab' fragments.
Chimeric antibody derivatives, i.e., antibody molecules that combine a non-
human
animal variable region and a human constant region are also contemplated
within the scope of
the invention. Chimeric antibody molecules can include, for example, the
antigen binding
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domain from an antibody of a mouse, rat, or other species, with human constant
regions.
Conventional methods may be used to make chimeric antibodies containing the
immunoglobulin variable region which recognizes the gene product of LPDL or
LPDLR
antigens of the invention (See, for example, Morrison et al., Proc. Natl Acad.
Sci. U.S.A.
81,6851 (1985); Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S.
Patent No.
4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European
Patent
Publication EP171496; European Patent Publication 0173494, United Kingdom
patent GB
2177096B). It is expected that chimeric antibodies would be less immunogenic
in a human
subject than the corresponding non-chimeric antibody.
Monoclonal or chimeric antibodies specifically reactive with a protein of the
invention
as described herein can be further humanized by producing human constant
region chimeras, in
which parts of the variable regions, particularly the conserved framework
regions of the
antigen-binding domain, are of human origin and only the hypervariable regions
are of non-
human origin. Such immunoglobulin molecules may be made by techniques known in
the art,
(e.g., Teng et al., Proc. Natl. ~Acad. Sci. U.S.A., 80, 7308-7312 (1983);
Kozbor et al.,
Immunology Today, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92, 3-16
(1982)), and PCT
Publication W092/06193 or EP 0239400). Humanized antibodies can also be
commercially
produced (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great
Britain.)
Specific antibodies, or antibody fragments, reactive against LPDL or LPDLR
proteins
may also be generated by screening expression libraries encoding
immunoglobulin genes, or
portions thereof, expressed in bacteria with peptides produced from the
nucleic acid molecules
of LPDL or LPDLR. For example, complete Fab fragments, VH regions and FV
regions can
be expressed in bacteria using phage expression libraries (See for example
Ward et al., Nature
341, 544-546: (1989); Huse et al., Science 246, 1275-1281 (1989); and
McCafferty et al.
Nature 348, 552-554 (1990)). Alternatively, a SCID-hu mouse, for example the
model
developed by Genpharm, can be used to produce antibodies or fragments thereof.
(iii) Antisense Oligonucleotides
Isolation of a nucleic acid molecule encoding LPDL or LPDLR enables the
production
of antisense oligonucleotides that can modulate the expression and/or activity
of LPDL and/or
LPDLR. Accordingly, the present invention provides an antisense
oligonucleotide that is
complimentary to a nucleic acid sequence encoding LPDL and LPDLR.
The term "antisense oligonucleotide" as used herein means a nucleotide
sequence that
is complimentary to its target.
The term "oligonucleotide" refers to an oligomer or polymer of nucleotide or
nucleoside monomers consisting of naturally occurring bases, sugars, and
intersugar
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(backbone) linkages. The term also includes modified or substituted oligomers
comprising
non-naturally occurring monomers or portions thereof, which function
similarly. Such
modified or substituted oligonucleotides may be preferred over naturally
occurring forms
because of properties such as enhanced cellular uptake, or increased stability
in the presence of
nucleases. The term also includes chimeric oligonucleotides which contain two
or more
chemically distinct regions. For example, chimeric oligonucleotides may
contain at least one
region of modified nucleotides that confer beneficial properties (e.g.
increased nuclease
resistance, increased uptake into cells), or two or more oligonucleotides of
the invention may
be joined to form a chimeric oligonucleotide.
The antisense oligonucleotides of the present invention may be ribonucleic or
deoxyribonucleic acids and may contain naturally occurnng bases including
adenine, guanine,
cytosine, thymidine and uracil. The oligonucleotides may also contain modified
bases such as
xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl
adenines, 5-halo
uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine,
pseudo uracil, 4-
thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl
adenines, 8-hydroxyl
adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-
thiol guanine, 8-
thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines,
other aza and deaza
uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl
uracil and 5-trifluoro
cytosine.
Other antisense oligonucleotides of the invention may contain modified
phosphorous,
oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl
intersugar
linkages or short chain heteroatomic or heterocyclic intersugar linkages. For
example, the
antisense oligonucleotides may contain phosphorothioates, phosphotriesters,
methyl
phosphonates, and phosphorodithioates. In an embodiment of the invention there
are
phosphorothioate bonds links between the four to six 3'-terminus bases. In
another
embodiment phosphorothioate bonds link all the nucleotides.
The antisense oligonucleotides of the invention may also comprise nucleotide
analogs
that may be better suited as therapeutic or experimental reagents. An example
of an
oligonucleotide analogue is a peptide nucleic acid (PNA) wherein the
deoxyribose (or ribose)
phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone
which is
similar to that found in peptides (P.E. Nielsen, et al Science 1991, 254,
1497). PNA analogues
have been shown to be resistant to degradation by enzymes and to have extended
lives in vivo
and in vitro. PNAs also bind stronger to a complimentary DNA sequence due to
the lack of
charge repulsion between the PNA strand and the DNA strand. Other
oligonucleotides may
contain nucleotides containing polymer backbones, cyclic backbones, or acyclic
backbones.
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For example, the nucleotides may have morpholino backbone structures (U.S.
Pat. Nol 5,034,
506). Oligonucleotides may also contain groups such as reporter groups, a
group for improving
the pharmacokinetic properties of an oligonucleotide, or a group for improving
the
pharmacodynamic properties of an antisense oligonucleotide. Antisense
oligonucleotides may
also have sugar mimetics.
The antisense nucleic acid molecules may be constructed using chemical
synthesis and
enzymatic ligation reactions using procedures known in the art. The antisense
nucleic acid
molecules of the invention or a fragment thereof, may be chemically
synthesized using
naturally occurnng nucleotides or variously modified nucleotides designed to
increase the
biological stability of the molecules or to increase the physical stability of
the duplex formed
with mRNA or the native gene e.g. phosphorothioate derivatives and acridine
substituted
nucleotides. The antisense sequences may be produced biologically using an
expression vector
introduced into cells in the form of a recombinant plasmid, phagemid or
attenuated virus in
which antisense sequences are produced under the control of a high efficiency
regulatory
region, the activity of which may be determined by the cell type into which
the vector is
introduced.
The antisense oligonucleotides may be introduced into tissues or cells using
techniques
in the art including vectors (retroviral vectors, adenoviral vectors and DNA
virus vectors) or
physical techniques such as microinjection. The antisense oligonucleotides may
be directly
administered in vivo or may be used to transfect cells in vitro which are then
administered in
vivo. In one embodiment, the antisense oligonucleotide of LPDL or LPDLR may be
delivered
to testis, hepatocytes and/or endothelial cells in a liposome formulation.
(iv) Diagnostic Assays
The finding by the present inventors that LPDL and LPDLR are involved in the
regulation of lipid and lipoprotein metabolism allows the detection of
conditions involving an
increase or decrease in LPDL or LPDLR activity or expression. Such conditions
include
disorders selected from the group consisting of lipase deficiency, lipoprotein
defects,
hypertriglyceridemia (primary genetic defect or secondary from other
diseases), fatty liver
diseases, cardiovascular disorders (including but not limited to
hypertriglyceridemia,
dyslipidemia, atherosclerosis, coronary artery disease, cerebrovascular
disease hypertension,
and peripheral vascular disease), body weight disorders (including but not
limited to obesity,
cachexia and anorexia), inflammation (including but not limited to sinusitis,
asthma,
pancreatitis, osteoarthritis, rheumatoid arthritis and acne), eczema,
Sjogren's syndrome,
gastrointestinal disorders, viral diseases and postviral fatigue, psychiatric
disorders, cancer,
cystic fibrosis, endometriosis, pre-menstrual syndrome, alcoholism, congenital
liver disease,
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Alzheimer's syndrome, hypercholesterolemia, autoimmune disorders, atopic
disorders, acute
respiratory distress syndrome, articular cartilage degradation, diabetes and
diabetic
complications. In preferred embodiments, the conditions are selected from the
group
consisting of lipase deficiency, atherosclerosis, fatty liver disease and
dyslipidemias, such as
hypercholesterolemia, hypertriglyceridemia, mixed (combined) dyslipidemia,
lipid or
lipoprotein deficient states, and/or any other tissue or plasma disorders of
lipid or lipoprotein
metabolism.
Accordingly, the present invention provides a method of detecting a condition
associated with increased or decreased LPDL or LPDLR expression or activity
(including an
absence) comprising assaying a sample for (a) a nucleic acid molecule encoding
a LPDL or
LPDLR protein or a fragment thereof or (b) a LPDL or LPDLR or a fragment
thereof. In one
embodiment, the condition associated with decreased LPDL or LPDLR expression
or activity
is hypertriglyceridemia.
(a) Nucleic acid molecules
The nucleic acid molecules encoding LPDL and LPDLR as described herein or
fragments thereof, allow those skilled in the art to construct nucleotide
probes for use in the
detection of nucleotide sequences encoding LPDL or LPDLR or fragments thereof
in samples,
preferably biological samples such as cells, tissues and bodily fluids. The
probes can be useful
in detecting the presence of a condition associated with LPDL or LPDLR or
monitoring the
progress of such a condition. Accordingly, the present invention provides a
method for
detecting a nucleic acid molecules encoding LPDL or LPDLR comprising
contacting the
sample with a nucleotide probe capable of hybridizing with the nucleic acid
molecule to form
a hybridization product, under conditions which permit the formation of the
hybridization
product, and assaying for the hybridization product.
Example of probes that may be used in the above method include the nucleic
acid
sequences shown in Figure 1 A (SEQ.1D.N0:1 ), Figure 2A (SEQ.ID.N0:3), Figure
2C
(SEQ.JI7.N0:5), Figure 3A (SEQ.ID.N0:7), Figure 4A (SEQ.)D.N0:9), Figure 4C
(SEQ.1D.N0:11), Figure 15 (SEQ.ID.N0.17), (SEQ.ID.N0.18), (SEQ.1D.N0.19),
(SEQ.ID.N0.20), (SEQ.ID.N0.21), (SEQ.>I7.N0.22), (SEQ.1D.N0.23),
(SEQ.ID.N0.24),
(SEQ.ID.N0.25),(SEQ.ID.N0.26);Figure 16-A (SEQ.ID.N0.27),(SEQ.ID.N0.28),
(SEQ.1D.N0.29),(SEQ.ID.N0.30),(SEQ.)D.N0.31), (SEQ.1D.N0.32),(SEQ.1D.N0.33),
(SEQ.ID.N0.34),(SEQ.ID.N0.35),(SEQ.1D.NO.36); Figure (SEQ.>D.N0:77),
21
(SEQ.ID.N0:78) or Figure 22 (SEQ.1D.N0:79), (SEQ.ID.N0:80) or fragments
thereof. A
nucleotide probe may be labelled with a detectable substance such as a
radioactive label which
provides for an adequate signal and has sufficient half life such as 32P, 3H,
14C or the like.
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Other detectable substances which may be used include antigens that are
recognized by a
specific labelled antibody, fluorescent compounds, enzymes, antibodies
specific for a labelled
antigen, and chemiluminescence. An appropriate label may be selected having
regard to the
rate of hybridization and binding of the probe to the nucleic acid to be
detected and the amount
of nucleic acid available for hybridization. Labelled probes may be hybridized
to nucleic acids
on solid supports such as nitrocellulose filters or nylon membranes as
generally described in
Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.). The
nucleotide
probes may be used to detect genes, preferably in human cells, that hybridize
to the nucleic
acid molecule of the present invention preferably, nucleic acid molecules
which hybridize to
the nucleic acid molecule of the invention under stringent hybridization
conditions as
described herein.
Nucleic acid molecules encoding a LPDL or LPDLR protein can be selectively
amplified in a sample using the polymerase chain reaction (PCR) methods and
cDNA or
genomic DNA. It is possible to design synthetic oligonucleotide primers from
the nucleotide
sequences shown in Figure lA (SEQ.ID.NO:1), Figure 2A (SEQ.ID.N0:3), Figure 2C
(SEQ.1D.N0:5), Figure 3A (SEQ.1D.N0:7), Figure 4A (SEQ.ID.N0:9), Figure 4C
(SEQ.1D.N0:11), Figure 15 (SEQ.ID.N0.17), (SEQ.ID.N0.18), (SEQ.1D.N0.19),
(SEQ.1D.N0.20), (SEQ.ID.N0.21), (SEQ.1D.N0.22), (SEQ.117.N0.23),
(SEQ.)D.N0.24),
(SEQ.1D.N0.25), (SEQ.ID.N0.26); Figure 16-A (SEQ.ID.N0.27), (SEQ.>D.N0.28),
(SEQ.1D.N0.29), (SEQ.ID.N0.30), (SEQ.1D.N0.31), (SEQ.ID.N0.32),
(SEQ.ID.N0.33),
(SEQ.>D.N0.34), (SEQ.ID.N0.35), (SEQ.ID.N0.36); Figure 21 (SEQ.ID.N0:77),
(SEQ.1D.N0:78) or Figure 22 (SEQ.B7.N0:79), (SEQ.ID.N0:80) for use in PCR. A
nucleic
acid can be amplified from cDNA or genomic DNA using oligonucleotide primers
and
standard PCR amplification techniques. The amplified nucleic acid can be
cloned into an
appropriate vector and characterized by DNA sequence analysis. cDNA may be
prepared from
mRNA, by isolating total cellular mRNA by a variety of techniques, for
example, by using the
guanidinium-thiocyanate extraction procedure of Chirgwin et al., Biochemistry,
18, 5294-5299
(1979). cDNA is then synthesized from the mRNA using reverse transcriptase
(for example,
Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or
AMV
reverse transcriptase available from Seikagaku America, Inc., St. Petersburg,
FL).
Genomic DNA may be used directly for detection of a specific sequence or may
be
amplified enzymatically in vitro by using PCR prior to analysis (Saiki et al.,
1985, Science,
230: 1350-1353 and Saiki et al., 1986, Nature, 324: 163-166). Reviews of this
subject have
been presented by Caskey C.T., 1989, Science, 236: 1223-1229 and by Landegren
et al., 1989,
Science, 242: 229-237. The detection of specific DNA sequence may be achieved
by methods
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such as hybridization using specific oligonucleotides (Wallace et al., 1986,
Cold Spring
Harbour Symp. Quant. Biol., 51: 257-261), direct DNA sequencing (Church et
al., 1988, Proc.
Natl. Acad. Sci., 81: 1991-1995, the use of restriction enzymes (Flavell et
al., 1978, Cell, 15:
25-41; Geever et al., 1981, Proc. Natl. Acad. Sci., 78: 5081-5085),
discrimination on the basis
of electrophoretic mobility in gels with denaturing reagent (Myers et al.,
1986, Cold Spring
Harbour Sym. Quant. Biol., 51: 275-284), RNase protection (Myers et al., 1985,
Science, 230:
1242-1246), chemical cleavage (Cotton et al., 1985, Proc. Natl. Acad. Sci.,
85: 4397-4401), and
the ligase-mediated detection procedure (Landegren et al., 1988, Science, 241:
1077-1080).
Using PCR, characterization of the level of or condition of the subject
polynucleotides present
in the individual may be made by comparative analysis.
With the characterization of the LPDL or LPDLR gene product and its function,
functional assays can also be used for LPDL or LPDLR gene diagnosis and
screening and to
monitor treatment. For example, enzymatic testing to determine levels of gene
function, rather
than direct screening of the LPDL or LPDLR gene or product, can be employed.
Testing of this
nature has been utilized in other diseases and conditions, such as in Tay-
Sachs.
The invention thus provides a process for detecting disease by using methods
known
in the art and methods described herein to detect changes in expression of or
mutations to the
subject polynucleotides. For example, decreased expression of a subject
polynucleotide can be
measured using any one of the methods well known in the art for the
quantification of
polynucleotides, such as, for example, PCR, RT-PCR, DNase protection, Northern
blotting
and other hybridization methods.
(b) Proteins
The LPDL or LPDLR protein may be detected in a sample using antibodies that
bind to
the protein as described in detail above. Accordingly, the present invention
provides a method
for detecting a LPDL or LPDLR protein comprising contacting the sample with an
antibody
that binds to LPDL or LPDLR which is capable of being detected after it
becomes bound to
the protein in the sample.
Antibodies specifically reactive with LPDL or LPDLR, or derivatives thereof,
such as
enzyme conjugates or labeled derivatives, may be used to detect LPDL or LPDLR
in various
biological materials, for example they may be used in any known immunoassays
which rely on
the binding interaction between an antigenic determinant of LPDL or LPDLR, and
the
antibodies. Examples of such assays are radioimmunoassays, enzyme immunoassays
(e.g.
ELISA), immunofluorescence, immunoprecipitation, latex agglutination,
hemagglutination and
histochemical tests. Thus, the antibodies may be used to detect and quantify
LPDL or LPDLR
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in a sample in order to determine its role in particular cellular events or
pathological states, and
to diagnose and treat such pathological states.
In particular, the antibodies of the invention may be used in immuno-
histochemical
analyses, for example, at the cellular and sub-subcellular level, to detect
LPDL or LPDLR, to
localise it to particular cells and tissues and to specific subcellular
locations, and to quantitate
the level of expression.
Cytochemical techniques known in the art for localizing antigens using light
and
electron microscopy may be used to detect LPDL or LPDLR. Generally,~an
antibody of the
invention may be labelled with a detectable substance and LPDL or LPDLR may be
localised
in tissue based upon the presence of the detectable substance. Examples of
detectable
substances include various enzymes, fluorescent materials, luminescent
materials and
radioactive materials. Examples of suitable enzymes include horseradish
peroxidase, biotin,
alkaline phosphatase, 13-galactosidase, or acetylcholinesterase; examples of
suitable fluorescent
materials include umbelliferone, fluorescein, fluorescein isothiocyanate,
rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a
luminescent material includes luminol; and examples of suitable radioactive
material include
radioactive iodine I-125, I-131 or 3-H. Antibodies may also be coupled to
electron dense
substances, such as ferritin or colloidal gold, which are readily visualised
by electron
microscopy.
Indirect methods may also be employed in which the primary antigen-antibody
reaction
is amplified by the introduction of a second antibody, having specificity for
the antibody
reactive against LPDL or LPDLR. By way of example, if the antibody having
specificity
against LPDL is a rabbit IgG antibody, the second antibody may be goat anti-
rabbit gamma-
globulin labelled with a detectable substance as described herein.
Where a radioactive label is used as a detectable substance, LPDL may be
localized by
autoradiography. The results of autoradiography may be quantitated by
determining the
density of particles in the autoradiographs by various optical methods, or by
counting the
grams.
(v) Screening for LPDL or LPDLR Mutations
Nucleic acid sequences of LPDL or LPDLR might be determined in order to assay
for
changes, preferably disease-causing mutations that may be used as indicators
of disease
prognosis or as aids to inform treatment of these diseases. Such diseases
include disorders
selected from the group consisting of lipase deficiency, lipoprotein defects,
hypertriglyceridemia (primary genetic defect or secondary from other
diseases), fatty liver
diseases, cardiovascular disorders (including but not limited to
hypertriglyceridemia,
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dyslipidemia, atherosclerosis, coronary artery disease, cerebrovascular
disease hypertension,
and peripheral vascular disease), body weight disorders (including but not
limited to obesity,
cachexia and anorexia), inflammation (including but not limited to sinusitis,
asthma,
pancreatitis, osteoarthritis, rheumatoid arthritis and acne), eczema,
Sjogren's syndrome,
gastrointestinal disorders, viral diseases and postviral fatigue, psychiatric
disorders, cancer,
cystic fibrosis, endometriosis, pre-menstrual syndrome, alcoholism, congenital
liver disease,
Alzheimer's syndrome, hypercholesterolemia, autoimmune disorders, atopic
disorders, acute
respiratory distress syndrome, articular cartilage degradation, diabetes and
diabetic
complications. In preferred embodiments, the conditions are selected from the
group
consisting of lipase deficiency, atherosclerosis, fatty liver disease and
dyslipidemias, such as
hypercholesterolemia, hypertriglyceridemia, mixed (combined) dyslipidemia,
lipid or
lipoprotein deficient states, and/or any other tissue or plasma disorders of
lipid or lipoprotein
metabolism.
The knowledge of the human LPDL and LPDLR sequences provides a method for
screening for diseases involving abnormally activated or inactivated LPDL or
LPDLR in
which the activity defect is due to a mutant LPDL or LPDLR gene. For example,
unregulated
Jak 3 kinase leads to tumorigenesis (Schwaller, J. et al., (1998), EMBO J., v.
17, p. 5321-33;
Lacronique et al., (1997), Science, v. 278, p. 1309-12; Peeters et al.,
(1997), Blood, v. 90, p.
2535-40). As discussed above, the LPDL and LPDLR proteins may play roles in
the
regulation of triglyceride activity and metabolism, lipoprotein metabolism,
energy
homeostatsis and other lipase related funtions. Patients may be screened
routinely using probes
to detect the presence of a mutant SAP gene by a variety of techniques.
Genomic DNA used
for the diagnosis may be obtained from body cells, such as those present in
the blood, tissue
biopsy, surgical specimen, or autopsy material. The DNA may be isolated and
used directly
for detection of a specific sequence or may be PCR amplified prior to
analysis. RNA or
cDNA may also be used. To detect a specific DNA sequence hybridization using
specific
oligonucleotides, direct DNA sequencing, restriction enzyme digest, RNase
protection,
chemical cleavage, and ligase-mediated detection are all methods which can be
utilized.
Oligonucleotides specific to mutant sequences can be chemically synthesized
and labelled
radioactively with isotopes, or non-radioactively using biotin tags, and
hybridized to individual
DNA samples immobilized on membranes or other solid-supports by dot-blot or
transfer from
gels after electrophoresis. The presence or absence of these mutant sequences
is then
visualized using methods such as autoradiography, fluorometry, or colorimetric
reaction.
Suitable PCR primers can be generated which are useful for example in
amplifying portions of
the subject sequence containing identified mutations.
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Direct DNA sequencing reveals sequence differences between normal and mutant
DNA. Cloned DNA segments may be used as probes to detect specific DNA
segments. PCR
can be used to enhance the sensitivity of this method. PCR is an enzymatic
amplification
directed by sequence-specific primers, and involves repeated cycles of heat
denaturation of the
DNA, annealing of the complementary primers and extension of the annealed
primer with a
DNA polymerase. This results in an exponential increase of the target DNA.
Other nucleotide
sequence amplification techniques may be used, such as ligation-mediated PCR,
anchored PCR
and enzymatic amplification as would be understood by those skilled in the
art.
As examples in amplifying LPDL and LPDLR genes, primers have been designed
within the adjacent intron regions of exons as listed in Figure 18-A for human
LPDL gene:
Primer 1F (SEQ.lD.N0.37), Primer 1R (SEQ.ID.N0.38), Primer 2F (SEQ.lD.N0.39),
Primer
2R (SEQ.ID.N0.40), Primer 3F (SEQ.ID.N0.41), Primer 3R (SEQ.ID.N0.42), Primer
4F
(SEQ.ID.N0.43), Primer 4R (SEQ.ID.N0.44), Primer SF (SEQ.>D.N0.45), Primer SR
(SEQ.ID.N0.46), Primer 6F (SEQ.ID.N0.47), Primer 6R (SEQ.ID.N0.48), Primer 7F
(SEQ.ID.N0.49), Primer 7R (SEQ.ID.NO.50), Primer 8F (SEQ.ID.NO.51), Primer 8R
(SEQ.>D.N0.52), Primer 9F (SEQ.ID.N0.53), Primer 9R (SEQ.1D.N0.54), Primer lOF
(SEQ.lD.N0.55), Primer l OR (SEQ.ID.N0.56), and in Figure 18-B for human LPDLR
gene:
Primer 1F (SEQ.1D.N0.57), Primer 1R (SEQ.ID.N0.58), Primer 2F (SEQ.>D.N0.59),
Primer
2R (SEQ.ID.N0.60), Primer 3F (SEQ.ID.N0.61), Primer 3R (SEQ.117.N0.62), Primer
4F
(SEQ.ID.N0.63),Primer (SEQ.ID.N0.64), 5F (SEQ.)D.N0.65), Primer
4R Primer SR
(SEQ.ID.N0.66),Primer (SEQ.ID.N0.67), 6R (SEQ.ID.N0.68), Primer
6F Primer 7F
(SEQ.ID.N0.69),Primer (SEQ.ID.N0.70), 8F (SEQ.ID.N0.71), Primer
7R Primer 8R
(SEQ.ID.N0.72),Primer (SEQ.ID.N0.73), 9R (SEQ.ID.N0.74), Primer
9F Primer lOF
(SEQ.ID.N0.75),Primer the primers could also
lOR be designed
(SEQ.ID.N0.76).
However,
elsewhere the introns,be disigned within
within the exons or
within the promoter
and
regulatory region.
Sequence alterations may also generate fortuitous restriction enzyme
recognition sites
that are revealed by the use of appropriate enzyme digestion followed by gel-
blot hybridization.
DNA fragments carrying the site (normal or mutant) are detected by their
increase or reduction
in size, or by the increase or decrease of corresponding restriction fragment
numbers. Genomic
DNA samples may also be amplified by PCR prior to treatment with the
appropriate restriction
enzyme and the fragments of different sizes are visualized under IJV light in
the presence of
ethidium bromide after gel electrophoresis.
Genetic testing based on DNA sequence differences may be achieved by detection
of
alteration in electrophoretic mobility of DNA fragments in gels. Small
sequence deletions and
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insertions can be visualized by high-resolution gel electrophoresis. Small
deletions may also
be detected as changes in the migration pattern of DNA heteroduplexes in non-
denaturing gel
electrophoresis. Alternatively, a single base substitution mutation may be
detected based on
differential primer length in PCR. The PCR products of the normal and mutant
gene could be
differentially detected in acrylamide gels.
Nuclease protection assays (S1 or ligase-mediated) also reveal sequence
changes at
specific locations. Alternatively, to confirm or detect a polymorphism
restriction mapping
changes ligated PCR, ASO, REF-SSCP and SSCP may be used. Both REF-SSCP and
SSCP
are mobility shift assays that are based upon the change in conformation due
to mutations.
DNA fragments may also be visualized by methods in which the individual DNA
samples are not immobilized on membranes. The probe and target sequences may
be in
solution or the probe sequence may be immobilized. Autoradiography,
radioactive decay,
spectrophotometry, and fluorometry may also be used to identify specific
individual genotypes.
According to an embodiment of the invention, the portion of the DNA segment
that is
informative for a mutation can be amplified using PCR. The DNA segment
immediately
surrounding a specific mutation acquired from peripheral blood or other tissue
samples from an
individual can be screened using constructed oligonucleotide primers. This
region would then
be amplified by PCR, the products separated by electrophoresis, and
transferred to membrane.
Labeled probes are then hybridized to the DNA fragments and autoradiography
performed.
(vi) LPDL and LPDLR uromoters and regulatory elements
The promoter and the regulatory elements of both LPDL and LPDLR genes might be
used to modify cellular process in controlling gene of choice for expression.
The controlled
gene expression can be tissue-specific. The inventors have described promoter
and regulatory
sequences of both LPDL or LPDLR gene as in Figure 21-A for mouse lpdl
(SEQ.l)7.N0:77);
21-B for human LPDL gene (SEQ.>D.N0:78); Figure 22-A for mouse lpdlr
(SEQ.ID.N0:79);
22-B for human LPDLR gene (SEQ.)D.N0:80); However, the regulatory sequences
may be
located upstream of the provided sequences, within the intron or exon
sequences or within the
3'UTR regrion. For example, the human HMGB1 promoter is modulated by a
silencer and an
enhancer-containing intron. With intron 1 included in the construct, the HMGB1
promter
activity can be increase at 2~3 folds (Lum et al. Biochim Biophys Acta, 1520,
79-84, 2001).
To use tissue specific promoter directing gene expression in desired tissue
have been
demonstrated be a successful strategy to modify cellular processes or achieve
targeted
therapeutic effect (Maxwell et al. Leuk Lymphoma. 7,457-62, 1992, Yu et al.
Cancer Gene
Ther., 8, 628-35, 2001). One example is to use prostate-specific antigen (PSA)
promoter to
direct cytotoxic gene diphtheria toxin (DT) to prostate tissue for prostate
cancer therapy. Such
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treatment preferentially kill PSA-positive prostate cancer cells in vitro, and
regressed tumor
growth and prolonged animal survival in vivo (Li et al. Cancer Res., 62, 2576-
82., 2002).
Since lpdl gene is strongly and specificly expressed in the primary
spermatocytes in
testis tissue, lpdl promoter can be employed to direct gene to testis tissue
to modify cellular
processes. One application is to target DT toxin gene to testis in eliminating
spermatocytes and
achieving male sterility. The LPDLR gene is expressed more widely in prostate,
testis, colon,
mammary and salivary gland. The LPDLR promoter can be further dissected to
identify the
tissue specific elements which can be used along or in combination with other
promoters in
targeting different tissues.
The delivery systems of promoter/fusion gene construct can be viral or non-
viral
delivery systems. The construct can be delivered to cells in vitro or in vivo
with either somatic
or stem cell treatment.
(vii) LPDL and LPDLR Modulators
In addition to antibodies and antisense oligonucleotides described above,
other
substances that modulate LPDL or LPDLR expression or activity may also be
identified.
Accordingly, the present invention includes the use of the nucleic acids
encoding LPDL and the
LPDLR and the respective proteins to develop or identify substances that
modulate LPDL or
LPDLR expression or activity.
(a) Substances that Bind LPDL or LPDLR
Substances that affect LPDL or LPDLR activity can be identified based on their
ability
to bind to either protein.
Substances which can bind with the LPDL or LPDLR of the invention may be
identified by reacting the LPDL or LPDLR with a substance which potentially
binds to LPDL
or LPDLR, and assaying for complexes, for free substance, or for non-complexed
LPDL or
LPDLR, or for activation of LPDL or LPDLR. In particular, a yeast two hybrid
assay system
may be used to identify proteins which interact with LPDL or LPDLR (Fields, S.
and Song, O.,
1989, Nature, 340:245-247). Systems of analysis which also may be used include
ELISA.
Accordingly, the invention provides a method of identifying substances which
can
bind with LPDL or LPDLR comprising the steps of:
- reacting LPDL or LPDLR and a test substance, under conditions which allow
for
formation of a complex between the LPDL or LPDLR and the test substance, and
- assaying for complexes of LPDL or LPDLR and the test substance, for free
substance or
for non complexed LPDL or LPDLR, wherein the presence of complexes indicates
that the
test substance is capable of binding LPDL. or LPDLR.
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The LPDL or LPDLR protein used in the assay may have the amino acid sequence
shown in Figure 1B or 2B may be a fragment, analog, derivative, homolog or
mimetic thereof
as described herein.
Conditions which permit the formation of substance and LPDL or LPDLR complexes
may be selected having regard to, factors such as the nature and amounts of
the substance and
the protein.
The substance-protein complex, free substance or non-complexed proteins may be
isolated by conventional isolation techniques, for example, salting out,
chromatography,
electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel
electrophoresis,
agglutination, or combinations thereof. To facilitate the assay of the
components, antibody
against LPDL or LPDLR or the substance; or labelled LPDL or LPDLR, or a
labelled substance
may be utilized. The antibodies, proteins, or substances may be labelled with
a detectable
substance as described above.
LPDL or LPDLR, or the substance used in the method of the invention may be
insolubilized. For example, LPDL or LPDLR or substance may be bound to a
suitable carrier.
Examples of suitable Garners are agarose, cellulose, dextran, Sephadex,
Sepharose,
carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic
film, plastic tube,
glass beads, polyamine-methyl vinyl-ether-malefic acid copolymer, amino acid
copolymer,
ethylene-malefic acid copolymer, nylon, silk, etc. The carrier may be in the
shape of, for
example, a tube, test plate, beads, disc, sphere etc.
The insolubilized protein or substance may be prepared by reacting the
material with a
suitable insoluble carrier using known chemical or physical methods, for
example, cyanogen
bromide coupling.
The proteins or substance may also be expressed on the surface of a cell using
the
methods described herein.
The invention also contemplates assaying for an antagonist or agonist of the
action of
LPDL or LPDLR.
It will be understood that the agonists and antagonists that can be assayed
using the
methods of the invention may act on one or more of the binding sites on the
protein or
substance including agonist binding sites, competitive antagonist binding
sites, non-competitive
antagonist binding sites or allosteric sites.
The invention also makes it possible to screen for antagonists that inhibit
the effects of
an agonist of LPDL or LPDLR. Thus, the invention may be used to assay for a
substance that
competes for the same binding site of LPDL.
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The invention further provides a method for assaying for a substance that
affects a
LPDL or LPDLR regulatory pathway comprising administering to a human or animal
or to a
cell, or a tissue of an animal, a substance suspected of affecting a LPDL or
LPDLR regulatory
pathway, and quantitating the LPDL or LPDLR protein or nucleic acids encoding
LPDL or
LPDLR, or examining the pattern and/or level of expression of LPDL or LPDLR,
in the human
or animal or tissue, or cell. LPDL or LPDLR may be quantitated and its
expression may be
examined using the methods described herein.
(b) Peptide Mimetics
The present invention also includes peptide mimetics of the LPDL or LPDLR of
the
invention. For example, a peptide derived from a binding domain of LPDL or
LPDLR will
interact directly or indirectly with an associated molecule in such a way as
to mimic the native
binding domain. Such peptides may include competitive inhibitors, enhancers,
peptide
mimetics, and the like. All of these peptides as well as molecules
substantially homologous,
complementary or otherwise functionally or structurally equivalent to these
peptides may be
used for purposes of the present invention.
"Peptide mimetics" are structures which serve as substitutes for peptides in
interactions
between molecules (See Morgan et al (1989), Ann. Reports Med. Chem. 24:243-252
for a
review). Peptide mimetics include synthetic structures which may or may not
contain amino
acids and/or peptide bonds but retain the structural and functional features
of a peptide, or
enhancer or inhibitor of the invention. Peptide mimetics also include
peptoids, oligopeptoids
(Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367), and peptide libraries
containing
peptides of a designed length representing all possible sequences of amino
acids corresponding
to a peptide of the invention.
Peptide mimetics may be designed based on information obtained by systematic
replacement of L-amino acids by D-amino acids, replacement of side chains with
groups
having different electronic properties, and by systematic replacement of
peptide bonds with
amide bond replacements. Local conformational constraints can also be
introduced to
determine conformational requirements for activity of a candidate peptide
mimetic. The
mimetics may include isosteric amide bonds, or D-amino acids to stabilize or
promote reverse
turn conformations and to help stabilize the molecule. Cyclic amino acid
analogues may be
used to constrain amino acid residues to particular conformational states. The
mimetics can
also include mimics of inhibitor peptide secondary structures. These
structures can model the
3-dimensional orientation of amino acid residues into the known secondary
conformations of
proteins. Peptoids may also be used which are oligomers of N-substituted amino
acids and can
be used as motifs for the generation of chemically diverse libraries of novel
molecules.
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Peptides of the invention may also be used to identify lead compounds for drug
development. The structure of the peptides described herein can be readily
determined by a
number of methods such as NMR and X-ray crystallography. A comparison of the
structures of
peptides similar in sequence, but differing in the biological activities they
elicit in target
molecules can provide information about the structure-activity relationship of
the target.
Information obtained from the examination of structure-activity relationships
can be used to
design either modified peptides, or other small molecules or lead compounds
that can be tested
for predicted properties as related to the target molecule. The activity of
the lead compounds
can be evaluated using assays similar to those described herein.
Information about structure-activity relationships may also be obtained from
co-
crystallization studies. In these studies, a peptide with a desired activity
is crystallized in
association with a target molecule, and the X-ray structure of the complex is
determined. The
structure can then be compared to the structure of the target molecule in its
native state, and
information from such a comparison may be used to design compounds expected to
possess.
(c) Modulation of the LPDL or LPDLR Promoter
As would be readily apparent to those skilled in the art, it is also possible
to modulate
LPDL or LPDLR through manipulation of their promoters. One or more alterations
to a
promoter sequence of LPDL or LPDLR may increase or decrease promoter activity,
or increase
or decrease the magnitude of the effect of a substance able to modulate the
promoter activity.
"Promoter activity" is used to refer to the ability to initiate transcription.
The level of
promoter activity is quantifiable for instance by assessment of the amount of
mRNA produced
by transcription from the promoter or by assessment of the amount of protein
product produced
by translation of mRNA produced by transcription from the promoter controlling
genes such as
reporter gene 13-galactosidase. The amount of a specific mRNA present in an
expression
system may be determined for example using specific oligonucleotides which are
able to
hybridize with the mRNA and which are labelled or may be used in a specific
amplification
reaction such as the polymerase chain reaction. In vivo promoter activity
assay can be
investigated by transgenic mouse system. In such embodiment, the LPDL promoter
controlled
f3-galactosidase constructs will be introduced into mouse embryoes and the
activity of the
reporter gene expression in different tissues including testis may be studied.
Substances which affect the LPDL or LPDLR promoter activity may also be
identified
using the methods of the invention by comparing the pattern and level of
expression of a
reporter gene, in cells in the presence, and in the absence of the substance.
Accordingly, a
method for assaying for the presence of an agonist or antagonist of LPDL or
LPDLR promoter
activity is provided comprising providing a cell containing a reporter gene
under the control of
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the promoter with a substance which is a suspected agonist or antagonist under
conditions
which permit interaction and assaying for the increase or decrease of reporter
gene product.
(d) Drug Screening Methods
In accordance with one embodiment, the. invention enables a method for
screening
candidate compounds for their ability to increase or decrease the activity of
a LPDL or LPDLR
protein. The method comprises providing an assay system for assaying LPDL or
LPDLR
activity, assaying the activity in the presence or absence of the candidate or
test compound and
determining whether the compound has increased or decreased LPDL or LPDLR
activity.
Accordingly, the present invention provides a method for identifying a
compound that
affects LPDL or LPDLR protein activity or expression comprising:
(a) incubating a test compound with a LPDL or LPDLR protein or a nucleic acid
encoding a LPDL or LPDLR protein, and
(b) determining an amount of LPDL or LPDLR protein activity or expression and
comparing with a control (i.e. in the absence of the test substance), wherein
a
change in the LPDL or LPDLR protein activity or expression as compared to
the control indicates that the test compound has an effect on LPDL or LPDLR
protein activity or expression.
In accordance with a further embodiment, the invention enables a method for
screening
candidate compounds for their ability to increase or decrease expression of a
LPDL or LPDLR
protein. The method comprises putting a cell with a candidate compound,
wherein the cell
includes a regulatory region of a LPDL or LPDLR gene operably joined to a
reporter gene
coding region, and detecting a change in expression of the reporter gene.
In one embodiment, the present invention enables culture systems in which cell
lines
which express the LPDL or LPDLR gene, and thus LPDL or LPDLR protein products,
are
incubated with candidate compounds to test their effects on expression. Such
culture systems
can be used to identify compounds which upregulate or downregulate LPDL or
LPDLR
expression or function, through the interaction with other proteins.
Such compounds can be selected from protein compounds, chemicals and various
drugs that are added to the culture medium. After a period of incubation in
the presence of a
selected test compound(s), the expression of LPDL or LPDLR can be examined by
quantifying
the levels of LPDL or LPDLR mRNA using standard Northern blotting procedure,
as described
in the examples included herein, to determine any changes in expression as a
result of the test
compound. Cell lines transfected with constructs expressing LPDL or LPDLR can
also be used
to test the function of compounds developed to modify the protein expression.
In addition,
transformed cell lines expressing a normal LPDL or LPDLR protein could be
mutagenized by
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the use of mutagenizing agents to produce an altered phenotype in which the
role of mutated
LPDL or LPDLR can be studied in order to study structure/function
relationships of the protein
products and their physiological effects.
Alternatively, rather than evaluating the levels of LPDL or LPDLR expression
in the
presence of a test compound, other proteins which interact with the LPDL or
LPDLR protein
products may be assessed through signal transduction assays, such as are well
known in the art.
Such assays would identify the impact of certain compounds on LPDL or LPDLR
function and
subsequent intracellular protein interaction and physiological effect.
The present invention also includes screening compounds for their ability to
affect the
interaction between LPDL or LPDLR and their binding partners.
Accordingly, the present invention provides a method for identifying a
compound that
affects the binding of an LPDL or LPDLR protein and an LPDL or LPDLR binding
protein
comprising:
(a) incubating (i) a test compound, (ii) an LPDL or LPDLR protein and (iii) an
LPDL or LPDLR binding protein under conditions which permit the binding of
LPDL or LPDLR protein to the LPDL or LPDLR binding protein, and
(b) assaying for complexes of LPDL or LPDLR protein and the LPDL or LPDLR
binding protein and comparing to a control (i.e. in the absence of the test
substance), wherein a reduction of complexes indicates that the compound has
an effect on the binding of the LPDL or LPDLR protein to an LPDL or
LPDLR binding protein.
All testing for novel drug development is well suited to defined cell culture
systems
which can be manipulated to express LPDL or LPDLR and study the result of LPDL
or
LPDLR protein signaling and gene transcription. Animal models are also
important for testing
novel drugs and thus may also be used to identify any potentially useful
compound affecting
LPDL or LPDLR expression and activity and thus physiological function.
(viii) Industrial Uses
Dietary fats have important effects on human health and disease. The efficient
digestion of dietary fats (triglycerides) can be achieved by a group of lipase
proteins which are
secreted into digestive tracts. They include lingual, gastric and pancreatic
lipases (Hamosh
Nutrition. 6, 421-8.,1990, Lowe, J Nutr. 127, 549-57. 1997). Although lipase
faction is
important for normal lipid metabolism, inhibition of its faction can also be
used in treating
disease state. For example, an inhibitor of gastric lipases, Ro 18-
tetrahydrolipstatin, was
identifed and tested in treating obese patients (Hauptman et al. Am J Clin
Nutr. 55, 3095-13S
1992). The regulation of dietary lipid in digestive system is complicated. For
example,
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Knochout of carboxyl ester lipase decreased intestinal absorption of dietary
cholesteryl ester
but retinyl ester absorption is normal (Weng et al. Biochemistry. 38, 4143-9.
1992).
Because LPDLR lipase is expressed in the colon, mammary and salivary glands,
it
functions in promoting dietary lipid digestion and energy intake. Accordingly,
increase of
LPDLR lipase activity in the digestive tracts should help energy intake and
gaining weight
while disruption of LPDLR function should have negative effect on body energy
metabolism
and inducing weight loss. In food industry, LPDLR lipase might be served as a
nutriment or
food additive in improving health conditions. Alternatively, modulation or
cancellation of
LPDLR lipase function may help controlling body weight for obese patients.
LPDLR lipase
treatment or modulation could be applyed alone or combined with other lipases
such as gastric
lipase or LPDL. In a similar manner, modulation of lipase function can be used
in meat
industry in controlling leanness of animals such as pig, cow and chiken.
In oil and waste management industry, lipase can be used for cleaning of lipid
contamination. For example, enzymatic kinetics of continuous hydrolysis of
palm oil
triglyceride in organic solvent using a source of immobilized lipase was
studied in packed bed
reactor (Min et al., Artif Cells Blood Substit Immobil Biotechnol. 27, 417-21,
1999). Another
group used continuous cultivation technique screening for lipase-producing
microorganisms
suitable for the degradation of domestic wastes and interesterification of
butter fat by lipase
isolates (Pabai et al. Can J Microbiol. 42, 446-52, 1996). The LPDL and LPDLR
lipases might be
used in oil industry or in waste management. They may be used alone or in
combination with
each other or with other mammalian or bacteria lipases.
(ix) Therapeutic Uses
As previously discussed, the LPDL or LPDLR proteins of the invention are
likely
involved in the regulation of triglyceride metabolism, lipoprotein metabolism
and energy
homeostasis. Accordingly, the present invention provides a method of
modulating triglyceride,
lipoprotein metabolism and energy homeostasis comprising of administering to a
cell or animal
in need thereof, an effective amount of agent that modulates LPDL or LPDLR
expression
and/or activity. Apart from applying LPDL and/or LPDLR genes or proteins, the
invention also
includes a use of an agent that modulates LPDL or LPDLR expression or activity
to modulate
triglyceride metabolism, lipoprotein metabolism and energy homeostasis, or to
prepare a
medicament to modulate triglyceride, lipoprotein metabolism and energy
homeostasis.
The term "agent that modulates LPDL or LPDLR expression and/or activity" means
any substance that can alter the expression and/or activity of LPDL or LPDLR
and includes
agents that can inhibit LPDL or LPDLR expression or activity and agents that
can enhance
LPDL or LPDLR expression or activity. Examples of agents which may be used to
modulate
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LPDL or LPDLR include nucleic acid molecules encoding LPDL or LPDLR, the LPDL
or
LPDLR protein as well as fragments, analogs, derivatives or homologs thereof,
antibodies,
antisense nucleic acids, peptide mimetics, substances isolated using the
screening methods
described herein or substances that modulate the interaction of LPDL or LPDLR
with LPDL
OR LPDLR associating or binding proteins.
The term "effective amount" as used herein means an amount effective, at
dosages and
for periods of time necessary to achieve the desired results.
The term "animal" as used herein includes all members of the animal kingdom,
including humans.
As stated previously, LPDL or LPDLR may be involved in modulating triglyceride
activity and metabolism and stimulators and inhibitors of LPDL or LPDLR may be
useful in
modulating disorders involving triglyceride activity such as
hypertriglyceridemia. For example,
substances that stimulate LPDL (for example, identified using the methods of
the invention)
may be used to prevent hypertriglyceridemia and the diseases caused by
hyertriglyceridemia,
such as atherosclerosis. Inhibitors could be used where increased triglyceride
levels would
advantageous.
In one embodiment, the invention provide a methods of treating lipase
deficiencies,
fatty livers, hypertriglyceridemia, lipoprotein metabolism defects, preventing
and treating
atheroscrelosis and cardiovascular diseases, by administering to a cell or
animal an effective
amount of an agent that modulates, preferably stimulate, the expression or the
biological
activity of LPDL or LPDL, such that there is a reduction in triglyceride
activity. The diseases
treated not only include those genetic defects in lipid metabolism but also
the secondary
diseases result from other primary defects such as the dyslipidemia from
diabetes or lipoprotein
defects.
In another embodiment, the invention provides methods of modifying the body
energy
homeostasis. For example, hormone sensitive lipase (HSL) hydrolyzes the
triglycerides in
white fat mass and increases the body energy consumption (Kahn Nature Genet.
25, 6-9, 2000).
One study demonstrated the lean and obese women responded very differently in
a
carbonhydrate load and the plasma triglycerides were significantly higher in
obese women than
in the lean controls (Dallongeville et al., J Nutr. 132, 2161-6., 2002). In
one example, LPDL
and LPDLR lipases can be used to increase the body energy metabolism,
accelerate
triglycerides comsumption and decrease the body weight or prevent the obese
people from
gaining more weight. In another mechanism, HSL, LPDL and/or LPDLR lipase
function can be
abolished or decreased to help lean body slowing down energy comsuption and
gain weight.
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The therapy includes but not limits to the following diseases: lipase
deficiency,
lipoprotein defects, hypertriglyceridemia (primary genetic defect or secondary
from other
diseases), fatty liver diseases, cardiovascular disorders (including but not
limited to
hypertriglyceridemia, dyslipidemia, atherosclerosis, coronary artery disease,
cerebrovascular
disease hypertension, and peripheral vascular disease), body weight disorders
(including but
not limited to obesity, cachexia and anorexia), inflammation (including but
not limited to
sinusitis, asthma, pancreatitis, osteoarthritis, rheumatoid arthritis and
acne), eczema, Sjogren's
syndrome, gastrointestinal disorders, viral diseases and postviral fatigue,
psychiatric disorders,
cancer, cystic fibrosis, endometriosis, pre-menstrual syndrome, alcoholism,
congenital liver
disease, Alzheimer's syndrome, hypercholesterolemia, autoimmune disorders,
atopic disorders,
acute respiratory distress syndrome, articular cartilage degradation, diabetes
and diabetic
complications. In preferred embodiments, the conditions are selected from the
group consisting
of lipase deficiency, lipoprotein defects, hypertriglyceridemia, high
cholesterol, atherosclerosis,
fatty liver disease, cardiovascular diseases, hyper triglyceride metabolism
and hypo triglyceride
metabolism.
In accordance with another embodiment, the present invention enables gene
therapy as
a potential therapeutic approach, in which normal copies of the LPDL or LPDLR
gene are
introduced into patients to successfully code for normal LPDL or LPDLR protein
in several
different affected cell types. Mutated copies of the LPDL or LPDLR gene, in
which the protein
product is changed, can also be introduced into patients.
Retroviral vectors can be used for somatic cell gene therapy especially
because of their
high efficiency of infection and stable integration and expression. The
targeted cells however
must be able to divide and the expression of the.levels of normal protein
should be high. The
full length LPDL or LPDLR gene can be cloned into a retroviral vector and
driven from its
endogenous promoter or from the retroviral long terminal repeat or from a
promoter specific
for the target cell type of interest. Other viral vectors which can be used
include lentivirus,
adenovirus, adeno-associated virus, vaccinia virus, bovine papilloma virus, or
a herpesvirus
such as Epstein-Barr virus. Gene transfer could also be achieved using non-
viral means
requiring infection in vitro. This would include calcium phosphate, DEAF
dextran,
electroporation, cationic or anionic lipid formulations (liposomes) and
protoplast fusion.
Although these methods are available, many of these are lower efficiency.
Transplantation of normal genes or mutated genes that code for an active LPDL
or
LPDLR into a targeted affected area of the patient can also be useful therapy
for any disorder in
which LPDL or LPDLR activity is deficient. In this procedure, a LPDL or LPDLR
gene is
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transferred into a cultivatable cell type such as hepatocytes and testis
cells. The transformed
cells are then injected into the patient.
The invention also provides a method for reversing a transformed phenotype
that
results from excessive expression from the LPDL or LPDLR human gene sequence,
and/or
hyper-activation of a LPDL or LPDLR protein product. Anti-sense based
strategies can be
employed to explore gene function, inhibit gene function and as a basis for
therapeutic drug
design. The principle is based on the hypothesis that sequence specific
suppression of gene
expression can be achieved by intracellular hybridization between mRNA and a
complementary anti-sense species. It is possible to synthesize anti-sense
strand nucleotides that
bind the sense strand of RNA or DNA with a high degree of specificity. The
formation of a
hybrid RNA duplex may interfere with the processing/transport/translation
and/or stability of a
target mRNA.
Hybridization is required for an antisense effect to occur. Antisense effects
have been
described using a variety of approaches including the use of antisense
oligonucleotides,
injection of antisense RNA, DNA and transfection of antisense RNA expression
vectors.
Therapeutic antisense nucleotides can be made as oligonucleotides or expressed
nucleotides. Oligonucleotides are short single strands of DNA which are
usually 15 to 20
nucleic acid bases long. Expressed nucleotides are made by an expression
vector such as an
adenoviral, retroviral or plasmid vector. The vector is administered to the
cells in culture, or to
a patient, whose cells then make the antisense nucleotide. Expression vectors
can be designed
to produce antisense RNA, which can vary in length from a few dozen bases to
several
thousand.
Antisense effects can be induced by control (sense) sequences. The extent of
phenotypic changes are highly variable. Phenotypic effects induced by
antisense are based on
changes in criteria such as biological endpoints, protein levels, protein
activation measurement
and target mRNA levels.
In the present invention, mammalian cells in which the LPDL or LPDLR gene is
overexpressed and which demonstrate an abnormal phenotype, can be transfected
with anti-
sense LPDL or LPDLR nucleotide DNA sequences that hybridizes to the LPDL or
LPDLR
gene in order to inhibit the transcription of the gene and reverse or reduce
the abnormal
phenotype. Expression vectors can be used as a model for anti-sense gene
therapy to target the
LPDL or LPDLR which is expressed in abnormal cells. In this manner abnormal
cells and
tissues can be targeted while allowing healthy cells to survive. This may
prove to be an
effective treatment for cell abnormalities induced by LPDL or LPDLR.
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Immunotherapy is also possible for the treatment of diseases associated with
excess
LPDL or LPDLR activity. Antibodies can be raised to a hyperactive LPDL or
LPDLR protein
(or portion thereof) and then be administered to bind or block the abnormal
protein and its
deleterious effects. An immunogenic composition may be prepared as
injectables, as liquid
solutions or emulsions. The LPDL or LPDLR protein may be mixed with
pharmaceutically
acceptable excipients compatible with the protein. Such excipients may include
water, saline,
dextrose, glycerol, ethanol and combinations thereof. The immunogenic
composition and
vaccine may further contain auxiliary substances such as emulsifying agents or
adjuvants to
enhance effectiveness. Immunogenic compositions and vaccines may be
administered by
subcutaneous or intramuscular injection. The immunogenic preparations and
vaccines are
administered in such amount as will be therapeutically effective, protective
and immunogenic.
Dosage depends on the route of administration and will vary according to the
size of the host.
The invention also makes it possible to screen for antagonists that inhibit
the effects of
LPDL or LPDLR. Thus, the invention may be used to assay for a substance that
anatagonizes
or blocks the action of the proteins.
Substances identified by the methods described herein, may be used for
modulating
LPDL or LPDLR activity or action and accordingly may be used in the treatment
of conditions
involving perturbation of the protein. In particular, the substances may be
particularly useful in
the treatment of disorders of hematopoietic cell proliferation.
(viii) Pharmaceutical Compositions
The above described substances including LPDL and LPDLR proteins, nucleic
acids
encoding LPDL or LPDLR proteins, antibodies, and antisense oligonucleotides as
well as
other agents that modulate LPDL or LPDLR may be formulated into pharmaceutical
compositions for administration to subjects in a biologically compatible form
suitable for
administration in vivo for the treatment of various conditions. Such
conditions include
disorders selected from the group consisting of eczema, cardiovascular
disorders (including
but not limited to hypertriglyceridemia, dyslipidemia, atherosclerosis,
coronary artery disease,
cerebrovascular disease hypertension, and peripheral vascular disease),
inflammation
(including but not limited to sinusitis, asthma, pancreatitis, osteoarthritis,
rheumatoid arthritis
and acne), Sjogren's syndrome, gastrointestinal disorders, viral diseases and
postviral fatigue,
body weight disorders (including but not limited to obesity, cachexia and
anorexia),
psychiatric disorders, cancer, cystic fibrosis, endometriosis, pre-menstrual
syndrome,
alcoholism, congenital liver disease, Alzheimer's syndrome,
hypercholesterolemia,
autoimmune disorders, atopic disorders, acute respiratory distress syndrome,
articular cartilage
degradation, diabetes and diabetic complications. In preferred embodiments,
the conditions are
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selected from the group consisting of high cholesterol, hypertriglyceridemia,
atherogenesis,
fatty liver disease, hyper triglyceride metabolism and hypo triglyceride
metabolism.
By "biologically compatible form suitable for administration in vivo" is meant
a form
of the substance to be administered in which any toxic effects are outweighed
by the
therapeutic effects. The substances may be administered to living organisms
including
humans, and animals.
Administration of a therapeutically active amount of pharmaceutical
compositions of
the present invention is defined as an amount effective, at dosages and for
periods of time
necessary to achieve the desired result. For example, a therapeutically active
amount of a
substance may vary according to factors such as the disease state, age, sex,
and weight of the
individual, and the ability of the substance to elicit a desired response in
the individual.
Dosage regimes may be adjusted to provide the optimum therapeutic response.
For example,
several divided doses may be administered daily or the dose may be
proportionally reduced as
indicated by the exigencies of the therapeutic situation.
An active substance may be administered in a convenient manner such as by
injection
(subcutaneous, intravenous, etc.), oral administration, inhalation,
transdermal~ application, or
rectal administration. Depending on the route of administration, the active
substance may be
coated in a material to protect the compound from the action of enzymes, acids
and other
natural conditions which may inactivate the compound. If the active substance
is a nucleic acid
encoding, for example, a modified LPDL or LPDLR it may be delivered using
techniques
known in the art.
The compositions described herein can be prepared by per se known methods for
the
preparation of pharmaceutically acceptable compositions which can be
administered to
subjects, such that an effective quantity of the active substance is combined
in a mixture with a
pharmaceutically acceptable vehicle. Suitable vehicles are described, for
example, in
Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack
Publishing
Company, Easton, Pa., USA 1985) or Handbook of Pharmaceutical Additives
(compiled by
Michael and Irene Ash, Gower Publishing Limited, Aldershot, England (1995)).
On this basis,
the compositions include, albeit not exclusively, solutions of the substances
in association with
one or more pharmaceutically acceptable vehicles or diluents, and may be
contained in buffered
solutions with a suitable pH and/or be iso-osmotic with physiological' fluids.
In this regard,
reference can be made to U.S. Patent No. 5,843,456. As will also be
appreciated by those
skilled, administration of substances described herein may be by an inactive
viral carrier.
The above disclosure generally describes the present invention. A more
complete
understanding can be obtained by reference to the following specific examples.
These
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examples are described solely for the purpose of illustration and are not
intended to limit the
scope of the invention. Changes in form and substitution of equivalents are
contemplated as
circumstances might suggest or render expedient. Although specific terms have
been employed
herein, such terms are intended in a descriptive sense and not for purposes of
limitation.
In one embodiment, transgenic cells containing genes of the invention can be
prepared
and used for introduction into a patient. In preparing cells for transfection
and subsequent
introduction into a patient's system, it is preferred to start with somatic
mammalian cells
obtained from the eventual recipient of the cell-based gene transfer treatment
of then present
invention. A wide variety of different cell types may be used, including
fibroblasts, endothelial
cells, smooth muscle cells, progenitor cells (e.g. from bone marrow, adipose,
or peripheral
blood), dermal fibroblasts, EPC (endothelial progenitor cells) or other
mesenchymal cells,
marrow stromal cells (MSC), and epithelial cells, and others. Dermal
fibroblasts are simply
and readily obtained from the patient's exterior skin layers, readied for in
vitro culturing by
standard techniques. Endothelial cells are harvested from the eventual
recipient, e.g. by
removal of a saphenous vein and culture of the endothelial cells. Progenitor
cells can be
obtained from bone marrow biopsies or isolated from the circulating blood, and
cultured in
vitro. The culture methods are standard culture techniques with special
precautions for
culturing of human cells with the intent of re-implantation.
The somatic gene transfer in vitro to the recipient cells, i.e. the genetic
engineering, is
performed by standard and commercially available approaches to achieve gene
transfer, as
outlined above. Preferably, the methods include electroporation, the use of
poly cationic
proteins (e.g. SUPERFECT*) or lipofection (e.g. by use of GENEFECTOR), agents
available
commercially and which enhance gene transfer. In particular, electroporation
provides a high
degree of transfection and does not require the use of any foreign material.
However, other
methods besides electroporation, lipofection and polycationic protein use,
such as viral
methods of gene transfer including adeno and retro viruses, may be employed.
These methods
and techniques are well known to those skilled in the art, and are readily
adapted for use in the
process of the present invention. Electroporation is the most preferred
technique, for use with
dermal fibroblast host cells, while the use of polycationic proteins is useful
for use with smooth
muscle cells.
The following non-limiting examples are illustrative of the present invention:
EXAMPLES
Example 1
Sequencing of the lpd locus and bioinformatic identification and cloning of a
novel lipase
(lpdl)
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Because mutant lpd homozygotes had hepatic steatosis and hypertriglyceridemia,
we
hypothesized that the murine lpd locus would encode a TG lipase. Since
analysis of the
junction sequences of the lpd transgene insertion locus did not yield any
lipase-related
sequences and the mouse genetic sequencing database was not available at that
time, we chose
to clone the entire wild-type lpd locus with bacteria artificial chromosomes
(BACs) that
encompassed the effected region. By screening a wild-type mouse genomic BAC
library with
two probes that flanked each side of the transgene, three BAC clones
containing the lpd locus
were identified. Using shotgun strategy, we sequenced one BAC clone (BAC#016)
with ~3-
fold redundancy. 719 sequences for a total of 449,373 base pairs (bp) were
randomly
sequenced. These sequences formed 93 contigs for a total contig length of
163,372 by (Figure
5A). We used the BLASTX engine (http://www.ncbi.nlm.nih.gov) to interrogate
all non-
redundant GenBank sequences with the 93 contigs and identified a fragment in
contig #6 that
had significant homology to a portion of both human and rat phosphatidylserine-
specific
phospholipase A1 (PS PLAI) with ~50% identity at the protein sequence level.
Following the identification of this lipase-related sequence, bioinformatic
gene prediction
tools were used to identify five putative exons from four contigs [#6 (Figure
6), #28 (Figure
7), #86 (Figure 8) and #98 (Figure 9)], which translated as a continuous
putative protein
sequence of 261 amino acid (designated as lpd lipase or lpdl) that includes
the highly
conserved Gly-Met-Ser-Leu-Gly lipase consensus sequences (Figure 5B&C).
With the availability of murine LPDL exon sequences, the inventors tried to
identify its
mouse and human ESTs in the published genetic databases but no ESTs with
significant
homology were found. Based on the predicted exon sequences of murine LPDL,
primers were
then designed to clone the cDNA fragment of murine LPDL and a 0.6 kb fragment
was
generated by RT-PCR from mouse testis sample cDNA. The upstream and downstream
sequences of the 0.6kb fragment were cloned by 5'RACE and 3'RACE (Rapid
Amplification
of cDNA End). All the cloned fragments are sequenced and assembled which
represents
mouse lpdl cDNA sequence of 2,056 by in length. Mouse LPDL gene encodes a LPDL
protein
of 423 amino acids (Figure 2B).
To clone the mouse full length lpdl gene, the inventors performed PCR cloning
in mouse
Marathon-Ready Testis cDNA (Clontech, Cat. No. 74551-1) with primers designed
from the
above assembled mouse lpdl cDNA sequences. The sequence of 5'-primer,
lpd5'UTR1, is:
CCGTCCTTCCCACTTGATTA, the sequence 3'-primer, lpd-Full-3R2, is:
GGTTGAAGATCTACCCTTGTTCC. A cDNA of 1,383 by (Figure 2C) was cloned which
encodes a lipase protein (lpdl2) of 407 amino acids (Figure 2D). Since the
human LPDL
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protein has 460 amino acids, the two mouse lpdl proteins identified here are
much shorter and
could be different spliced isoforms of the lpdl gene.
Example 2
Gene expression of LPDL and LPDLR gene
To identify the gene expression pattern, the inventors completed a mouse
multiple
tissue Northern blot analysis. With probes generated from predicted exon
sequences of the
genomic BAC clone, the inventors successfully detected a 2 kb band in testis
RNA but not in
any other adult mouse tissues examined including heart, brain, spleen, lung,
liver, skeleton
muscle and kidney (Fig. lOAa). By non-radiation RNA in situ hybridization with
DIG-labeled
anti-sense probe (Schaeren-Wiemers et al. 1993), mouse lpdl expression was
strongly detected
in testis (Figure 10 B-b) and weakly in the liver (Figure 10 B-d) in two week-
old mice as
compared to control tissue sections hybridized with sense probe (Figure 10 B-
a&c) In adult
mice, lpdl expression was detected in the cytoplasm of primary spermatocytes
but not in the
matured sperm or Leydig cells between the seminiferous tubules (Figure 10 B-
d).
With human LPDL gene as probe, Northern blotting also showed that human LPDL
was expressed in testis (Figure 10 A-b). However, for the human LPDLR gene,
Northern blot
hybridization suggested that it was highly expressed in colon, prostate and
testis with four
different sized isoforms ranging from 2 to 4 kb. The strongly expressed
isoform in colon was
~3 kb, whereas in testis the ~4 kb transcript was highly expressed. The ~3 and
4 'kb transcripts
were expressed at comparable levels in prostate (Figure 10 A-c).
Like LPDL, other lipases such as EL and HSL are also highly expressed in the
testis,
which may reflect higher TG energy metabolism (Hirata et al., 1999, Mairal et
al. 2002,
Haemmerle et al 2002). Hepatic expression of LPDL in two week-old mice
supports the
hepatic phenotype in the lpd mutant mice. Apart from the expression in testis
and prostate,
human LPDLR is expressed in colon, and ESTs of mouse lpdlr had also been
identified from
salivary gland and mammary gland suggesting a role in digesting exogenous
dietary TG.
Example 3
Cloning of human LPDL cDNA
Using mouse lpdl gene fragments as probes screening a human testis large
insert
cDNA library (Cat. No. HL5503u, Clontech, Palo Alto, California), four
positive clones have
been identified. Sequencing of these clones revealed a cDNA of 1685 by in
length. The open
reading frame (O1RF) was found to be 1383 by in size (Figure lA) with a start
codon (ATG) at
nucleotide 78 and stop codon (TAG) at nucleotide 1640. The ORF encodes a human
LPDL
protein of 460 amino acids (Figure 1B and Figure 11). A hydrophobic leader
sequence with a
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putative cleavage site after amino acid residue 15 was predicted by SPScan
program of
SeqWeb Wisconsin GCG Package. The lipase consensus sequence GXSXG was found
with an
active serine at amino acid residue 159. Sequence analysis suggested the
existence of two
additional active residues, Asp-183 and His-258 that are predicted to form a
catalytic triad
with Ser-159 (Emmerich et al. 1992). A lipase lid sequence has also been
identified between
two cysteine residues at 238 and 251 and likely functions to determine
substract specificity
(Dugi et al. 1995). Seven conserved cysteine residues at amino acid 55, 238,
251, 275, 286,
289 and 297 that could participate in disulfide bridge formation (van
Tilbeurgh et al. 1994)
were also found. Other conserved cysteines include Cysl l and Cys455 (Figure
11).
Example 4
Identification of a New LPDL-Related Lipase LPDLR
Using lpdl protein sequence to BLAST-search against the translated EST
database, the
inventors identified a mouse EST (BG868436, from salivary grand) which
translated to
another novel lipase with significant homology to lpdl. However, the homology
is only at the
protein level but not at nucleotide level. The inventors named this novel
lipase as lpdl-related
lipase or lpdlr. Sequencing of BG868436 revealed a cDNA of 2,155 by in length
with ORF
starting from nucleotide 78 and stopping at nucleotide 1640. The ORF encodes a
mouse lpdlr
protein of 451 amino acids (Figure 3 and Figure 12). The lipase consensus
sequence GxSxG
was found with an active serine at amino acid residue 154. Alignment analysis
suggested that,
for the putative catalytic triad, Asp 178 and Ser154 were conserved (Emmerich
et al. 1992), but
the normally conserved histidine residue within the triad was replaced by
Tyr253 (Figure 12).
A lipase lid sequence was also identified between two cysteine residues at 233
and 246 with
12 amino acid and demonstrates good structural similarity as LPDL proteins and
PS-PLA1.
Eight conserved cysteine residues at amino acid 12, 233, 246, 270, 281, 284,
292 and 446 that
could participate in disulfide bridge formation (van Tilbeurgh et al. 1994).
The human LPDLR
cDNA were sequenced as 2481 by in length which translate into a protein of 451
amino acids
(Figure 4B).
Examule 5
LPDL and LPDLR are new members of conserved lipase gene family
Alignment of human LPDL and mouse lpdlr protein sequences with other human
lipases revealed significant structural conservation (Figure 13). By algorithm
analysis to
compare two sequence alignment (Henikoff, et al. 1992), human LPDL exhibits 71
% identity
to mouse lpdl protein and 44% identity to mouse lpdlr. Comparing to other
human lipases,
LPDL exhibited 36%, 34%, 32%, 31 % and 31 % amino acid identity to human
endothelial
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derived lipase (EDL or LIPG), pancreatic lipase (PNLIP), hepatic lipase (HL),
pancreatic
lipase related protein 1 (LIP1) and lipoprotein lipase (LPL). Interestingly,
human LPDL also
demonstrates very high sequence homology to the phospholipase PS-PLA1 with an
amino acid
identity of 34%. The "catalytic triad" as well as the lipase consensus
sequences GxSxG are
conserved in all triglyceride lipases and PS-PLA1. Among the ten highly
conserved cysteine
residues required in triglyceride lipase for tertiary structure formation,
seven appeared to be
conserved in the LPDL protein (Figure 13).
The lid domain plays a crucial role in determining lipase substrate
specificity (Dugi et
al. 1995, Lowe 1997). The lid in both human LPDL and mouse lpdlr is composed
of 12 amino
acids, which is much shorter than those found in human PNLIP, LIP1, EDL, LPL
and HL (23,
23, 19, 22 and 22 residues, respectively) (Figure 13). Interestingly, both
hL,PDL and mLPDLR
lid sequences show higher homology to the lid of PS-PLAT which is also 12
amino acids in
length (Figure 13). However, the LPDL and lpdlr proteins does not contain the
phosphatidylserine-binding peptide motif that exists in PS-PLAT and functions
for
phosphatidylserine selectivity (Igarashi et al. 1995). Phylogenetic analysis
shows that LPDL,
LPDLR and PS PLA1 share higher structural conservation (Figure 14), suggesting
they form a
subfamily within the lipase gene family.
Example 6
Genomic Structure of Human LPDL Gene and LPDLR Gene
Using the mouse lpdl exon sequences to BLAST search against the nucleotide
genetic
database in GenBank, the inventors identified a genomic sequence of 340 kb
(AP001660) on
chromosome 21 q with significant homology to the mouse lpdl gene. Ten DNA
fragments from
this genomic sequence were further characterized as exons of the human LPDL
gene. The
exon/intron boundaries were determined using a combination of analysis with
exon/intron
consensus sequences, bioinformatic gene prediction tools and alignment with
the cloned
human cDNA sequences (Table 1). The exon sizes of human LPDL gene range from
90 to 386
by and they span a genomic region >100 kb. The largest intron, intron 9, spans
35.5 kb and the
smallest intron, intron 4, spans ~l kb (Table 1). Start and stop codons are
located in exons 1
and 10, respectively, and lipase consensus sequence GXSXG is in exon 3. Exons
4, 5 and 6
span the most conserved regions, including the lid sequences and two of three
active residues
within the triad structure for catalytic activity. The exon sequences and
partial intron
sequences of human LPDL gene is shown in Figure 15. Similarly, the exon
sequences of
human LPDLR gene were identified and shown in Figure 16.
Table 1 . Intron-axon boundaries of human LPDL
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Exon 5' boundary 3' boundary Intron size
size kb
by
1 106 ..GAGTTACGGA GTGAGATCTGgtaagatatt1 21
2 386 cttatttcagATAATAAAAGAAATCTTTTGgtaagtctgg2 3
3 109 ttaattgcagAAGCATGGTGAGAATAACAGgtaaaattat3 9
4 102 tgctttccagGTCTTGACCCGACTCCAATGgtaacaaatc4 15
5 90 ttcttctcagGTTTAGGCATATTTTCTCAGgtatactgac5 1
6 168 aacccttaagGAATTCAATTCCTCGGCTGGgtaagagaga6 2
7 105 caaatctcagGTTATCAAGCCCATTCTGTAgtaagttatt7 10
8 112 tattttgtagCCTATTATTTGGCTTTATGAgtaagtaaaa8 10
9 177 ttctctctagAAAGAACAAAACCCAGAAAGgtaagaaaat9 35
10 206 tctttctcagACCACCACTTCTATTTCTTGG...
Example 7
Genetic Disruption in mutant lpd locus
Using 5' RACE, sequencing, searching mouse genetic databases and bioinformatic
gene prediction, the inventors identified a continuous mouse genomic fragment
of 110 kb,
which contained all 10 exons of the mouse lpdl gene. When aligning the mouse
lpdl sequences
with the human genomic LPDL sequences, 9 of 10 exons were within the conserved
peaks
with >75% sequence identity (Figure 17). Within the ~5 kb region before exon
1, there was
another cluster of peaks with sequence identity of 50 to 75% which may
represent the
conserved promoter sequences for the gene and regulatory elements.
Since the identified lipase-like gene was a logical candidate for the lpd
phenotype, the
inventors next confirmed that the transgene insertion in the lpd locus
disrupted the lpdl lipase
gene. The inventors mapped the transgene junction clones relative to the gene
structure of the
mouse lpdl gene. One junction clone (D3) was mapped before mouse exon 10 while
the other
junction clone (3A) mapped after exon 10 (Figure 17), indicating that exon 10
of the lpdl gene
was deleted in the mutant lpd locus. It is perhaps of interest that ~7 kb
upstream of the lpdl
gene, there were five conserved peaks (with >75% identity) designated as
Conserved
Nucleotide Sequences (CNS), which may represent another gene (Figure 17).
The finding that the exon 10 of lpdl is deleted in the lpd mutant suggests
that C-
terminal sequences may participate in the substrate specificity during TG
hydrolysis. Besides
deletion of exon 10, other genetic rearrangements could not be ruled out
because of the
complexity of gene mutations, especially transgene-induced mutations. For
example, the most
recently characterized mutation fld (fatty liver dystrophy) is characterized
by a deletion of 2 kb
sequences eliminating exon 2 and 3, and inversion of 40 genomic sequences plus
a duplication
of 0.5 kb segments in 3'UTR(Peterfy et al., 2001).
Example 8
Human LPDL and LPDLR SNPs and association with plasma lipoproteins
Since the mouse lpd mutation had both disrupted lpdl and high plasma TG, the
inventors considered that LPDL gene variation in humans might contribute to
dyslipidemia.
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The inventors addressed this hypothesis in two ways. First, from genomic DNA
we directly
sequenced LPDL exons of 60 non-diabetic Caucasians with moderate to severe
hypertriglyceridemia (mean~SEM untreated TG 12.1+8.5 mmol/L, age 55+12 years)
who had
no obvious secondary cause of hyperlipidemia, and 10 matched normolipidemic
Caucasian
controls (untreated TG 1.1+0.3 mmol/L). The primer sequences used for
amplifying LPDL
exons are listed in Figure 18A. The hypertriglyceridemic subjects had
previously been shown
to have no mutation in LPL, HL or EDL. For newly discovered SNPs, allele
frequencies were
determined in subjects from 80 Caucasian subjects. The inventors found six non-
transcribed
and seven transcribed SNPs (Figure 19), including the nonsynonymous SNPs CSSY,
G364E,
E431K and D444E (Table 2). Genotype frequencies for each SNP did not deviate
significantly
from Hardy-Weinberg expectations in all samples. Mild to moderate pairwise
linkage
disequilibrium was observed for about half of the pairwise comparisons of LPDL
SNP
genotypes in Caucasians (data not shown). Two SNPs were further characterized
in several
additional samples of 80 individuals each: in African, East Indians, Chinese,
Inuit and
Amerindian, the frequencies for K431 were 0.57, 0.24, 0.05, 0.31 and 0.20,
respectively, and
the frequencies for E444 were 0.53, 0.51, 0.58, 0.69 and 0.51, respectively.
Allele frequencies
of six coding SNPs in 186 hypertriglyceridemic Caucasian subjects (TG>10
mmol/L) and 232
matched Caucasian controls (TG<1 mmol/L) were compared, and none was found to
be
significantly different between samples. However, heterozygosity for CSSY was
found only in
the Caucasian hypertriglyceridemic patients (2/186 vs 0/232), suggesting that
this might be a
rare mutation associated with hypertriglyceridemia.
Table 2 . LPDL SNPs and allele frequencies
A. Non-transcribed
LPDL SNPs
location nucleotide allele frequencies
(Caucasian
Intron 1 41 nt 5' to exon T: 0.14
2 C>T
Intron 2 74 nt 5' to exon T: 0.25
3 C>T
Intron 4 49 nt 3' to exon G: 0.32
4 A>G
Intron 16 nt 5' to exon C: 0.02
5 6 T>C
Intron 9 46 nt 3' to exon A: 0.35
9 G>A
3' to ORF nt +146 G>T T: 0.18
B. Transcribed
LPDL SNPs
location amino acidnucleotideallele frequencies (Caucasian
Exon 1 -54C>T T: 0.20
Exon 2 ~ CSSY 1646>A only in hypertriglyceridemic
subjects
Exon 3 S 159 477C>T T: 0.25
Exon 8 G364E 10916>A A: 0.03
Exon 9 E431 K 1291 G>A A: 0.34
Exon 10 D444E 1332C>A A: 0.55
3' UTR +806>A A: 0.43
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Next, the inventors tested for associations of SNP genotypes with plasma
lipoproteins
in three unrelated samples using our established approach (Hegele et al.,
1994, 2001a and
2001b). Two independently ascertained, unrelated samples of healthy,
normolipidemic
Caucasians (174 and 161 individuals) and a well-characterized sample of
healthy Inuit (208
subjects) (Hegele et al., 2001b) were studied. The first sample of 174
Caucasians was 48.3%
male and had mean (~SEM) age 50.1+4.3 years. The second sample of 161
Caucasians was
42.0% male and had mean (~SEM) age 53.7+5.8 years. In ANOVA, dependent
variables were
the four plasma lipoprotein traits (TG, total, HDL and LDL cholesterol),
appropriately
transformed to give distributions that were not significantly different from
normal. Correction
was made for age, sex and body mass index by including these as independent
covariates,
along with the genotype for the coding SNPs only, assuming dominant, co-
dominant and
recessive models for each minor allele, as described (Hegele et al., 2001b).
Seven significant
associations were found with plasma lipoproteins (Figure 20). At least one
LPDL SNP
genotype was associated with variation in HDL cholesterol in all three
samples. Also, LPDL
SNP genotypes were associated with variation in LDL cholesterol in both
Caucasian samples
(Figure 20).
While 7 transcribed SNPs were discovered, only one putative mutation was
identified, namely CSSY, which was present only in hypertriglyceridemic
subjects. C55 is an
important residue in LPDL, which is predicted to participate disulfide bridge
formation and in
determining lipase tertiary structure (van Tilbeurgh et al. 1994, Lowe 1997).
C55 is also
conserved in both mouse lpdlr and human PS-PLAT (Fig. 13). Therefore, the CSSY
substitution may affect function.
Replication in three independent normolipidemic samples strengthens the case
for
association between LPDL SNPs and HDL cholesterol, although linkage
disequilibrium with
unmeasured variants at another gene remains possible. Variation in HDL
cholesterol has been
previously been associated with SNPs in other lipases, specifically in LPL
(reviewed in Busch
and Hegele 2000), HL (Cohen et al. 1999) and EDL (deLemos et al. 2002). The
mechanisms
underlying these associations are unknown, but LPDL appears to be a fourth
lipase that is
associated with variation in plasma HDL cholesterol. The absence of
concomitant association
of LPDL SNPs with plasma TG is compatible with observations from other
experiments and
model systems in which TG and HDL metabolism are uncoupled (Hegele 2001 ).
Using a similar strategy, the inventors amplified the ten exons of human LPDLR
gene
in 30 non-diabetic Caucasians with moderate to severe hypertriglyceridemia
(untreated TG>10
mmol/L) with primers listed in Figure 18B. The hypertriglyceridemic subjects
had previously
been shown to have no mutation in LPL, HL or EDL. For newly discovered SNPs,
allele
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frequencies were determined in subjects from 80 Caucasian subjects. The
inventors found
three non-transcribed and two transcribed SNPs, (Table 3). The two coding SNPs
appeared to
be silent without amino acid substitution. The reference sequences for LPDLR
SNP screening
is the assembled exon sequences and corresponding protein sequences as listed
in Figure 16B.
A larger scale screening for LPDLR SNPs is expected.
Table 4 . LPDLR SNPs and allele frequencies
A. Non-transcribed LPDLR SNPs
location nucleotide allele frequencies (Caucasian
Intron 1 90 nt 3' to exon I G>A A: 0.01
Intron 8 29 nt 3' to exon 8 C>T T: 0.46
5' UTR nt -56 C>T T: 0.01
B. Transcribed LPDLR SNPs
location amino acid nucleotide allele frequencies Caucasian
Exon 6 C284 934C>T T: 0.087
Exon 9 K372 1198A>G A: 0.46
Example 9
Gene promoter and regulatory sequences in LPDL and LPDLR gene
Since the LPDL gene is highly expressed in testis and weakly expressed in the
liver but
not in any other tissues examined, LPDL promoter activity is very tissue
specific. When
aligning 100 the mouse and human LPDL genomic sequences together, the
inventors
identified ~4 kb region before exon-1 (Figure 17), which represents the
promoter and
regulatory region. The inventors then cloned the promoter region upto ~6 kb
(SEQ.ID.N0.77)
from the BAC#16 DNA of mouse lpdl gene. In defining the regulatory region and
identify the
tissue specific elements, primers were designed to clone differently-sized
fragments in the
promoter region (Figure 21). A serious of promoter/reporter gene constructs
ranging from ~6
kb (P1F-P7R) to ~2 kb (PSF-P7R) were constructed. One fragment of 341 by with
minimum
promoter sequences served as experimental control. Human LPDL promoter
sequence is
shown in Figure 21B (SEQ.)D.N0.78). With computing analysis of the promoter
sequences
using the online TESS software (http://www.cbil.upenn.edu/cgi-
bin/tess/tess33), the inventors
also identified potential binding sites for variety of transcription factors
within the ~6 kb
promoter region. To demonstrate it in principle, Figure 22 shows transcription
factors that
potentially bind to the 200 by region of murine lpdl promoter. The promoter
sequences of
human and murine LPDLR gene (SEQ.1D.N0.79 and SEQ.117.N0.80) are shown in
Figure 23
A and B, respectively. Since LPDLR gene is expressed in different tissues such
as prostate,
testis, colon, mammary and salivary gland, different tissue specific
regulatory elements are
predicted. Similar studies are being conducted in characterizing LPDLR
promoter. The
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characterized promoter and regulatory element could be employed to direct gene
expression in
desired tissues to modulate cellular processes and define the drug target.
Example 10
Expression recombinant LPDL and LPDLR protein
In investigation of the function of the gene product, the recombinant LPDL
protein is
expressed in baculovirus expression system using Invitrogen Bac-to-Bac HT
Baculovirus
Expression System (Invitrogen Cat. No. 10608016, Carlsbad, CA). A 6xHis tag is
engineered
into the construct for purification of the recombinant protein (anti-His
antibody is
commercially available). Recombinant baculovirus were generated and the
recombinant
proteins will be expressed in High Five cells (Invitrogen). Recombinant His6-
tagged LPDL
protein is purified from dialyzed culture media by immobilized metal-ion-
affinity
chromatography on Ni-nitrilotriacetic acid (Ni-NTA)-Sepharose (Qiagen Inc.).
The LPDL
protein is used in generating a monoclonal antibody. As the SNP in exon 2
(1646>A, CSSY)
is only found in hypertriglyceridemic subjects, a site-specific mutagenesis is
conducted in
human LPDL cDNA to create a mutant gene and express the mutant form of
protein, and its
function be analyzed. Similarily, LPDLR is being expressed in the same
baculovirus system.
Example 11
Generation of viral vectors for LPDL gene therapy in murine models
Since adenoviruses have a wide spectrum of tissue tropism, the inventors made
a first
generation adenovirus vector that carries the human LPDL gene and mouse lpdlr
gene,
respectively, for proof of principle in gene therapy. We used the AdMAXTM
adenovirus vector
system for the study. Since this system employs a Cre/LoxP site-specific
recombination
mechanism, the efficiency in rescuing the recombinant adenovirus is 30 folds
higher than the
regular strategy by homologous recombination (Ng et al. 1999). Northern blot
analysis and
RT-PCR have confirmed the virus-derived gene expression. To determine the
expression of
LPDL at protein level, we had generated an antibody against LPDL based on its
peptide
sequences and Western Blot analysis was performed. Apart from lpd null
function mice, there
are also other transgenic mouse models with hypertriglyceridemia could be used
for the LPDL-
adenovirus gene therapy study to test the hypothesis that overexpression of
LPDL and lpdlr
within the plasma compartment might ameliorate hypertriglyceridemia and
modulate lipid
processing. APOC3 transgenic mice and LPL deficient mice each develop marked
hypertriglyceridemia on a chow diet and could serve as models of
hypertriglyceridemia. Also,
the APOE knockout mouse has a component of hypertriglyceridemia due to
accumulation of
TG-rich lipoproteins and remnants, which might also serve as substrates for
circulating LPDL,
expressed from an adenoviral vector construct. In addition, the lpd mouse had
substantial TG
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accumulation in hepatocytes, suggesting deficiency of an intracellular lipase
activity. Since
the LAL deficient mouse develops liver TG accumulation, without
hypertriglyceridemia, and
given the tropism of adenovirus for the liver, the LAL deficient mouse might
also serve as an
instructive model for LPDL-adenoviral mediated gene therapy. We propose first
to test a first-
s generation adenovirus vector in proof of principle studies. As the first
generation adenovirus
is highly immunogenic and gene expression will be relatively transient, we
will explore use of
the helper-dependent third-generation adenovirus system. Since the immunogenic
viral genes
have been removed in the helper-dependent vector, the system will not generate
severe
immuno-toxicity and gene expression will be prolonged (Schiedner et. al.
1997). An AAV
viral vector may also be generated for in vivo studies of long term lpdl
replacement in a
knockout mouse model.
While the present invention has been described with reference to what are
presently
considered to be the preferred examples, it is to be understood that the
invention is not limited
to the disclosed examples. To the contrary, the invention is intended to cover
various
modifications and equivalent arrangements included within the spirit and scope
of the
appended claims.
All publications, patents and patent applications are herein incorporated by
reference
in their entirety to the same extent as if each individual publication, patent
or patent
application was specifically and individually indicated to be incorporated by
reference in its
entirety.
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SEQUENCE LISTING
<110> Wen Dr., Xiao-Yan
Steward Dr., A. Keith
Tsui, Lap-Chee
Hegele, Robert A.
<120> Lipase Genes and Proteins
<130> 43158-0001
<140> filed herewith
<141> 2002-12-23
<150> 60/346,603
<151> 2002-O1-10
<150> 60/341,786
<151> 2001-12-O1
<160> 80
<170> PatentIn Ver. 2.1
<210> 1
<211> 1683
<212> DNA
<213> Homo sapiens
<400> 1
ccattatggc cgggggagtt acggaatttc ctttttggtg aattgagtgc tgtttttgct 60
tttctcagat tccaaatgag agtatacatt tttctttgtt tgatgtgctg ggtgagatct 120
gataataaaa gaccatgcct tgaattctct cagctaagtg taaaggattc cttcagagat 180
ttatttattc cgagaataga gaccattctg atgatgtata caaggtacaa cctaaactgt 240
gctgagccac tgtttgaaca aaataactca cttaatgtta atttcaacac acaaaagaaa 300
acagtctggc ttattcacgg atacagacca gtaggctcca tcccattatg gcttcagaac 360
ttcgtaagga ttttgctgaa tgaagaagat atgaatgtaa ttgtagtaga ctggagccgg 420
ggtgctacaa cttttattta taatagagca gttaaaaaca ccagaaaagt tgctgtgagt 480
ttgagtgtgc acattaaaaa tcttttgaag catggtgcat ctcttgacaa ttttcatttc 540
ataggtgtga gcttaggggc tcatatcagt ggatttgttg gaaagatatt tcatggtcaa 600
cttggaagaa taacaggtct tgaccctgct gggccaaggt tctccagaaa accaccatat 660
agcagattag attacacgga tgcaaagttt gtggatgtca tccattctga ctccaatggt 720
ttaggcattc aagagccctt gggacatata gatttttatc caaatggagg aaataaacaa 780
cctggctgtc ctaaatcaat tttctcagga attcaattca ttaaatgcaa ccaccagaga 840
gcagttcact tgttcatggc atctttagaa acaaactgca attttatttc atttccttgt 900
cgttcataca aagattacaa gactagctta tgtgtggact gtgactgttt taaggaaaaa 960
tcatgtcctc ggctgggtta tcaagccaag ctatttaaag gtgttttaaa agaaaggatg 1020
gaaggaagac ctcttaggac cactgtgttt ttggatacaa gtggtacata tccattctgt 1080
acctattatt ttgttctcag tataattgtt ccagataaaa ctatgatgga tggctcgttt 1140
tcatttaaat tattaaatca gcttggaatg attgaagagc caaggcttta tgaaaagaac 1200
aaaccatttt ataaacttca agaagtcaag attcttgttc aattttataa tgactttgta 1260
aatatttcaa gcattggttt gacatatttc cagagctcaa atctgcagtg ttccacatgc 1320
acatacaaga tccagagtct catgttaaaa tcacttacat acccagaaag accaccactt 1380
tgcaggtata atattgtact taaagacaga gaggaagtgb ttcttaatcc aaacacatgt 1440
acaccaaaga acacataaga tgccttcttc catcaaatgc acttgcttgt gaattaatgg 1500
acttgtaaat gaaacaatgc aatcagtctt ttataatgca ctgttcaatt tgagattcaa 1560
gtatttctat ttcttggaaa aaattttaag aatcaaaaat aaagaaaata aaaaatgcat 1620
acagttaaac atttaaaaaa aaaaaaaaaa aaaaaaaaaa ccttgtcggc cgcctcggcc 1680
cag 1683
1/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
<210> 2
<211> 460
<212> PRT
<213> Homo Sapiens
<400> 2
Met Arg Val Tyr Ile Phe Leu Cys Leu Met Cys Trp Val Arg Ser Asp
1 5 10 15
Asn Lys Arg Pro Cys Leu Glu Phe Ser Gln Leu Ser Val Lys Asp Ser
20 25 30
Phe Arg Asp Leu Phe Ile Pro Arg Ile Glu Thr Ile Leu Met Met Tyr
35 40 45
Thr Arg Tyr Asn Leu Asn Cys Ala Glu Pro Leu Phe Glu Gln Asn Asn
50 55 60
Ser Leu Asn Val Asn Phe Asn Thr Gln Lys Lys Thr Val Trp Leu Ile
65 70 75 80
His Gly Tyr Arg Pro Val Gly Ser Ile Pro Leu Trp Leu Gln Asn Phe
85 90 95
Val Arg Ile Leu Leu Asn Glu Glu Asp Met Asn Val Ile Val Val Asp
100 105 110
Trp Ser Arg Gly Ala Thr Thr Phe Ile Tyr Asn Arg Ala Val Lys Asn
115 120 125
Thr Arg Lys Val Ala Val Ser Leu Ser Val His Ile Lys Asn Leu Leu
130 135 140
Lys His Gly Ala Ser Leu Asp Asn Phe His Phe Ile Gly Val Ser Leu
145 150 155 160
Gly Ala His Ile Ser Gly Phe Val Gly Lys Ile Phe His Gly Gln Leu
165 170 175
Gly Arg Ile Thr Gly Leu Asp Pro Ala Gly Pro Arg Phe Ser Arg Lys
180 185 190
Pro Pro Tyr Ser Arg Leu Asp Tyr Thr Asp Ala Lys Phe Val Asp Val
195 200 205
Ile His Ser Asp Ser Asn Gly Leu Gly Ile Gln Glu Pro Leu Gly His
210 215 220
Ile Asp Phe Tyr Pro Asn Gly Gly Asn Lys Gln Pro Gly Cys Pro Lys
225 230 235 240
Ser Ile Phe Ser Gly Ile Gln Phe Ile Lys Cys Asn His Gln Arg Ala
245 250 255
Val His Leu Phe Met Ala Ser Leu Glu Thr Asn Cys Asn Phe Ile Ser
260 265 270
Phe Pro Cys Arg Ser Tyr Lys Asp Tyr Lys Thr Ser Leu Cys Val Asp
275 280 285
2/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
Cys Asp Cys Phe Lys Glu Lys Ser Cys Pro Arg Leu Gly Tyr Gln Ala
290 295 300
Lys Leu Phe Lys Gly Val Leu Lys Glu Arg Met Glu Gly Arg Pro Leu
305 310 315 320
Arg Thr Thr Val Phe Leu Asp Thr Ser Gly Thr Tyr Pro Phe Cys Thr
325 330 335
Tyr Tyr Phe Val Leu Ser Ile Ile Val Pro Asp Lys Thr Met Met Asp
340 345 350
Gly Ser Phe Ser Phe Lys Leu Leu Asn Gln Leu Gly Met Ile Glu Glu
355 360 365
Pro Arg Leu Tyr Glu Lys Asn Lys Pro Phe Tyr Lys Leu Gln Glu Val
370 375 380
Lys Ile Leu Val Gln Phe Tyr Asn Asp Phe Val Asn Ile Ser Ser Ile
385 390 395 400
Gly Leu Thr Tyr Phe Gln Ser Ser Asn Leu Gln Cys Ser Thr Cys Thr
405 410 415
Tyr Lys Ile Gln Ser Leu Met Leu Lys Ser Leu Thr Tyr Pro Glu Arg
420 425 430
Pro Pro Leu Cys Arg Tyr Asn Ile Val Leu Lys Asp Arg Glu Glu Val
435 440 445
Phe Leu Asn Pro Asn Thr Cys Thr Pro Lys Asn Thr
450 455 460
<210> 3
<211> 2056
<212> DNA
<213> murine
<400> 3
ccatatggtc gacctgcagg cggccgcact agtgattgac cacgcgtatc gatgtcgact 60
tttttttttt tttttctctc ctccttttct ttgtcttctc atggttattg ctaatttgtc 120
gaggctaacc ttgaactgga gttattcctg cttgaattat atgccataga aggaatttta 180
tactacttta aatttatgag caaactggta ctctactatt attattcatg ttaatatgga 240
taatggtgag ataatatttt aatatgtaaa tgcacttctg taaagctttt gtaaacattc 300
acaatgtatt tacctttcct gccgtccttc ccacttgatt agttgtaaca atggcttatg 360
tcagagccat tttattttat tttaaaagaa tgtttatgac ttggaaccta ccacatttgt 420
tcatatttgt cattattatt tttattactt ctaatttagg aagtcttttt gcctggtcca 480
tgtacagctc taactttagc tgtgctcagt aaatcatgtt aatgtgcaaa taaaacagaa 540
ttctcccatt cagttgtata aacctatacc aacaaaacgg cactgttacc ctaatgctca 600
gattgaattt ttatcttttc agagaagaag agaacatgtc ttgaattcac aaagttaagt 660
gcaatgaata gcttaaaaga tttattttgt cccaaagtaa agataaatct gctgatgtac 720
tcaaggggca acgccaagtg tgcagagcca ctctttgaat ctaataactc actcaacacc 780
cgtttcaacc cagcgaagaa aaccgtctgg attattcatg ggtacaggcc ctttggttcc 840
accccagtgt ggctttctag gtttactaaa gcctttttga aacaggaaga tgtgaatctc 900
attgttgtag actggaacca gggcgctacg actttcatgt attctagagc ggttagaaat 960
accagaagag ttgctgagat tttgagagaa accattgaga atcttttgat ccatggggca 1020
tcacttgaca attttcattt cattggcatg agcttagggg ctcatattag tggatttgta 1080
ggaaagatat ttcatggtca acttggaaga attacaggtc ttgacccagc tggaccacaa 1140
3/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
ttttctagaa agccatcgaa tagcagatta tattacacag atgcaaagtt tgtagatgtc 1200
atccacactg atatcaaaag tttgggtatt ggagagccat cggggcacat tgatttttat 1260
ccaaatggag gaaaacatca gccaggttgt cctacatcaa ttttttcagg aaccaatttt 1320
attaaatgcg accatcagag agccatttac ttgttcctcg cagcttttga aacaagctgc 1380
aactttgttt catttccttg tcgttcctac aaagattaca agaatggttt gtgtgtggac 1440
tgtgggaatc tttacaaaga ctcctgccca agacttggtg agagaagttg tgtttattta 1500
ggtaatcaag ctaaactatg gaaagaagag ttgaagaaaa aaacagaaga atggcctctt 1560
agaaccactg catttttgga tactagtagc caaaatccat tttgtagtaa gttatttgca 1620
ttgtttgtag cctattattt tgctctcaat atagttgcct tgagtgaaac tatgagaaat 1680
ggctcaattt catttggttt attaaatgac cttggagatt tggaatactc aacactctat 1740
gagttactta ggaagagcaa gccatttgat aatcttcaag aagtcaagat tcttgttcag 1800
ttcgtaaatg acattgtgag catttcgcgc atttgcttga catactttca gtccacaaat 1860
ccttactgtg ccgcgtgcca gtacaaaata cagagccttg tgttaaaatc actcacatat 1920
ccagaaaggt gttaaaatca ctcacatatc cagaaagatg tttccctctt ttcttctctt 1980
tcaaagacca ccaatttgta agtataattt tgtattgaaa gaaggaacaa gggtagatct 2040
tcaaccagat gaatgt 2056
<210> 4
<211> 423
<212> PRT
<213> murine
<400> 4
Met Asn Ser Leu Lys Asp Leu Phe Cys Pro Lys Val Lys Ile Asn Leu
1 5 10 15
Leu Met Tyr Ser Arg Gly Asn Ala Lys Cys Ala Glu Pro Leu Phe Glu
20 25 30
Ser Asn Asn Ser Leu Asn Thr Arg Phe Asn Pro Ala Lys Lys Thr Val
35 40 45
Trp Ile Ile His Gly Tyr Arg Pro Phe Gly Ser Thr Pro Val Trp Leu
50 55 60
Ser Arg Phe Thr Lys Ala Phe Leu Lys Gln Glu Asp Val Asn Leu Ile
65 70 75 80
Val Val Asp Trp Asn Gln Gly Ala Thr Thr Phe Met Tyr Ser Arg Ala
85 90 95
Val Arg Asn Thr Arg Arg Val Ala Glu Ile Leu Arg Glu Thr Ile Glu
100 105 110
Asn Leu Leu Ile His Gly Ala Ser Leu Asp Asn Phe His Phe Ile Gly
115 120 125
Met Ser Leu Gly Ala His Ile Ser Gly Phe Val Gly Lys Ile Phe His
130 135 140
Gly Gln Leu Gly Arg Ile Thr Gly Leu Asp Pro Ala Gly Pro Gln Phe
145 150 155 160
Ser Arg Lys Pro Ser Asn Ser Arg Leu Tyr Tyr Thr Asp Ala Lys Phe
165 170 175
Val Asp Val Ile His Thr Asp Ile Lys Ser Leu Gly Ile Gly Glu Pro
180 185 190
4/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
Ser Gly His Ile Asp Phe Tyr Pro Asn Gly Gly Lys His Gln Pro Gly
195 200 205
Cys Pro Thr Ser Ile Phe Ser Gly Thr Asn Phe Ile Lys Cys Asp His
210 215 220
Gln Arg Ala Ile Tyr Leu Phe Leu Ala Ala Phe Glu Thr Ser Cys Asn
225 230 235 240
Phe Val Ser Phe Pro Cys Arg Ser Tyr Lys Asp Tyr Lys Asn Gly Leu
245 250 255
Cys Val Asp Cys Gly Asn Leu Tyr Lys Asp Ser Cys Pro Arg Leu Gly
260 265 270
Glu Arg Ser Cys Val Tyr Leu Gly Asn Gln Ala Lys Leu Trp Lys Glu
275 280 285
Glu Leu Lys Lys Lys Thr Glu Glu Trp Pro Leu Arg Thr Thr Ala Phe
290 295 300
Leu Asp Thr Ser Ser Gln Asn Pro Phe Cys Ser Lys Leu Phe Ala Leu
305 310 315 320
Phe Val Ala Tyr Tyr Phe Ala Leu Asn Ile Val Ala Leu Ser Glu Thr
325 330 335
Met Arg Asn Gly Ser Ile Ser Phe Gly Leu Leu Asn Asp Leu Gly Asp
340 345 350
Leu Glu Tyr Ser Thr Leu Tyr Glu Leu Leu Arg Lys Ser Lys Pro Phe
355 360 365
Asp Asn Leu Gln Glu Val Lys Ile Leu Val Gln Phe Val Asn Asp Ile
370 375 380
Val Ser Ile Ser Arg Ile Cys.Leu Thr Tyr Phe Gln Ser Thr Asn Pro
385 390 395 400
Tyr Cys.Ala Ala Cys Gln Tyr Lys Ile Gln Ser Leu Val Leu Lys Ser
405 410 415
Leu Thr Tyr Pro Glu Arg Cys
420
<210> 5
<211> 1383
<212> DNA
<213> murine
<400> 5
tataccaaca aaacggcact gttatcctaa tgctcagatt gaatttttat cttttcagag 60
aagaagagaa catgtcttga attcacaaag ttaagtgcaa tgaatagctt aaaagattta 120
ttttgtccca aagtaaagat aaatctgctg atgtactcaa ggggcaacgc caagtgtgca 180
gagccactct ttgaatctaa taactcactc aacacccgtt tcaaccc,~gc gaagaaaacc 240
gtctggatta ttcatgggta caggcccttt ggttccaccc cagtgtggct ttctaggttt 300
actaaagcct ttttgaaaca ggaagatgtg aatctcattg ttgtagactg gaaccagggc 360
gctacgactt tcatgtattc tagagcggtt agaaatacca gaagagttgc tgagatttcg 420
agagaaacca ttgagaatct tttgatccat gggacatcac ttgacaattt tcacttcatt 480
5/40 . ,
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
ggcatgagct taggggctca tattagtgga tttgtaggaa agatatttca tggtcaactt 540
ggaagaatta caggtcttga cccagctgga ccacaatttt ctagaaagcc atcgaatagc 600
agattatatt acacagatgc aaagtttgta gatgtcatcc acactgatat caaaagtttg 660
ggtattggag agccatcggg gcacattgat ttttatccaa atggaggaaa acatcagcca 720
ggttgtccta catcaatttt ttcaggaacc aattttatta aatgcgacca tcagagagcc 780
atttacttgt tcctcgcagc ttttgaaaca agctgcaact ttgtttcatt tccttgtcgt 840
tcctacaaag attacaagaa tggtttgtgt gtggactgtg ggaatcttta caaagactcc 900
tgcccaagac ttggtaatca agctaaacta tggaaagaag agttgaagaa aaaaacagaa 960
gaatggcctc ttagaaccac tgcatttttg gatactagta gccaaaatcc attttgtacc 1020
tattattttg ctctcaatat agttgccttg agtgaaacta tgagaaatgg ctcaatttca 1080
tttggtttat taaatgacct tggagatttg gaatactcaa cactctatga gaagagcaag 1140
ccatttgata atcttcaaga agtcaagatt cttgttcagt tcgtaaatga cattgtgagc 1200
atttcgcgca tttgcttgac atactttcag tccacaaatc cttactgtgc cgcgtgccag 1260
tacaaaataa agagccttgt gttaaaatca ctcacatatc cagaaagatg tttccctcct 1320
taatcactag tgaattcgcg gccgcctgca ggtcgaccat atgggagagc tcccaacgcg 1380
ttg 1383
<210> 6
<211> 407
<212> PRT
<213> murine
<400> 6
Met Asn Ser Leu Lys Asp Leu Phe Cys Pro Lys Val Lys Ile Asn Leu
1 5 10 15
Leu Met Tyr Ser Arg Gly Asn Ala Lys Cys Ala Glu Pro Leu Phe Glu
20 25 30
Ser Asn Asn Ser Leu Asn Thr Arg Phe Asn Pro Ala Lys Lys Thr Val
35 40 45
Trp Ile Ile His Gly Tyr Arg Pro Phe Gly Ser Thr Pro Val Trp Leu
50 55 60
Ser Arg Phe Thr Lys Ala Phe Leu Lys Gln Glu Asp Val Asn Leu Ile
65 70 75 80
Val Val Asp Trp Asn Gln Gly Ala Thr Thr Phe Met Tyr Ser Arg Ala
85 90 95
Val Arg Asn Thr Arg Arg Val Ala Glu Ile Ser Arg Glu Thr Ile Glu
100 105 110
Asn Leu Leu Ile His Gly Thr Ser Leu Asp Asn Phe His Phe Ile Gly
115 120 125
Met Ser Leu Gly Ala His Ile Ser Gly Phe Val Gly Lys Ile Phe His
130 135 140
Gly Gln Leu Gly Arg Ile Thr Gly Leu Asp Pro Ala Gly Pro Gln Phe
145 150 155 160
Ser Arg Lys Pro Ser Asn Ser Arg Leu Tyr Tyr Thr Asp Ala Lys Phe
165 170 175
Val Asp Val Ile His Thr Asp Ile Lys Ser Leu Gly Ile Gly Glu Pro
180 185 190
-- 6/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
Ser Gly His Ile Asp Phe Tyr Pro Asn Gly Gly Lys His Gln Pro Gly
195 200 205
Cys Pro Thr Ser Ile Phe Ser Gly Thr Asn Phe Ile Lys Cys Asp His
210 215 220
Gln Arg Ala Ile Tyr Leu Phe Leu Ala Ala Phe Glu Thr Ser Cys Asn
225 230 235 240
Phe Val Ser Phe Pro Cys Arg Ser Tyr Lys Asp Tyr Lys Asn Gly Leu
245 250 255
Cys Val Asp Cys Gly Asn Leu Tyr Lys Asp Ser Cys Pro Arg Leu Gly
260 265 270
Asn Gln Ala Lys Leu Trp Lys Glu Glu Leu Lys Lys Lys Thr Glu Glu
275 280 285
Trp Pro Leu Arg Thr Thr Ala Phe Leu Asp Thr Ser Ser Gln Asn Pro
290 295 300
Phe Cys Thr Tyr Tyr Phe Ala Leu Asn Ile Val Ala Leu Ser Glu Thr
305 310 315 320
Met Arg Asn Gly Ser Ile Ser Phe Gly Leu Leu Asn Asp Leu Gly Asp
325 330 335
Leu Glu Tyr Ser Thr Leu Tyr Glu Lys Ser Lys Pro Phe Asp Asn Leu
340 345 350
Gln Glu Val Lys Ile Leu Val Gln Phe Val Asn Asp Ile Val Ser Ile
355 360 365
Ser Arg Ile Cys Leu Thr Tyr Phe Gln Ser Thr Asn Pro Tyr Cys Ala
370 375 380
Ala Cys Gln Tyr Lys Ile Lys Ser Leu Val Leu Lys Ser Leu Thr Tyr
385 390 395 400
Pro Glu Arg Cys Phe Pro Pro
405
<210> 7
<211> 2155
<212> DNA
<213> murine
<400> 7
agaaagccag atcttcaggg tgggacgcgg gaacgcggca acgccaggct ctgtgcggag 60
tgcgacacgt cctcgtgttt ggggcacatg atctcttgag gttccctatg ctgaggctat 120
gttttttcat cagtttcatg tgcttggtga aatcagacac ggatgaaaca tgcccgtcct 180
tcaccagact gagcttccac agcgcggtgg ttggtacagg actctccgtg agactgatgc 240
tctacacaca gagagaccag acctgtgcac agatcatcaa ctccacagct ctggggagct 300
taaatgtgac caagaaaacc acttttataa tccacggttt ccggccaaca ggctcccctc 360
cggtttggat agaggagctg gtacagagtt tgatcagtgt acaagagatg aatgtagtgg 420
ttgttgattg gaatcgagga gcgacaactg tgatataccc ccatgcgtct agcaagacca 480
gacaagtagc ttctattttg aaggaattta ttgaccagat gttggtcaaa ggagcttctc 540
tggacaacat ttacatgatt ggagtaagtc taggagccca catagctgga tttgttggcg 600
agtcgtatga ggggaagctg gggagagtca caggtcttga ccctgcaggc cctttattca 660
7/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
actggagacc tcctgaggag agattagacc ccagcgacgc actgttcgtg gacgtcatcc 720
actctgacac tgacgcacta ggctacaagg aagccctagg acacatagac ttctacccga 780
atggaggact agatcaaccc ggttgcccca agacaatatt tggaggtata aagtacttca 840
agtgtgacca ccagatgtcc gtctacctgt accttgcatc cctgcaaaat aactgctcca 900
tcacggccta tccctgcgac tcctatcggg attataggaa tggcaagtgt gtcagctgtg 960
gcgctggaca aattgtgcca tgtccccgtg taggctacta tgctgacagc tggaaagagt 1020
acttatggga cagagatcct ccgatgacga aggcgttctt cgacacagct gagacaaagc 1080
catactgcat gtatcattat tttgtggaca ttgtatcttg gaacaagagc gtaagaagag 1140
ggtttattac aatcaaactg agaggcgaag atggcaatat cacagaatcc aaaatcgatc 1200
atgagccatc tgcatttgag aaataccatc aagtgagtct cctcgcaaga tttaatcgag 1260
atctggataa ggtggcagag atttccttgt tgttctccac agggtctgta gtgggcccaa 1320
agtacaagct cagggtcctg cagatgaagc tgaggtctct ggcccatcca gacaggcctc 1380
acttgtgtcg atatgatctc gtccttatgg agaatgttga gacatccttc caacctatcc 1440
tgtgctctca gcagcagatg tagctgctgt taagacctgt gggcagtgat gacatcaaga 1500
tctctacagc tataggcttt cagtgcctca ccctatagcg tcctgaaggc ttccaaagca 1560
tggggaagaa aaccaaggtt attttcggct gtgttccaag gatgtcgcgt cacaaaaatg 1620
tcaaaggacc tgcgattatg aaacaccact gggagggggg gggcgcccta actagggcaa 1680
gacagaaata gcccagctcc ctacagcaaa gcactttgtc tggctgcgtc ccgaggcccc 1740
acttgttccc cactgtcgaa tctgctgcct gtcataccac accctcaggg ccgtggggca 1800
agcatctaaa tggtccctgt gcccaatcct ctgccatctc ggcgggaacc gaatggagaa 1860
tgaaccagac tacctcacag ggactgtggg tggggagggc caggctgtga agggtttgga 1920
aatcccttgg gtcccttggg tggactactg tgaacatgct tttcttttga tagggtttgg 1980
ctcttacgta aagacacaca atctctcttt ttcaacacca gctttcctcc tggactgttt 2040
gcctcggggc gacaagaagg ctctggctta gtccagactc ttctgaagtg atgctgatgg 2100
ccagaggtat tgggctgggc tcatgccctg aggcctgtgc agctaagtgg aagaa 2155
<210> 8
<211> 451
<212> PRT
<213> murine
<400> 8
Met Leu Arg Leu Cys Phe Phe Ile Ser Phe Met Cys Leu Val Lys Ser
1 5 10 15
Asp Thr Asp Glu Thr Cys Pro Ser Phe Thr Arg Leu Ser Phe His Ser
20 25 30
Ala Val Val Gly Thr Gly Leu Ser Val Arg Leu Met Leu Tyr Thr Gln
35 40 45
Arg Asp Gln Thr Cys Ala Gln Ile Ile Asn Ser Thr Ala Leu Gly Ser
50 55 60
Leu Asn Val Thr Lys Lys Thr Thr Phe Ile Ile His Gly Phe Arg Pro
65 70 75 80
Thr Gly Ser Pro Pro Val Trp Ile Glu Glu Leu Val Gln Ser Leu Ile
85 90 95
Ser Val Gln Glu Met Asn Val Val Val Val Asp Trp Asn Arg Gly Ala
100 105 110
Thr Thr Val Ile Tyr Pro His Ala Ser Ser Lys Thr Arg Gln Val Ala
115 120 125
Ser Ile Leu Lys Glu Phe Ile Asp Gln Met Leu Val Lys Gly Ala Ser
130 135 140
8/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
Leu Asp Asn Ile Tyr Met Ile Gly Val Ser Leu Gly Ala His Ile Ala
145 150 155 160
Gly Phe Val Gly Glu Ser Tyr Glu Gly Lys Leu Gly Arg Val Thr Gly
165 170 175
Leu Asp Pro Ala Gly Pro Leu Phe Asn Trp Arg Pro Pro Glu Glu Arg
180 185 190
Leu Asp Pro Ser Asp Ala Leu Phe Val Asp Val Ile His Ser Asp Thr
195 200 205
Asp Ala Leu Gly Tyr Lys Glu Ala Leu Gly His Ile Asp Phe Tyr Pro
210 215 220
Asn Gly Gly Leu Asp Gln Pro Gly Cys Pro Lys Thr Ile Phe Gly Gly
225 230 235 240
Ile Lys Tyr Phe Lys Cys Asp His Gln Met Ser Val Tyr Leu Tyr Leu
245 250 255
Ala Ser Leu Gln Asn Asn Cys Ser Ile Thr Ala Tyr Pro Cys Asp Ser
260 265 270
Tyr Arg Asp Tyr Arg Asn Gly Lys Cys Val Ser Cys Gly Ala Gly Gln
275 280 285
Ile Val Pro Cys Pro Arg Val Gly Tyr Tyr Ala Asp Ser Trp Lys Glu
290 295 300
Tyr Leu Trp Asp Arg Asp Pro Pro Met Thr Lys Ala Phe Phe Asp Thr
305 310 315 320
Ala Glu Thr Lys Pro Tyr Cys Met Tyr His Tyr Phe Val Asp Ile Val
325 330 335
Ser Trp Asn Lys Ser Val Arg Arg Gly Phe Ile Thr Ile Lys Leu Arg
340 345 350
Gly Glu Asp Gly Asn Ile Thr Glu Ser Lys Ile Asp His Glu Pro Ser
355 360 365
Ala Phe Glu Lys Tyr His Gln Val Ser Leu Leu Ala Arg Phe Asn Arg
370 375 380
Asp Leu Asp Lys Val Ala Glu Ile Ser Leu Leu Phe Ser Thr Gly Ser
385 390 395 400
Val Val Gly Pro Lys Tyr Lys Leu Arg Val Leu Gln Met Lys Leu Arg
405 410 415
Ser Leu Ala His Pro Asp Arg Pro His Leu Cys Arg Tyr Asp Leu Val
420 425 430
Leu Met Glu Asn Val Glu Thr Ser Phe Gln Pro Ile Leu Cys Ser Gln
435 440 445
Gln Gln Met
450
9/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
<210> 9
<211> 2481
<212> DNA
<213> Homo Sapiens
<400> 9
ctagtggatc cccgggctgc aggattcggc asagaaaatc ccaaaatgga aactcttaac 60
ctctgcgaat aaatcattct tgtgaatgtg acacacgatc tctccagttt ccatatgttg 120
agattctact tattcatcag tttgttgtgc ttgtcaagat cagacgcaga agaaacatgt 180
ccttcattca ccaggctgag ctttcacagt gcagtggttg gtacgggtct aaatgtgagg 240
ctgatgctct acacaaggaa aaacctgacc tgcgcacaaa ccatcaactc ctcagctttt 300
gggaacttga atgtgaccaa gaaaaccacc ttcattgtcc atggattcag gccaacaggc 360
tcccctcctg tttggatgga tgacttagta aagggtttgc tctctgttga agacatgaac 420
gtagttgttg ttgattggaa tcgaggagct acaactttaa tatataccca tgcctctagt 480
aagaccagaa aagtagccat ggtcttgaag gaatttattg accagatgtt ggcagaagga 540
gcttctcttg atgacattta catgatcgga gtaagtctag gagcccacat atctgggttt 600
gttggagaga tgtacgatgg atggctgggg agaattacag gcctcgaccc tgcaggccct 660
ttattcaacg ggaaacctca ccaagacaga ttagatccca gtgatgcgca gtttgttgat 720
gtcatccatt ccgacactga tgcactgggc tacaaggagc cattaggaaa catagacttc 780
tacccaaatg gaggattgga tcaacctggc tgccccaaaa caatattggg aggatttcag 840
tattttaaat gtgaccacca gaggtctgta tacctgtacc tgtcttccct gagagagagc 900
tgcaccatca ctgcgtatcc ctgtgactcc taccaggatt ataggaatgg caagtgtgtc 960
agctgcggca cgtcacaaaa agagtcctgt ccccttctgg gctattatgc tgataattgg 1020
aaagaccatc taagggggaa agatcctcca atgacgaagg cattctttga cacagctgag 1080
gagagcccat tctgcatgta tcattacttt gtggatatta taacatggaa caagaatgta 1140
agaagagggg acattaccat caaattgaga gacaaagctg gaaacaccac agaatccaaa 1200
atcaatcatg aacccaccac atttcagaaa tatcaccaag tgagtctact tgcaagattt 1260
aatcaagatc tggataaagt ggctgcaatt tccttgatgt tctctacagg atctctaata 1320
ggcccaaggt acaagctcag gattctccga atgaagttaa ggtcccttgc ccatccggag 1380
aggcctcagc tgtgtcggta tgatcttgtc ctgatggaaa acgttgaaac agtcttccaa 1440
cctattcttg gcccagagtt gcagttgtaa ctgtggccag gacacatggc cataaataat 1500
agaaagaaag ctacaaccac aggctgtttg aaagcttcac ctcacctttc tgcaaagcag 1560
aaaaagtatg aaaaaaccaa ggcttttttc agtagcgtcc tatggatgtc acattgtaca 1620
tcaaacaacc ttgtgattat aaaacgatcc tgggaaggag cccctaacta gggcaagtca 1680
gaaatagcca ggctcgcagc agcgcagcgc tgtgtctgct gtgtcctggg gcctcccttg 1740
ttccgacctg tcaattctgc tgcctgtcac gcgggtggtt ctgcccatcg cggctgcggg 1800
tcaagcatct tcaagggaag gacggactgg aggcctcacc gtggactcaa ctctgcattc 1860
tccgtgccac attcctccag ttcccacacg tagaagggaa cgaaactgac gtctacctca 1920
tggggctgct gtgtgggttt gggaggcaaa aatctatgaa gggttttttg aaatcccata 1980
ggtgccacat ctatgagatg tttgataaat gtgaatatgc ttttacattt gggcttatct 2040
aatttgcaat aagagagcct ctctctatca acaccagctt ctctctcggg ctgtttgctc 2100
agggaaggca agaaagccac gtgctggccc tctgccttct ctaaagtgct gttggagcat 2160
ggaggagctg gaggagatgg ggatggactg acagctaaga gggcggctgc tgggactaga 2220
tagtggatga agaaagaagg acgaggaagc cgtggggcag cctcttcaca tggggacagg 2280
ggatggagca tgaggcaggg gaaggaaaag cagagcttat ttttcaccta aggtggagaa 2340
ggatcacttt acaggcaacg ctcattttaa gcaaccctta agaaatgttt atttttcttt 2400
attaccaatk ctttttatga ttattgaaga aatttagaaa atgcgtagat acaaaaaaaa 2460
aaaaaaaaaa ckcgaggggg c 2481
<210> 10
<211> 451
<212> PRT
<213> Homo Sapiens
<400> 10
Met Leu Arg Phe Tyr Leu Phe Ile Ser Leu Leu Cys Leu Ser Arg Ser
1 5 10 15
10/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
Asp Ala Glu Glu Thr Cys Pro Ser Phe Thr Arg Leu Ser Phe His Ser
20 25 30
Ala Val Val Gly Thr Gly Leu Asn Val Arg Leu Met Leu Tyr Thr Arg
35 40 45
Lys Asn Leu Thr Cys Ala Gln Thr Ile Asn Ser Ser Ala Phe Gly Asn
50 55 60
Leu Asn Val Thr Lys Lys Thr Thr Phe Ile Val His Gly Phe Arg Pro
65 70 75 80
Thr Gly Ser Pro Pro Val Trp Met Asp Asp Leu Val Lys Gly Leu Leu
85 90 95
Ser Val Glu Asp Met Asn Val Val Val Val Asp Trp Asn Arg Gly Ala
100 105 110
Thr Thr Leu Ile Tyr Thr His Ala Ser Ser Lys Thr Arg Lys Val Ala
115 120 125
Met Val Leu Lys Glu Phe Ile Asp Gln Met Leu Ala Glu Gly Ala Ser
130 135 140
Leu Asp Asp Ile Tyr Met Ile Gly Val Ser Leu Gly Ala His Ile Ser
145 150 155 160
Gly Phe Val Gly Glu Met Tyr Asp Gly Trp Leu Gly Arg Ile Thr Gly
165 170 175
Leu Asp Pro Ala Gly Pro Leu Phe Asn Gly Lys Pro His Gln Asp Arg
180 185 190
Leu Asp Pro Ser Asp Ala Gln Phe Val Asp Val Ile His Ser Asp Thr
195 200 205
Asp Ala Leu Gly Tyr Lys Glu Pro Leu Gly Asn Ile Asp Phe Tyr Pro
210 215 220
Asn Gly Gly Leu Asp Gln Pro Gly Cys Pro Lys Thr Ile Leu Gly Gly
225 230 235 240
Phe Gln Tyr Phe Lys Cys Asp His Gln Arg Ser Val Tyr Leu Tyr Leu
245 250 255
Ser Ser Leu Arg Glu Ser Cys Thr Ile Thr Ala Tyr Pro Cys Asp Ser
260 265 270
Tyr Gln Asp Tyr Arg Asn Gly Lys Cys Val Ser Cys Gly Thr Ser Gln
275 280 285
Lys Glu Ser Cys Pro Leu Leu Gly Tyr Tyr Ala Asp Asn Trp Lys Asp
290 295 300
His Leu Arg Gly Lys Asp Pro Pro Met Thr Lys Ala Phe Phe Asp Thr
305 310 315 320
Ala Glu Glu Ser Pro Phe Cys Met Tyr His Tyr Phe Val~Asp Ile Ile
325 330 335
11/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
Thr Trp Asn Lys Asn Val Arg Arg Gly Asp Ile Thr Ile Lys Leu Arg
340 345 350
Asp Lys Ala Gly Asn Thr Thr Glu Ser Lys Ile Asn His Glu Pro Thr
355 360 365
Thr Phe Gln Lys Tyr His Gln Val Ser Leu Leu Ala Arg Phe Asn Gln
370 375 380
Asp Leu Asp Lys Val Ala Ala Ile Ser Leu Met Phe Ser Thr Gly Ser
385 390 395 400
Leu Ile Gly Pro Arg Tyr Lys Leu Arg Ile Leu Arg Met Lys Leu Arg
405 410 415
Ser Leu Ala His Pro Glu Arg Pro Gln Leu Cys Arg Tyr Asp Leu Val
420 425 430
Leu Met Glu Asn Val Glu Thr Val Phe Gln Pro Ile Leu Gly Pro Glu
435 440 445
Leu Gln Leu
450
<210> 11
<211> 2436
<212> DNA
<213> Homo sapiens
<400> 11
aaaatcccac agtggaaact cttaagcctc tgcgaagtaa atcattcttg tgaatgtgac 60
acacgatctc tccagtttcc atatgttgag attctactta ttcatcagtt tgttgtgctt 120
gtcaagatca gacgcagaag aaacatgtcc ttcattcacc aggctgagct ttcacagtgc 180
agtggttggt acgggactaa atgtgaggct gatgctctac acaaggaaaa acctgacctg 240
cgcacaaacc atcaactcct cagcttttgg gaacttgaat gtgaccaaga aaaccacctt 300
cattgtccat ggattcaggc caacaggctc ccctcctgtt tggatggatg acttagtaaa 360
gggtttgctc tctgttgaag acatgaacgt agttgttgtt gattggaatc gaggagctac 420
aactttaata tatacccatg cctctagtaa gaccagaaaa gtagccatgg tcttgaagga 480
atttattgac cagatgttgg cagaaggagc ttctcttgat gacatttaca tgatcggagt 540
aagtctagga gcccacatat ctgggtttgt tggagagatg tacgatggat ggctggggag 600
aattacaggc ctcgaccctg caggcccttt attcaacggg aaacctcacc aagacagatt 660
agatcccagt gatgcgcagt ttgttgatgt catccattcc gacactgatg cactgggcta 720
caaggagcca ttaggaaaca tagacttcta cccaaatgga ggattggatc aacctggctg 780
ccccaaaaca atattgggag gatttcagta ttttaaatgt gaccaccaga ggtctgtata 840
cctgtacctg tcttccctga gagagagctg caccatcact gcgtatccct gtgactccta 900
ccaggattat aggaatggca agtgtgtcag ctgcggcacg tcacaaaaag agtcctgtcc 960
ccttctgggc tattatgctg ataattggaa agaccatcta agggggaaag atcctccaat 1020
gacgaaggca ttctttgaca cagctgagga gagcccattc tgcatgtatc attactttgt 1080
ggatattata acatggaaca agaatgtaag aagaggggac attaccatca aattgagaga 1140
caaagctgga aacaccacag aatccaaaat caatcatgaa cccaccacat ttcagaaata 1200
tcaccaagtg agtctacttg caagatttaa tcaagatctg gataaagtgg ctgcaatttc 1260
cttgatgttc tctacaggat ctctaatagg cccaaggtac aagctcagga ttctccgaat 1320
gaagttaagg tcccttgccc atccggagag gcctcagctg tgtcggtatg atcttgtcct 1380
gatggaaaac gttgaaacag tcttccaacc tattctttgc ccagagttgc agttgtaact 1440
gttgccagga cacatggcca taaataatag aaagaaagct acaaccacag gctgtttgaa 1500
agcttcacct cacctttctg caaggcagaa aaagtatgaa aaaaccaagg cttttttcag 1560
tagcgtccta tggatgtcac attgtacatc aaacaacctt gtgattataa aacgatcctg 1620
ggaaggagcc cctaactagg gcaagtcaga aatagccagg ctcgcagcag cgcagcgctg 1680
tgtctgctgt gtcctggggc ctcccttgtt ccgacctgtc aattctgctg cctgtcacgc 1740
12/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
gggtggttct gcccatcgcg gctgcgggtc aagcatcttc aagggaagga cggactggag 1800
gcctcaccgt ggactcaact ctgcattctc cgtgccacat tcctccagtt cccacacgta 1860
gaagggaacg aaactgacgt ctacctcatg gggctgctgt gtgggtttgg gaggcaaaaa 1920
tctatgaagg gttttttgaa atcccatagg tgccacatct atgagatgtt tgataaatgt 1980
gaatatgctt ttacatttgg gcttatctaa tttgcaataa gagagcctct ctctatcaac 2040
accagcttct ctctcgggct gtttgctcag ggaaggcaag aaagccacgt gctggccctc 2100
tgccttctct aaagtgctgt tggagcatgg aggagctgga ggagatgggg atggactgac 2160
agctaagagg gcggctgctg ggactagata gtggatgaag aaagaaggac gaggaagccg 2220
tggggcagcc tcttcacatg gggacagggg atggagcatg aggcaaggga aggaaaagca 2280
gagcttattt ttcacctaag gtggagaagg atcactttac aggcaacgct cattttaagc 2340
aacccttaag aaatgtttat gtttctttat taccaatgta atctatgatt attgaaggaa 2400
atttagaaaa tgcgtagata caaaattaaa aaaaaa 2436
<210> 12
<211> 451
<212> PRT
<213> Homo Sapiens
<400> 12
Met Leu Arg Phe Tyr Leu Phe Ile Ser Leu Leu Cys Leu Ser Arg Ser
1 5 10 15
Asp Ala Glu Glu Thr Cys Pro Ser Phe Thr Arg Leu Ser Phe His Ser
20 25 30
Ala Val Val Gly Thr Gly Leu Asn Val Arg Leu Met Leu Tyr Thr Arg
35 40 45
Lys Asn Leu Thr Cys Ala Gln Thr Ile Asn Ser Ser Ala Phe Gly Asn
50 55 60
Leu Asn Val Thr Lys Lys Thr Thr Phe Ile Val His Gly Phe Arg Pro
65 70 75 80
Thr Gly Ser Pro Pro Val Trp Met Asp Asp Leu Val Lys Gly Leu Leu
85 90 95
Ser Val Glu Asp Met Asn Val Val Val Val Asp Trp Asn Arg Gly Ala
100 105 110
Thr Thr Leu Ile Tyr Thr His Ala Ser Ser Lys Thr Arg Lys Val Ala
115 120 125
Met Val Leu Lys Glu Phe Ile Asp Gln Met Leu Ala Glu Gly Ala Ser
130 135 140
Leu Asp Asp Ile Tyr Met Ile Gly Val Ser Leu Gly Ala His Ile Ser
145 150 155 160
Gly Phe Val Gly Glu Met Tyr Asp Gly Trp Leu Gly Arg Ile Thr Gly
165 170 175
Leu Asp Pro Ala Gly Pro Leu Phe Asn Gly Lys Pro His Gln Asp Arg
180 185 190
Leu Asp Pro Ser Asp Ala Gln Phe Val Asp Val Ile His Ser Asp Thr
195 200 205
Asp Ala Leu Gly Tyr Lys Glu Pro Leu Gly Asn Ile Asp Phe Tyr Pro
13/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
210 215 220
Asn Gly Gly Leu Asp Gln Pro Gly Cys Pro Lys Thr Ile Leu Gly Gly
225 230 235 240
Phe Gln Tyr Phe Lys Cys Asp His Gln Arg Ser Val Tyr Leu Tyr Leu
245 250 255
Ser Ser Leu Arg Glu Ser Cys Thr Ile Thr Ala Tyr Pro Cys Asp Ser
260 265 270
Tyr Gln Asp Tyr Arg Asn Gly Lys Cys Val Ser Cys Gly Thr Ser Gln
275 280 285
Lys Glu Ser Cys Pro Leu Leu Gly Tyr Tyr Ala Asp Asn Trp Lys Asp
290 295 300
His Leu Arg Gly Lys Asp Pro Pro Met Thr Lys Ala Phe Phe Asp Thr
305 310 315 320
Ala Glu Glu Ser Pro Phe Cys Met Tyr His Tyr Phe Val Asp Ile Ile
325 330 335
Thr Trp Asn Lys Asn Val Arg Arg Gly Asp Ile Thr Ile Lys Leu Arg
340 345 350
Asp Lys Ala Gly Asn Thr Thr Glu Ser Lys Ile Asn His Glu Pro Thr
355 360 365
Thr Phe Gln Lys Tyr His Gln Val Ser Leu Leu Ala Arg Phe Asn Gln
370 375 380
Asp Leu Asp Lys Val Ala Ala Ile Ser Leu Met Phe Ser Thr Gly Ser
385 390 395 400
Leu Ile Gly Pro Arg Tyr Lys Leu Arg Ile Leu Arg Met Lys Leu Arg
405 410 415
Ser Leu Ala His Pro Glu Arg Pro Gln Leu Cys Arg Tyr Asp Leu Val
420 425 430
Leu Met Glu Asn Val Glu Thr Val Phe Gln Pro Ile Leu Cys Pro Glu
435 440 445
Leu Gln Leu
450
<210> 13
<211> 3145
<212> DNA
<213> murine
<400> 13
tgctattttc ttttaaaagc atccatttat tgtaactcat atatgtaacc ttagaattta 60
cagggcaaaa tagacaggat tattctgagt ttggggccag gcttcagtta catagtgaat 120
tctagggtat tttgagctgt gaatatgcat atatatctat tatggatgta tctttgaaaa 180
gggacaaaag tcacggcagg aagaaggagg gagtggggag aaggagagtt ggagagaagg 240
gttgctggag acagaagaaa ctaacaaaga tagaacaatt tctgaagacc aaggttaatg 300
gattttagca gatattttca ttatatacag ttgctcaaaa cattttaaaa gctctatgat 360
14/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
ttcatcagtg ctttcttcct aggtttgggt attggagagc catcggggca cattgatttt 420
tatccaaatg gaggaaaaca tcagccaggt tgtcctacat caattttttc aggtatatga 480
aaaaagtatt ttcattgttt ttttttcttt tgttgaaact agattttttt tcatacaata 540
tattctgatt acagttatac agggtccaaa atgatatggg aaattaattt atcttttggc 600
tacttaatct gtgggcaaga ttttgatatc agaatttttg ctattgactt aaaagagtta 660
ctgacaagaa ttaatttaaa ttgatactac ttaatatact aaggttacta gcatatacaa 720
caactagtca tgggtgaaac ctgtttggtg ttgtttcaac aattttagtt cattatttta 780
accataaaga acaattttct attatctaga aaaacacatc taactcaagt atcctagaat 840
acttctagaa tatgagtttg atcacaagat ttagcactta caataccaca tatacacaga 900
tttgacaaca ctggccactt ctgtgtccca cacatggggt tattcatggc ttctgagtgt 960
ggaatggggc cctcttaagc acggtagcgg ataagcgcat gaccaaattc cactgtgact 1020
aagagaagat tgaaccaacg aaagaaaata tgaaaaactg gggaagggaa gtgacataag 1080
tgcattttaa caacacttgt ctccatagac atagttgaca ttgcatcagt attttaggga 1140
tatagaacta gaaaagagaa attctataat tgcaaacact gttgcaatgt gttagtatag 1200
cctgttaaat tgaaacagct atgctttccg gattattatt tgatgaaaat gttttattaa 1260
tttcatgaaa aagtcatgct caatttaaaa atttgatggg acttttttta ttggatattt 1320
tatttattta catttcaaat ataccccttt cctggtttct cctccagaaa ctacctatcc 1380
catcctccct tctcctgctt ctatgagggt gctccccccc ccactcctgc ctccccacct 1440
gggaattccc ctacactggg gcatccagcc ttcacaaggc caagggctca tttcccatta 1500
atgcccaaaa aggccatcct ctgctacatg tgtggctgaa gccatgggtc tctccatgta 1560
tactctttgg ttagtggttt aatctctggg agttctggtg ggtctggctg gttggtattg 1620
ttgttcttcc tatgggttgc aaactccttc agctccttta gtcctttctc taactcttcc 1680
aatggggacc ctgtgctcag tccaatggtt gaactataac tgtattttca gtgctctgta 1740
tacataagaa acagaattcg gtataactta atggtcaggt ttttcccttg atcttgattt 1800
catatattta cagttaattt tgcttgtttt tctcacacct aaggaaccaa ttttattaaa 1860
tgcgaccatc agagagccat ttacttgttc ctcgcagctt ttgaaacaag ctgcaacttt 1920
gtttcatttc cttgtcgttc ctacaaagat tacaagaatg gtttgtgtgt ggactgtggg 1980
aatctttaca aagactcctg cccaagactt ggtgagagaa gttgtgttta tacaattcct 2040
tttgaagttg agtaagacca tcagggtagt attttaggtt ggatgctaaa gtgtgtttta 2100
tattaatctg ctactacatt acagtttggg gaaaatttat caacagagta atggcagatt 2160
cactgtatac attctgtatc atttttttgt ctaaggaatt ttccttcctg tcttcctatg 2220
tctagaggca gaatgtctac attctttgtg cactggaaac tgtttttttt cctgtctttt 2280
aacaggaagt tttaggtaca agtgaagagg tgtaccttgt agctagctag ctttggattt 2340
tctctcagca agtcatcaag aatcctgtat agttcatgcc ttctatatca gtaaaacata 2400
aaaagggttg ttatgttggc agcttttgaa aatgaaaacc taccagtgaa aatagagaga 2460
aaaataaata actcctaaaa gatgggatga agtaggagca aataaagagt acaatggggg 2520
tataatcaaa gtgcactatt tatattgcct tggttggaat tttgttgctg tgaggagaca 2580
ccatgaccac agtaactctt acaaacaaac aaacaaacaa acaaacaaac aacccactca 2640
aacctttgag tggggcttgt ttaccattca gagatgtagt gcattattat catggaggga 2700
cgcgtagtgg catgcaggca ggcatggtgc tggagaagta gctgagagtt tcaaatctgg 2760
acagcaggca gcagaaagag agagtgacac tgggcccgat gtgagcattt gagacttcaa 2820
agttcactcc caggaacaca ctccttccaa caaggccaca gctcctaata ttggccttcc 2880
ctacagctct acaagggccc atttcatcca aacctccaaa ttcatattat ctatatagat 2940
aaaattgtga agtaataaat aaataattac aagatggtca agtaaatagt ttaaaagcag 3000
aagacttaaa agtattccag aggcagttct atatttccca aagagaattc caggtctcaa 3060
catattataa acttatcaat atctgcttaa tatcatggaa agatattcca gaaatgcttg 3120
caaagtctaa ggtgctctag gagta 3145
<210> 14
<211> 2933
<212> DNA
<213> murine
<400> 14
ttcatctact ctttagataa atttggtaaa atatcatatc aatattttgt tgaaacatct 60
ttcctgtgtt ttgaatgcct ttgaagggtg ctgataaata ttcaaataat gaaagtactt 120
gtgtttgtca aatttatttt aattgtaaat atgttgggac agatttaaac taaaattgca 180
agtgcttatg tttataattg aagatcaggc aagtgtaaaa tagagcaagc tagtaaaata 240
taactcaatt gacagttttt gttttgcatt tcaggtcttg acccagctgg accacaattt 300
15/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
tctagaaagc catcgaatag cagattatat tacacagatg caaagtttgt agatgtcatc 360
cacactgata tcaaaagtaa aataataagt atctatgaga tttgtgtttc aaagcaaaat 420
gtgaaatgta gatcccaagt ttgaggttgt aagatgaaga agagttgcag ataaaactgt 480
ttgggattta attctccatt ctgtgggcag tcactgactt tacaatactt ctttggcatg 540
ttcaatcctg ctatttagct ttctttccca cctgtaatgc atggcaagta gaactgagca 600
tagcactaaa aacacagata aggaatattt tctataatta ggcattagtt ctctattgat 660
ttaaatagtc tttataaaga ccctctctaa cagggtttgg atggcgttgg ttataaaaag 720
gtcgacaaag gcattagaag agctatcgag actgtcctga ggcccttttg attgaatgaa 780
taagatgagt cagttggaga gagacagaga gagacagaga tacacacaca cacacacaca 840
cacacacaca cacacacaca cagagagaga gagagagaga gagagagaga gggagagaga 900
gaggtagata gatagataga tggatagata gatagataga tagatagaga cagacagaca 960
gacagacaga cagagtttaa tgaccttctg gatttgatgg ggtaattcta acactgactg 1020
aactcttcat atatatcagg gagactcttg attacttacc aaactcactt caatgttttt 1080
ctgcatgtat gctatataaa catgtttata gcagcactcc ttaaaatact aaggacacag 1140
aagcagtcta gatgcccacg aatagataaa catacctgga aatttatata gccataaaga 1200
aagatgaaat cacaacattt tcaggaaatg gagtgaaact tggaaccatc cttttaagca 1260
agccaagcca aagtatagac aaatatccct gtcctttttg ttttcacaat cagcacaccg 1320
ggtgctgctg agaaggctct gtggttcaga tcatgcactg cccagagttt gattttcatc 1380
acttataaca tgtggctcat aactccagat ctagggagta ttaagtagtt ctggccttca 1440
cagacacccg tagttatgtg cacatgcccc acacagacac aaccacgtac ggatcattaa 1500
aaacaataaa aaaatctcta aagttatgtt cttttgtgta tgtacatatg tgtacatatg 1560
gcaggactgc ctcaaggacc atttgtggat tcacaataag taccagttgt ttcttcctga 1620
tgactgagta gctgtgttgt ttttatcctg acccatcggc aggtgggaca tctgtctcaa 1680
gaggtgcttc ttgcttttta gctttcatgc tgctctgaga catggagagg tttcagagat 1740
ttctccaaat agccgacttt cttctagggc actggttctc agccttccta attctgtgac 1800
ccttcaatac agtttctcat tttgcagcaa cccataacca attatgttca ttgctacttc 1860
atgactgcca ttttgctact gttatgaatt ataatgcaaa tatctgtgtt ttctaacggt 1920
cttaggtgaa tcctgtgaaa agatcaattg accctcacag ggatggtgac cccataggct 1980
gagaaccact gttctagggt catattttaa ataatggcaa ttctttgctt aaattcaact 2040
aagtgagtac attataatgc taaaggaggg attccccacg gctacattga aggcacactt 2100
gcttctatgg agtctgagga cttgtaatga aatgacttaa accggttttc atctttttac 2160
cttgaacaca cttttatctg ctcctcatgc tgtgtatgcc cccattttat ggctctttat 2220
gatatcatga caccttactg gattctgttg tatgctctac ctaaattaaa agtgctgcct 2280
cttgatccat atctggacac taatattatc tcaagaattc agtaacacga aattgcttgt 2340
accctcagct aaataatttc aatcttatct ggcagtgaaa tttcacatct aagtatagtc 2400
ccaagttttc tgagattgat atgcctttca tgctcccttt ggttatagag taaaatattg 2460
gtaatagatt cagaattgga accgtctcca gagccctggg aatctagaag caaatagaag 2520
cacatgaatc ataggcacat ggctcatgcc atcctaactc aagttgtaat cggtggagga 2580
agcaagttgt tgtagatttt gctactgagt aaagctagca tcagcacgcc caaggcaccg 2640
tcatccaaac agggagaaaa gcatcacact gccctgacag ctgtatgtga aatacctaat 2700
aatatttcac cttgtgctga tccttgccca ttgtatctta ggcagtttct ttgtggaccc 2760
tagtgtgctc ccagtatact gttgactcac tgcctcccac tcttctcaca accacttaag 2820
ttgtcaaact tcagagtgag aaattctcta gccaagctac attaaaaagc tttccagttt 2880
taacttccat tttaaaaaag tattcaagga ttatccttgc tgctaggtgt ttt 2933
<210> 15
<211> 3470
<212> DNA
<213> murine
<400> 15
aagtgactaa aataattttt tattaatttt ttttaaatct ccaattttaa aaacattggg 60
actcaggacg gacgttgaga taacaggcac tacatactgt ggaaagggtg agctaagtag 120
agaattttag gaaaaaatca tggcattttg cagggagaga agtagccaaa ccagagcttt 180
gtggtttgga cagcaactcc ctcctctttt ttatacgcca ttgctttaag ggatttgaag 240
aacagaattg ctttttttgc tgggaacgtc cctgccttgc tctgcaatag tggtgaaaaa 300
tgatcatcat tttatgagct tcaaacatca ctgtgtaatc tgcatcatca ctattagctc 360
cctgggagtc ggccaaaaaa tagacaccca gagagctccc aaaccttctt acttgatgta 420
gatcagatgt ctaaaccttg tccttcgatt gatttgctat actatctaac ccgtgctctt 480
16/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
ggaacccacc ttgagtccat agacttcatc atagcatttc aaaagtaaga gtgacttata 540
cacttttacc agttctgcta ggggctttgt tataccctga agggaagaca gaaggctagc 600
atatatttcg taatcaaaca gctccagcct tgggtcttag tccgtttgag ccactgcatg 660
acaaactctt taaactgacc ttatgtacaa acacattcat gtctcatggt tctgcaacga 720
cctggtggag tctggctctg gtgagggcca gttcttggct catagtttga atcctttctc 780
agcattctaa catcatggaa gatccaaatg agcttcttta ggttttgcct gtcaggacat 840
tagccccatt cttgaatgct ccaactttgt gacaccattg cattctctgt gcccacccta 900
tttcctgcag aggccataaa tattaaggtt tagaattttg gaggcacaaa tattcagaat 960
tcagtgactt agaatacatc tttgtatgta gctttgtata tgtctttaac ttctgttttc 1020
ttagtaaaat ggatatataa cagaatgtta tttagttaat aattttagca tagggttact 1080
tttaaataat aagattgaaa tggcaaacat gaatatgaaa tataaagcat aaaacacata 1140
ttaaggtagc acaaaataag cacacagtca tgacaattat tagtatttgt tcattatatg 1200
tctcagtaat taaatgttac ttttacatat ctcactctgt atatgggtga ttaaaacttt 1260
aaatttgtac taaacataaa tttaaacttt caagtaaagt ttaatgttgt tattggaaat 1320
ggatagagaa agatcagata ttttgtggtg caagcagaac aaacccatat ctttttacag 1380
ttataagtcc tcttccttat gctccaaact tatatgaatt atatgaagcc aaaatcaata 1440
atggatttca gaatgtcagt atgctaggaa ttatgtgtga ttatccaaaa ttaaagttat 1500
tacattgtat ttttcattta tagatccatg gggcatcact tgacaatttt catttcattg 1560
gcatgagctt aggggctcat attagtggat ttgtaggaaa gatatttcat ggtcaacttg 1620
gaagaattac aggtaaaaca atctatttct tttgtaaaat tatcttttta cctggatgaa 1680
atggacaatt ttcttttttt tttctttcca ttttttatta ggtatttagc tcatttacat 1740
ttccaatgct ataccaaaat tcccccatac ccacccaccc ccactcccct accacccact 1800
cccccttttt ggccctggcg ttcccctgta ctggggcata taaagtttgc ctgaccaatg 1860
ggcctctctt tgcagtgatg gccgactagg catcttttga tacatatgca gctagagtca 1920
agagctccgg ggtactggtt agttcataat gttgttccac ctatagggtt gcagatctct 1980
ttagctcctt gggtgctttc tctagttcct ccattggggg ccctgtgttc cattcaataa 2040
ctgactgtga gcatccactt ctgtgtttgc taggcccccg catagtctca caagagacag 2100
ctatatctgg gtcctttcac aaaatcttgc tagtgtatgc aatggtgtca gcgtttggaa 2160
gctgatcatg ggatggatct ctggatatgg caatcactag atggtccatc ctttcgtcac 2220
acttccaaat tctgtctctg taactccttc catgggtgtt ttgtttccta ttctaagaag 2280
gggcaaagtg tccacacttt ggtcttcgtt ctcttgagtt taatgtgttt agcaaattgt 2340
atcttatatc ttgggtatcc taagtttctg ggctaatata cacttatcag tgagtacata 2400
ttgtgagagt tcctttgtgg ttgggttacc tcactcagga tgatgccctc cagatccatc 2460
catttgccta ggaatttcat aaatttattc tttttaacag ctgagtagta ctccattgtg 2520
taaatgtacc acattttctg tatccattcc tctgttgagg ggcatctagg ttctttccag 2580
cttctggcta ttataaataa ggctgctatg aacatagtgg agcatgtgtc cttcttaccg 2640
gttggggtat cttctggata tatgcccaga agaggtattg ctggatcttc tggtagtact 2700
atgtccaaaa tgcttcctat accaccttaa aatcccaagc ctcagacata ttaaaagaat 2760
aaactttact tgattggttg ggttgttttg ttttggtttt tttgtttttt cattagatac 2820
tttatttaca tttcaaatgt tatccccttt cctaatttcc cccctgaaaa ccctctatcc 2880
cttccttccc tctccctact caccaaccca cccattcctg cttcctggcc ctggcattca 2940
cctatactgg ggcatagaac cttcagagga ccaaagcctt ctcctccaat tgattacaga 3000
tcaggccatc ttctactaca tatgcagctg gagccatgag ttccaccatg tgttttcttt 3060
gtctgatgat ttagtctcag ggagctctga aggtactggt tagtccatat tgttattcct 3120
cctatggggc tgcaaacccc ttcagctcct tgggtccttc tctagcttct tcattgggga 3180
ccctgtgctc tgtccaatgg atggctgtga gcatccactt atgtatttat taggcactgc 3240
agagcccctc aggagacagc tatatcaggc tcctgtcagc aagcatttgt taacatgcac 3300
aaccgtgtct agatttggtg gttgacaatg ggatggatcc ccagatggga cagtcaatgg 3360
atggtccttc cttctctgta actccttcca tgggtacttt gttcccactt ctaagaagga 3420
ccgaagtatc cacactttgg tcttctttct tgagtttcat gtgttttgca 3470
<210> 16
<211> 905
<212> DNA
<213> murine
<400> 16
caatgtattt acctttcctg ccgtccttcc cacttgatta gttgtaacaa tggcttatgt 60
cagagccatt ttattttatt ttaaaagaat gtttatgact tggaacctat cacatttgtt 120
17/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
catatttgtc attattattt ttattacttc taatttagga agtctttttg cctggtccat 180
gtacagctct aactttagct gtgctcagta aatcatgtta atgtgcaaat aaaacagaat 240
tctcccattc agttgtataa acctatacca acaaaacggc actgttatcc taatgctcag 300
attgaatttt tatcttttca gagaagaaga gaacatgtct tgaattcaca aagttaagtg 360
caatgaatag cttaaaagat ttattttgtc ccaaagtaaa gataaatctg ctgatgtact 420
caaggggcaa cgccaagtgt gcagagccac tctttgaatc taataactca ctcaacaccc 480
gtttcaaccc agcgaagaaa accgtctgga ttattcatgg gtacaggccc tttggttcca 540
ccccagtgtg gctttctagg tttactaaag cctttttgaa acaggaagat gtgaatctca 600
ttgttgtaga ctggaaccag ggcgctacga ctttcatgta ttctagagcg gttagaaata 660
ccagaagagt tgctgagatt ttgagagaaa ccattgagaa tcttttggta agttggaaaa 720
atttaaggta gacccatgat gtacaatata tggtttctta tgtgacacca tgaaatgaat 780
actttttccg taagcagttg gaactttaac ttagacatag aaattttaag ctatattcca 840
tctactctaa ataataattc catatttcat tttcatattt ttaaacatga gaaacctttt 900
atttt 905
<210> 17
<211> 506
<212> DNA
<213> Homo sapiens
<400> 17
aattcaactt gtgtagctga aggtttgttt gtgacttatt acagagcctg tgacttaaaa 60
atccttccca caaccacaag ctaaagtggg agaaaacaaa ctacctcacc ttttcaacca 120
agagggagga gcaaaaatca gtgaactttt acagaagaac ctgccagcct gtgatgatcc 180
taccaaagag aaacctcaat gagttacgga atttcctttt tggtgaattg agtgctgttt 240
ttgcttttct cagattccaa atgagagtat acatttttct ttgtttgatg tgctgggtga 300
gatctggtaa gatattaatt aataatttga tttatcttta aaaattgcat ataaaatagt 360
gtggtctgta ttctgggata aagaatgtta tgatataatg ggataaaaat atgttagatt 420
attggtacta attataacaa acattactat taagaaaact tctgtgtgtg tacgtgtgtg 480
tgtgcatgtg tttgtttaat aagttg 506
<210> 18
<211> 786
<212> DNA
<213> Homo sapiens
<400> 18
ttccattact tttggttcag aaaatctttt tacataatta atacattacc ttcaacattg 60
cagttgctca gtaaattatg ttgaagggct catgaaatga aatcctccct accctccaca 120
tttaattctg ttctttgcaa tagcaacagg aataaaatac tatcccagta ctcagcttga 180
ctgacatttt cttatttcag ataataaaag accatgcctt gaattctctc agctaagtgt 240
aaaggattcc ttcagagatt tatttattcc gagaatagag accattctga tgatgtatac 300
aaggaacaac ctaaactgtg. ctgagccact gtttgaacaa aataactcac ttaatgttaa 360
tttcaacaca caaaagaaaa cagtctggct tattcacgga tacagaccag taggctccat 420
cccattatgg cttcagaact tcgtaaggat tttgctgaat gaagaagata tgaatgtaat 480
tgtagtagac tggagccggg gtgctacaac ttttatttat aatagagcag ttaaaaacac 540
cagaaaagtt gctgtgagtt tgagtgtgca cattaaaaat cttttggtaa gtctgggaat 600
tttatgtatt atacatgcta tacaatatat tatacgtgct atacaatatg cagtgtgtta 660
cattcaatac cacaaagtga taataaacta tatacttttc cccataagca ttaacaatta 720
ctggtttgat ataccaatgt atgcagccat cttttctttg tctgcctaaa tgaataattc 780
tatacc 786
<210> 19
<211> 509
<212> DNA
<213> Homo sapiens
18/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
<400> 19
ataaagaaaa atcaaatatt ttgtggcaaa agttgaacaa atatatagtc atcttgaaat 60
tataaagtct ctttcccgat atccccaaaa tgaatcaagc cagaaaaata atgaattttg 120
aaatgtcgat atatgagggg ggcatgattt gttattactt gaaatgaata ctgtaatatt 180
gcctttctct ttaattgcag aagcatggtg catctcttga caattttcat ttcataggtg 240
tgagcttagg ggctcatatc agtggatttg ttggaaagat atttcatggt caacttggaa 300
gaataacagg taaaattatt tttataaaat tattgcttta gcatactttt aagtatcaga 360
agttactttg ctaatttttt tttttttgag acagagtctc gctctgttgc ccaggctgca 420
gtgcagtggt gcgatctcgg ctcactgcaa cctctgcttc gtgggcttaa gtgattctcc 480
tgcctcagcc tcctgagtag ctgggatta 509
<210> 20
<211> 502
<212> DNA
<213> Homo Sapiens
<400> 20
ctgaaggtta gtagtattat tcaaatactg aagttacctg ctattcattc acttccattt 60
gtttttagat atgttcttgg ataggtttaa actaaaagta taaatgcttt ttatgtttat 120
aatgcagtaa taatggaagt atgcaatgta aggagctctg tggacttgat gagactgatt 180
atttctttct tgctttccag gtcttgaccc tgctgggcca aggttctcca gaaaaccacc 240
atatagcaga ttagattaca cggatgcaaa gtttgtggat gtcatccatt ctgactccaa 300
tggtaacaaa tcaggattta ttacctaatt atttgaaaat gaaaaggaaa atgtagacct 360
tgggcaagag gagggcataa gaagaaaaaa catcatggat aaaatgattt taaatctgta 420
attatagaat gataatattt cccaagcaat atttcttagg gttagtgatt tcctgcattg 480
tcaaatggca ctctttgact tc 502
<210> 21
<211> 490
<212> DNA
<213> Homo Sapiens
<400> 21
ggcaaatata ttaaattaat gaaatatact tttcatgaag gaattataga agaaaaactt 60
atctttataa aaacagcact tattttcaat tctaatattt aaagagtaac tttcaatgga 120
ttttataggc.tattttgttt tatgcaagat actcacctga ttatatacat gttgtgattc 180
ttgggtgcct ttcttctcag gtttaggcat tcaagagccc ttgggacata tagattttta 240
tccaaatgga ggaaataaac aacctggctg tcctaaatca attttctcag gtatactgac 300
agtatttttg aatgaactac ataatattcg aaatgatgaa agaaaataac ttacttttcc 360
cctcttaatc tgtaggcagt gttttgattt catcatcatt gcccatgact caggagagtc 420
attggttaac aatggtttaa tttgaccatg cttagttcat aaaggttacc agtagacatc 480
cattaggtca 490
<210> 22
<211> 568
<212> DNA
<213> Homo Sapiens
<400> 22
tctcatgcaa aagacatact aaatttaagt tttcattggt actatttgtt tagtggtttg 60
acataaaact gtgtattata gtcagtaata ttcataaggt acatagtatt tagcaaaact 120
tcatctttac tttgttttta aatgtacttg aacatggctt aatacatcta cagttctttg 180
tttttttttt aacccttaag gaattcaatt cattaaatgc aaccaccaga gagcagttca 240
cttgttcatg gcatctttag aaacaaactg caattttatt tcatttcctt gtcgttcata 300
caaagattac aagactagct tatgtgtgga ctgtgactgt tttaaggaaa aatcatgtcc 360
tcggctgggt aagagagatt attatagtta ctatcattct tatttaatat gaataacata 420
attgttagta ttttctgctg gaagctaaac tgtgcactct gtttacttgt taatacatga 480
19/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
ccattttatg gaaaaatcat ctgtagagat gtggtacaga gtaaatattt cattccctac 540
taaacatttt taatatattt aaagagaa 568
<210> 23
<211> 505
<212> DNA
<213> Homo Sapiens
<400> 23
aatcctgaat ttaagagttg ttattgtaat taaaataaat ggaatgctac attatttgaa 60
aaaatgcttt ttaagttatc aaaatccatg atgatcatta tgatgattat atgcactgat 120
cttttagtta ctttagtgat ttgacatttt tgttatttga tggaaatcca atttctaatg 180
ttctatttct caaatctcag gttatcaagc caagctattt aaaggtgttt taaaagaaag 240
gatggaagga agacctctta ggaccactgt gtttttggat acaagtggta catatccatt 300
ctgtagtaag ttattaacaa ttttaacaaa taaacaaatt tttagttttt tttgcactaa 360
tctacatttg attcaaagtg ttctttgcac ccactcaaat tccatcatgt tctttcaggc 420
aaatttaaaa tttgctatag ccagtgggac aaattcaact aaggacttga tcttttttta 480
gataaaatgg agagacgctt gggta 505
<210> 24
<211> 512
<212> DNA
<213> Homo Sapiens
<400> 24
gtagtcaaaa attgacaaac aaaaggtaaa atatattacc catttaccaa atgagtcctc 60
tcaataatat tatttgcata tgtaatatga gcctagatta catatctatc tatacataca 120
catatatata tatctgaata tgtaccaaaa ccaaaaatta aaaaataatg tgagttgtat 180
tctatatttt tattttgtag cctattattt tgttctcagt ataattgttc cagataaaac 240
tatgatggat ggctcgtttt catttaaatt attaaatcag cttggaatga ttgaagagcc 300
aaggctttat gagtaagtaa aatttactat tttcttctct catatattca atgtttgctt 360
tcttcagtat cccatccaac agataattta aatagtgtag tttatttttt aatctagaga 420
gtcttaaaat gttaaatatt aaaaggatct catatgttat attctaattc ctttatttta 480
cagagaaaga taacatatgt taaagaggca cc 512
<210> 25
<211> 577
<212> DNA
<213> Homo Sapiens
<400> 25
ttattatttt gcatttatta ttttgttctg ttataaatta cggttttaat tattttaata 60 .
aatactgcaa taatattttg tttggccttt tccctataat tttgttctct gtggcctccc 120
ttattaggat tatattgatt ttagaattga.gttatgtttc aaattattaa tatgctgcgt 180
gtatcaaatt ttctctctag aaagaacaaa ccattttata aacttcaaga agtcaagatt 240
cttgctcaat tttataatga ctttgtaaat atttcaagca ttggtttgac atatttccag 300
agctcaaatc tgcagtgttc cacatgcaca tacaagatcc agagactcat gttaaaatca 360
cttacatacc cagaaaggta agaaaattaa attttgattt caacatgtta tcttttaaac 420
ttgaaaatat tggtttctta agtaatggtt taaaaattca tgtattcaaa tatctgttta 480
ttgtgtattt cagactatat tttacataaa tattagaatc tcaaaaaggt ataaaggttt 540
ttttggcttt aataaaattt atttgggaat ctcattg 577
<210> 26
<211> 606
<212> DNA
<213> Homo Sapiens
20/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
<400> 26
aagcattatg tctatttcat ccctacacag ccaaatgaga tagaatggaa atgatcccat 60
atatttgttc caactcccta catttagaga actagctagt tacaggcatg gactattcta 120
atattaatga gagaaggaaa tcaactctgt tgttttacta atagttattt ttttgttctc 180
tccatttttc tctttctcag accaccactt tgcaggtata atattgtact taaagacaga 240
gaggaagtgt ttcttaatcc aaacacatgt acaccaaaga acacataaga tgccttcttc 300
catcaaatgc acttgcttgt gaattaatgg acttgtaaat gaaacaatgc aatcagtctt 360
ttataatgca ctgttcaatt tgagattcaa gtatttctat ttcttggaaa aaattttaag 420
aatcaaaaat aaagaaaata aaaaatgcat acagttaaac attccaaata tattctgaaa 480
gttgtttatt tctgtttact ttttctgctt cctcaattta atacttatca ttgactgttt 540
accttaatta tttattaata atgaataaat gccttttgaa aaataggcaa gtttagtgaa 600
tacttc 606
<210> 27
<211> 730
<212> DNA
<213> Homo Sapiens
<400> 27
cacaagaacc aaggggaaaa gatctcaagt cacacttggt ttcaacacac caagccaaac 60
aactaataaa ccgcccacga agaatgaaac tattctgaac tcgtcattct gcagatgcag 120
attataatat taagatatgc acaaaattgt ataaagtaga caatgagtga ccttttattg 180
aaaagaggag gagtcaaaga tcctgaaagg gttgggactg cctgcagtca gtccctaggg 240
aacttcctgt tgtcaccaca cctctgagtc gtctgagctc actgtgagca aaatcccaca 300
gtggaaactc ttaagcctct gcgaagtaaa tcattcttgt gaatgtgaca cacgatctct 360
ccagtttcca tatgttgaga ttctacttat tcatcagttt gttgtgcttg tcaagatcag 420
gtaggttatt tacaaactgg ttttcttaac tgcttaaaaa atagaattag ttgtgttgtc 480
attttgcatg tcatcattcc acttccttcg gtgaacttaa gtccatagag tcgtttttaa 540
ggaaaaagat ataggaagcc tgtatcattt ccttaacatt tccttaaaaa aaaaaaaaaa 600
agatacaggt tagatgaact ttctcatcct gaaaaaaatt tctcaataat aaaagaagct 660
attatgtctt tgaattaatt tcttcttatt tacaacaatc aaatatgctg aaaaatctag 720
aagtcttatt 730
<210> 28
<211> 969
<212> DNA
<213> Homo sapiens
<400> 28
gatggcatgc acctgtagtc ccagctactc aaggaggctg aggagggaga attgcttgag 60
cctgggaggt ggaagttata gtgagctgat attgcgccac tgcactccag cttgggtgag 120
agtgagaccc tgtctcaaag tatgtatata tgattttgca acttcattca actggatcca 180
gcccttagcc aggtcctatc aggaacataa gaagaactcc tacaaatata gaacagcact 240
cttgcttgat gcccagctaa ggaaagacct gtcgtgatgc catctaattt ttattttcag 300
acgcagaaga aacatgtcct tcattcacca ggctgagctt tcacagtgca gtggttggta 360
cgggactaaa tgtgaggctg atgctctaca caaggaaaaa cctgacctgc gcacaaacca 420
tcaactcctc agcttttggg aacttgaatg tgaccaagaa aaccaccttc attgtccatg 480
gattcaggcc aacaggctcc cctcctgttt ggatggatga cttagtaaag ggtttgctct 540
ctgttgaaga catgaacgta gttgttgttg attggaatcg aggagctaca actttaatat 600
atacccatgc ctctagtaag accagaaaag tagccatggt cttgaaggaa tttattgacc 660
agatgttggt aagagaattc tcttagatga ttcagagcta agttgaaact ggcccattat 720
ctgagggctt aaagccaaga gatagctaca gtgagcagga ggcctatatt gcaacatttt 780
gcaaatgtga ataagcccca gggaaggtgg gatgaggggc tgtgcaaaat gtccataggt 840
atgtttgtta ggctgtgcac tgcacagggc acccatctga gggagcaccc ttgtcattgt 900
gacattatag atttatatat ttattatgat aaatttccac cagatgatga caaagtgtct 960
tgaggaagg 969
21/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
<210> 29
<211> 708
<212> DNA
<213> Homo Sapiens
<400> 29
gggctgggat tacaagcgtg ggccactgtg cccagcagac agactatttt gaatcctgct 60
ctgctctctc cccagctgcc tgatctgggg cagatgactt cagcttaggg cctcagtttc 120
ctcatctgca taatcaaaag aatagtattt actttgcatg gatgttgtga cttttaaatg 180
agataataga tgtaaggtgc ttagaattac ttctccaaag tcaacagcca ggaaatttct 240
acaaatgtta attccttggc cccttacaat ttacaatgat ggacctcttg aaattccagg 300
cagaaggagc ttctcttgat gacatttaca tgatcggagt aagtctagga gcccacatat 360
ctgggtttgt tggagagatg tacgatggat ggctggggag aattacaggt aagtgcctgt 420
gaatgggcat gggttggtag cctgggcagg gtgttcactg ggggagtatt atgtaggcca 480
atccaactcc ttgggttcaa agccagctcc aattacctaa cctctctgag tgtgggtcat 540
tgcatgcaaa aggaagatca aaataatatt gacctcaggc tattatgtaa taaaacataa 600
gtaaagcatt ttgtaaattg tgtatactat agaaaagatg agtagtgact ttatttctat 660
tatggctgga aaaacagcta tgtcccattc aaatctgtgt gccgcaaa 708
<210> 30
<211> 701
<212> DNA
<213> Homo Sapiens
<400> 30
ccccacccct tgaacctcat acacgtgtac gttccctact gttgagctct gcatgacaca 60
tgcccctgat ggagtctgga tgccattctc aggggcacag ccagggtaac cgtctccact 120
gtgggaacaa gctgcagagc catggaaagg agtcctggcc aagtgggacg ggagtgtgag 180
cgctcaccca cctgagggtg ccctgcagcc tgggagccac cctcatccag aaggatcaaa 240
ggcccggctg caggtgactc atggtgtggg gctgccaagt gactctgttt tccttccagg 300
cctcgaccct gcaggccctt tattcaacgg gaaacctcac caagacagat tagatcccag 360
tgatgcgcag tttgttgatg tcatccattc cgacactgat ggtaacgctc ctttccttgt 420
gggtcagtga caccgccagg ctcctaaaga gtgtcccctg gggagagata atcatgtagg 480
agcagatcag gttcctctag attctaactt ttttttccag gcagggttcc aaatccaccg 540
aaatattgaa gggaagctgg tttaatgtag gcacccagga ccttgtgcta gagcgtggat 600
gattatccca cggtctcttc atatttttat ttttttaatt ttttttgaga cagagtctcg 660
ctctgtcgcc caggctggag tgcagtggcg caatctcgac t 701
<210> 31
<211> 690
<212> DNA
<213> Homo Sapiens
<400> 31
aaaaaaaaaa gagaagaaat atatcatttt attagctttg cagtggatga gtgactaaac 60
tccctagggt tgttagaaat attggaagta aattccttac catcttcaca gttaacctag 120
ttggaaatca ttgccttcta actaactagc tgggtgactt tgggcaaaac tcttacatag 180
gtataacaat aatgataaac aacaataata atacaattcc tgcttatacc tggcaggcaa 240
atgagctgtg agataggaaa aattcaaagg agtataaaag cagaatgtgt tctcttttag 300
cactgggcta caaggagcca ttaggaaaca tagacttcta cccaaatgga ggattggatc 360
aacctggctg ccccaaaaca atattgggag gtaagccttt atattttgta aaatagtggt 420
ataaaaaggc atatttggag gctgtgcatg gtggctcatg cctgtaatcc cagcactttg 480
ggaggccgag acgggtggat cacctgaggt caggagtttg agacgagcct ggccaacata 540
gtgaaacccc atctctacta aatacaaaaa attatccagg tgtggtgacg catgcctgta 600
gtcccagcta ctcgggaggc tgaggcaaga gaatctcttg aacctgggag acagaggttg 660
cggtgagccg agttcacgcc actgcactcc 690
22/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
<210> 32
<211> 768
<212> DNA
<213> Homo sapiens
<400> 32
cctgcttcgg cctcctaaag tgctgggatt acaggcgtga gccactgcac ctggccctgg 60
gtctttcacc aaagcatatt tgctctaatt cttgccacaa ccttaatttg atttcatctt 120
ttctcctccc acttgccagc atgaattctt atctatggga ttttataaag attaaatgaa 180
ataaaataca ctgaaagagc gcagggccat atgagaaatt gtgtgtgtat agaatatgtg 240
atatagtatt actaaatgct tcaaccgctc tactctttca tcacttcttt tacaaccaag 300
gatttcagta ttttaaatgt gaccaccaga ggtctgtata cctgtacctg tcttccctga 360
gagagagctg caccatcact gcgtatccct gtgactccta ccaggattat aggaatggca 420
agtgtgtcag ctgcggcacg tcacaaaaag agtcctgtcc ccttctgggt aagtctaacc 480
agcagtttcc tctctgattc acaaagaact aatttttggt atgaatccca gaaccacttt 540
atcatgtaaa aactggcccc ataattgtat taaaaacttg tttttctgct gggcacagtg 600
gctcatgcct gtaatcccag cactttggga ggttgaagca gctggatcac ttgaggtcaa 660
gagtttgaga ccagcctggg caaaatggtg aatccctgtc tctacaaaaa tacaaaaagt 720
agccgggtgt ggtggtgcgt gcctgtagtc ccagctactc gagaggag 768
<210> 33
<211> 697
<212> DNA
<213> Homo sapiens
<400> 33
atgtcagtgt tatttttagt ccacaccagt gtgtgccaca ccccacacct ctccaccctg 60
ctactgagaa ccagtaaagg gggaaaacaa ctggagggtc ctgttgccct tcagctctta 120
gggacagggt tggccagcaa gatgggtcac cctgcaggcc ctcaccatgg gggatggagg 180
tgggatggag tttctctcag aagtggtgga tacagtcaaa ctcaatgttc ttgctacctc 240
agttgctttt atgtttgtat aatacatatt aatcttacaa ttttttaaac ttgtttcata 300
ggctattatg ctgataattg gaaagaccat ctaaggggga aagatcctcc aatgacgaag 360
gcattctttg acacagctga ggagagccca ttctgcagtg agtggtgaat gtggttgttg 420
gcttttgcct aactctcatt ttggggatgg ctgagtctca gcttcctggc tacttcttgg 480
caaataagag ccatgatgaa gcatcaactt tggagctgga agggcctcgg ttgactcctg 540
agtctaccac ataatagctg tgggaccttg gtgaagtcat ttgacttctc tgagtctcaa 600
tgtcctgact ggtcaaatgg gggtaataat gcctgtattc tgggatatta ggtttaatct 660
gatgccattt aaaaatatta atttaataaa atctcaa 697
<210> 34
<211> 712
<212> DNA
<213> Homo sapiens
<400> 34
gatgtgaacc acactcagcc ataagcccta agacttcaca atacctggaa acactggttc 60
aacgatgatg cctgaccatg gtgactggaa accaatgaac tctgctatca ctcatgccat 120
ctttaccaaa gagaagagaa gggaatatta gaaacttctg ccctggatct ttgcagagaa 180
accagagaga aagagaagaa gagactcagt gattagggag gttcagtttt taggaaccag 240
cccttcaggg gatcagttgg tagtatctct ctcttaccgt gttctctctc tgttttgcag 300
tgtatcatta ctttgtggat attataacat ggaacaagaa tgtaagaaga ggggacatta 360
ccatcaaatt gagagacaaa gctggaaaca ccacagaatc caaaatcaat cagtaagttg 420
agttacaatt gtttgcagtg cgctatttcc ctttttgctc agagaaatta aatcacaaat 480
ccacctactt tcttatttat agatctatgc ttttccttgt tctataaaag atataagggg 540
aactattgct tagttgcttc cctggttctt gatctcccag ggtctttaca aatgggattg 600
gtgcaagggt tttctttcct tccctccttc tcttcttcct ttctttgctt cctccctttt 660
tCCttCCttt CCtCCCtCtt tCCCtCtCtt tCtCCCtttg ttCCtCtCCt CC 712
23/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
<210> 35
<211> 774
<212> DNA
<213> Homo Sapiens
<400> 35
tggtgtgtac taggcagtca ttagcatcgt gcctctctaa tgtcattttg ggtcacttac 60
atcgcccttt ggagtcagca gactttgccc atcaaagttg actagttctt gggtggttac 120
tacactccag cctgggcaac agtgtgaacc ctgtctcaga aaaaaaaaaa aaaaaaaagt 180
cagctggtgc aggctatggg gtgaaatagt taccagctta ccagtgactt gcaggcttca 240
aaactgcttt ctctcattag tagtctttat ccatttttca gtattacttc ttccttccag 300
tgaacccacc acatttcaga aatatcacca agtgagtcta cttgcaagat ttaatcaaga 360
tctggataaa gtggctgcaa tttccttgat gttctctaca ggatctctaa taggcccaag 420
gtacaagctc aggattctcc gaatgaagtt aaggtccctt gcccatccgg agaggtgagc 480
tgagggtacg gtttgacaaa acgctgtttt ccaaagatgt tgctggatcc tgtccaatct 540
gcttggtctg atgtgttaat aagactctat tattcacaat gagggacatg atgggttttc 600
caaaaatagt ttttattata aacaagtagc ccaggccaag tgtagtggct caagcctata 660
atctcagcac tttgggaggc caagctggga ggattgcttc agctcaggag ttctagccag 720
cctgggcaac gtatggagac cctgtctcta caaaaaaaga aaaaaaaaac aacc 774
<210> 36
<211> 1684
<212> DNA
<213> Homo Sapiens
<400> 36
ggaaccacag acacgcacta ccacacccag ctaatttttg tatttttagt agagacgggg 60
tttagccatg ttggccaggc tggtcttgaa ctcctggcct caagtatctg cccgcctcgg 120
cctcttaaaa tgctgggtgg gattacaggc atgagtcacc gtgcctggcc accgtaagtg 180
ttttctatat taaaaacaat tgctaaataa ttttgtatct taaggatata atattattta 240
ctagaccatt gcatttttat aaattcattt acaaggatat tttgtcttat tttcccaggc 300
ctcagctgtg tcggtatgat cttgtcctga tggaaaacgt tgaaacagtc ttccaaccta 360
ttctttgccc agagttgcag ttgtaactgt tgccaggaca catggccata aataatagaa 420
agaaagctac aaccacaggc tgtttgaaag cttcacctca cctttctgca aggcagaaaa 480
agtatgaaaa aaccaaggct tttttcagta gcgtcctatg gatgtcacat tgtacatcaa 540
acaaccttgt gattataaaa cgatcctggg aaggagcccc taactagggc aagtcagaaa 600
tagccaggct cgcagcagcg cagcgctgtg tctgctgtgt cctggggcct cccttgttcc 660
gacctgtcaa ttctgctgcc tgtcacgcgg gtggttctgc ccatcgcggc tgcgggtcaa 720
gcatcttcaa gggaaggacg gactggaggc ctcaccgtgg actcaactct gcattctccg 780
tgccacattc ctccagttcc cacacgtaga agggaacgaa actgacgtct acctcatggg 840
gctgctgtgt gggtttggga ggcaaaaatc tatgaagggt tttttgaaat cccataggtg 900
ccacatctat gagatgtttg ataaatgtga atatgctttt acatttgggc ttatctaatt 960
tgcaataaga gagcctctct ctatcaacac cagcttctct ctcgggctgt ttgctcaggg 1020
aaggcaagaa agccacgtgc tggccctctg ccttctctaa agtgctgttg gagcatggag 1080
gagctggagg agatggggat ggactgacag ctaagagggc ggctgctggg actagatagt 1140
ggatgaagaa agaaggacga ggaagccgtg gggcagcctc ttcacatggg gacaggggat 1200
ggagcatgag gcaagggaag gaaaagcaga gcttattttt cacctaaggt ggagaaggat 1260
cactttacag gcaacgctca ttttaagcaa cccttaagaa atgtttatgt ttctttatta 1320
ccaatgtaat ctatgattat tgaaggaaat ttagaaaatg cgtagataca aaattaaaaa 1380
aaaatactgt ccacgatcct attagaggta attaatgtta gccttttgga acaaggctgt 1440
cacctatttt gccaacacgt gaattcaaaa catgaaccgg tttgcttttg gagaatctga 1500
agactccagt ttgaggaatc ctttgcttcc ctggaggtag atgctgtctg caaatctaga 1560
atgacagcag gagtccagtc aagaggtcct gtcaggccaa ggccagaaag aagggaggac 1620
aatccctggg gccagatgcc cagtgtgagg ggaggcatga tctgtcccat ggctgtggcc 1680
actg 1684
24/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
<210> 37
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 37
aaatccttcc cacaaccaca 20
<210> 38
<211> 22
<212> DNA
<213> Homo sapiens
<400> 38
tcccagaata cagaccacac to 22
<210> 39
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 39
accttcaaca ttgcagttgc ~ 20
<210> 40
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 40
cactgcatat tgtatagcac g 21
<210> 41
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 41
aaagtctctt tcccgatatc c 21
<210> 42
<211> 19
<212> DNA
<213> Homo sapiens
<400> 42
gcaaagtaac ttctgatac 19
<210> 43
<211> 21
<212> DNA
<213> Homo sapiens
<400> 43
25/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
tatgcaatgt aaggagctct g 21
<210> 44
<211> 20
<212> DNA
<213> Homo sapiens
<400> 44
agtgccattt gacaatgcag 20
<210> 45
<211> 20
<212> DNA
<213> Homo sapiens
<400> 45
ttatgcaaga tactcacctg 20
<210> 46
<211> 20
<212> DNA
<213> Homo sapiens
<400> 46
agtgccattt gacaatgcag 20
<210> 47
<211> 19
<212> DNA
<213> Homo sapiens
<400> 47
aaatgtactt gaacatggc 19
<210> 48
<211> 20
<212> DNA
<213> Homo sapiens
<400> 48
acagtttagc ttccagcaga 20
<210> 49
<211> 22
<212> DNA
<213> Homo sapiens
<400> 49
gatggaaatc caatttctaa tg 22
<210> 50
<211> 21
<212> DNA
26/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
<213> Homo Sapiens
<400> 50
atttgagtgg gtgcaaagaa c 21
<210> 51
<211> 22
<212> DNA
<213> Homo sapiens
<400> 51
catttaccaa atgagtcctc tc 22
<210> 52
<211> 23
<212> DNA
<213> Homo sapiens
<400> 52
ttgtatggga tactgaagaa agc 23
<210> 53
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 53
attttgttct ctgtggcctc 20
<210> 54
<211> 22
<212> DNA
<213> Homo Sapiens
<400> 54
gtctgaaata cacaataaac ag 22
<210> 55
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 55
ccatatattt gttccaactc 20
<210> 56
<211> 21
<212> DNA
<213~> Homo Sapiens
<400> 56
tggaatgttt aactgtatgc a 21
27/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
<210> 57
<211> 21
<212> DNA
<213> Homo sap,iens
<400> 57
caacacacca agccaaacaa c 21
<210> 58
<211> 24
<212> DNA
<213> Homo sapiens
<400> 58
gttaaggaaa tgatacaggc ttcc 24
<210> 59
<211> 22
<212> DNA
<213> Homo Sapiens
<400> 59
gcaacttcat tcaactggat cc 22
<210> 60
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 60
gctcactgta gctatctctt g 21
<210> 61
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 61
ttctccaaag tcaacagcca g , 21
<210> 62
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 62
ctttgaaccc aaggagttgg a 21
<210> 63
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 63
28/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
gagtctggat gccattctca g 21
<210> 64
<211> 23
<212> DNA
<213> Homo Sapiens
<400> 64
gaatctagag gaacctgatc tgc 23
<210> 65
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 65
gccttctaac taactagctg g 21
<210> 66
<211> 22
<212> DNA
<213> Homo Sapiens
<400> 66
aaactcctga cctcaggtga tc 22
<210> 67
<211> 21
<212 > DNA
<213> Homo sapiens
<400> 67
gctctaattc ttgccacaac c 21
<210> 68
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 68
tgctgggatt acaggcatga g 21
<210> 69
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 6.9
tctctcagaa gtggtggata c 21
<210> 70
<211> 21
<212> DNA
29/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
<213> Homo Sapiens
<400> 70
gttgatgctt catcatggct c 21
<210> 71
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 71
ctttgcagag aaaccagaga g 21
<210> 72
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 72
aaccagggaa gcaactaagc a 21
<210> 73
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 73
taccagctta ccagtgactt g 21
<210> 74
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 74
atcagaccaa gcagattgga c 21
<210> 75
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 75
gattacaggc atgagtcacc g 21
<210> 76
<211> 22
<212> DNA
<213> Homo sapiens
<400> 76
acaatgtgac atccatagga cg 22
30/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
<210> 77
<211> 6660
<212> DNA
<213> murine
<400> 77
tttcatctta ccgccggcgg tggtggcgca tgccttcaat tctagcactt gggaggcaga 60
aacgggcaga tttctgagtt cgaggcaagt ctggtctaca gagtgagttc taggacagcc 120
aggactacac agagaaaccc tgtctcgaaa aacaaatcat cttaccccag tcagaagggc 180
caagataaat aagacaagta acaaaacatg gtggcaaggc tgtgggtgaa gaacaacact 240
tatccattgc ttgtgggagt gacaactggt cctgccatta tggacatcag tgtggtggtt 300
ccttagaaag gtctacctca aggtccagct atgtgactct tcaatatgtg cccaaaggat 360
cctactacag aggcgtccct gtttgtcgct gctctattcg tgataaacag aaattggaaa 420
cagtctagat gttcacgaat ggatgaacag ataacgaaat taacacaacg gaatattatt 480
cagctgtgaa aaaaaatgaa actaggggga atttcaggta aatagatgta actagaggca 540
attatcctga gtgaggtaac ccgtacacag aaggcaaatt agatactgtg tgttttctct 600
tatatgtgcc tgtgagcttt caagctaaga gtcaggctag taagggaaca gtagagaaga 660
aaggatttta caaggaaggg ggagtagaat gtagtgttat ctggtgagat ggctcagcag 720
gtaagagcac tgactgttct tcgggaggtc atgagttcaa atctcagcaa ccacatggtg 780
gctcacaacc atccttaatg agaaacaaat aaaaaaccct ataggccaga gcgagcagtg 840
ctggagtgaa agggaagtgg ggaaaagggg ggaaaaagaa tgtagtgtta ttaagaggta 900
aagaaaaagc cagaataggg aaaggcaaga gagcagggca aaggagcata taattgaggg 960
gtaactaaca gctaacacca actgagaaac cacctaacaa cctactactg taggaccttg 1020
ctaacccttt ttcacatatg tgaaatgaat ttaaattgtc accaaatgac aggtgagaca 1080
atgcttcacc tagacgtctc atgtcaccaa gtaaccttcc cccccccccc atatagggat 1140
ggattgcagg ttttggagtc attgagcaaa ggggtttcca catctccccc cccccccaaa 1200
aaaaaaaatc acaggctttt accaagttta ttgatttctc accacagctt gataaggtcc 1260
tattgacatt tatatcacca aactcgaagc ttagctgcta cccagctaaa agcttaaccc 1320
cttctgaata gcattcacag tactagaagt tactcgctgg actaccagag gaaaaaagta 1380
atttcaccca gctacagacc ccgtgaccta caatcatgaa ctatccacaa tatataccgg 1440
tgcaaccata gcacaaatgt tattgaagta accaaccgtt ttttgattgg acataaggac 1500
cacctcctga gatgggacac agatctgaca ctgttacagt gggcaagaat ccgagaatag 1560
cccatggacc cagggagaga cattttactt ttattttgct aaagaaacat agcgataaaa 1620
tgactccaaa tgacatgttg ctgtacccat cgaccagtgg ctccttccag cctcatcaga 1680
gaagcccttt ctttgtagaa gatgaaaatc catctcccag agatccacaa ctgggcaatg 1740
agcagagagt aagagacttg acaagcagtc ggttctaaat tgaggttttt caaacccaat 1800
ctgcttgaga ctcattgatc tatgagggag aggtggaaga aagaattgta agcgccaagg 1860
atggtgtacg atgccaagga aacagtatca tccatatacc ataggattga cacatgcatg 1920
aaattacaga aactagagca acatgcaaag gatctgcaca ggcccaaact aacaagggcc 1980
cagcactgag agctggagtt gacagggtcc cacacctaaa caagaaagga ttcacacttg 2040
gtaactgcta gcaaagagaa attcactttt ctccaatgga tcatcactga gtctatcaac 2100
cacactccaa ggcagcccta tgcccaagag tgactgtcaa aggctttctg ttatggtttt 2160
ttttttcatt ttttgtcttt tttgcttgtt tgttttttat ttgcttttcg tatttttgtt 2220
gttttatata tttaaagttt ttttttgttt gtttgttttt tttgagaaat gaagtcagag 2280
cagcagaggg gtggaaagga tctgggagga gttggggagg agaaagcatc atcaaaataa 2340
attgtgtgag gatattttag taaaatacac attttaggaa aatctcattt ctcttttaac 2400
tctgacatac ttagctgtgt gagttgggca aatagcttga cttttgtttt tttttttttt 2460
gttgttgttg tttgtttgtt tgtttttttt gtttttattt tttaagaaac agattagacc 2520
aaatagagct attttatcca atatttattt ttgaattgct tttaggaagt caggaattgt 2580
ttttagtcct ggcaacaaag gccaatatag aagtctggat agcgggatgt cagtagcttt 2640
agtggttact tttggttacc tttgcttatt actgctcaag tctatgaata ttgtctctct 2700
ttgcttcatc atccaaataa atacagtgca gtaagaagta agtacatgct agagagcaaa 2760
tgtatgtcgt tcaaaataat ggacacttga aactttctat tcagtgaaca tctcttacat 2820
atttgtactt tttttatttc aaaaaatcta attcaaaact atttttctct gtagcatata 2880
tacatattca gtgcttacat aaaagtttgt atttccaaat aattaaaggt tgtattgtat 2940
tatattatat tgttctatga aatgtattct attttctgta cctatttgta aattgtctta 3000
gttccttaac acttctacgc tcttttggct ttatatacat ttatgctatt tattttttag 3060
atttcttatt catcattttg aaacagctca tatgcttgga tatataccta gcgtttgatc 3120
tcaacagata gctcttttta cttaagtaca ctgtattttc taatttgaat cgaatgcatg 3180
tctctcagaa atgtgaaaaa gaagtgtgtt tgttttcggt gtttactagc ccattaatca 3240
31/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
catataaagc tttaatggga acatgtgcaa tactccacaa agaatttgac tatttttatc 3300
agaatgtttt atacttctcc ttgcacgaac ataccaaagt gtaaaaatat acatgtgtat 3360
aaatatggtt ttgcgtgcat gtaaaatcct ggagttgaca gactgctagt tatatagata 3420
tgcagatttc tggtttcact tcagtagctc atcccagaaa gtgtattctg ctgggatttc 3480
ctctactgtg caatctagaa tctctggctg ccccacatac gtagtaacat aattttctag 3540
ttatagtttt caacccctct ttattttgaa ctagtgctgc ataatcaaat ataaacatcc 3600
aaggcagaac ccgacacact tgtgactata taataaatac ctgttgaaat tgtatctctg 3660
aaaaaaatga gcatctctaa ggtgtatccc aaaaactaca tctatggatc tcaggagatg 3720
cttagtaaaa gttgtataaa tgaacttgca tatgaatgaa ttaatgaatg ggagaggttg 3780
gtaatggatg tttgctttaa ctacaaataa tgatacatat gcattattaa tcttcacagt 3840
atacacaatt agagaaattc tgtcccatta gacatttcta ttttccattt gatgccacgc 3900
tagtgatgtt gagatgaatt cttttgttct cattgttttt tttttttatt ttgcatttgt 3960
gattttatta tggaatgaaa ggagcttgca gttctgtaac aatgtccttt ggaggtctaa 4020
ttgctgattc agaggttaat attttcgaaa tcttgtcact gtctggcctc aaacttgcaa 4080
tgtagccatg gctgatcttg aaagtctttt gttctcatct tctcctgatg gcaagattta 4140
caactcattt atgtggtatt agaaatctaa tctagcgtct ggaccagcct gtgtgcacac 4200
tttactaaca gagctacagt cctcagactt tattaaatat ttttaccaga attatataga 4260
ttaaataaaa gttcatccca aaatacattc tgaaaattga cttccctagt aaatactgct 4320
tagctcttat aattagtgtt caagtgctca gaaacagggc tgatgaacag aacttcatca 4380
ccctacagag tttgtaactc ctcacccaca taatctcctt tgcgacattg tttactttgt 4440
acatgtatcg atatgattct tactctcata ctgtatttct gagtaagaat aaaataaaca 4500
cacagaatac taaaaccatt agtatatttt acattatatt atcttccact gacattccac 4560
taggcattag taaccaaatg ttctcatgct gaaagctagt aaaatatatg ataatgaagg 4620
aatgccattt tatgttttga gactctgcaa aatactctat tggctaaaac cccacattca 4680
tatgagcagg tcagaatgat gactataaaa atatcatcat aaaacaaaca aacaaacaaa 4740
aaacaggaaa aaggtaaatt atgtcttttg gaaggttttt ctcctagtct attaagtatc 4800
atctcattaa aaccacattt tacacgtttt acactgtgtt tctcccatct caattaacac 4860
ggttacacgc ttttcatttt gagttttaca gctattaaca gttgtgtgtg tggaggtgtc 4920
tgcactcatc cacattctta aaaacacagc ctcaataaaa cttgagcact tcaaattgaa 4980
gaggtttatc agcagttacg tgacctggtt tcaaaagaag tttttcatgt ctgggggttt 5040
ctaaaccatc tctccagact ttcagattag tggctgatga cctgtgctgt aaataaaaca 5100
ttccatgttt tactgtatta ttgaaataat tccaagaaac atattttcta aaaacaagtt 5160
ttgttttgaa cctatgctct gtgtatgaac agttattgat ttttttcttg ctttctcaac 5220
attcttgcct ggaaagaaaa tagaagaaaa actaaataag cattaaagag aaaactagca 5280
atgcctttaa aatgtttttt ttttttaata ttaaaaccaa agagattttt ttttcttttc 5340
aaattttagc tccaagagac aagcaagaac ataagtccag acgttattcc cctccaggtc 5400
tgccctccca cagcacttcc tcatcccatt actcctaccc tgtctctgag aggatgtccc 5460
cacccagccc tcgtgtcctc ccccctccct ggggcctcaa gtctctgaaa ggttaggcct 5520
accttctctg actgaggcca gaccaggctg tcctctgctg tacatgtgtt gggggccttg 5580
taccagcgag tgtatgctgc ctggttggtg gttcagtgtc tgagaggtct aaaatagcca 5640
ctgtgttttg gatgcataat gacatgcata aaaacccagg cagtttcatg ggtgtggatt 5700
ttgttattga tctcaggtct gtgaagaact cagtttataa tcaaaatgat cataaaacac 5760
aacttaccct ggctctttct atttctggaa tttctagtaa catttttttt ttcgcctgtg 5820
ctttccacaa acatatcctc ccatgccact tctaaaattc tgaggttaag gaatgaagac 5880
atctacaatt aggtttctca ttcccttaaa atgttatttg tgtagctgaa catttgtgag 5940
ttattacgaa gtctttcttt ggcttagaca cgctctctta acacacgcta gtggataata 6000
acagactgct tcaccttttc cagccggggg aggagcaaga gtcattttta caagaaagct 6060
tgccggactg agacaatcct atcacacaag aaccttaacg aattgcagaa atttctggtg 6120
aatcccactg tttctttcac actttctgat accaaatgag aatatacatt tttctgtgtt 6180
tgatatgctg ggcaagattt ggtaagctgt ttattaataa cttatcatta acatgcctgt 6240
aaaataatta atatggttta tattatctta tgccatgata gtttgaaaaa cttactagga 6300
ttatggtatt tataacagac aagattgcta tcatgaaggt aggtatgtgc tcatatatac 6360
atatgtgggt ttgctaaata ggagttttat gatgtattta tttgtcagaa gattaatagc 6420
tggaaaaaat atgcaaacag ggagtaagta gaaatattca agattaaaaa gtaattaagg 6480
agtaaggtat tagttcagtg tctgaaaatt atgacgtatt atgtaagttg cttccatttc 6540
atttgtctga aaagtctaac atttgaagaa aattattgag taggtgattt atcatttgca 6600
aagcaaacca aatatcaatg caggaggagg tctttctgtc tctgtctctc tctctgtctc 6660
<210> 78
32/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
<211> 5040
<212> DNA
<213> murine
<400> 78
tttggttagc tggttcagaa gagcgagaac tccctttgaa agaaacagag taacaggtta 60
cttcatggta catttttttt tgagttgtaa tattaaacca taaaatgact caaaatattt 120
taggacgtag tagtgatttt aaaattctgt actcagagaa gatatatgca tatctgacct 180
ctcaaatcat atttatagat tgtttacagt actatatata tatcaatgaa ttaaaatcaa 240
cctttgtcta aatcaatgac tgtaaataag aatgacattg acgttggact ttgtaagata 300
catgatggtt tgttccagac acctgtaaac taaaaatggc aaataaacaa gtgttatttg 360
tttccagtgt acaaggcagg agtttaaccc tcatggacaa aagccaattc aatcttatac 420
atgatgtgcc agaacataat ataccctggc ctattctaca gacattaaaa gtaatggcaa 480
aaccgcaatt acttttgcac taacctaata ccaggttgtg taagttaacc agaaagtacc 540
agagagcatg atgcaaccat gtattacaaa agaaaggaag aaaaaaagaa atgcaaagag 600
acgcactttt gctttgcaat aaagcattgt ttttgttatt agtatttgtt tgcttttagt 660
atagcaagag gaatttaaaa accgacaagt gacagaccaa taagcctgac cacttagagg 720
aggctgcgta catattgcaa gaattaccag atggcctcta ttagtctcct gaaacatgaa 780
agagctagag taatggtata acattctctt ggcagctaat catgaatgat ctgttcctga 840
tatgcgtgca ggagaagttg ttgaggccat tcttgtcagg aagttttttc cgccactact 900
aagggtaact ctacgttgcc tttcaccact ttgctcttac aggggctaaa atcctgtgEa 960
acatgtatag aaagatttcc ccagaaggac taaatgccct ctctctatct tccaaatagt 1020
acagattcag taccatacat cagcagtaac aagattagta aatacccttg ctatataaat 1080
gttacagttg tgtgacccca gttcattcag atttgctcaa tttataaggg ccaatagcaa 1140
ccatcatagc tacttgctgg ctgatactaa aacgattatc ttacatttta tttttagaaa 1200
agatgctata ggtttgtcaa gactaattaa ctatataaca ttatgaatta gacacgtgtt 1260
ctgagaaaat ttaacaatga ccagaaggta gaaggtactt ttttttgttt tggtcttata 1320
ctacatagga aacaaattgc ttacaactct gagaatagta ccaaccacaa aacactttga 1380
agattaatca gtgacaggac ctaccaaatc gatactgcac gttaattggt cttccatata 1440
aacgaattcc attcagcaaa gctatggcat aagacaccga ttctgggtgt ttaaagcaga 1500
caaatccaaa agactttggc tttccttctc tgtctttgca tatagtcact ttggttagtg 1560
gccccgccta taagaaactt aaaaattatt ttctcataaa tccaaagatt ttttttatac 1620
tcaaataagt aaattcaact ttagactaaa gaaatgtgtt agcaatatct ttatgtagct 1680
ctgctaataa tgtatctttc taataataaa tattacactt cctcacactt tctttgtatt 1740
ttctggctca ttggggaaac atacatggtg ttaagaacag gaaattctag gtattagttt 1800
caaactgtaa aagaaatggt agactatcct tctgttaggc ttaacaaacc aatttagaaa 1860
acatataaat ctaagattaa aatactactt tgtctccata cacattcaca gtgttgaatt 1920
caaaaaataa ctgaaatgag aaattcaatg ctggttgagt tatcctcaaa aaagctggcg 1980
atgttatatc atagtttaaa ttttctgtca tgaagattaa atatcttttc aagaataaat 2040
aaatttatca tgcattttga tttggaaact ccatctggta aatttccaaa ggaaaaatgt 2100
ttaaatatgt tcaagacttt taagtatatt gcattgttta taagtgtgag aaattagaga 2160
ggctctaaat ctaacaactg gagatattat tccatggtgt gttactaaaa cggaataaaa 2220
tataaattat aaatattcag attatattga tagaaaaacc tataatatgc aatgaagatt 2280
taaaaacaca gagccaaata tcatagaaaa ttgttaatta caactaacga aaaattacat 2340
cacagccatg ataatgattg gaggaggcac ttacaaacac aaacaactga tttaatgctg 2400
aatggttcag actttaataa ctgattattt ttccctgtag agttgcataa ataaaaaaat 2460
tgcttttact ctaaagaaaa atattaatga tcatctttaa aataattctg tgtgttaaga 2520
tttagtcttt tcctttcatt ctctgtgctc tcccagggac tagcttccct tcccttctgg 2580
cttcaaggac cacctcatca acaaatttac ttttcccacc atgtctcttt catggagaca 2640
cctcctggag atgtctatgt ttttgtactt caatctcaac atatttgaaa ctgaattcac 2700
catattttct ctaaatctca actctagtat taaaagtgta atgataataa atctgatttt 2760
ctcatttcca agtataatca gaaatttgaa agaaaatagt aaaaggataa atatactgta 2820
atataaaaac caataaaatt ggtgattttt atacagatat taaatagatg taatcactat 2880
gggacagaac atgcttacgt gatttgaatt ttgacataaa tttagataat tcaaagcatt 2940
tgtaaagata aaagtcaaca tgaagactta cttttagcaa caatcacact atcttacagt 3000
attttttaaa tcacaatgaa atacatagtg tgatagttgt caagttttgc tgggtgttga 3060
agtctaaagt ggaagagaaa ctccactttg gcaattcctt tcaataccta gacaaaaata 3120
agaacttatg cttcttacct gatctattaa ataaaattta tattttgctt aggtggaaga 3180
aggaactgat aaagaatgag acgataatat atagtcttgt ggatccacac aactgatatt 3240
gatcgagtcc tgattgacta aaaataatgg ttattggata atgcaatccc accccagtct 3300
33/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
tccactctcc tttttttgga ggctcaaagt gaaagaagtc ttctggagaa aaataactct 3360
aagtttactt tatatttctt tcggtttttg ttgttgtttt ttttaactta atatagtaag 3420
ttttaaatga tactgaatga aaaaacagat atggattaga gttcataaaa aaggaggcaa 3480
gtttaaaaag atgaattaaa aggatgaacc tgaaagtctt aaaatcaata atatttttca 3540
aataataaaa aagttttacg tccaggaaag gaaactgtat tcctggaaag aaccctctct 3600
accaaattgt atctacaatt aaaagagggt tgctttagtt aagtttcgag acaaaagtaa 3660
gtcctttcac aaccaaataa aggacccctt tgacccattc aatcctgaaa atttagttct 3720
gtaattgcct ccctcggaac tttcccttat tctgatagga gaaagtaact attgatttct 3780
cttgatgtta gtattattac taataataac agcagtacca ttagcagtag tagcggtagc 3840
aaacagcgag tgctagactc agctgtagcc attttaccat taggccacat attccgatag 3900
ccacattata ctgcagatac tactactctc ttttgacata taaggaaact gaagcactga 3960
gagttcattg acaggctcag agtcacactg ctgacctcag agttcgcacg cggtaaccac 4020
ttttctatct atgtcccttc tctttttata gattatcgta aacattgtaa ttttaagttc 4080
tccctgttag gaagataaaa ggatgtctcg tcaatttgtt ttcccagcac cctcagtatt 4140
tgccccgtaa ttccggatca ttacacggtt agcggaatgg ctgaatgagt gtatcttttt 4200
ggcaagtgat acctagctgt ataggacaat acgataagtg agcgtgtata caggacacag 4260
caggcttaat cctagaggta ccaggtctca aagtttccga aggtcctctt ggtatgaatt 4320
aaagggcctt gtccagggaa tcacctggtc acagtcacgg taagttggct gccaaatcat 4380
ctcaaatctc acttttcatg ctggaataaa aatactgtcc gcataaaagg gcttgaagga 4440
aggagcaagg tgtaaaagga aacattataa aagcgacccc tctccttttg ggggatgtca 4500
ggcgtgctta ctacttgacg gagaacagtg accaggctgt tttcggttct gcccgcgcac 4560
acccgaggcc caggcagcct ctgggaagcc cttaatcaga aacgggggtt gaaggaacca 4620
cgtccctgcc caaccccagg actctgcagc gcggatgggg atggggaacg cgccctcatt 4680
tcactcccca gccgctcacc tggagacagg tgcgccaaga ggaaacgaaa ttaaaacccc 4740
tgctcaggat gggacggaag ggggccgggc tccgggtggt ccaggtgcgc gctatgtttc 4800
cggcccgacg tgctccgccc ctccgccgcc ccttcccaga gacggtacct gaaggaacag 4860
ctcgtacaga atctcttccc gaactcgggc ctctaaattc ccaacaaaca cggtcctgtc 4920
ggcctcctcc tgacagggta acatcctccg gtctcggccg tagaatgaag taggagctga 4980
gaccccgccc cttctccggc tttacggcgc cgcctgggtg agggggcggg gccggcgccg 5040
<210> 79
<211> 4351
<212> DNA
<213> murine
<400> 79
ctctgtatag ccttggctaa cttcaaactc cggatcctca tgcctcaggg gttaaaggta 60
tgcaccatga cactcagcca attttaatta ctacactctc atgctttcct gtttgtttgt 120
ttgtttgttt gtttggttgg tttggtgtgg ttttgttttt gttgttgttg ttattgttgt 180
tttgttttgt tttgttttgt tttgttttgt tttgttttgt ttgagacagg ggtttttaag 240
tgtagccctg gttgtcctgg aactcactat gtagatcagg ctagccccaa actcagagat 300
ctgcttgcct ctgcctctca agttctggaa ataaagatgt gtgccaccac tgcctggctg 360
gaatccatgc tttctaaaca tataatttga tgccatgaga aaaaccatta gtcccccaac 420
ctccaacgaa gtaagattct tcactccctg tgttgtctgg gctgggaatt cagagtggtc 480
ctgacctcaa attgtcctcc tgttgtaacc atcatgtcta gaaacaatgt ttagctttct 540
ttgtggttga tattatgagg cctgcggtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 600
gtgcgtgtgt aattgtgtga ttgtgttgta tgtgggcaca agtgtcctct gggtcagagg 660
aggacatgct ccttgcctta ttccttcaag acaaagtatc tcatgaagtc tgctactttt 720
cttcctattc atcctattca tctacctttt cttcctattt gctcagaagt ctgctctctt 780
ttcttcctat tcatttgtct ctgtccacaa ctttggtttt acaggtgtgt gactctatgc 840
acatctgact tttaacatgg ggactggaga cttgaactca ggccctcgtg tctgcagaac 900
aagtgctctt acccactaag cccagaaaaa tacttttaaa gcattttatt attactttat 960
tattgtcatt gttattgcta ttattattat tatttgcacc catgcatgtg gaggtcaaag 1020
gacaactttt aggagttctc ttcttccact gtgagtttca gtcagttcat ctgcttgtta 1080
agaaatgtgg ggaagaggag gagaaaaaag aagaaaggaa gggagggagg gcaagaggga 1140
agaaaggcat agtttgggga gtataaatct aacctctctg ttggatgagt cgaggaaact 1200
gactcatctt tctgaacttg tattccaagt gagagacagc aggttggcaa gagcgtgtta 1260
cagtgagtca tacggctact tggccgggac acaggtcacc tctcctggct gtcaaataga 1320
gaataatcaa actacctttc cccagtacct tcacacccgc atacctgccc tcccagtcca 1380
34/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
tgcactagaa gctggactgc ccctttccac cctgctgtct gtggtgagtg gggagaagag 1440
gaagcagaac aagtaagcac acagcacact gtaggctgga gaggcagctg tggtgattca 1500
cacatagaga gagttccagc aattgggagc ctgaggcagg agtacaggaa gttcattgcc 1560
tgcaagttcg acaccaggtg aggctctgaa tgaaaaaact acactaaata aagatggtga 1620
tgctaaaagg ctgggctgag gacaatagtg acatttgtga attatccctt aatagggcca 1680
taggaattga gagcttcttg atcagctggt ggagaaaaag atgggggctt cctcagaatt 1740
cctttaaatt aatgctcttt cagtgactgt aagagttgac ccatttttgt aagttttaaa 1800
aacacatttt actttgaaat aattattggt tggcaagaca tttcaaaagc agtagaaggt 1860
ccttgtgtgt acacacccag tttcccatca gcgcaacatc ttatgaaagt agtaacattc 1920
taccaaaacc agaaaatagt agacattggt acaacccaga gactctgtca gcaccacatg 1980
tgcacgtgtg tgtctctgtg tgtgtttctc tgtaatctca ctgcatggat ttgtatggat 2040
accacccaat taaagtacag aagtattcct ccattgaagg atcccttgca gggctgggga 2100
ggcaggcagc ctggtggtag agcacctgct tagcccaagg tcctggatcc catctccacc 2160
cactaaacag ataagagtcc tgtgctgact cacatctaca ctcctcttct cccccacctt 2220
tggagggttt tgttttccta ttgcacagtt ggtacttgtt attacactgt caatatttct 2280
tactcctcta aaggagaaag aatggattcc tccattgctt ctctgctgca tttaatgctg 2340
gaatgaaaac tgagcaccca gccatgaggt taggggaact tctgtgctct aaccattaat 2400
aacatctaga agaccaccct gcagagtgac ccttattcct gtaactgagg atcggatagt 2460
ttcgccccta aagtacattg gctaggtcca gataagcact ttagactgat gggaactcaa.2520
caaatactta tcaggtgggt acactgaccc agcagggagc ggcacattcc agggcaggtt 2580
cattaaagag acagaacaac tctctgtcct ccagagccct tgttttcaaa ggagcaaggc 2640
ttagagcaag tgcaaagcag aaaagcgggt tgagttgggt ttcctgttta cagttcctta 2700
attcatttcc cgacaggaca gaaaagcaaa gcccactaca gttctcaaaa ggtctaggag 2760
gccctcattc ctcaagttcc tcctgaagcc cccttctcag gttttctttg ttttgttttg 2820
gtttggtttt tcaagacacc gggtttctct ctatagccct ggctgtcctg gaactcactt 2880
tgtagaccag gctggcctcc caccactgcc cggctcccct tctctgttct atagcaagtc 2940
ttcagagggg tggggacagt gtcagctgtg tgacctggaa gatagtacct ggagttagca 3000
agaggcagga gcaattactt tggcagcagg catagtgttg aagacttgag ctcacatacc 3060
tcatttagtc tgcataaccc cgtgagtcaa gagatcttat cttcatttca tgattgagaa 3120
agttgagaca cacagaacat aaatgtttta tttattgagg ccacatacct ctgtcaagaa 3180
gcacttgaca caggcacctc gacaatgcta aatgactaat ggcttatgcc tgatgtttga 3240
gtgaaaatgt ggggtttttt tgttgttagg ttttgtcctt actattttta aggttttcaa 3300
atacccagaa acttatgatt aggggaattt tcactacaaa gagggttaca tcactttgca 3360
gtgtatcctg tcattagtga cctgcctttc catctaggct aggtacactc tttactgcca 3420
aagtaccctc acccacaata acctcacaga gcagaaggat tggttacttc tagtgtaggc 3480
cttagaaagg ttggaaagat gtagatgtgt tcttcccctt taagacgaca actggatggg 3540
tgagattgca cagcgggtaa aggtgcttgt ccccagcctg accccagact atgcacggaa 3600
ggacagggac tgcnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3660
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnccacact 3720
cacctgccac actcagtaaa taaaatgcaa aaataaaaac tcagttatga gggagaccag 3780
gactctggat gagaagtaag caagctaagt acaactctga agcatagaga gattcgtaaa 3840
ttaggctgta tagctgaact caatgaaatg tttttaataa aagtctaaag acagctctgt 3900
ggagaccttg gagttaacat gaacgtggct ttgtatacat agtggcgata ggagaatact 3960
gacccggatt tgtcatccgg gattcattca taaaggaaaa ggattccaaa tgaggttgat 4020
ttcaacacac caagccaaac agctaggaaa ccgcccacga agaaggaaac cttttactcg 4080
gcatccttca aatgcaaatg acacattaac atatgcacca attgtgcaag cagaaagtcc 4140
cagacctttt attgcaaaga ggaggagcca aagatcctga caggactaac agggccacag 4200
agcctccctc ggggacttcc tgttgtcacg gactcactcg cttcgagaaa gccagacttc 4260
agggtgggac gcgggaacgc gggaacgcca ggctctgtgc ggagtgcgac acgtcctcgt 4320
gtttggggca catgatctct tgaggttccc t 4351
<210> 80
<211> 15000
<212> DNA
<213> Homo sapiens
<400> 80
ttgcctttac tataccagcc ataaataata aagaaccagc caccaggttt cagtggaaaa 60
gtgttacctc agggaatgct taatagtcca actatttgtc agacttttgt aggtcgagct 120
35/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
cttcaaccag ttagagaaaa gttttcagac tgttatatta ttcattatat tgatgatatt 180
ttatgtgctg cagaaacgaa agataaatta attgactgtt atacatttct gcaagcagag 240
gttgccaatg ctggactggc aatagcatct gataagatcc aaacctctac tccttttcat 300
tatttaggga tgcagataga aaatagaaaa attaagccac aaaaaataga aataagaaaa 360
gacacattaa aaacactaaa tgattttcaa aaattactag gagatattaa ttggattcgg 420
ccaactctag gcattcctac ttatgccatg tcaaatttgt tctctatatt aagaggagac 480
tcagacttaa atagtaaaag aatattaacc ccagaggcaa caaaagaaat taaattagtg 540
gaagaaaaaa ttcagtcagc gcaaataaat agaatagatc ccttagcccc actccaactt 600
ttgatttttg ccactgcaca ttctccaaca ggcatcatta ttcaaaatac tgatcttgtg 660
gagtggtcat tccttcctca cagtacagtt aagactttta cattgtactt ggatcaaata 720
gctacattaa tcggtcagac aagattacga ataataaaat tatgtggaaa.tgacccagac 780
aaaatagttg tccctttaac caaggaacaa gttagacaag cctttatcaa ttctggtgca 840
tggcagattg gtcttgctaa ttttgtggga attattgata atcattaccc aaaaacaaag 900
atcttccagt tcttaaaaat gactacttgg attctaccta aaattaccag acgtgaacct 960
ttagaaaatg ctctaacagt atttactgat ggttccagca atggaaaagc agcttacaca 1020
gggccgaaag aacgagtaat caaaactcca tatcaatctg ctcaaagagc agagttggtt 1080
gcagtcatta cagtgttaca agattttgac caacctatca atattatatc agattctgcc 1140
tatgtagtac aggctacaag ggatgttgag acagctctaa ttaaatatag catggatgat 1200
cagttaaacc ngctattcaa tttattacaa caaactgtaa gaaaaagaaa tttcccattt 1260
tatattactc atattcgagc acacactaat ttaccagggc ctttgactaa agcaaatgaa 1320
gaagctgact tactggtatc atctgcactc ataaaagcac aagaacttca tgctttgact 1380
catgtaaatg cagcaggatt aaaaaacaaa tttgatgtca catggaaaca ggcaaaagat 1440
attgtacaac attgcaccca gtgtcaagtc ttacacctgc ccactcaaga ggcaggagtt 1500
aatcccagag gtctgtgtcc taatgcatta tggcaaatgg atgtcacgca tgtaccttca 1560
tttggaagat tatcgtatgt tcatgtaaca gttgatactt attcacattt catatgggca 1620
acttgccaaa caggagaaag tacttcccat gttaaaaaac atttattgtc ttgttttgct 1680
gtaatgggag ttccagaaaa aatcaaaact gacaatggac caggatattg tagtaaagct 1740
ttccaaaaat tcttaagtca gtggaaaatt tcacatacaa caggaattcc ttataattcc 1800
caaggacagg ccatagttga aagaactaat agaacactca aaactcaatt agttaaacaa 1860
aaagaagggg gagacagtaa ggagtgtacc actcctcaga tgcaacttaa tctagcactc 1920
tatactttaa attttttaaa catttataga aatcagacta ctacttctgc agaacaacat 1980
cttactggta aaaagaacag cccacatgaa ggaaaactaa tttggtggaa agataataaa 2040
aataagacat gggaaatagg gaaggtgata acgtggggga gaggttttgc ttgtgtttca 2100
ccaggagaaa atcagcttcc tgtttggata cccactagac atttgaagtt ctacaatgaa 2160
cccgtcggag atgcaaagaa aagggcctcc acggaggtag taacaccagt cacatggatg 2220
gataatccta tagaagtata tgttaatgat agtgtatagg gtacctggcc ccatagataa 2280
tcgctgccct gccaaacctg aggaagaaag gatgatgata aatatttcca ttaggtatcg 2340
ttatcctcct atttgcctag ggagagcacc aagatgttta atgcctgcag tccaaaattg 2400
gttggtagaa gtacctactg tcagtcccat cagtagattc acttatcaca tggtaagcag 2460
gatgtcactc aggccacagg taaattattt acaagacttt tcttatcaaa gatcattaaa 2520
atttagacct aaagagaaac cttgccccaa ggaaattccc aaagaatcaa aaaatacaga 2580
agttttagtt tgggaagaat gtgtggccaa tagtgcggtg atattacaaa acaatgaatt 2640
cggaactatt atagattggg tacctcgagg tcaattctac cacaattgct caggacaaac 2700
tcagtcgtgt ccaagtgcac aagtgagtcc agctgttgat agcgacttaa cagaaagttt 2760
agacaaacat aagcataaaa aattgcagtc tttctaccct tgggaatggg gagaaaaagg 2820
aatctctacc ccaagaccaa aaataataag tcctgtttct ggtcctgaac atccagaatt 2880
atggaggctt actgtggcct cacaccacat tagaatttgg tctggaaatc aaactttaga 2940
aacaagagat cgtaagccat tttatactgt cgacctaaat tccagtccag cagttccttt 3000
acaaagttgc gtaaagcccc cttatatgct agttgtagga aatatagtta ttaaaccaga 3060
ctctcaaact ataacctgtg aaaattgtag attgcttact tgcattgatt caacttttaa 3120
ttggcaacac cgtattctgc tggtgagagc aagagagggc gtgtggatcc ctgtgtccat 3180
ggaccgaccg tgggaggcct caccatccgt ccatattttg actgaagtat taaaaggtgt 3240
tttaaataga tccaaaagat tcatttttac tttaattgca gtgattatgg gattaattgc 3300
agtcacagct acggctgctg tagcaggagt tgcattgcac tcttctgttc agtcagtaaa 3360
ctttgttaat cattggcaaa aaaattctac aagattgtgg aattcacaat ctagtattga 3420
tcaaaaattg gcaaatcaaa ttaatgatct tagacaaact gtcatttgga tgggagacag 3480
actcatgagc ttagaacatc gtttccagtt acagtgtgac tggaatacgt cagatttttg 3540
tattacaccc caaatttata atgagtctga gcatcactgg gacatggtta gacgccatct 3600
acagggaaga gaagataatc tcactttaga catttccaaa ttaaaagaac aaattttcga 3660
agcatcaaaa gcccatttaa atttggtgcc aggaactgag gcaattgcag gagttgctga 3720
36/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
tggcctcgca aatcttaacc ctgtcacttg ggttaagacc attggaagta ctacgattat 3780
aaatctcata ttaatccttg tgtgcctgtt ttgtctgttg ttagtctgca ggtgtaccca 3840
acagctccga agagacagcg accatcgaga acgggccatg atgacgatgg cggttttgtc 3900
gaaaagaaaa gggggaaatg tggggaaaag caagagagat cagattgtta ctgtgtctgt 3960
gtagaaagaa gtagacatag gagactccat tttgttatgt actaagaaaa attcttctgc 4020
cttgagattc tgttaatcta taaccttacc cccaaccctg tgctctctga aacatgtgct 4080
ctgtcaactc agagttaaat ggattaaggg cggtgcaaga tgtgctttgt taaacagatg 4140
cttgaaggca gcatgctcct taagagtcat caccactccc taatctcaag tacccaggga 4200
cacaaaaact gcggaaggcc gcagggacct ctgcctagga aagccaggta ttgtccaagg 4260
tttctcccca tgtgatagtc tgaaatatgg cctcgtggga agggaaagac ctgaccgtcc 4320
cccagcccga cacccgtaaa gggtctgtgc tgaggaggat tagtaaaaga ggaaggaatg 4380
cctcttgcag ttgagacaag aggaaggcat ctgtctcctg cccgtccctg ggcaatggaa 4440
tgtctcggta taaaacccga ttgtatgctc catctactga gatagggaaa aaccgcctta 4500
gggctggagg tgggacctgc gggcagcaat actgctttgt aaagcattga aatgtttatg 4560
tgtatgcata tctaaaagca cagcacttaa tcctttacct tgtctatgat gcaaagacct 4620
ttgttcacgt gtttgtctgc tgaccctctc cccacaattg tcttgtgacc ctgacacatc 4680
cccctcttcg agaaacaccc acagatgatc aataaatact aagggaactc agaggctggc 4740
gggatcctcc atatgctgaa cgctggttcc ccgggtcccc ttctttcttt ctctatactt 4800
tgtctctgtg tctttttctt ttccaaatct ctcgtcccac cttacgagaa acacccacag 4860
gtgtgtaggg gcaacccacc cctacaggta cataacataa aatacagaaa tggcaaaaga 4920
taaagcctac ctgttaaact aaggcatgga agggatggat attttaagtg atttttacta 4980
tattttggcc cttgaaagtc attagaatgt gactgcaagt cgccaggata ataaacctgc 5040
attgagttag cttaagtgtg tagaaaacag aaaaaaaaaa aaaaaaactt taaaaaatgt 5100
cctttggtgt tggccatttt cctgcatcag cctttagctg gaatgtctta gaagtgagct 5160
cgggaaagta gggttgtggc tggtttcttt ttattcgttc tgggctcctt aggcagcttc 5220
tatttttaat agcctgccac ttgttgctgt ccttaccctg agaggaagga aggaagacta 5280
caagtggcag taagaggcta gagaatgaca gggtggtaaa gtgtgtgtgt aagcgcggag 5340
gctggaggca agagagtaac aggctgggag aaaaccactc cccttatctt ctaggccacc 5400
tacgcctgga gccaaccctg ttaggtaagg tactgaaaat gttcttttta ttatttttac 5460
cacccccaag cagaggggct taggggagga gcacacatgc agcacagggt ctgctttgaa 5520
gtttcttagc cacagtgttt ttaaacattt tataaaaaag caaaacaaac attccttact 5580
gctgtgtctg tcttctgaag atgacagcag ctggcagagc tcctccacct tagcctagtt 5640
agcatttaat atatggctac aggagagcgc cccttattag cgggctcctc ccccagggct 5700
ctgtactcta ggctgcttca cactcctttg cccaccttag gctagtaagg ttggggtgca 5760
catggactgt tttttaccgc tttccctagg gggccaggga agacaacaaa ttgcttgtca 5820
actgagacat gcgaggggtg gcagggaagg gcagaagtag gaaaagcagc agcaggccac 5880
ttcctgggca cagttactaa ggcacgtgac cgtggtgagg cggatcctca ggacctggct 5940
ggagctgcct gctctgtgcc ttttggcctc ccctccgtca ccataaaccc agaaaggggt 6000
aacctgccca gactgccagt gaccaaatgg gcactccctg gcatcttagc aactctcctg 6060
ccccaggcct ctgtaggcag caagaaagct acagctagct ggaataaaac ccaggaggca 6120
gcatgcaaac tcccctcgta ctccagaaaa acagaaaaaa gacaaggcca gaaaggctga 6180
tgctggaaat aggaaatggg aacttgcata ttaggaggac aatcataatc agccactttc 6240
cttccctcct ggaggcaccc ctccagcagg gaagttggtt gagagacgtg gtcacccgat 6300
cataccgatg ttcgctgacc agggggtctt atctaagaac tgtcccttgg gcggcctcta 6360
gtctccttaa gggggatggg aggtgctgtt ggccgaacga acggggccaa caaacgcagg 6420
tcccttgaag ggtggaggaa aggagggcaa tgggttttct atagggggag gaaaaggctc 6480
cggtcgcctt atccttacta gaaaaaatgt cctccttagg cctctctaca gaagggaatg 6540
aagtagagag aggagcactg ccccgccccc gctgttctgg ttcctgtctt tcctcactag 6600
aaaaaatctc ttccttattc cccatagaag ggaatgaagt ggagagagga atccctccct 6660
ttaaatcttc tgaattattt tcaggtggag ataatgggcc gtctaaccgc gatctggcag 6720
ataaagagga tacaacgcag agtgtaccag cgcccaggtg gtcaaaatag taaaattagt 6780
aaaatgacct tgctcatatc ctcttttcag gcagcgacct acctgctccc atagctctaa 6840
atctaaggtt ccttgatcag gaaaccaagg acattcctgc cgaattaaaa gcatgagctt 6900
gtgtagagct ccaggctcta cagtgcactg gacagcctta agtagctgtt gcactgtttt 6960
aaaaatactt tctgggcccg ggcgcagtgg ttcacacctg taatcccagc actttgggag 7020
gcggaggcgg gcggatcacg aggtcgggag gtcgagatca ctctggctaa cgcggtgaaa 7080
ccccgtctct actaggctgg caggcgcctg tagtcccagc tactcgggag gctgaggcag 7140
gagaatggtg tgaacccggg aggcggagct tgcagtgagc ggagatcgcg ccactgcact 7200
ccagcctggg cggcagagcg agactccata tcaaaacaaa caaacaaaca aaaaaaactt 7260
tctgttcttt ggtcatttcc tgacccatga taactcaaca cctgatattt tacgtgtggg ?320
37/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
tcgtgtcctc ctgataggag tcaggaatct ctccctttaa atcttctgaa ttcttttcag 7380
gtggagattc accgaaaatt gatgagctct cctccttcac acgtaacttt aaggcgatca 7440
tgtcggggac accacttgca gagcttatag tttacaatga atgccacgaa atgggttctc 7500
agacaattcc agctgagcgg ggcggggggc agctcttatt tattttttaa ctatacttta 7560
agttttgggt tacatgtgca gaacgtgcag ttttgttaca caggtataca cgtgccctgg 7620
tttgctgcac ccatcaaccc gtcacctaca ttaggtattt ctcctaatgt tatccctccc 7680
ctagcccccc actccaccac aggccccgat gtgtgaacag acgcttctca aaagaagaca 7740
tttatgcagc caacangaca cgagaaaaga tcatcactgg tcattagaga aatgcaaatc 7800
aaaaccacga tgagatacca tctcacgcca gttagaatgg ggggtggctc ttcttttttt 7860
tttttttttt ttgagacgga gtcgctctgt tgcccaggct ggagtgcagt ggcatgatct 7920
cggctcactg caacctctgc ctcctgggtt caagcaattc tctgcctcag cctcccgagt 7980
agctgggatt acaggcacct gccaccacgc ctggctaatt tttttgtatt tttagtagag 8040
acggggtttc atcatcttga ccaggctggt cttgaatcgt gatccacccc tctccgtctc 8100
ccaaagtgct aggattacag gcgtgagcca ccgcgcccgg cattgcgggg tagctcttct 8160
aagagagtcc gccccggcat ccgtccgcca agtatttatt gaaagggctt gttaaaccac 8220
aaacatccac tagatggcgt ttttgaggtc gggtcgtgag gcacctgtgg ccttgtaaaa 8280
gcactcaaac tgcattctca ggaggctgtt ttcagcgttc cttatcacac cacacactcc 8340
actccctgtc ctgttttcag ggtcgagttt cattctcatg cacaaataac atacacacag 8400
cgcctcagta tttttccatg ccccgacctc aaatgccttg tacataagct tgactatgtt 8460
gccttgcgcc ccccacagtt ggcatcacag gcatgagcca ccatgctggc ctatattagc 8520
attattatta ccattttcat tgttaatgtg tctatcttct tgagggaagg agcatgttct 8580
atttcttcct tatttcttgt actgagagct ccagaaatgt tccataaatg agggaatgat 8640
gaaatgaaac aaataaatgg aattggggaa agttaccctt atctctttgc aggatatgaa 8700
ttgaagaaac ccacttatct ttccaaactt ggactgcaag tgagaaatgg cagggtcact 8760
gatgtggaag ggaagaccaa agggtgttat agtgagtcat ataccagctt ggagccctag 8820
acctgggcgg gtggggacgt aggtcacctt tcttggctgt tgaaacaaat gagagttata 8880
aaatctccac cccttagttc cattacactt catacctgtt tttcctgtcc atgcgtttgg 8940
aagcttgcct gtacctttta aatatgttct ctgtagcaag tgacttctag gtttaataaa 9000
gaagaagaaa aacccatagg tttctagatt ggaaactagg aaaactgtgg tattcttgag 9060
ttgtaatcag gaggtcatag aattgagaaa atagtaatta actgattgga aatgaagaaa 9120
tgctttttct tagaatactt ttgaataaat tatttcaaat gattatgagt tgactcatct 9180
tttaaaattg tggaaaagtt cttttttttt ttttttgaga ctgagtcttg ctctgttgtg 9240
cagactggaa tgcagtggcg caatctcggt tcactgcaac ctccacctcc cgggttcaag 9300
cgattctcct gcctcagcct cccgagtagc tgggattaca ggcgcctgcc acgatgccca 9360
gctaatttgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtatat ttgagacgga 9420
gtttcactct tgtttcccag gctggagtgc aatggcacaa cctcagctca ccacaacctc 9480
tgcctcccaa cctctacctc ctgggttcaa gcaattctcc tgtctcagcc tcccaagtag 9540
ctgggattat aggcataagc caccacacct ggtgaatttt tttgtatttt tagtagagac 9600
gggatttcat catattgggt gaggctggtc tcgaactcct gacctcaggt gatccaccca 9660
cctcagcctc ccaaagtgct gggattacag acataagcca ccacgcccag cctggaaaac 9720
tttattttga aataattttt gggtcgggcc tggtggcata cacctgtaat cccaggactt 9780
tgggaggccg aggagagtgg atcacctgag tccaggagtt tgagaccagc ctggccaaca 9840
tggcaaaacg ctgtctctac taaaaaatac aaaaattagc tctgcgtggt ggcacatgcc 9900
tgtaatccca gctactcagg aggctgaggg gcaggaggat agctagaaac tgggaggcgg 9960
aggttgtggt gaaccgaggt tgcaccacta cactctagcc tgggctacag agcaagactg 10020
tcacaaaaaa aaaaaaagaa aactaaaaga aaataaaaat aattattggt tctcagaaag 10080
ttacaaaaat aggtgcagtg gcttgtgcct atggtcccag ctactcagga ggctgaggtg 10140
gaggatcagt tgagcccaag agttggaggt tgtggtgggt gacagggtga gaccttgtct 10200
caaaaaaata aaaaatcaat ataaaaataa gcaaaacaaa aaaattgacc ctggttgaca 10260
atccacagat ctcatttaga gtttcacatg taaatgtgtg tagttctgtg ctattttatc 10320
acctgtccac caccacatag ccactaccac aattaagata cacaattatt tcatcactaa 10380
aagatgattt tttttttttt aagatggagt cttgctcttg tcacccaggc tggagtgcaa 10440
tggcacaata tcggctcact gcaacctcta cctcctgggt tcaagcaatt cttctgcctc 10500
agccttccga gtagttggga ttacaggcac ctgccaccac acccagctaa cttttgtatt 10560
tttagtagag tcggggtttc accatgttgg ccaggctcgt ctccaactcc tgacaaaggt 10620
gatccacctg cctcggcctt ccaaagtgct gggattacag gcatgagcct ggccaactaa 10680
aagattcttc tacagtcaaa cccattctcc tctcctccct gcctctcctc tccccattct 10740
cgggaatgtt ttatgttttc ttatatttga tatttctcac tattgcaaaa aaataaggga 10800
cagattctcc catcgatttt tgtgctgttc tgaaagccta aaatagaaag gagcacgtta 10860
agaagttaga gaccttctgt gcccaccagc aggaaaatcg ttaatagtaa cacaaagagg 10920
38/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
ccaggcacgg tggctcatgc ctgtaatccc agcactttga gaggctaagg cgggaggact 10980
gcttgagtcc aggggtttga gaccagcctg ggcaacactg tgagacacca tctctacaaa 11040
aacattaaaa aattagcagg gcatggtgac atgtgcctgt gaccccagct cctcaggagg 11100
ctgaggtggg agaatccctt aagcccagga ggtcaagacc agtgagctgt gatggagtca 11160
ctgcactcaa gtctggatga cagagtgaga ccctgtttca aaaacaacaa caacaacaaa 11220
aagtaacaca aagaatcctc ccaacagaaa tgattccctt tcctataatt ggatagtttc 11280
caggaaaatg tacattagct ctgtgtagat aagcacttgg catggtactt aaaactcaca 11340
ggacctaaca aatatttatc aggcaagtac actgattgaa cagggagcaa ggaattccct 11400
aacaggttcc taaaagagat gtgaaacaag tctccatttg ttccagaggg cagttgtttt 11460
caaattagta aagcttacag caataaaagc aaatttttct ctaaactaaa caaaagctga 11520
gcgaaaaata gacaggctaa caggccatct atttggtttt tctttttttt ttgttgtcaa 11580
gacagagtct cactctgtcg cccaggctgg agtgcagtgg cgcgatcttg gctcactgca 11640
acctccacct cccagattca agcgattctc ctgcctcagc cttccaagta gctgggatta 11700
taggcgtttg ccaccacgcc tggctcactt ttgtattttt agtaaagatg aggtttcccc 11760
atgttggcca ggctggtgtc gaactcctga cctcaggtga tccgcccgcc ttggcctccc 11820
aaagtgttgg aattacaggc ctgggccatc acacccgacc tatttggttt cacttattca 11880
cagttcctta atttatttct ccaagcagga gacaaaacca aaaccaaaaa caggctgaag 11940
tggtgactga agtctctcac ctgaagaccc aagacttagg gtgcccccag tcctaaaagt 12000
cctgtttaag tctctcttct caacattgtg cagcctgcct tctgctggat aggaaaggta 12060
cagatgtgtg gccagctaga taataacaac agttatcaag ctgccagagc gctcacctta 12120
gcgcagtgct aaggacttta gctgtaatat ctcatttagt ccgtataaca accctgtgaa 12180
atgtgtgatt ttattatctt cattttacag ctaagaaaac agactttaag tacctaaatg 12240
ttgggtcagg cacggtgact catgcctgta atctcaacac tttgggaggc tgaggtggga 12300
atatcacttg aatccagaag ttctgagacc agcctgggca atgtagcaag tatcccacct 12360
ctacaatcac aaaattagcc gtgcgtggtg gcatacacct gtagttctag ggaagctgag 12420
gcagggggat cacttgagcc taggagttcg aggttacagt gagcttgcac cactgcactc 12480
cagcctgggt ggtagagtga gactttgtct gaaaaaaaaa aaaaaagaaa agaaaagaaa 12540
gcggcgggcc gggcgcggtg gctgacgcct gtaatcccag cactttggga ggccgaggcg 12600
ggcggatcac gaggtcagga gatgagacca tcctggctaa cacggtgaaa cctcgtctct 12660
actaaaaata caaaaaaatc agccgggcgt ggtggcgggc gcctgtagcc ccagctactc 12720
gggaggctga ggcgggagaa tggcgtgaac ccgggaggtg gagcctgcag tgagccgaga 12780
tcgcgccact gcattccagc gtgtgcgaca gtgagactcc gtctcccaaa aaaaaaaaaa 12840
aaaaaaaaaa aaaaaaagaa cttaaatgtt ttgcctaagt ctacacaact aagctagtgg 12900
aagacaaggg atttgaactc aggttagtat aataagtcag ccctaggcag tcttggtatt 12960
cattaggtct gatgtgtgac acagaaatct gcacacagta cttttaaaag cccccaggtg 13020
attctgactt actgacttct tgggaagcat tgctagaatc tacttcctgc aaaggaatct 13080
cctgctactc aaatggggtg ggaacatgaa gagaactaat agataaactt atatgagcgg 13140
tactattact tcacaaaccc atacttcagg tagacaatgc taaatgacta attgcttttg 13200
cctgatgctt tggatgaaat gttttcttaa gttgcgtctt tattatttat ttatttattt 13260
atttagagac ggggtctcac tctgtcaccc aggctggagt gcagtggtat gattttggct 13320
catggcaact tcctgcttcc tgggctcagg tgatcctccc acttcagcct ccttagtagc 13380
tgggaccaca ggtgcactaa agaatgtctg tatttttttt cagagatggg tttttgccat 13440
gttgcccaag ctggactgga actcctgggc tcaagcgatc cacccccttc agcctcccaa 13500
agtgctgaga ttacaggcat gagccaccgc gcccagcata ttaatgtttt ttattatttt 13560
ttatttattt attttttttg agacggagtc tcgctcggtt gcacaggctg gagtgcagtg 13620
gagcgatctt ggctcactgc aagctccgcc tcccaggttc acgccattct cccgcttcag 13680
cctcccgagt agctggcagg cgcccgccac cacgcccggc taattgtatt tttagtagag 13740
agggggtttc accgtgttag ccaggatggt ctcgatctcc tgacctcgtg atccgcccgc 13800
ctcggcctcc caaagtgctg ggattacagg tttgagccac cgcgcccggc ttattaattg 13860
atttttaagc tcatcagacc actagaagcc tgagttagtg ttgcaagcac tctcgccaga 13920
aagggaatta aattaccttg gagctatgaa gcacaagttt agttacctct atccctgaga 13980
tacatagatt ctggcatggg gttctatttg aaagtttttc atttattttt cctttttatt 14040
tatgtattta ttgtttgaga cagtcactct gtcgcccagg ctgaagtgca gtgatgtgac 14100
ctggactcat tgcaacctct gcctcccggg ttcaagcaat tcttgtgcct cagcctcctg 14160
aggtagctgg gattacaggt gcgtgtcacc acacccggct aatttttgta attttagtag 14220
agacggtttc gccatgttgg ccaggctggt ctcaaactcc tgacctcaag cagtccaccc 14280
gcctcagcct cccaaagtgt tgggattaca ggcgtgagcc accgtgccgg cctgtttttc 14340
ttttttaaaa aagacctatt agagctgtag aaattctcgg taggaagaaa tacaaataaa 14400
gatatataat accaaaaaaa gtttctttta atctgaagca tacatatatt ttggcaaatg 14460
tagatgtata attggaccta atgaaatgtt cttaaaatct aaagactgcc ctatgatgaa 14520
39/40
CA 02471119 2004-06-18
WO 03/055995 PCT/CA02/01998
tgagtgtatg aattaacata aaaactggtt ttgtatatac aggccaacag atacaaactg 14580
gatttgttat ttcattcaca agaaccaagg ggaaaagatc tcaagtcaca cttggtttca 14640
acacaccaag ccaaacaact aataaaccgc ccacgaagaa tgaaactatt ctgaactcgt 14700
cattctgcag atgcagatta taatattaag atatgcacaa aattgtataa agtagacaat 14760
gagtgacctt ttattgaaaa gaggaggagt caaagatcct gaaagggttg ggactgcctg 14820
cagtcagtcc ctagggaact tcctgttgtc accacacctc tgagtcgtct gagctcactg 14880
tgagcaaaat cccacagtgg aaactcttaa gcctctgcga agtaaatcat tcttgtgaat 14940
gtgacacacg atctctccag tttccatatg ttgagattct acttattcat cagtttgttg 15000
40/40
