Note: Descriptions are shown in the official language in which they were submitted.
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
HUMAN LYASES AND ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences. of human
lyases and associated
proteins and to the use of these sequences in the diagnosis, treatment, and
prevention of reproductive
and neurological disorders, inflammatory disorders, and cell proliferative
disorders, including cancer,
and in the assessment of the effects of exogenous compounds on the expression
of nucleic acid and
amino acid sequences of human lyases and associated proteins.
BACKGROUND OF THE INVENTION
Lyases are a class of enzymes that catalyze the cleavage of C-C, C-O, C-N, C-
S, C-(halide),
P-O, or other bonds without hydrolysis or oxidation to form two molecules, at
least one of which
contains a double bond (Stryer, L. (1995) Biochemistry, W.H. Freeman and Co.,
New York NY,
p.620). Under the International Classification of Enzymes (Webb, E.C. (1992) E
Nomenclature 1992: Recommendations of the Nomenclature Committee of the
International Union of
Biochemistry and Molecular Biolo~v on the Nomenclature and Classification of
Enzymes, Academic
Press, San Diego CA), lyases form a distinct class designated by the numeral 4
in the first digit of the
enzyme number (i.e., EC 4.x.x.x).
Further classification of lyases reflects the type of bond cleaved as well as
the nature of the
cleaved group. The group of C-C lyases includes carboxyl-lyases
(decarboxylases), aldehyde-lyases
(aldolases), oxo-acid-lyases, and other lyases. The C-O lyase group includes
hydro-lyases, lyases
acting on polysaccharides, and other lyases. The C-N lyase group includes
ammonia-lyases, amidine-
lyases, amine-lyases (deaminases), and other lyases.
Lyases are critical components of cellular biochemistry, with roles in
metabolic energy
production, including fatty acid metabolism and the tricarboxylic acid cycle,
as well as other diverse
enzymatic processes.
Phosphoenolpyruvate carboxykinase (ATP) (EC 4.1.1.49) is a lyase involved in
gluconeogenesis, the production of glucose from storage compounds in the body.
This enzyme
catalyzes the decarboxylation of oxaloacetate to form phosphoenolpyruvate,
accompanied by
hydrolysis of ATP. (See, e.g., Matte, A. et al. (1997) J. Biol. Chem. 272:8105-
8108; Medina, V. et
al. (1990) J. Bacteriol. 172:7151-7156.)
L-rhamnose and D-fucose are 6-deoxyhexoses found in complex carbohydrates in
bacterial
cell walls. One of the steps in the pathways leading to the synthesis of these
carbohydrates is the
conversion of dTDP-D-glucose to an unstable 4-keto-6-deoxy intermediate, a
reaction catalyzed by
the lyase dTDP-D-glucose 4,6-dehydratase (EC 4.2.1.46). (See, e.g., Tonetti,
M. et al. (1998)
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
Biochimie 80:923-931; Yoshida, Y. et al. (1999) J. Biol. Chem. 274:16933-
16939.)
Isocitrate lyase (EC 4.1.3.1) is involved in the glyoxylate cycle, a
modification of the citric
acid cycle. The glyoxylate cycle occurs in bacteria, fungi, and plants.
Isocitrate lyase catalyzes the
cleavage of isocitrate to yield succinate and glyoxylate. (See, e.g.,
Beeching, J.R. (1989) Protein Seq.
Data Anal. 2:463-466; Atomi, H. et al. (1990) J. Biochem. 107:262-266.)
Aldolases are lyases which catalyze aldol condensation reactions. Fructose 1,6-
bisphosphate
aldolase (FBP-aldolase; EC 4.1.2.13) catalyzes the reversible cleavage of
fructose 1,6-bisphosphate to
yield dihydroxyacetone phosphate, a ketose, and glyceraldehyde 3-phosphate, an
aldose. Class I
FBP-aldolases are found in higher organisms, and exist as homotetramers. Class
II FBP-aldolases
tend to be dimeric, occur in yeast and bacteria, and have an absolute
requirement for a divalent canon
for catalytic activity. (See, e.g., Hall, D.R. et al. (1999) J. Mol. Biol.
287:383-394.)
Pseudouridine is an isomer of uridine which helps to maintain the specific
tertiary structures
of certain rRNAs, tRNAs, and small nuclear and nucleolar RNAs. Pseudouridine
is not directly
incorporated into these RNAs, but is synthesized by pseudouridine synthases
(EC 4.2.1.70), lyases
which act on specific uridine residues within these RNAs. The Rlu family of
pseudouridine synthases
includes Escherichia coli ribosomal large subunit synthase A, which
synthesizes pseudouridine at
position 746 in 23S rRNA and Escherichia coli ribosomal large subunit synthase
C, which
synthesizes pseudouridine at positions 955, 2504, and 2580 in 23S rRNA. (See,
e.g., Conrad, J. et al.
(1998) J. Biol. Chem. 273:18562-18566.)
Fumarate lyases are a group of lyases which share limited sequence homology
and use
fumarate as a substrate. These enzymes include fumarase (EC 4.2.1.2),
aspartase (EC 4.3.1.1),
arginosuccinase (EC 4.3.2.2), and adenylosuccinase (EC 4.3.2.2). (See, e.g.,
Woods, S.A. et al.
(1988) Biochim. Biophys. Acta 954:14-26; Woods, S.A. et al. (1988) FEMS
Microbiol. Lett. 51:181-
186; Zalkin, H. and J.E. Dixon (1992) Prog. Nucleic Acid Res. Mol. Biol.
42:259-287.)
The glyoxalase system is involved in gluconeogenesis, the production of
glucose from
storage compounds in the body. It consists of the lyase glyoxalase I (EC
4.4.1.5), which catalyzes the
formation of S-D-lactoylglutathione from methylglyoxal, a side product of
triose-phosphate energy
metabolism, and glyoxalase II, which hydrolyzes S-D-lactoylglutathione to D-
lactic acid and reduced
glutathione. Glyoxalases are involved in hyperglycemia, non-insulin-dependent
diabetes mellitus, the
detoxification of bacterial toxins, and the control of cell proliferation and
microtubule assembly.
(See, e.g., Thornalley, P.J. (1993) Mol. Aspects Med. 14:287-371.)
Aconitase (EC 4.2.1.3) is a lyase which carries out a crucial step in the
tricarboxylic acid
cycle. Aconitase catalyzes the reversible transformation of citrate into
isocitrate through a cis-
aconitate intermediate. Two forms of aconitase are found in mammalian cells, a
cytosolic aconitase
(Kennedy, M.C. et al. (1992) Proc. Nat). Acad. Sci. USA 89:11730-11734) and a
mitochondria)
2
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
aconitase (Mirel, D.B. et al. (1998) Gene 213:205-218).
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) is a
lyase which
carries out a crucial step in the Calvin cycle during photosynthesis. Rubisco
catalyzes the covalent
incorporation of carbon dioxide into the 5-carbon sugar ribulose 1,5-
bisphosphate along with the
simultaneous cleavage of this molecule into two molecules of 3-
phosphoglycerate. (See, e.g.,
Hartman, F.C. and M.R. Harpel (1994) Annu. Rev. Biochem. 63:197-234.) Specific
methyltransferases (EC 2.1.1.43) catalyze the methylation of amino groups near
the N-termini of the
small and large subunits of Rubisco (Ying, Z. et al. (1998) Acta Biol. Hung.
49:173-184; Klein, R.R.
and R.L. Houtz (1995) Plant Mol. Biol. 27:249-261).
Proper regulation of lyases is critical to normal physiology. For example,
mutation induced
deficiencies in the uroporphyrinogen decarboxylase can lead to photosensitive
cutaneous lesions in
the genetically-linked disorder familial porphyria cutanea tarda (Mendez, M.
et al. ( 1998) Am. J.
Genet. 63:1363-1375). It has also been shown that adenosine deaminase (ADA)
deficiency stems
from genetic mutations in the ADA gene, resulting in the disorder severe
combined
immunodeficiency disease (SCID) (Hershfield, M.S. (1998) Semin. Hematol.
35:291-298).
The discovery of new human lyases and associated proteins and the
polynucleotides encoding
them satisfies a need in the art by providing new compositions which are
useful in the diagnosis,
prevention, and treatment of reproductive and neurological disorders,
inflammatory disorders, and cell
proliferative disorders, including cancer, and in the assessment of the
effects of exogenous compounds
on the expression of nucleic acid and amino acid sequences of human lyases and
associated proteins.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, human lyases and associated
proteins, referred to
collectively as "HLYAP" and individually as "HLYAP-1," "HLYAP-2," "HLYAP-3,"
"HLYAP-4,"
"HLYAP-5," "HLYAP-6," "HLYAP-7," "HLYAP-8," "HLYAP-9," and "HLYAP-10." In one
aspect, the invention provides an isolated polypeptide comprising an amino
acid sequence selected from
the group consisting of a) an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
10, b) a naturally occurring amino acid sequence having at least 90% sequence
identity to an amino
acid sequence selected from the group consisting of SEQ ID NO:1-10, c) a
biologically active fragment
of an amino acid sequence selected from the group consisting of SEQ ID NO:1-
10, and d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
10. In one alternative, the invention provides an isolated polypeptide
comprising the amino acid
sequence of SEQ ID NO:1-10.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising an
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
amino acid sequence selected from the group consisting of a) an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-10, b) a naturally occurring amino acid
sequence having at least
90% sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
10, c) a biologically active fragment of an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-10, and d) an immunogenic fragment of an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-10. In one alternative, the polynucleotide encodes a
polypeptide selected
from the group consisting of SEQ ID N0:1-10. In another alternative, the
polynucleotide is selected
from the group consisting of SEQ ID NO:11-20.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-10, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-10, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
NO:1-10, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:l-10. In one alternative, the invention provides a
cell transformed with the
recombinant polynucleotide. In another alternative, the invention provides a
transgenic organism
comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an
amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-10, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-10, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
NO:1-10, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-10. The method comprises a) culturing a cell under
conditions suitable for
expression of the polypeptide, wherein said cell is transformed with a
recombinant polynucleodde
comprising a promoter sequence operably linked to a polynucleotide encoding
the polypeptide, and b)
recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino acid
sequence selected from the group consisting of SEQ ID NO:1-10, b) a naturally
occurring amino acid
sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-10, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-10, and d) an immunogenic fragment of
an amino acid
4
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
sequence selected from the group consisting of SEQ ID NO:1-10.
The invention further provides an isolated polynucleotide comprising a
polynucleotide sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID NO:11-20, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID NO:11-20, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to b), and e)
an RNA equivalent of a)-d). In one alternative, the polynucleotide comprises
at least 60 contiguous
nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide comprising a
polynucleotide sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID N0:11-20, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:11-20, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to b), and e)
an RNA equivalent of a)-d). The method comprises a) hybridizing the sample
with a probe comprising
at least 20 contiguous nucleotides comprising a sequence complementary to said
target polynucleotide
in the sample, and which probe specifically hybridizes to said target
polynucleotide, under conditions
whereby a hybridization complex is formed between said probe and said target
polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and
optionally, if present, the amount thereof. In one alternative, the probe
comprises at least 60 contiguous
nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample, said
target polynucleotide having a sequence of a polynucleotide comprising a
polynucleotide sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID NO:11-20, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID NO:11-20, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to b), and e)
an RNA equivalent of a)-d). The method comprises a) amplifying said target
polynucleotide or
fragment thereof using polymerase chain reaction amplification, and b)
detecting the presence or
absence of said amplified target polynucleotide or fragment thereof, and,
optionally, if present, the
amount thereof.
The invention further provides a composition comprising an effective amount of
a polypeptide
comprising an amino acid sequence selected from the group consisting of a) an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-10, b) a naturally occurring
amino acid sequence
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
having at least 90% sequence identity to an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-10, c) a biologically active fragment of an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-10, and d) an immunogenic fragment of an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-10, and a pharmaceutically acceptable
excipient. In one
embodiment, the composition comprises an amino acid sequence selected from the
group consisting of
SEQ ID N0:1-10. The invention additionally provides a method of treating a
disease or condition
associated with decreased expression of functional HLYAP, comprising
administering to a patient in
need of such treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising an amino acid sequence selected from the
group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:1-10, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-10, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-10, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-10. The method
comprises a)
exposing a sample comprising the polypeptide to a compound, and b) detecting
agonist activity in the
sample. In one alternative, the invention provides a composition comprising an
agonist compound
identified by the method and a pharmaceutically acceptable excipient. In
another alternative, the
invention provides a method of treating a disease or condition associated with
decreased expression of
functional HLYAP, comprising administering to a patient in need of such
treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising an amino acid sequence selected from
the group consisting
of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-
10, b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-10, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-10, and d) an
immunogenic fragment
of an amino acid sequence selected from the group consisting of SEQ ID N0:1-
10. The method
comprises a) exposing a sample comprising the polypeptide to a compound, and
b) detecting
antagonist activity in the sample. In one alternative, the invention provides
a composition comprising
an antagonist compound identified by the method and a pharmaceutically
acceptable excipient. In
another alternative, the invention provides a method of treating a disease or
condition associated with
overexpression of functional HLYAP, comprising administering to a patient in
need of such treatment
the composition.
The invention further provides a method of screening for a compound that
specifically binds
6
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
to a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-10, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-10, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-10, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-10. The method
comprises a) combining
the polypeptide with at least one test compound under suitable conditions, and
b) detecting binding
of the polypeptide to the test compound, thereby identifying a compound that
specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-10,
b) a naturally
occurnng amino acid sequence having at least 90% sequence identity to an amino
acid sequence
selected from the group consisting of SEQ ID NO:l-10, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-10, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-10. The
method comprises a) combining the polypeptide with at least one test compound
under conditions
permissive for the activity of the polypeptide, b) assessing the activity of
the polypeptide in the
presence of the test compound, and c) comparing the activity of the
polypeptide in the presence of the
test compound with the activity of the polypeptide in the absence of the test
compound, wherein a
change in the activity of the polypeptide in the presence of the test compound
is indicative of a
compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID NO:11-20, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide comprising a polynucleotide
sequence selected from the
group consisting of i) a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:11-20, ii) a naturally occurring polynucleotide sequence having at least
90% sequence identity to
a polynucleotide sequence selected from the group consisting of SEQ ID N0:11-
20, iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to ii),
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions
whereby a specific
hybridization complex is formed between said probe and a target polynucleotide
in the biological
sample, said target polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of i) a polynucleotide sequence selected from the group consisting
of SEQ ID N0:11-20,
ii) a naturally occurring polynucleotide sequence having at least 90% sequence
identity to a
polynucleotide sequence selected from the group consisting of SEQ ID NO:11-20,
iii) a
polynucleotide sequence complementary to i), iy) a polynucleotide sequence
complementary to ii),
and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide
comprises a fragment of
a polynucleotide sequence selected from the group consisting of i)-v) above;
c) quantifying the
amount of hybridization complex; and d) comparing the amount of hybridization
complex in the
treated biological sample with the amount of hybridization complex in an
untreated biological
sample, wherein a difference in the amount of hybridization complex in the
treated biological sample
is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs),
clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments
used to assemble full-
length sequences encoding HLYAP.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of HLYAP.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-
specific expression
patterns of each nucleic acid sequence as determined by northern analysis;
diseases, disorders, or
conditions associated with these tissues; and the vector into which each cDNA
was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA clones
encoding HLYAP were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same meanings
as commonly understood by one of ordinary skill in the art to which this
invention belongs. Although
any machines, materials, and methods similar or equivalent to those described
herein can be used to
practice or test the present invention, the preferred machines, materials and
methods are now described.
All publications mentioned herein are cited for the purpose of describing and
disclosing the cell lines,
protocols, reagents and vectors which are reported in the publications and
which might be used in
connection with the invention. Nothing herein is to be construed as an
admission that the invention is
not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"HLYAP" refers to the amino acid sequences of substantially purified HLYAP
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, marine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
HLYAP. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of HLYAP either by
directly interacting with
HLYAP or by acting on components of the biological pathway in which HLYAP
participates.
An "allelic variant" is an alternative form of the gene encoding HLYAP.
Allelic variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times in
a given sequence.
"Altered" nucleic acid sequences encoding HLYAP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as HLYAP or a
polypeptide with at least one functional characteristic of HLYAP. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding HLYAP, and improper or unexpected hybridization to
allelic variants, with
a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding HLYAP.
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
The encoded protein may also be "altered," and may contain deletions,
insertions, or substitutions of
amino acid residues which produce a silent change and result in a functionally
equivalent HLYAP.
Deliberate amino acid substitutions may be made on the basis of similarity in
polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues, as long as the
biological or immunological activity of HLYAP is retained. For example,
negatively charged amino
acids may include aspartic acid and glutamic acid, and positively charged
amino acids may include
lysine and arginine. Amino acids with uncharged polar side chains having
similar hydrophilicity
values may include: asparagine and glutamine; and serine and threonine. Amino
acids with
uncharged side chains having similar hydrophilicity values may include:
leucine, isoleucine, and
valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid sequence
to the complete native amino acid sequence associated with the recited protein
molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well known
in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity of
HLYAP. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of HLYAP either by
directly interacting with HLYAI? or by acting on components of the biological
pathway in which
HLYAP participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments thereof,
such as Fab, F(ab')2, and Fv fragments, which are capable of binding an
epitopic determinant.
Antibodies that bind HLYAP polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used
to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from
the translation of RNA, or
synthesized chemically, and can be conjugated to a carrier protein if desired.
Commonly used carriers
that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and keyhole
limpet hemocyanin (KLH). The coupled peptide is then used to immunize the
animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies which
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
bind specifically to antigenic determinants (particular regions or three-
dimensional structures on the
protein). An antigenic determinant may compete with the intact antigen (i.e.,
the immunogen used to
elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic HLYAP, or
of any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding HLYAP or fragments
of HLYAP may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(Applied Biosystems,
Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which
has been assembled from
one or more overlapping cDNA, EST, or genomic DNA fragments using a computer
program for
11
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison
WI) or Phrap
(University of Washington, Seattle WA). Some sequences have been both extended
and assembled to
produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of the
side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide. Chemical
modifications of a polynucleotide sequence can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
12
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
A "fragment" is a unique portion of HLYAP or the polynucleotide encoding HLYAP
which is
identical in sequence to but shorter in length than the parent.sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide)
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID NO:11-20 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID NO:11-20, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID NO:11-20 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:11-20 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID NO:11-20 and the region of SEQ ID NO:11-20 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO:1-10 is encoded by a fragment of SEQ ID NO:11-20. A
fragment
of SEQ ID NO:1-10 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-10. For example, a fragment of SEQ ID NO:1-10 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:1-10.
The precise length of
a fragment of SEQ ID NO:1-10 and the region of SEQ ID NO:1-10 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the liagment.
A "full-length" polynucleotide sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full-
length" polynucleotide sequence encodes a "full-length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer to
the percentage of residue matches between at least two polynucleotide
sequences aligned using a
13
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps in
the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence
alignment program. This program is part of the LASERGENE software package, a
suite of molecular
biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in
Higgins, D.G.
and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992)
CABIOS 8:189-191.
For pairwise alignments of polynucleotide sequences, the default parameters
are set as follows:
Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted"
residue weight table is
selected as the default. Percent identity is reported by CLUSTAL V as the
"percent similarity" between
aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms is
provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment Search
Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which
is available from several
sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off:' SO
Expect: 10 ,
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example, as
14
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
defined by a particular SEQ ID number, or may be measured over a shorter
length, for example, over
the length of a fragment taken from a larger, defined sequence, for instance,
a fragment of at least 20, at
least 30, at least 40, at least 50, at least 70, at least 100, or at least 200
contiguous nucleotides. Such
lengths are exemplary only, and it is understood that any fragment length
supported by the sequences
shown herein, in the tables, figures, or Sequence Listing, may be used to
describe a length over which
percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes in
a nucleic acid sequence can be made using this degeneracy to produce multiple
nucleic acid sequences
that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default parameters
of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e
sequence alignment
program (described and referenced above). For pairwise alignments of
polypeptide sequences using
CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3,
window=5, and
"diagonals saved"=5. The PAM250 matrix is selected as the default residue
weight table. As with
polynucleotide alignments, the percent identity is reported by CLUSTAL V as
the "percent similarity"
between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12
(Apr-21-2000) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
Open Gap: 1l and Extension Gap: 1 penalties
Gap x drop-off.' S0
Expect: l0
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence, for
example, as defined by a particular SEQ ID number, or may be measured over a
shorter length, for
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for instance,
a fragment of at least 15, at least 20, at least 30, at least 40, at least 50,
at least 70 or at least 150
contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment length
supported by the sequences shown herein, in the tables, figures or Sequence
Listing, may be used to
S describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule. in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the stringency
of the hybridization process, with more stringent conditions allowing less non-
specific binding, i.e.,
binding between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for
annealing of nucleic acid sequences are routinely determinable by one of
ordinary skill in the art and
may be consistent among hybridization experiments, whereas wash conditions may
be varied among
experiments to achieve the desired stringency, and therefore hybridization
specificity. Permissive
annealing conditions occur, for example, at 68°C in the presence of
about 6 x SSC, about 1 % (w/v)
SDS, and about 100 ~eJml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (T"~ for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of the
target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and conditions
for nucleic acid hybridization are well known and can be found in Sambrook, J.
et al., 1989, Molecular
Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Press,
Plainview NY; specifically
see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1 % SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration may
16
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salmon sperm DNA at about 100-200 ~g/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A hybridization
complex may be formed in solution (e.g., Cot or Rot analysis) or formed
between one nucleic acid
sequence present in solution and another nucleic acid sequence immobilized on
a solid support (e.g.,
paper, membranes, filters, chips, pins or glass slides, or any other
appropriate substrate to which cells
or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide sequence
resulting in the addition of one or more amino acid residues or nucleotides,
respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression of
various factors, e.g., cytokines, chemokines, and other signaling molecules,
which may affect cellular
and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of HLYAP
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment of
HLYAP which is useful in any of the antibody production methods disclosed
herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides, polypeptides,
or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of HLYAP. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or immunological properties of HLYAP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
17
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an HLYAP may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in the
art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary by
cell type depending on the enzymatic milieu of HLYAP.
"Probe" refers to nucleic acid sequences encoding HLYAP, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule. Typical
labels include radioactive isotopes, ligands, chemiluminescent agents, and
enzymes. "Primers" are
short nucleic acids, usually DNA oligonucleotides, which may be annealed to a
target polynucleotide by
complementary base-pairing. The primer may then be extended along the target
DNA strand by a DNA
polymerase enzyme. Primer pairs can be used for amplification (and
identification) of a nucleic acid
sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers may
be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual,
2°d ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo , Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
18
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas South
West Medical Center, Dallas TX) is capable of choosing specific primers from
megabase sequences
and is thus useful for designing primers on a genome-wide scope. The Primer3
primer selection
program (available to the public from the Whitehead Institute/MIT Center for
Genome Research,
Cambridge MA) allows the user to input a "mispriming library," in which
sequences to avoid as primer
binding sites are user-specified. Primer3 is useful, in particular, for the
selection of oligonucleotides for
microarrays. (The source code for the latter two primer selection programs may
also be obtained from
their respective sources and modified to meet the user's specific needs.) The
PrimeGen program
(available to the public from the UK Human Genome Mapping Project Resource
Centre, Cambridge
UK) designs primers based on multiple sequence alignments, thereby allowing
selection of primers that
hybridize to either the most conserved or least conserved regions of aligned
nucleic acid sequences.
Hence, this program is useful for identification of both unique and conserved
oligonucleotides and
polynucleotide fragments. The oligonucleotides and polynucleotide fragments
identified by any of the
above selection methods are useful in hybridization technologies, for example,
as PCR or sequencing
primers, microarray elements, or specific probes to identify fully or
partially complementary
polynucleotides in a sample of nucleic acids. Methods of oligonucleotide
selection are not limited to
those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
19
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions (UTRs).
Regulatory elements interact with host or viral proteins which control
transcription, translation, or RNA
stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding HLYAP, or fragments thereof, or HLYAP itself, may comprise a
bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding molecule. For
example, if an antibody is specific for epitope "A," the presence of a
polypeptide comprising the epitope
A, or the presence of free unlabeled A, in a reaction containing free labeled
A and the antibody will
reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides by
different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell type
or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods well
known in the art, and may rely on any known method for the insertion of
foreign nucleic acid sequences
into a prokaryotic or eukaryotic host cell. The method for transformation is
selected based on the type
of host cell being transformed and may include, but is not limited to,
bacteriophage or viral infection,
electroporation, heat shock, lipofection, and particle bombardment. The term
"transformed" cells
includes stably transformed cells in which the inserted DNA is capable of
replication either as an
autonomously replicating plasmid or as part of the host chromosome, as well as
transiently transformed
cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants, and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook, J. et al. (1989),
supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having at
least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of the
nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of nucleic acids may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or
at least 98% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an "allelic"
(as defined above), "splice," "species," or "polymorphic" variant. A splice
variant may have significant
identity to a reference molecule, but will generally have a greater or lesser
number of polynucleotides
due to alternative splicing of exons during mRNA processing. The corresponding
polypeptide may
21
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
possess additional functional domains or lack domains that are present in the
reference molecule.
Species variants are polynucleotide sequences that vary from one species to
another. The resulting
polypeptides generally will have significant amino acid identity relative to
each other. A polymorphic
variant is a variation in the polynucleotide sequence of a particular gene
between individuals of a given
species. Polymorphic variants also may encompass "single nucleotide
polymorphisms" (SNPs) in
which the polynucleotide sequence varies by one nucleotide base. The presence
of SNPs may be
indicative of, for example, a certain population, a disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having at
least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of the
polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of polypeptides may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human lyases and associated
proteins
(HLYAP), the polynucleotides encoding HLYAP, and the use of these compositions
for the diagnosis,
treatment, or prevention of reproductive and neurological disorders,
inflammatory disorders, and cell
proliferative disorders, including cancer.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
HLYAP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs)
of the polypeptide
and nucleotide sequences, respectively. Column 3 shows the clone IDs of the
Incyte clones in which
nucleic acids encoding each HLYAP were identified, and column 4 shows the cDNA
libraries from
which these clones were isolated. Column 5 shows Incyte clones and their
corresponding cDNA
libraries. Clones for which cDNA libraries are not indicated were derived from
pooled cDNA libraries.
In some cases, GenBank sequence identifiers are also shown in column 5. The
Incyte clones and
GenBank cDNA sequences, where indicated, in column 5 were used to assemble the
consensus
nucleotide sequence of each HLYAP and are useful as fragments in hybridization
technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
shows homologous sequences as identified by BLAST analysis; and column 7 shows
analytical methods
and in some cases, searchable databases to which the analytical methods were
applied. The methods of
column 7 were used to characterize each polypeptide through sequence homology
and protein motifs.
22
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding HLYAP. The first column of Table
3 lists the nucleotide
SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1.
These fragments are
useful, for example, in hybridization or amplification technologies to
identify SEQ ID NO:11-20 and
to distinguish between SEQ ID NO:11-20 and related polynucleotide sequences.
The polypeptides
encoded by these fragments are useful, for example, as immunogenic peptides.
Column 3 lists tissue
categories which express HLYAP as a fraction of total tissues expressing
HLYAP. Column 4 lists
diseases, disorders, or conditions associated with those tissues expressing
HLYAP as a fraction of total
tissues expressing HLYAP. Column S lists the vectors used to subclone each
cDNA library.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding HLYAP were isolated. Column 1 references the
nucleotide SEQ ID
NOs, column 2 shows the cDNA libraries from which these clones were isolated,
and column 3 shows
the tissue origins and other descriptive information relevant to the cDNA
libraries in column 2.
SEQ ID N0:13 maps to chromosome 1 within the interval from 213.2 to 222.7
centiMorgans.
SEQ ID N0:19 maps to chromosome 14 within the interval from 112.6 to 116.3
centiMorgans.
The invention also encompasses HLYAP variants. A preferred HLYAP variant is
one which
has at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the HLYAP amino acid sequence, and which contains at
least one functional or
structural characteristic of HLYAP.
The invention also encompasses polynucleotides which encode HLYAP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected from
the group consisting of SEQ ID NO:11-20, which encodes HLYAP. The
polynucleotide sequences of
SEQ ID NO:l 1-20, as presented in the Sequence Listing, embrace the equivalent
RNA sequences,
wherein occurrences of the nitrogenous base thymine are replaced with uracil,
and the sugar backbone
is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
HLYAP. In
particular, such a variant polynucleotide sequence will have at least about
80%, or alternatively at least
about 90%, or even at least about 95% polynucleotide sequence identity to the
polynucleotide sequence
encoding HLYAP. A particular aspect of the invention encompasses a variant of
a polynucleotide
sequence comprising a sequence selected from the group consisting of SEQ ID
NO:l 1-20 which has at
least about 80%, or alternatively at least about 90%, or even at least about
95 % polynucleotide
sequence identity to a nucleic acid sequence selected from the group
consisting of SEQ ID NO:11-20.
Any one of the polynucleotide variants described above can encode an amino
acid sequence which
contains at least one functional or structural characteristic of HLYAP.
23
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the genetic
code, a multitude of polynucleotide sequences encoding HLYAP, some bearing
minimal similarity to the
polynucleotide sequences of any known and naturally occurring gene, may be
produced. Thus, the
invention contemplates each and every possible variation of polynucleotide
sequence that could be made
by selecting combinations based on possible codon choices. These combinations
are made in
accordance with the standard triplet genetic code as applied to the
polynucleotide sequence of naturally
occurring HLYAP, and all such variations are to be considered as being
specifically disclosed.
Although nucleotide sequences which encode HLYAP and its variants are
generally capable of
hybridizing to the nucleotide sequence of the naturally occurring HLYAP under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding HLYAP or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding HLYAP and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater half life,
than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode HLYAP
and
HLYAP derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell systems
using reagents well known in the art. Moreover, synthetic chemistry may be
used to introduce
mutations into a sequence encoding HLYAP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
NO:I 1-20 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of the
embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment of
DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia
Biotech, Piscataway
NJ), or combinations of polymerases and proofreading exonucleases such as
those found in the
ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably, sequence
24
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
preparation is automated with machines such as the MICROLAB 2200 liquid
transfer system
(Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI
CATALYST
800 thermal cycler (Applied Biosystems). Sequencing is then carried out using
either the ABI 373 or
377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA
sequencing system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences are
analyzed using a variety of algorithms which are well known in the art. (See,
e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and Biotechnolo~y, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding HLYAP may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising a
known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al.
(1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al.
(1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction
enzyme digestions and
ligations may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 Primer Analysis software
(National Biosciences,
Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides
in length, to have a
GC content of about 50% or more, and to anneal to the template at temperatures
of about 68°C to
72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T) library
does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence into 5'
non-transcribed regulatory regions.
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
Capillary electrophoresis systems which are commercially available may be used
to analyze the
size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode HLYAP may be cloned in recombinant DNA molecules that direct expression
of HLYAP, or
fragments or functional equivalents thereof, in appropriate host cells. Due to
the inherent degeneracy of
the genetic code, other DNA sequences which encode substantially the same or a
functionally equivalent
amino acid sequence may be produced and used to express HLYAP.
The nucleotide sequences of the present invention can be engineered using
methods generally
Irnown in the art in order to alter HLYAP-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction sites,
alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of HLYAP, such as its biological or
enzymatic activity or its ability
to bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
26
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding HLYAP may be synthesized, in whole
or in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-
232.) Alternatively,
HLYAP itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide
synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g.,
Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated
synthesis may be achieved
using the ABI 431A peptide synthesizer (Applied Biosystems). Additionally, the
amino acid sequence
of HLYAP, or any part thereof, may be altered during direct synthesis and/or
combined with sequences
from other proteins, or any part thereof, to produce a variant polypeptide or
a polypeptide having a
sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active HLYAP, the nucleotide sequences
encoding HLYAP or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in a
suitable host. These elements include regulatory sequences, such as enhancers,
constitutive and
inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
encoding HLYAP. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
HLYAP. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding HLYAP and its initiation codon and upstream regulatory
sequences are inserted
into the appropriate expression vector, no additional transcriptional or
translational control signals may
be needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted,
exogenous translational control signals including an in-frame ATG initiation
codon should be provided
by the vector. Exogenous translational elements and initiation codons may be
of various origins, both
natural and synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers
appropriate for the particular host cell system used. (See, e.g., Scharf, D.
et al. (1994) Results Probl.
Cell Differ. 20:125-162.)
27
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding HLYAP and appropriate transcriptional
and translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding HLYAP. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, su ra; Van Heeke,
G. and S.M. Schuster
_ (1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al. (1987) Methods
Enzymol. 153:516-544;
Scorer, C.A. et al. (1994) Bio/Technology 12:181-184; Engelhard, E.K. et al.
(1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-
1945; Takamatsu,
N. (1987) EMBO J. 6:307-311; Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;
Brogue, R. et al.
(1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105; The
McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York
NY, pp.
191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-
3659; and Harnngton,
J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from
retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
The invention is not umited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding HLYAP. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding HLYAP can be
achieved using a
multifunctional E. cou vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or
PSPORT1 plasmid
(Life Technologies). Ligation of sequences encoding HLYAP into the vector's
multiple cloning site
disrupts the lacZ gene, allowing a colorimetric screening procedure for
identification of transformed
bacteria containing recombinant molecules. In addition, these vectors may be
useful for in vitro
28
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
transcription, dideoxy sequencing, single strand rescue with helper phage, and
creation of nested
deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster
(1989) J. Biol. Chem.
264:5503-5509.) When large quantities of HLYAP are needed, e.g. for the
production of antibodies,
vectors which direct high level expression of HLYAP may be used. For example,
vectors containing
the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of HLYAP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, su ra;
Bitter, supra; and Scorer, supra.)
Plant systems may also be used for expression of HLYAP. Transcription of
sequences
encoding HLYAP may be driven viral promoters, e.g., the 35S and 19S promoters
of CaMV used alone
or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, su ra; Brogue, supra; and Winter,
su ra.) These
constructs can be introduced into plant cells by direct DNA transformation or
pathogen-mediated
transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y
(1992) McGraw Hill,
New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding HLYAP
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses HLYAP in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers,
or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
HLYAP in cell lines is preferred. For example, sequences encoding HLYAP can be
transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
29
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include,
but are not limited to, the herpes simplex virus thymidine kinase and adenine
phosphoribosyltransferase
genes, for use in tk- and apr cells, respectively. (See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232;
Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or
herbicide resistance can be
used as the basis for selection. For example, dhfr confers resistance to
methotrexate; neo confers
resistance to the aminoglycosides neomycin and G-418; and als and pat confer
resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g.,
Wigler, M. et al. (1980)
Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J.
Mol. Biol. 150:1-14.)
Additional selectable genes have been described, e.g., trpB and hisD, which
alter cellular requirements
for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins
(GFP; Clontech),13
glucuronidase and its substrate B-glucuronide, or luciferase and its substrate
luciferin may be used.
These markers can be used not only to identify transformants, but also to
quantify the amount of
transient or stable protein expression attributable to a specific vector
system. (See, e.g., Rhodes, C.A.
(1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding HLYAP is inserted within a marker gene sequence, transformed
cells containing
sequences encoding HLYAP can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding HLYAP under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding HLYAP
and that express
HLYAP may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
Immunological methods for detecting and measuring the expression of HLYAP
using either
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques include
enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and
fluorescence
activated cell sorting (FACS). A two-site, monoclonal-based immunoassay
utilizing monoclonal
antibodies reactive to two non-interfering epitopes on HLYAP is preferred, but
a competitive binding
assay may be employed. These and other assays are well known in the art. (See,
e.g., Hampton, R. et
al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN,
Sect. IV; Coligan, J.E.
et al. (1997) Current Protocols in Immunolo~y, Greene Pub. Associates and
Wiley-Interscience, New
York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa
NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled hybridization
or PCR probes for detecting sequences related to polynucleotides encoding
HLYAP include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, the sequences encoding HLYAP, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety of
commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for ease
of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents, as
well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding HLYAP may be
cultured under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors containing
polynucleotides which encode HLYAP may be designed to contain signal sequences
which direct
secretion of HLYAP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of the
polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which cleaves a
"prepro" or "pro" form of the
protein may also be used to specify protein targeting, folding, and/or
activity. Different host cells
which have specific cellular machinery and characteristic mechanisms for post-
translational activities
(e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type
Culture
Collection (ATCC, Manassas VA) and may be chosen to ensure the correct
modification and processing
31
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding HLYAP may be ligated to a heterologous sequence resulting
in translation of a
fusion protein in any of the aforementioned host systems. . For example, a
chimeric HLYAP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of HLYAP
activity. Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available affinity
matrices. Such moieties include, but are not limited to, glutathione S-
transferase (GST), maltose
binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-
His, FLAG, c-myc, and
hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their
cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate resins,
respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
these epitope tags. A fusion protein may also be engineered to contain a
proteolytic cleavage site
located between the HLYAP encoding sequence and the heterologous protein
sequence, so that HLYAP
may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein
expression and purification are discussed in Ausubel (1995, supra, ch. 10). A
variety of commercially
available kits may also be used to facilitate expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled HLYAP may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These systems
couple transcription and translation of protein-coding sequences operably
associated with the T7, T3, or
SP6 promoters. Translation takes place in the presence of a radiolabeled amino
acid precursor, for
example, 35S-methionine.
HLYAP of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to HLYAP. At least one and up to a plurality of test
compounds may be
screened for specific binding to HLYAP. Examples of test compounds include
antibodies,
oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
HLYAP, e.g., a ligand or fragment thereof, a natural substrate, a structural
or functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which HLYAP
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express HLYAP,
either as a secreted
32
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing HLYAP or cell membrane fractions which contain HLYAP
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either HLYAP or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fiuorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
HLYAP, either in
solution or affixed to a solid support, and detecting the binding of HLYAP to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to a
solid support.
HLYAP of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of HLYAP. Such compounds may include agonists,
antagonists, or partial
or inverse agonists. In one embodiment, an assay is performed under conditions
permissive for
HLYAP activity, wherein HLYAP is combined with at least one test compound, and
the activity of
HLYAP in the presence of a test compound is compared with the activity of
HLYAP in the absence
of the test compound. A change in the activity of HLYAP in the presence of the
test compound is
indicative of a compound that modulates the activity of HLYAP. Alternatively,
a test compound is
combined with an in vitro or cell-free system comprising HLYAP under
conditions suitable for
HLYAP activity, and the assay is performed. In either of these assays, a test
compound which
modulates the activity of HLYAP may do so indirectly and need not come in
direct contact with the
test compound. At least one and up to a plurality of test compounds may be
screened.
In another embodiment, polynucleotides encoding HLYAP or their mammalian
homologs
may be "knocked out" in an animal model system using homologous recombination
in embryonic
stem (ES) cells. Such techniques are well known in the art and are useful for
the generation of animal
models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding
region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-IoxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
33
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding HLYAP may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding HLYAP can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a
region of a polynucleotide encoding HLYAP is injected into animal ES cells,
and the injected
sequence integrates into the animal cell genome. Transformed cells are
injected into blastulae, and
the blastulae are implanted as described above. Transgenic progeny or inbred
lines are studied and
treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress HLYAP, e.g., by secreting HLYAP
in its milk, may
also serve as a convenient source of that protein (Janne, J. et al. (1998)
Biotechnol. Annu. Rev. 4:55-
74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of HLYAP and human lyases and associated proteins. In
addition, the expression of
HLYAP is closely associated with reproductive and nervous tissue,
inflammation, cell proliferation,
and cancer. Therefore, HLYAP appears to play a role in reproductive and
neurological disorders,
inflammatory disorders, and cell proliferative disorders, including cancer. In
the treatment of disorders
associated with increased HLYAP expression or activity, it is desirable to
decrease the expression or
activity of HLYAP. In the treatment of disorders associated with decreased
HLYAP expression or
activity, it is desirable to increase the expression or activity of HLYAP.
Therefore, in one embodiment, HLYAP or a fragment or derivative thereof may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of HLYAP. Examples of such disorders include, but are not limited to,
a reproductive
disorder, such as a disorder of prolactin production, infertility, including
tubal disease, ovulatory
defects, and endometriosis, a disruption of the estrous cycle, a disruption of
the menstrual cycle,
polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial
or ovarian tumor, a
uterine fibroid, autoimmune disorders, an ectopic pregnancy, and
teratogenesis, cancer of the breast,
librocystic breast disease, and galactorrhea, a disruption of spermatogenesis,
abnormal sperm
34
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
physiology, cancer of the testis, cancer of the prostate, benign prostatic
hyperplasia, prostatitis,
Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia;
a neurological
disorder, such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral
neoplasms, Alzheimer's
disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease
and other extrapyramidal
disorders, amyotrophic lateral sclerosis and other motor neuron disorders,
progressive neural
muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis
and other demyelinating
diseases, bacterial and viral meningitis, brain abscess, subdural empyema,
epidural abscess,
suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral
central nervous system
disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and polymyositis,
inherited, metabolic,
endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental
disorders including
mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD),
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial
frontotemporal dementia; an inflammatory disorder, such as acquired
immunodeficiency syndrome
(AIDS), Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
(APECED),
bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis,
diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,
multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma,
Sjogren's syndrome,
systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of cancer,
hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal,
and helminthic infections, and
trauma; and a cell proliferative disorder, such as actinic keratosis,
arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD),
myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and a cancer,
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma,
and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow,
brain, breast, cervix,
gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,
muscle, ovary, pancreas,
parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus.
In another embodiment, a vector capable of expressing HLYAP or a fragment or
derivative
thereof may be administered to a subject to treat or prevent a disorder
associated with decreased
expression or activity of HLYAP including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
HLYAP in
conjunction with a suitable pharmaceutical carrier may be administered to a
subject to treat or prevent a
disorder associated with decreased expression or activity of HLYAP including,
but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of HLYAP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or activity
of HLYAP including, but not limited to, those listed above.
In a further embodiment, an antagonist of HLYAP may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of HLYAP.
Examples of such
disorders include, but are not limited to, those reproductive and neurological
disorders, inflammatory
disorders, and cell proliferative disorders, including cancer, described
above. In one aspect, an antibody
which specifically binds HLYAP may be used directly as an antagonist or
indirectly as a targeting or
delivery mechanism for bringing a pharmaceutical agent to cells or tissues
which express HLYAP.
In an additional embodiment, a vector expressing the complement of the
polynucleodde
encoding HLYAP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of HLYAP including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made by
one of ordinary skill in the art, according to conventional pharmaceutical
principles. The combination
of therapeutic agents may act synergistically to effect the treatment or
prevention of the various
disorders described above. Using this approach, one may be able to achieve
therapeutic efficacy with
lower dosages of each agent, thus reducing the potential for adverse side
effects.
An antagonist of HLYAP may be produced using methods which are generally known
in the
art. In particular, purified HLYAP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind HLYAP.
Antibodies to HLYAP may
also be generated using methods that are well known in the art. Such
antibodies may include, but are
36
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies,
Fab fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with HLYAP or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral gels
such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants
used in humans, BCG
(bacilli Calmette-Guerin) and Corynebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
HLYAP have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches of
HLYAP amino acids may be fused with those of another protein, such as KLH, and
antibodies to the
chimeric molecule may be produced.
Monoclonal antibodies to HLYAP may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not limited
to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-
hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
HI.YAP-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton,
D.R. (1991) Proc. Natl. Acad.-Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
37
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for HLYAP may also be
generated.
For example, such fragments include, but are not limited to, F(ab~2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of the
F(ab~2 fragments. Alternatively, Fab expression libraries may be constructed
to allow rapid and easy
identification of monoclonal Fab fragments with the desired specificity. (See,
e.g., Huse, W.D. et al.
(1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
HLYAP and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering HLYAP epitopes is generally used, but a competitive
binding assay may also be
employed (Pound, su ra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for HLYAP. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of HLYAP-antibody
complex divided by the
molar concentrations of free antigen and liee antibody under equilibrium
conditions. The I~ determined
for a preparation of polyclonal antibodies, which are heterogeneous in their
affinities for multiple
HLYAP epitopes, represents the average affinity, or avidity, of the antibodies
for HLYAP. The I~
determined for a preparation of monoclonal antibodies, which are monospecific
for a particular HLYAP
epitope, represents a true measure of affinity. High-affinity antibody
preparations with Ka ranging from
about 109 to 10'2 L/mole are preferred for use in immunoassays in which the
HLYAP-antibody complex
must withstand rigorous manipulations. Low-affinity antibody preparations with
Ka ranging from
about 106 to 10' L/mole are preferred for use in immunopurification and
similar procedures which
ultimately require dissociation of HLYAP, preferably in active form, from the
antibody (Catty, D.
(1988) Antibodies. Volume I: A Practical Approach, IRL Press, Washington DC;
Liddell, J.E. and A.
Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
the quality and suitability of such preparations for certain downstream
applications. For example, a
polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of HLYAP-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and
38
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
Coligan et al., supra.)
In another embodiment of the invention, the polynucleotides encoding HLYAP, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
modifications of gene
expression can be achieved by designing complementary sequences or antisense
molecules (DNA, RNA,
PNA, or modified oligonucleotides) to the coding or regulatory regions of the
gene encoding HLYAP.
Such technology is well known in the art, and antisense oligonucleotides or
larger fragments can be
designed from various locations along the coding or control regions of
sequences encoding HLYAP.
(See, e.g., Agrawal, S., ed. (1996) Antiseiise Therapeutics, Humana Press
Inc., Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Moms, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding HLYAP may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-linked
inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe
combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988)
Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA.
93:11395-11399),
39
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trmanosoma cruzi). In the
case where a genetic deficiency in HLYAP expression or regulation causes
disease, the expression of
HLYAP from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
HLYAP are treated by constructing mammalian expression vectors encoding HLYAP
and introducing
these vectors by mechanical means into HLYAP-deficient cells. Mechanical
transfer technologies for
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii) ballistic
gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-
mediated gene transfer, and (v)
the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev.
Biochem. 62:191-
217; Ivies, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. R~cipon (1998)
Curr. Opin. Biotechnol.
9:445-450).
Expression vectors that may be effective for the expression of HLYAP include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen,
Carlsbad CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). HLYAP may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
(RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes), (ii) an
inducible promoter (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998)
Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the
ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding HLYAP from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
respect to HLYAP expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding HLYAP under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in an
appropriate vector producing cell line (VPCL) that expresses an envelope gene
with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses a
method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et
al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al.
(1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding HLYAP to cells which have one or more genetic
abnormalities with respect to
the expression of HLYAP. The construction and packaging of adenovirus-based
vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to
be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999) Annu.
Rev. Nutr. 19:511-544; and Verma, LM. and N. Somia (1997) Nature 18:389:239-
242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding HLYAP to target cells which have one or more genetic
abnormalities with
respect to the expression of HLYAP. The use of herpes simplex virus (HSV)-
based vectors may be
especially valuable for introducing HLYAP to cells of the central nervous
system, for which HSV has a
tropism. The construction and packaging of herpes-based vectors are well known
to those with
41
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (
1999) Exp. Eye
Res.169:385-395). The construction of a HSV-1 virus vector has also been
disclosed in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use
of recombinant HSV
d92 which consists of a genome containing at least one exogenous gene to be
transferred to a cell under
the control of the appropriate promoter for purposes including human gene
therapy. Also taught by this
patent are the construction and use of recombinant HSV strains deleted for
ICP4, ICP27 and ICP22.
For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and
Xu, H. et al. (1994) Dev.
Biol. 163:152-161, hereby incorporated by reference. The manipulation of
cloned herpesvirus
sequences, the generation of recombinant virus following the transfection of
multiple plasmids
containing different segments of the large herpesvirus genomes, the growth and
propagation of
herpesvirus, and the infection of cells with herpesvirus are techniques well
known to those of ordinary
skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding HLYAP to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based on
the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol. 9:464-
469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full-length
genomic RNA, resulting
in the overproduction of capsid proteins relative to the viral proteins with
enzymatic activity (e.g.,
protease and polymerase). Similarly, inserting the coding sequence for HLYAP
into the alphavirus
genome in place of the capsid-coding region results in the production of a
large number of HLYAP-
coding RNAs and the synthesis of high levels of HLYAP in vector transduced
cells. While alphavirus
infection is typically associated with cell lysis within a few days, the
ability to establish a persistent
infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis
virus (SIN) indicates that
the lytic replication of alphaviruses can be altered to suit the needs of the
gene therapy application
(Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of
alphaviruses will allow the
introduction of HLYAP into a variety of cell types. The specific transduction
of a subset of cells in a
population may require the sorting of cells prior to transduction. The methods
of manipulating
infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and
performing alphavirus infections, are well known to those with ordinary skill
in the art.
Oligonucleotides derived fiom the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can
42
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and Immunoloaic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA by
preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding HLYAP.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by
any method known in the art for the synthesis of nucleic acid molecules. These
include techniques for
chemically synthesizing oligonucleotides such as solid phase phosphoramidite
chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA sequences
encoding HLYAP. Such DNA sequences may be incorporated into a wide variety of
vectors with
suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA
constructs that
synthesize complementary RNA, constitutively or inducibly, can be introduced
into cell lines, cells, or
tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine, and
wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms
of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
43
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding HLYAP.
Compounds which may be effective in altering expression of a specific
polynucleotide may include,
but are not limited to, oligonucleotides, antisense oligonucleotides, triple
helix-forming
oligonucleotides, transcription factors and other polypeptide transcriptional
regulators, and non-
macromolecular chemical entities which are capable of interacting with
specific polynucleotide
sequences. Effective compounds may alter polynucleotide expression by acting
as either inhibitors or
promoters of polynucleotide expression. Thus, in the treatment of disorders
associated with increased
HLYAP expression or activity, a compound which specifically inhibits
expression of the
polynucleotide encoding HLYAP may be therapeutically useful, and in the
treament of disorders
associated with decreased HLYAP expression or activity, a compound which
specifically promotes
expression of the polynucleotide encoding HLYAP may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properkies of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding HLYAP is exposed to at least one test compound thus
obtained. The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
HLYAP are assayed
by any method commonly known in the art. Typically, the expression of a
specific nucleotide is
detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding HLYAP. The amount of hybridization may be
quantified, thus
forming the basis for a comparison of the expression of the polynucleotide
both with and without
exposure to one or more test compounds. Detection of a change in the
expression of a polynucleotide
exposed to a test compound indicates that the test compound is effective in
altering the expression of
the polynucleotide. A screen for a compound effective in altering expression
of a specific
polynucleotide can be carried out, for example, using a Schizosaccharomyces
pombe gene expression
system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al.
(2000) Nucleic Acids
Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
Res. Commun. 268:8-13). A particular embodiment of the present invention
involves screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
44
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable for
use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells taken
from the patient and clonally propagated for autologous transplant back into
that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of such
therapy, including, for example, mammals such as humans, dogs, cats, cows,
horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition which
generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and
proteins. Various
formulations are commonly known and are thoroughly discussed in the latest
edition of Remin~ton's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of HLYAP,
antibodies to HL,YAP, and mimetics, agonists, antagonists, or inhibitors of
HI.YAP.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the case
of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-acting
formulations is well-known in the art. In the case of macromolecules (e.g.
larger peptides and proteins),
recent developments in the field of pulmonary delivery via the alveolar region
of the lung have enabled
the practical delivery of drugs such as insulin to blood circulation (see,
e.g., Patton, J.S. et al., U.S.
Patent No. 5,997,848). Pulmonary delivery has the advantage of administration
without needle
injection, and obviates the need for potentially toxic penetration enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination of
an effective dose is well within the capability of those skilled in the art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising HLYAP or fragments thereof. For example, liposome
preparations
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, HI.YAP or a fragment thereof may be joined to a
short cationic N-
terminal portion from the HIV Tat-1 protein. . Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, monkeys,
or pigs. An animal model may also be used to determine the appropriate
concentration range and route
of administration. Such information can then be used to determine useful doses
and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example HLYAP
or fragments thereof, antibodies of I-B.YAP, and agonists, antagonists or
inhibitors of HLYAP, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the subject
requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the active
moiety or to maintain the desired effect. Factors which may be taken into
account include the severity
of the disease state, the general health of the subject, the age, weight, and
gender of the subject, time
and frequency of administration, drug combination(s), reaction sensitivities,
and response to therapy.
Long-acting compositions may be administered every 3 to 4 days, every week, or
biweekly depending
on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~cg to 100,000 ~cg, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
46
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind HLYAP may be used
for the
diagnosis of disorders characterized by expression of HLYAP, or in assays to
monitor patients being
treated with HLYAP or agonists, antagonists, or inhibitors of HLYAP.
Antibodies useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for HLYAP include methods which utilize the antibody and a
label to detect HLYAP
in human body fluids or in extracts of cells or tissues. The antibodies may be
used with or without
modification, and may be labeled by covalent or non-covalent attachment of a
reporter molecule. A
wide variety of reporter molecules, several of which are described above, are
known in the art and may
be used.
A variety of protocols for measuring HLYAP, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
HLYAP expression. Normal
or standard values for HLYAP expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibody to HLYAP under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of HLYAP
expressed in
subject, control, and disease samples from biopsied tissues are compared with
the standard values.
Deviation between standard and subject values establishes the parameters for
diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding HLYAP may
be used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect and
quantify gene expression in biopsied tissues in which expression of HLYAP may
be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
HLYAP, and to monitor regulation of HLYAP levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding HLYAP or closely related
molecules may be used to
identify nucleic acid sequences which encode HLYAP. The specificity of the
probe, whether it is made
from a highly specific region, e.g., the 5'regulatory region, or from a less
specific region; e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding HLYAP, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the HLYAP encoding sequences. The hybridization
probes of the subject
47
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:11-20 or from
genomic sequences including promoters, enhancers, and introns of the HLYAP
gene.
Means for producing specific hybridization probes for DNAs encoding HLYAP
include the
cloning of polynucleotide sequences encoding HLYAP or HLYAP derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a variety
of reporter groups, for example, by radionuclides such as 32P or 35S, or by
enzymatic labels, such as
alkaline phosphatase coupled to the probe via avidin/biotin coupling systems,
and the like.
Polynucleotide sequences encoding HLYAP may be used for the diagnosis of
disorders
associated with expression of HLYAP. Examples of such disorders include, but
are not limited to, a
reproductive disorder, such as a disorder of prolactin production,
infertility, including tubal disease,
ovulatory defects, and endometriosis, a disruption of the estrous cycle, a
disruption of the menstrual
cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an
endometrial or ovarian
tumor, a uterine fibroid, autoimmune disorders, an ectopic pregnancy, and
teratogenesis, cancer of the
breast, fibrocystic breast disease, and galactorrhea, a disruption of
spermatogenesis, abnormal sperm
physiology, cancer of the testis, cancer of the prostate, benign prostatic
hyperplasia, prostatitis,
Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia;
a neurological
disorder, such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral
neoplasms, Alzheimer's
disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease
and other extrapyramidal
disorders, amyotrophic lateral sclerosis and other motor neuron disorders,
progressive neural
muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis
and other demyelinating
diseases, bacterial and viral meningitis, brain abscess, subdural empyema,
epidural abscess,
suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral
central nervous system
disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and polymyositis,
inherited, metabolic,
endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental
disorders including
mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD),
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial
48
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
frontotemporal dementia; an inflammatory disorder, such as acquired
immunodeticiency syndrome
(AIDS), Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
(APECED),
bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis,
diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,
multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma,
Sjogren's syndrome,
systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of cancer,
hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal,
and helminthic infections, and
trauma; and a cell proliferative disorder, such as actinic keratosis,
arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD),
myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and a cancer,
including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma,
and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow,
brain, breast, cervix,
gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,
muscle, ovary, pancreas,
parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus . The
polynucleotide sequences encoding HLYAP may be used in Southern or northern
analysis, dot blot, or
other membrane-based technologies; in PCR technologies; in dipstick, pin, and
multiformat ELISA-like
assays; and in microarrays utilizing fluids or tissues from patients to detect
altered HLYAP expression.
Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding HLYAP may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding HLYAP may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a suitable
incubation period, the sample is washed and the signal is quantified and
compared with a standard
value. If the amount of signal in the patient sample is significantly altered
in comparison to a control
sample then the presence of altered levels of nucleotide sequences encoding
HLYAP in the sample
indicates the presence of the associated disorder. Such assays may also be
used to evaluate the efficacy
of a particular therapeutic treatment regimen in animal studies, in clinical
trials, or to monitor the
treatment of an individual patient.
49
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
In order to provide a basis for the diagnosis of a disorder associated with
expression of
HLYAP, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding HLYAP, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from normal
subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values obtained
from samples from patients who are symptomatic for a disorder. Deviation from
standard values is
used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding HLYAP
may involve the use of PCR. These oligomers may be chemically synthesized,
generated enzymatically,
or produced in vitro. Oligomers will preferably contain a fragment of a
polynucleotide encoding
HLYAP, or a fragment of a polynucleotide complementary to the polynucleotide
encoding HLYAP, and
will be employed under optimized conditions for identification of a specific
gene or condition.
Oligomers may also be employed under less stringent conditions for detection
or quantification of
closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding HLYAP may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic disease
in humans. Methods of SNP detection include, but are not limited to, single-
stranded conformation
polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP,
oligonucleotide primers
derived from the polynucleotide sequences encoding HLYAP are used to amplify
DNA using the
polymerase chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the secondary
and tertiary structures of PCR products in single-stranded form, and these
differences are detectable
using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of
individual
overlapping DNA fragments which assemble into a common consensus sequence.
These computer-
based methods filter out sequence variations due to laboratory preparation of
DNA and sequencing
errors using statistical models and automated analyses of DNA sequence
chromatograms. In the
alternative, SNPs may be detected and characterized by mass spectrometry
using, for example, the high
throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
Methods which may also be used to quantify the expression of HLYAP include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C. et
al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple
samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described in Seilhamer, J.J. et al.,
"Comparative Gene Transcript
Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference. The
microarray may also be
used to identify genetic variants, mutations, and polymorphisms. This
information may be used to
determine gene function, to understand the genetic basis of a disorder, to
diagnose a disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic profile.
In another embodiment, antibodies specific for HLYAP, or HLYAP or fragments
thereof may
be used as elements on a microarray. The microarray may be used to monitor or
measure protein-
protein interactions, drug-target interactions, and gene expression profiles,
as described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
51
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at a
given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a
signature similar to that of a compound with known toxicity, it is likely to
share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain
expression information
from a large number of genes and gene families. Ideally, a genome-wide
measurement of expression
provides the highest quality signature. Even genes whose expression is not
altered by any tested
compounds are important as well, as the levels of expression of these genes
are used to normalize the
rest of the expression data. The normalization procedure is useful for
comparison of expression data
after treatment with different compounds. While the assignment of gene
function to elements of a
toxicant signature aids in interpretation of toxicity mechanisms, knowledge of
gene function is not
necessary for the statistical matching of signatures which leads to prediction
of toxicity. (See, for
example, Press Release 00-02 from the National Institute of Environmental
Health Sciences, released
February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.)
Therefore, it is
important and desirable in toxicological screening using toxicant signatures
to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological sample
52
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
containing nucleic acids with the test compound. Nucleic acids that are
expressed in the treated
biological sample are hybridized with one or more probes specific to the
polynucleotides of the
present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a proteome
can be subjected individually to further analysis. Proteome expression
patterns, or profiles, are
analyzed by quantifying the number of expressed proteins and their relative
abundance under given
conditions and at a given time. A profile of a cell's proteome may thus be
generated by separating and
analyzing the polypeptides of a particular tissue or cell type. In one
embodiment, the separation is
achieved using two-dimensional gel electrophoresis, in which proteins from a
sample are separated by
isoelectric focusing in the first dimension, and then according to molecular
weight by sodium dodecyl
sulfate slab gel electrophoresis in the second dimension (Steiner and
Anderson, supra). The proteins are
visualized in the gel as discrete and uniquely positioned spots, typically by
staining the gel with an agent
such as Coomassie Blue or silver or fluorescent stains. The optical density of
each protein spot is
generally proportional to the level of the protein in the sample. The optical
densities of equivalently
positioned protein spots from different samples, for example, from biological
samples either treated or
untreated with a test compound or therapeutic agent, are compared to identify
any changes in protein
spot density related to the treatment. The proteins in the spots are partially
sequenced using, for
example, standard methods employing chemical or enzymatic cleavage followed by
mass spectrometry.
The identity of the protein in a spot may be determined by comparing its
partial sequence, preferably of
at least 5 contiguous amino acid residues, to the polypeptide sequences of the
present invention. In
some cases, further sequence data may be obtained for definitive protein
identification.
A proteomic profile may also be generated using antibodies specific for HLYAP
to quantify the
levels of HLYAP expression. In one embodiment, the antibodies are used as
elements on a microarray,
and protein expression levels are quantified by exposing the microarray to the
sample and detecting the
levels of protein bound to each array element (Lueking, A. et al. (1999) Anal.
Biochem. 270:103-111;
Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a variety of
methods known in the art, for example, by reacting the proteins in the sample
with a thiol- or amino-
reactive fluorescent compound and detecting the amount of fluorescence bound
at each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and should
53
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
be analyzed in parallel with toxicant signatures at the transcript level.
There is a poor correlation
between transcript and protein abundances for some proteins in some tissues
(Anderson, N.L. and J.
Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures
may be useful in the
analysis of compounds which do not significantly affect the transcript image,
but which alter the
proteomic profile. In addition, the analysis of transcripts in body fluids is
difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated biological
sample are separated so that the amount of each protein can be quantified. The
amount of each protein
is compared to the amount of the corresponding protein in an untreated
biological sample. A difference
in the amount of protein between the two samples is indicative of a toxic
response to the test compound
in the treated sample. Individual proteins are identified by sequencing the
amino acid residues of the
individual proteins and comparing these partial sequences to the polypeptides
of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared with
the amount in an untreated biological sample. A difference in the amount of
protein between the two
samples is indicative of a toxic response to the test compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are well
known and thoroughly described in DNA Microarravs: A Practical Approach, M.
Schena, ed. (1999)
Oxford University Press, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding HLYAP
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a multi-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial P1
54
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop genetic
linkage maps, for example, which correlate the inheritance of a disease state
with the inheritance of a
particular chromosome region or restriction fragment length polymorphism
(RFLP). (See, e.g.,
Larder, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic map
data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, su ra, pp. 965-968.)
Examples of genetic map
data can be found in various scientific journals or at the Online Mendelian
Inheritance in Man (OMIM)
World Wide Web site. Correlation between the location of the gene encoding
HLYAP on a physical
map and a specific disorder, or a predisposition to a specific disorder, may
help define the region of
DNA associated with that disorder and thus may further positional cloning
efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse, may
reveal associated markers even if the exact chromosomal locus is not known.
This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely localized
by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia
to 11q22-23, any sequences
mapping to that area may represent associated or regulatory genes for further
investigation. (See, e.g.,
Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the
instant invention may
also be used to detect differences in the chromosomal location due to
translocation, inversion, etc.,
among normal, carrier, or affected individuals.
In another embodiment of the invention, HLYAP, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between HLYAP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geyser, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with I-ILYAP,
or fragments thereof,
and washed. Bound HLYAP is then detected by methods well known in the art.
Purified HLYAP can
also be coated directly onto plates for use in the aforementioned drug
screening techniques.
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
Alternatively, non-neutralizing antibodies can be used to capture the peptide
and immobilize it on a
solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding HLYAP specifically compete with a test compound
for binding HLYAP.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with HLYAP.
In additional embodiments, the nucleotide sequences which encode HLYAP may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. No. 60/172,307, are hereby expressly incorporated by
reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some tissues
were homogenized and lysed in guanidinium isothiocyanate, while others were
homogenized and lysed
in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life
Technologies), a monophasic
solution of phenol and guanidine isothiocyanate. The resulting lysates were
centrifuged over CsCI
cushions or extracted with chloroform. RNA was precipitated from the lysates
with either isopropanol
or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
56
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-1000
bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
pcDNA2.1 plasmid
(Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
Recombinant
plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-
BlueMRF, or SOLR
from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo excision
using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were
purified using at least
one of the following: a Magic or WIZARD Minipreps DNA purification system
(Promega); an AGTC
Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8
Plasmid, QIAWELL
8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L.
PREP 96 plasmid
purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1 ml of
distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in 384-
well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-
200 thermal cycler
(MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides were
57
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the ABI
PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with
standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel, 1997,
supra, unit 7.7). Some of the cDNA sequences were selected for extension using
the techniques
disclosed in Example VI.
The polynucleotide sequences derived from eDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 5 summarizes the tools, programs, and algorithms used and provides
applicable descriptions,
references, and threshold parameters. The first column of Table 5 shows the
tools, programs, and
algorithms used, the second column provides brief descriptions thereof, the
third column presents
appropriate references, all of which are incorporated by reference herein in
their entirety, and the fourth
column presents, where applicable, the scores, probability values, and other
parameters used to evaluate
the strength of a match between two sequences (the higher the score, the
greater the homology between
two sequences). Sequences were analyzed using MACDNASIS PRO software (Hitachi
Software
Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and
polypeptide sequence alignments were generated using the default parameters
specified by the clustal
algorithm as incorporated into the MEGALIGN multisequence alignment program
(DNASTAR), which
also calculates the percent identity between aligned sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA sequences
and by masking ambiguous bases, using algorithms and programs based on BLAST,
dynamic
programing, and dinucleotide nearest neighbor analysis. The sequences were
then queried against a
selection of public databases such as the GenBank primate, rodent, mammalian,
vertebrate, and
eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation
using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled
into full
length polynucleotide sequences using programs based on Phred, Phrap, and
Conned, and were screened
for open reading frames using programs based on GeneMark, BLAST, and FASTA.
The full length
polynucleotide sequences were translated to derive the corresponding full
length amino acid sequences,
and these full length sequences were subsequently analyzed by querying against
databases such as the
GenBank databases (described above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM,
Prosite,
and Hidden Markov Model (HMM)-based protein family databases such as PFAM. HMM
is a
probabilistic approach which analyzes consensus primary structures of gene
families. (See, e.g.,
Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide and
58
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
NO:11-20. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies were described in The Invention section above.
IV. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a gene
and involves the hybridization of a labeled nucleotide sequence to a membrane
on which RNAs from a
particular cell type or tissue have been bound. (See, e.g., Sambrook, supra,
ch. 7; Ausubel, 1995,
su ra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
search can be modified to determine whether any particular match is
categorized as exact or similar.
The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
, 5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the length
of the sequence match. The product score is a normalized value between 0 and
100, and is calculated
as follows: the BLAST score is multiplied by the percent nucleotide identity
and the product is divided
by (5 times the length of the shorter of the two sequences). The BLAST score
is calculated by
assigning a score of +5 for every base that matches in a high-scoring segment
pair (HSP), and -4 for
every mismatch. Two sequences may share more than one HSP (separated by gaps).
If there is more
than one HSP, then the pair with the highest BLAST score is used to calculate
the product score. The
product score represents a balance between fractional overlap and quality in a
BLAST alignment. For
example, a product score of 100 is produced only for 100% identity over the
entire length of the shorter
of the two sequences being compared. A product score of 70 is produced either
by 100% identity and
70% overlap at one end, or by 88% identity and 100% overlap at the other. A
product score of 50 is
produced either by 100% identity and 50% overlap at one end, or 79% identity
and 100% overlap.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding HLYAP occurred. Analysis involved the categorization
of cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease~condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
59
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
sequence of interest was counted and divided by the total number of libraries
across all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in Table
3.
V. Chromosomal Mapping of HLYAP Encoding Polynucleotides
The cDNA sequences which were used to assemble SEQ ID N0:11-20 were compared
with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID NO:11-20 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 5). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Gen~thon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
The genetic map locations of SEQ ID N0:13 and SEQ ID N0:19 are described in
The
Invention as ranges, or intervals, of human chromosomes. The map position of
an interval, in
centiMorgans, is measured relative to the terminus of the chromosome's p-arm.
(The centiMorgan
(cM) is a unit of measurement based on recombination frequencies between
chromosomal markers.
On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans,
although this can
vary widely due to hot and cold spots of recombination.) The cM distances are
based on genetic
markers mapped by G~n~thon which provide boundaries for radiation hybrid
markers whose
sequences were included in each of the clusters. Human genome maps and other
resources available
to the public, such as the NCBI "GeneMap'99" World Wide Web site
(httt3:;; wvvw.nchi.nlni.nih. ~ovf~enemat~; ), can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
VI. Extension of HLYAP Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:11-20 were produced by
extension of an
appropriate fragment of the full length molecule using oligonucleotide primers
designed from this
fragment. One primer was synthesized to initiate 5' extension of the known
fragment, and the other
primer, to initiate 3' extension of the known fragment. The initial primers
were designed using OLIGO
4.06 software (National Biosciences), or another appropriate program, to be
about 22 to 30 nucleotides
in length, to have a GC content of about 50% or more, and to anneal to the
target sequence at
temperatures of about 68°C to about 72°C. Any stretch of
nucleotides which would result in hairpin
structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one extension
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)ZS04,
S and (3-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerise (Stratagene), with the following
parameters for primer
pair PCI A and PCLB: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68 °C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~cl to 10 ~cl aliquot of the reaction mixture was
analyzed by electrophoresis
on a 1 % agarose mini-gel to determine which reactions were successful in
extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%) agarose
gels, fragments were excised, and agar digested with Agar ACE (Promega).
Extended clones were
religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector
(Amersham
Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in
restriction site overhangs,
and transfected into competent E. coli cells. Transformed cells were selected
on antibiotic-containing
media, and individual colonies were picked and cultured overnight at
37°C in 384-well plates in LB/2x
carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham
Pharmacia Biotech) and Pfu DNA polymerise (Stratagene) with the following
parameters: Step 1:
94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min;
Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4
repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C.
DNA was quantified by PICOGREEN
reagent (Molecular Probes) as described above. Samples with low DNA recoveries
were reamplified
using the same conditions as described above. Samples were diluted with 20%
dimethysulfoxide (1:2,
61
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the
DYENAMIC
DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator
cycle
sequencing ready reaction kit (Applied Biosystems).
In like manner, the polynucleotide sequences of SEQ ID NO:11-20 are used to
obtain 5'
regulatory sequences using the procedure above, along with oligonucleotides
designed for such
extension, and an appropriate genomic library.
VII. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID NO:l 1-20 are employed to screen
cDNAs, genomic
DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about
20 base pairs, is
specifically described, essentially the same procedure is used with larger
nucleotide fragments.
Oligonucleotides are designed using state-of the-art software such as OLIGO
4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~Ci of [y-
32P] adenosine
triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase
(DuPont NEN, Boston
MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superfine size
exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot
containing 10' counts per
minute of the labeled probe is used in a typical membrane-based hybridization
analysis of human
genomic DNA digested with one of the following endonucleases: Ase I, Bgl II,
Eco RI, Pst 1, Xba I, or
Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5 %
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VIII. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), su ra). Suggested
substrates include silicon, silica, glass slides, glass chips, and silicon
wafers. Alternatively, a procedure
analogous to a dot or slot blot may also be used to arrange and link elements
to the surface of a
substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be
produced using available methods and machines well known to those of ordinary
skill in the art and may
contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470;
62
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson
(1998) Nat. Biotechnol.
16:27-31.)
Full length cDNAs, Expressed Sequence Tags (SSTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The array
elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described in
detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA. sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~.xl oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/~1 RNase inhibitor, 500 i.iM dATP, 500 ~M dGTP, 500
NM dTTP, 40 ~M
dCTP, 40 NM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37 °C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85 °C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 ~.il SX SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element is
amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
63
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
fig. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 ~.xl of the array
element DNA, at an average
concentration of 100 ng/~.~1, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60
°C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 i.xl of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65 °C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of
140 Erl of SX SSC in a corner of the chamber. The chamber containing the
arrays is incubated for
about 6.5 hours at 60 °C. The arrays are washed for 10 min at 45
°C in a first wash buffer (1X SSC,
0.1% SDS), three times for 10 minutes each at 45 °C in a second wash
buffer (0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20~micrometers.
64
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater N.>) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra fiom both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are differentially
expressed, the calibration is done by labeling samples of the calibrating cDNA
with the two
fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
IX. Complementary Polynucleotides
Sequences complementary to the HLYAP-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring HLYAP. Although
use of oligonucleotides
comprising from about 15 to 30 base pairs is described, essentially the same
procedure is used with
smaller or with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of HLYAP. To
inhibit transcription, a
complementary oligonucleotide is designed from the most unique 5' sequence and
used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is
designed to prevent ribosomal binding to the HLYAP-encoding transcript.
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
X. Expression of HLYAP
Expression and purification of HLYAP is achieved using bacterial or virus-
based expression
systems. For expression of HLYAP in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express HLYAP upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of HLYAP in eukaryotic cells is
achieved by infecting insect
or mammalian cell lines with recombinant Autographica californica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding HLYAP by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera fru~iperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K. et
al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther.
7:1937-1945.)
In most expression systems, HLYAP is synthesized as a fusion protein with,
e.g., glutathione S-
translerase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosoma iaponicum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Pharmacia
Biotech). Following purification, the GST moiety can be proteolytically
cleaved from HLYAP at
specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification
using commercially available monoclonal and polyclonal anti-FLAG antibodies
(Eastman Kodak). 6-
His, a stretch of six consecutive histidine residues, enables purification on
metal-chelate resins
(QIAGEN). Methods for protein expression and purification are discussed in
Ausubel (1995, supra,
ch. 10 and 16). Purified HLYAP obtained by these methods can be used directly
in the assays shown in
Examples XI and XV.
XI. Demonstration of HLYAP Activity
Lyase activity of HLYAP is demonstrated through a variety of specific enzyme
assays. In
general, HLYAP is incubated with its substrates) under conditions suitable for
the enzymatic reaction
being assayed. After a suitable period of time, the reaction is terminated,
and the formation of the
66
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
products) are monitored spectrophotometrically, chromatographically,
fluorometrically, or by some
other appropriate method. Lyase activity is proportional to the amount of
products) formed, or the rate
of product formation. Some examples of specific lyase activity assays are
described below.
Glyoxalase I activity of HLYAP is measured by monitoring the formation of
glutathione
thioester from methylglyoxal and glutathione. HLYAP is incubated with 2mM
methylglyoxal and 2
mM reduced glutathione in 0.1 M sodium phosphate, pH 7.0, at 30°C.
Formation of the glutathione
thioester is monitored spectrophotometrically at a wavelength of 240 nm.
Glyoxalase I activity of
HLYAP is proportional to the rate of formation of the glutathione thioester.
(See, e.g., Ridderstrom, M.
et al. (1998) J. Biol. Chem. 273:21623-21628.)
dTDP-D-glucose 4,6-dehydratase activity of HLYAP is measured by monitoring the
formation
of dTDP-4-keto-6-deoxy-D-glucose from dTDP-D-glucose. HLYAP is incubated with
50 mM Tris-
HCl, pH 7.6, 12 mM MgCl2, 4 mM dTDP-D-glucose, 0.9 unit of inorganic
pyrophosphatase, and 8
mM NADPH for 3 hours at 37°C. The sugar components in the mixture are
coupled with 2-
aminopyridine and then analyzed chromatographically using an anion-exchange
column. Dehydratase
activity is proportional to the amount of dTDP-4-keto-6-deoxy-D-glucose
formed. (See, e.g., Yoshida,
1999, supra.)
Aconitase activity of HLYAP is measured in an assay coupled to isocitric
dehydrogenase.
HLYAP is incubated with isocitric dehydrogenase, NADP, and citrate, and the
reduction of NADP is
monitored fluorometrically. Aconitase activity is proportional to the rate of
NADP reduction. (See,
e.g., Costello, L.C. et al. (1997) J. Biol. Chem. 272:28875-28881; Costello,
L.C. et al. (1996) Urology
48:654-659.)
XII. Functional Assays
HLYAP function is assessed by expressing the sequences encoding HLYAP at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include pCMV SPORT plasmid (Life Technologies) and pCR3. l plasmid
(Invitrogen), both of which
contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are
transiently transfected into a
human cell line, for example, an endothelial or hematopoietic cell line, using
either liposome
formulations or electroporation. 1-2 ~cg of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate the
67
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
apoptotic state of the cells and other cellular properties. FCM detects and
quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events include
changes in nuclear DNA content as measured by staining of DNA with propidium
iodide; changes in
cell size and granularity as measured by forward light scatter and 90 degree
side light scatter; down-
s regulation of DNA synthesis as measured by decrease in bromodeoxyuridine
uptake; alterations in
expression of cell surface and intracellular proteins as measured by
reactivity with specific antibodies;
and alterations in plasma membrane composition as measured by the binding of
fluorescein-conjugated
Annexin V protein to the cell surface. Methods in flow cytometry are discussed
in Ormerod, M.G.
(1994) Flow Cytometry, Oxford, New York NY.
The influence of HLYAP on gene expression can be assessed using highly
purified populations
of cells transfected with sequences encoding HLYAP and either CD64 or CD64-
GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind to
conserved regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success NY).
mRNA can be purified from the cells using methods well known by those of skill
in the art. Expression
of mRNA encoding HLYAP and other genes of interest can be analyzed by northern
analysis or
microarray techniques.
XIII. Production of HLYAP Specific Antibodies
HLYAP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the HLYAP amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, su ra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-Aldrich,
St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS) to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with
the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-HLYAP
activity by, for example, binding the peptide or HLYAP to a substrate,
blocking with 1 % BSA, reacting
with rabbit antisera, washing, and reacting with radio-iodinated goat ants-
rabbit IgG.
68
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
XIV. Purification of Naturally Occurring HLYAP Using Specific Antibodies
Naturally occurring or recombinant HLYAP is substantially purified by
immunoaffinity
chromatography using antibodies specific for HLYAP. An immunoaffinity column
is constructed by
covalently coupling anti-HLYAP antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing HLYAP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of HLYAP (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/HLYAP binding (e.g., a buffer of pH 2 to pH 3, or a high
concentration of a chaotrope, such
as urea or thiocyanate ion), and HLYAP is collected.
XV. Identification of Molecules Which Interact with HLYAP
HLYAP, or biologically active fragments thereof, are labeled with '251 Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated with the
labeled HLYAP, washed,
and any wells with labeled HLYAP complex are assayed. Data obtained using
different concentrations
of HLYAP are used to calculate values for the number, affinity, and
association of HLYAP with the
candidate molecules.
Alternatively, molecules interacting with HLYAP are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song (1989, Nature 340:245-246), or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
HLYAP may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S. Patent
No. 6,057,101 ).
Various modifications and variations of the described methods and systems of
the invention will
be apparent to those skilled in the art without departing from the scope and
spirit of the invention.
Although the invention has been described in connection with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are obvious
to those skilled in molecular biology or related fields are intended to be
within the scope of the following
claims.
69
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
_ _ r, _ _
-' -. x --
N M r1 c-I r1
O O N O .-I r1
O
~O~o W~ ~ O~ w M~ x
OD (Y., N ~ O LC7 d~ N
L~ U' U
~I1
CO ~ LC7 U l0 L~ L(1
a N O (~
.-i
O~ ~ O ~', l0 M M
O ~-1 01 H
~
l~ .-I l~ N O1 O1 O
U a ' Ul N ,.'~.,
M
O1 01 -~- t0 W M N I~
'-' '-' "
b1 d~
N ~-1 00 v-1 N M
r1
c-i O~ i-i r1 c-I _
c-i O c-I .-I
' .
x x H x x -' _ -. _
-~ x
'M 'I~ ~to M N
N Ul H l0
-~-NO~N~ ~QONO N 00 r1
r1 H I~ N r1 O H O
.~ U O H M
H
0 o ~ o m M H O H
0~ O m
~
H H u1 H ov ~ Z U
~ ~ oo z M
H
o~c~ ~~oa xo~a H h~ x
N r1 '-' ~ N N ~ H Z
~ ~ to
'J_a (~'l0 'W w~ U7_ H
H H ~ .-. E-1 ~. U1
~' E-I l0
-.-
~M '~N V
~
O~ O ~ .00
o~o
Hx H~ HHf1-~ ~o f=-.H
goo .~w~ ~xwa. xw ~x x
a~ xw~o xHw.~ ~, off
M '
~
L!1 t0 [~ M H C7 Lll O\ M
w d 01 O ~,u~Q, ~,
,~a~,~~,~ ~~~~ ~a
~
L~ a-I O V N 00 M
H ~o a ~ r~ ~r W ~o
l~ - a ao ' ~o
~o x
x
~
l0 _ O~ '-' M N l0
'-' d~ 'r '-' rl '-'
M N
c-I ri M ~O N N
,'
r1 l0 O e-I l0 01 '
_ _ c-I
_x~.._wH 'xrx-- _~ ...x~_
.-. -- L~ ~. t!1 .-. 0~
l0 W d~ I~ 10 O
N N
r1 .-I r1 CO r-1 O r1
O N CL DO O d~ o0
O O
o O [~ O ri O H O
[~ [' r1 H O ~1
E-I H
H a1 .~ H m N H O P0
~ ~ ~ ~
O SC
o~z o~ o~.~oH o~ z~H
a
z~a zoM~ z x~.a
N H H N N N N ~
U V7
x '~' H
a H ~. .-. ~ ~ .~ v,
., p~ r. v
~ v v
~' d, '~ r1 ~ N
di N r1
,1 r1
O O .-I O O lO O r-i O
r1 N O O
r1
~ ~ H ~ H H ~ x ~
H >C fs.' H H H
x H p4 x
xo~ xo~x rxHxa,H wN min wo
o N ~ a1 M ~ ~
l H H m ~C .~ z
~ M ~ ~1 ~ O~ M
l0 I~ 01 z c1 ~ U
H H 00 O
al
r d~ U7 I~ H O , r1
L~ r1 ~ M Pa , z
U1 ~ ~ u,x M ow
01 orx~ a.x o ,'fin
~oxM Lfl
.~x~o
~o M PO ~o W ~ ~ o~
P4 ao PO ~o H H W
~ W W a,
r1 O '~ N " '-' V~ N .-I
'~ r1 N ~ v 'w' ~--
M -- 111
M N N 01 r1
5y O O O O O O
H H H H H H
rd O W D ~ O U
z C~.~ H H z
H cn ~ z
a H w x H
a a w H H
~r
N ~ 111 L~ M
H
O r1 LC1 N M M
H ~
r1 LI1 ov V~ H 00
U ~ ao o M ~o ~o
d N N N N
''d
O
-~
z
O r1 N M d~ Lf1 ~O
Gl
N r1 w-1 c1 u-I v-1 c-I
H
r~
U
O~
~
W
z~
z
r1 N M d~ Lf1 l0
O~
~
L~
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
v-1 c-I ' M r1
~O
O 10 O 01 r1 O
O r1
z~~
o
~
r1 r1 N ~ ~O M
O W U7 PG O
o U ~r Wo 0.1 ~
ao H m
x
~--N-- oo~- M aN--
N M O '-' H
.--I u-I Ov - i-1
l0 t0 .--I
. r-I
w--x w ,~-.x-- .x--
~MO~ ~N -~I~O100.-iN0
~ o~ ,--i vo N ~
H d~ o H ~ M
H cx
O u-i O O 00 H
O M l0 O ~ d~
~ ~ ~ H
o ~
H ~1
O
H ~ H ~ z t
~t t o zoaa
oM om~~U
H a N z M a H
M r1 N ~ M
U1 U1 Cu U1 H FC
W H
p .H .o, O fx
. C7 ~ ~
D4 P4
O _ _
.-. _ P4
l0 fx ~ ~.
~
~ ~ ~ ~
O 10 O ~-i l0 O
O r1 O r-I w-i
H CT-~ H o fl.~ ~
H o H x H
x
~ O ~ ~ H co H
d~ ~ N ~ O
O FC M
x pz wHw r~o~~~ ~r~N
o~Mz ~H~ ~~~z
W ~ '
a1 (Y.,M ~ PG O
l~ r1 H O ,~
W PO M 00
~O W ~ d~ W ov
M 0.~ ~ W O
L N
rl~Nv N'-'r-INUN'-'u1Nvd~
~ 00 --
p4
r-I l0 - .--I 10
. r-I 10
.H--x .wN .,~~x_. ,~
~ M ~ ~ H L~ ~
M M dl c-1 N
d~ .-I
r1 l0 r1 r1 l~ .-~
O N O O c-i M
.-i O O
o,~Ht~ oino oo,H~ ooH
H o ~1 H ao H
D .~ t~ ~ ~ o
FC ~ M
O .~ z O N H fa
H o ~ o H ~
W C~-~
NHOO HMd, ,"Z,MHt~fl.'NOH
1 l ~
U7
~- W O a
~ N r ~ N ~ N
Ov
x -PO G7 H ,(p ~
. , .O ~N
.
H ., r~ x -.
~ .. ,.
v ,~ v
v
N 0~ N N
fx L~
O r1 O O r1 O
O l0 O r1 r1
~ H ~ ~ H C~.~.~
x H H H x H
t~ x
xo~no wx~ xoNxm xo~
~z pH~ ~ ~.H~ Mzp
.-.-i oo ~ m ~ v~
~ U H C9 U
o ~ ~o
v t o N ~
N W r.~ M ~;
M z o,
d~ a
~r o
.o
y o p; ~ o~ O OD
r1 .~ o U1 U
W W u7 l
M ~ ~O M U ~1 L~
rl f~ w N 'J
C4 F~, N ~ rl Lfl
d~ ~ .-1 v M d~
N ~ v ~
U7 M
r1 N r1 r1
H H H
H ~
z
z
a w
w a o r~
M d~ 00 O
N N M r1 111
~ L N r1 N
O r1 O 10 0~
r-I O M Lf1 Lf1
H
U 00 r1 N L
N M M d~
..
'O
O
.'"
1~
O l~ CO 01 O
Ca
N ri r1 r1 N
H
r~
U
OI
~
W
z~
z
a
W
o
'~
w
71
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
U ~ U U
~
-r-I U1 U7
~
'd
Ll W v
j ' '
, cn ~ cn m ~ u~
, ~ H ~wxrxw ~ ,
~ -a
,
w-
w~ W ~
~
Ca
C H rn H
-~ o ~ ~ H O o
~
a
~P ~~ ~~ ~
W
a P
vC
7
U
W
tn u1 a~ tn o,
N
N
O U7 r-1 Lh
b7 ~ .U -rl O
~
0
r O b7 S-i ,S~
-I b1
~
'
-I
~-1 r
,,f.,
d~ ~-1 U1
~-~ o~
~ ~
,~
~l0 N N N
~ ~
.
-~ U ~
U1 1J ~ d~ ~
~ N
U
~ u7 W io o ..
O1 M
W
u~ rd E-i '-~
~ q ~
~ _~
~
~
b1 S~ ~ L(1 N .. ~ U7
f~ 1.1
q
' '~
~ -~
~ I
~ '~ ~a ~1
~ r-
N ~ ' .~~~ ~~~ ~ ~ a~
., O ~
-~a, o ~ L~ -1
l0
O
N ~ ~ v N M ~
_~ ~ ~C9 W ~~'~M ~yo
'1
~ ~
NaCov N m~ ~'~EI
E-t~E~ ~d'~N ~ ~ ~~ria~,~[9
~~
CO
'
'
t71 CJ ~ ~ S~ ~ N .r-I
~ O <i ~ ~ '~ ~ N M ~
~ U7
'
W -ri U7 .~. r~ c~ F-~ U
uW .~ ~ or,x
w~~ o ~,~~'~ ~ar~~C ~,~
~~
~-~
E-~ o ~ , ~n
rd r-C f~ m o -~ .
~
FC ~ w U z a~ ~ Wn H H
~r
o
N
(~ M
~
-.-i
(~
fly
1~
r1
~' Lf1
.~'I
~
N U1 di
-r-I
~ o z
~n
O U o~ O
Pa N o~ a0
'r
N M c-1
z z z
o a, ao
d' ~ N
d~ e-I M O N c-I
Id
td
.-I N U1 r1 ~ E-~
-r-I n7 r1 w-I
r-I
U~
~r C1l E-I Lfl E-I O 01
~
.S', M O0 U1
~-I
l~
J M 00 M 01 r1
L
' U1
.- ~ H ~ H
,
,
l0 OD N
O O1 M L~ O O~ N ~
CO
~ ~ H H H
N
M L!1 lD
-r-I d~ N r1
U
_~
N d~ N
W q c-i N M
z
72
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
ro
U ~ '
U " U
U
'
x
) U7 U7 ~.
7 U1 r 7 C/~
-1 7 U7 ~
, I ~,
I
.,
wxww w-~I H ~aHOwxww ~
~ ~
H O H H H O r~ O H O H H U~
~
aaao o ~I vaxaaaoa~
mwoo~ ~a~ ooocnwoowr~~~n
U
-r-I I r'] 00
r-1 N OI
(O N O O~ ~ M
~
.1.1 U LC7
N
N
i W
~
'L ?~ U b~
.~
-r-I .. U +~ '-'
O ~ ~ U1 L~ ~-I S-I ~i U7
d~
~a
~
~
'
~
~'
~n
w
~,
w cn ~r ~ m ~, v
is
v
u~ U cn ' U
..
O
o
N ~ ~ N ~ S-I
U
'
U1 1~ ~ U1 1.1 U ~ q h
CS
i
~ ~ W ~' a
N v v a
' '
o ~ ~I v -- ~n m
~ (~ o ~, >~
''l~ - '~ -r1 H b1 -~-'
j' r-I (~ -r1
O ~
i O
r 'r r-I U7 -r-I M
. (!j ~r
.,
r-I N ~ -r-I
L~
r
S-y ri N U7 U r1 ~
0 N ~'d - f~
~ N
\ ~ ~ H ~ ~
/ l S N ~ H
~ -- ~ ~ Ilf ~ ~ 11
S H
~ ~
. r -I M
M J.J H .-I ~ ri Lf1
-I ~ ~ I v ~.,' a
-1 H N
H c-i I ~
N ~ ~ I
'~
-
M
N ~ -r-I. 111 ~ ~ . I ~ . . o~ ~.
~ I -,1 I ,~ FC
O . y~ I l
~ Ll1
~ I
O
'
U] N ' S-1 ~ O
4-1 ''~ d~ f~ ~
(~ M r
M M , r1
N U7 N ~ ,~
~ N lf'W -I
, r-I
d~
U7 (~ O ~
~ 5~r Gu' ~
w W U7 O .
~H xo
a
~
~
,~ ~ ~~
~ Nw ~ -b,
~' ~~
' ~r!
- - U ~r C7 d~ x s~
~ w m cn
U d~ cx t~
-- N
H
a
0
-r-I
(~
U7
+~
~I
v
s~
~
+~
v ~n r1
-r-I
~ o Z
cn
o U
w ~.,
z
a
0
N N N
-r-I U7 U7 d~ H
r-1
U7
1~
~, ~
N
.>~. vO d~ d
~i
1J
v O
-r-I
H H
O f~ v1 H v1
~L
u1
O M r-1 N d~
l
0 M v-i 01
~H N H
N
v
~ ~ ~
-r-1 d~ I~
U ..-1 l -I
~ M .
4)
W
~ Z ~ In io
73
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
s~
O u7
' '
' ~
~
'y , I ~; U7 rn I I ~,
V7 ~ tn U7 U7 U7
I ~ I f~,,
, I V7 r~
H ~HOwxocw ra H ~aHHOwxwHxw.~l
H cncnu~u~a~U~z Hw
~
~n
nm
U
a~Uw
a~ow a v~axaaa~o ~,
a ~axa
, wa~ww
oo~cnr~a
w
ovc~mwww ~~n ~
,
a~
to m o
fa ~ r0 ~ o
I oo ~ o N
w U .o ~a x ~
H W o ~ o
U
_
'
l0 N r-I ~7 M M
~ .~a
r-I O ~ (l1 U7 ' ~1
~ ~-1
J.J ~
l J J
J l0 O (!j
~ ~: -~
x ~ ~n s~
~a
ri O ''t'~ c-I ..-1 C~ N
.~ .~
~ O
~
~ , U ,
N
td
~ ~ W
U1 b~'C ~ ~C
r1 00 10 . O L~ N l0 00 N M
O
Ul L~ lf1 CO ~ M 00 d~ L~ OD O O1
lf1 00 M
r1 M N M LIl r1 N N N V~ M
V~ I~
~
~~7zw ..o~z~aa~7o1,1
- O I I I tf1 C
W
I I I I I I I I
y
~
. ..
~ r1 O M L~ y,~ U 1 L~ M O~ M L~
~ r1 O CO N
x ~ I O v0 O1 Lll
Lfl CO d~ c0
O N L ~ ~
.,~ L
I ri N N . .~
~ ~ ~ rl r1 N N M M
d~ L!1
,
" d
,~xww ~ ~ y
.,~ ~o~xcn~aaac~~
" ~..~ ~ , .~ ~ s~ H a U o
(($ d~ L(1 r1 00 ~ ~ w-i l0 N ~ r1 01
~ M N W ~ N
~
~ ~ w .
- Ll1 M 10 Ld L(1
O 01 L~ O~ r1 c-I l0
OD Lf1 00
Q ~
'?;j \ U7 N
~
~
U ~a ~ (a Lll r1 ~ 01 10 r1 r1 N Lll
N N M ~ M M d
~xF"ILIZ ~ ~ ~ L
ao d~NC7a~G1a~U7U' ~C
f"i
1W S N~
W N NJJ ~ ~
U~ I I I I N ~C7 I I I I I I I I
I
~ ~~ ~ ~ ~.~
l I L~ 00 O~ CO ~M
(~ I I M M C~ O I~ O O
Op
N
N ~, ~ ~ ~ ~ ~ ~
r ~ ~ .
I M N l0 00 01 l~ d~
l 01 d~ Op M l0
l
~
'
'
~ _,1 ~
~ O r .r1 ~-I
r Lfl
' ,
f~ ~ ' l0 L~ r1 r1 ri N M
(~ -r1 M d~ d~
d~ r1 c-I N M
r1
U - -~. ~ ~ ~
V ~ ~ ,
/ ~ ~aaw~na ~ ~ ~
~ a~zwwt~r~H ova
a. o
, ~n , N
~ .
,
z ~~~
N
0
r-1
.r-1
-r-I L~ N Ll1
(a
U7
1~ pp ,-1 1~1
r-i
~
.f"-~ M l0 I~
'y
1-1
zzz
mom
O U r1 umo o~
L~ ~ r d~ I_n
?~
M d~ L~ L~
zzzz
a
O M N l0 U1 c-I d~ l0 M
Lh O l0 01 N w-I l0 l0 O ~ r1
r-I l0 M M r-i M OO If1 L~ LC7 L(1 l0
J~ Ll1
-~ '~ N U1 O~ U7 00 H E~ N E-i E-i
r-I ~ M E-i
~ U7 00
M L~ CJ) N O U7 r1 L~ L~ r1 N
~ OO O 00 d~ O~ t!1 M L~ M M
N O N N M r1 r1 r1 c<' d~ L~ ~ d~ l0 d~
.r1
N ~ H v~ ao cn ~ N E-~ N H ~n ~
5, E~ ~n
H ~n
P, M Lll M O O1 00 O N O1
O O O O L~ c-I LCl CO O l0 r1 1D l0
M ~
10 N M M r1 M ~D M Lf1 l0
M M (
~ ~ H
(n
N
.
'., di O
~
,~
.f
r-1 I~ 00
U
_~
~ M L~
Pr'
w q c~ 00
Z
74
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
~ O
U
_ N
W
" ~
~ O
(a I ~ I .~., U1
, U7 C/~
I U7
' ' ~
~ W
F( ~t,
, H O
E-I E'~
~
'
P4 C7 ~ PI1 C7 Pa
L~
~,
v H
td N v
.j"., I U1 a0
U
U7 U v z (~ Id U1 M
W bWl S-a U -rl dl
p U I 1~ v .-. W
v
Lf1 v w~ W -r-I 00
M r-I
~ "
~ p
r-I c1 (~ U1 \ O v .~ tJl
~ )-
.,,
"
Q I ,L JJ U7 (a
, f~ ~ ,~
f~ c-I fa
,~" ~ Q~ r-I 01 (~ ~ ,L;
~ ~-I -r-I
l0
U7 U7 M UI ~-I r1 L(1
l~ c-I U ~.1 U7
M
~C ~ ~
~
~ X ~ !~ (~
~ ,-a y, N
c", M
Qa ,~ ' r-I -ri O N
,L", -r1 rJl
O
,~ U7 ~-I ~ ~ V~ Id
.-I c0 t~ v
U f.2, dl
-~I -~I r~ -.-i ~--I
~ ~ W ~n
M
P: ~2 U d~ U7 b1 dl
U7 ~ v '-' ~ ~
b1
u1
~
-r-I
..
O U ~ I~ di
q ~
/~ ~ W ~
~
v
U1 W
>=i
H
(~ -~ FC V~
..l N U7 N v
O
~
~w ~"d~
l ~ ..A4
N ~ -ri ~ '~ ~, N
~ ~ I
~ l
~ '~ CO N
~ ~
- r
O
--I~n~ ~~x~ ~,~x
s~
o M
r-I r.,
-ri
~ r1 oo ~o
v
v ~n N z
-~I
~ o
~n
O U r1 o~
C~ l~ l0 N
't
H N Lf1 r1
U zz z
>~
O r1 M M GO
O 01 ~ l0
1 M L(1
l J l
l
r ~ M d
- Ill 01 r1
d
U
1
N En E-I E-I
H N
E-I H
'~ d~ N c-I 01
a0 M d~ ~
N O d0 M V~ Lf1 r1
--rl Ll1 M dl
M
u1 H u1 u~ E-I
~ H ~
O In ~ o ~ ~
r1 dl ~
Ill M ~ ~ N
Ill N M If1
H ~ ~ H In
~
N
dl a~
-r-I av a~
U Lf1 N
_~
a,
~
f~.'
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
H
w
H
p U U P4 U U U
~ z o z z z
V W H ~ H H H
O
W
r1 [~ Lf1 N ~ O
.~ ~. .-. ~ L
01 M O O W O~ 00 O
01 O -I 10
OD
N N ~ L~ M 00 N N
N O a0 r1
1J r1 N ri r1 M r1
~ O O O O O O
.r1
O ~O v0 ~ O v0 O v0
~
U ~ ~ ~ O ~ O ~ O
O O O O ~
?-I td t6 t~ -r-Itd -~ ~ -r-I
-~-I r-t -~ ~
O ~ ~ ~ aJ ?-I +~ 7.r
~ +-~ W ~ ~-I 1~
-- -~ H -- -- -~ -- E-~
E-~ H t6 H ~ r0
~ r6 c~ E-i
N O \ c-i 01 ~ O \
WI \ j..~ \ ~1 ~I
,4 ,<I \
(p u1 O >-'r t~ d~ N >:",
U ~.,' ~", ~ N >_.' v v
N N N i:
d~ o O M V~ d~ ~ O
O O w O W w
W w w O
.
-,...I
O O J-1 O O O 1~
11 JJ O r-I JJ r-I r-I
r-I r-I r-I 1-1
.,1 .-. ~- Id '-' '-' " '' l~
G4 ~ rtf O rd O O
p O O (~
'
Pa G1~ Pa W 0.~ W
~ ~ ~ ~ ~ ~
U U r-I U U U r1
r1 r-I U r1 r1 r1 r1
r-I r-I r1 rl
-j; .~., W .~'.,j"., ~"., .~'.,
w w ri w r-I w r1
r-I r-I r-I V--~
~a ~ s~ ra ~s rt r~ ~
s~ >~ v ~ v v
v v v r~
U U H U U U U H
H H U H U U
U U U H
M
4J
W n t~ t~ o
LC1 t0 l0 N
H H H
O O O O O O
(~
.r-I ~ .~ N
.!J
U7 l0 LW O O .-1
O
m ~ mo N ~ N ~. vo
E~ M N v
d~
N '~ ~ ~ '~ ~
N ~ O
'~ 0
?-I N ~ O 0
w
M N N O c-I ~ d~ O
O ~ ~ ~ ,--I
. . O
~
W O O O H O H O H
.j".,.-. --- H ~ (a
[~
0 ~- ~ '~ S-1 '-' \ ~ ~I
~ ~.1 \ l11 \ L~ \ ~',
~ ~1 l0
v o W rd U U U cd U
-rl u0 u1 rd vo -~
r1
+~ v v v ri v r-1 v r-I
r-1 r-I .-I -r-I r-I -r-I
.--1 ,-1 r-I .!J
U7 'J 'J 'J ~ ''r J.-~ ~ ~
U ~ 1~ J-~ 11
~ O U7
U7 -r-I -rl -rl -.-i N rl U
f~ O U U N v O N N
O O U --
r-I J-~ 1~ JJ U1 .t~ -r-I 1~ U7
~-t ~ U) -r-1 -ri -~ -r-I
~-- v U1 J-~
H U U U cd U O U (~
G4 (~ O O U O >~
N
~~n ~~m ~5f.~~ ~Ra~ ~~w ~~t~,~,~
'd '~ ''d ''d O '~ O
~ O O O O ~ O O
~ ~ O b~
O O O -rl O +~ O -.-I
O -~ +~ J~ O 1.~
O O -rt O S-I
Biro s~~ ~I ~a ~a
~ ~ ~ ~a~ a~
~ N ~ ~ N N
N N ~ N ~
~ ~
~
v N ~ ~ r
~ I d
c
a
xz xUz xUxz xxU xzx rxUxc~
In ~ c~ ,~ oo o a,
J d ~ O ~ l0 M
L~
M
J N 0 C V~ d~ ~ ~O
dW i ~ 00 O~
U I I I 1 I I ~ I
v I
y -1 O M I~ d~ ~ ~ 111
M
~1 00 d~ M LC1 M r1 O1
O~
(~ M r1 d~ L~ d~ d~ LCl
00
Cu
'~
O
-~
z
O c-i N M d~ Lf1 l0
C-1
v .-I r1 r1 ri r1 ri
H
r~
U
O~
~
W
z~
76
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
H
O
U U ~ U
z z o z
V H H W H
O d~ Lfl Lfl
01 rl l0 01
-rl M M l~ M M O
M t~
. . ,~ . . N
H H
O O . O p
. .
--o ~-o --o
w ~ fa rtS rd
U -c", ~ ~
O O O O ~
~ ~ O
_ - - -
O
~H ~E-I ~H ~H
~ (d c0 cd
O M \ 01 d~
\ ~1 \ \
~-1 ,~-I ,~-1
U7 O Cr N M Fi
w F"-, ~,' ~,' N
N N N
Ll1 V~ d~ d~
O O O O
W W 4-1 W
Id
(p O O 1~ O O 11
~ 1J r1 JJ r-I
CT-ir-I - r1 '~
'r ~ d
S
-r-I(d (a f( (
0 O O O
P~ 0.~ L4 W
~ ~ ~ ~
U U rl U U rl
r1 r1 r1 r-I
r-I r-I
.(-'-,~'-, ~', F'.,
W W w w
ri r1 r-I r-I
(~ ra (If td
>~ ~ >~ >~
N ~ N N
U U H U U H
H U H U
U U
U
M
O O N r1 M
(a
-ri ~ L(1 Lf1 l0
J-~
u~ ~ ~
H yo M O O\ l0
N O O
~',
~-1
~-I N M lO L(1
w ~
k O N N N
0 N
~ H H H
W O O ~. O O ~
>-i H (a ~ (a
(~
O ~- -r ~ 'r
\ O N ~O
~I ~', ~' ~,'
N U r1 O 00
-r-I~ -r-I -r-I -r-I
1.1 ~ (v N N a-i
.,1 N N JJ
r-I .1J 1.1
U7 ,~ ~ 'J ~
U 1~ U7 U7
~ U7
U7 -rl -r-I -r-I r1
(~ N O o O
U N ~ N
-r-1JJ J-~ J-1 J-W--
~-1 -r-I v ~-- J-~
U7 J-~ 1~
H U U >~ U U >~
fs-iO .t'-,
l0
~ U7 ~ ~ U7
-r-I UI -r-1
-r-I
''~ 'd '~ ''!~
O ~ ~ ~
O O O O
O O O O O O
w S-I O l-I
-r1 S-r
S-I ~I ~-I ~I
(~ ~J '~ ~J
'~ 1J 1J 1.1
~ C~ , S~
~ ~-I S-I S-I
!-I U7 U7 N
N N N N N N
N td N ~
Id ~
P4 PG rx x z
x z z ~
U ~ c~
U1 N
eD
J 11 N
(~
O
O
O
1 1 ~
'
~ 111 N
, I ~ a0
U r1
I
I
I
y -1 ~ d~ r
c0
oD
i l
r1 l0
a Lh
N
-I
L~
O
.
H
CV
''d
O
-~
z
o ~ co o, o
~1
H r1 r1 r1 N
H
U
OI
~
W
z~
77
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
I
v ~ ~ ''O ''d N U
O r1 N ~"., d~ -ri v
U ~t u7
U7 W6 -rl ~ ~ (d f~ H
N ''~ f~
cn w ~, o o 3 ~ r~ o ~n
~ v ~ ? rt
~ ~ v U
~
.R o ~ ~ u~ ~n
y ~ ~ v ~ w v ~ u7 ~ m -~I ~I
-~I r6 'L~ ~ v v
v ~I U ~I o o o ~,
,~ -~I
x ~
~-1 ~ ~ N ((j '~ 1J j"-, ~-I '~ -ri
U r-I r-I U1 .-1
N
v O O U ~ v N U E~ w ~
N 'C U
f-i ~ O 'O ~ ~ N -~, O ''O N
~'-, ~ N t~
.-1 w N (d O U7 ,~ 'Z~ ~,' U7
S-I -.-I -r-I ,~ -r-I
fa'
.1J
r-i ~-I ,~ ~ U7 O N ''~ O
(d S-I (a '~
zi w , w N -~ ~ ~ >~ s~
7, N ?i - 'd
N v N O N ~-1 J-1 J-J ri (~ ~I
~ O ~1 ~-I N H v
,i,''J ~ ~,' O '~ -r1 (a r~ N
(O O U7 H 1.)
1-1 O U7 T'-. ?-W 'Lj -r1 1~ ?, ,~
1-I -1 O J-~ f1~ (0
ri ~ f~
U1 O O -rl O .R ~ (d
N U1 O b1 U7 O v v
v
N -rl -rl ~ O ~-I O O U U
~ (~ ~'-, -r1 U7 ~-I
- ~ ~ ~ U7 1J U 1J
~ J i -
~ " l
O ~ ~ + , 7 N
S-i O r ~ , r >~.,
1 ~ 'r ,~' N U O
U7 ~ 'i~
-rl ,t', J--i
U7 C~
w N S-I U W ~'.. ~ N -T'-,
L; O t~ O
~r
O SC S-~ 1~ 1W UI r1
U -rl -I O ~.r
N 'd
'1~ U1 ~., v (~ r-I f~ U1 O ~-I
-.-1 -r1 r-I '~
.f"-, (U ~
N U1 ~ +-~ r-i ''~ (~ -rl U O
~ ~ O r-I N
~ f(S
w -.1 1~ bm0 ~ N S-i 1~ U U
-t"-,(d 1~ ~ O ~ ''d
f~ 1~ >:'., U U1 v ml U
O 1~ ~ O -~',
~', f1-
r-I -r-I~ -,~ -,~ ~a ~ ~ u~ ~n
u~ o ~ v v r-I
~a ~
O b11-i.-i ~ 1-~ N r(i >~ ~r -~-I
to ~ U ''d (d N U
~
u1 ~", ~ ~ 1J Ul U1 r-I v -r-I ~
fff -.1 v (a 'Lj ~ 'J S~,
?,77 ((f
r-I ~ f~ ''C N -r-I ~-I ~ v
~ N U -ri r-I O O -r-I
-.i -r-I N
r1 L~ ~ J~ '~ -f-',.-i O
U7 ~-I L; '~ O ~ ~-1
r1 Ul
1W C N v r~ U ~ N f~ 'd U
r6 f~ rl
~ ~ r-I ~ ~ +-~ 1~ ~
b1 N U ~ 'r v N O
~ O O 'd O
d O ~ Id O 7-
S'
O O r S -, I S-I
~ -I f~ ~ s ~-I I JJ
~ N WIi U ~ v
~ 1~ S
~ !
-I r - - , w
w -I . w w -.-i , <n
~n ., r1 ~
w ~ r1 w ~ r1
U S~
~
-r 'r~ 'd (f3 ''d 't~ C ''~ ~ 'Cf
U -I N ~ U S-a ~ ,~ f,2,''d ,~
v +-~
U7 v v ~ b~ v v -r-I v ~ v v
v 'd v -~I Sa
~
y -r w tn +~ w ~.I w -.-I +~
g r-I w 'O ''~ +~
~ rd
I~c~~~6r1~~ ~~~w ~ v~ ~d>~
r-I r-I U rl r-i 't~ r1 r1 r-I
o f-,' r-I b~ O ''~ N
U r1 ~.,
N O O ~ ''C O O O O td O
N v >~ -.-1 ~ U -rl
S-a
u7 u1 rtS u1 m N r1 u1 ~ u7
N f,1 ~ to r1 ~ w
r1 r~
N -ri .--i r1 -r-I r-I O -r1
1J U +J ~ r-I U -rl (d
O ~ -("-,
a ~ r6 r~ N ~ ,~ ~ f~
7. o o
FC '~ r-C r-C ~ FC -~ FC
U s.~ b1 +-~ ?W-I
r1 -~I z >~ z
v o s~ v ~ v ~a ~I
z z o
rd v ~ ~ ~ ~ N
O rd r1 w LL U7 O
''d '~ U
1~
~ -I I i (a ~ r-I
- ' ~1 v
S U7 J
-I r ~., -I - b1
J-~ ~1'~f( ~1 b7 ~"., bl .~, (6
~' b1 ~-I ~ -ri ~
-r-I ' ~ U7 U ~
~-I ' ' -I
-~ i
> -I ~
' -
'
U1 .f (a ~ r ?-I -. a
-y 31 ~ > ~-1 -I -r .
-r-I-r-I , , (~ S 7
O v -r-I ~ . .
~ .S -r -r-I
-r-I -r-I
r-I ~-1
O c~ v ,f.,'
v +~
J-J U7 t!) ~ u1 U7 U1 UI ~ U1
d ,~".,(d ~r v -rl b1 O 'O
~ l ~ r-I ~ ~ s~
3 ~ I o ~ f; ?W
~ r~
~n ~
+~
r r 10 U -.-i ~ r1 O
00 r-I U (~ v
't~
-r-I
't~ ''~ Ll1 'Z~ 'C~ ~ 't~ N '[j
- O N ~ U -rl U1 -r-I I
~ w r-I
v v N ,~ N N rd N r-I v
U7 'd 7C ~ ~ U ~.I
1.i 1.~ .N N a .~.~ .N (~ +-~
N ~ ~ ~ ~ U N U1 u7 ~
G~a r1 v
U U U ~ '-I U U O .~',U ~ ~ U
r1 J.-i (~ f~ O N
~ ~ w W ~ ~ '-d ~ O ?-i ~
v U W N w ~r
+~
~.I S-i O ~ ~ ri ~ -r1 ~I
w !~ s~ u1 'a~ v I
1~ 1~ 1~ -rl .1J JJ O 1~ ~ 1-~
O f~ O f6 C'., M
O ~ L~,
U7 U1 U7 N U7 U1 I U7 -rl U1
'd N F,' U .S', v O I-c7
r-I f~" -rl J~
-f L; -~,, W', .~., ~".,
, H ,~ O ~ S-1 U1 ~
~ ~r J- W ,~ -rl
(0
O O O O -r1 O O ~ ~'-,O ~ W O
O (a S-i f~ J~ f~ ~
U U
U U U r-I U U N C2,-~-1U U ra U
I ~ U7 O U N -
~ ~
F.~ N v J--~ W 'r ~ ~ S-I
S- a 3 U !~
U1 U7 U7 r1 U1 U1 I N (d U7
(~ w r-I 'O .f'-, ~, O O
N .1J U O
to td r~ ~ r~ ~ co t~ U 0
N -rl ~-I ~-I ~ ~ ~-I
cn 'd cd w
3 3 3~~~,~ 3 3~ v 3 0 ~X, 3w
?. ~ 0~ U ~n ~
?,o,?,~ ~,o~wo ~~~ ~ ~N~ ~~~NN ~~o
~
~ s~ ~ ~I ~I ~ -~I ~ o o ~
d~ ~n bW ?, c~ -~I +~ v
o ~I >~
c~ w c~ ~ O U c~ I r~
~ N v O ~ ~ ~
r~ ?~ ~ -~I
S-a ?-I ~-I -r1 S-I ~-I .~.,~-I ~-1 ~-I
f~ U -rl ,fir -r1 ~ r0 O
,~ ~-1 O ~-1 v U
,~ N .R ,~ O ,~ td S~
~ w I ~ u7 -n ~ ~
Sa ~ ~r ~ ~
~ v c -~ v -I
-I ~ ~ v c~ v
~ rd
-
-r-I-~-I-r-I -I -7 'y-n ~
w ~ ,~ v r n . U ,W -7
~l ~l c~ w ~7 - ~I
O U ~l w M . ,~ U
~ W v w 1.1
~0 ~ W
r1 M N N ~ H
~y O O O O O O O
En H En H H H E-~
N ~ z
z w N z
H ~ ~ z U
~ ~
a H N
~
O
-~
z
.-I N M d~ 111 10 L~
N c-I c-I r1 r1 r1 r1 r1
H
r~
U
OI
~
W
z~
78
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
w
~ ~ N~
~
~~
~ ~ ~.~Nw N
~ w
-I , rd
~ -
d -
s~ >~ ~ - ~
~I r w ~ o ~I
'd ~ O + O ((f ~-1 ~ +~
r0 r b~
~ O ?-I U
N R,.~ ,~ ,-I ~ ~ r1 ~ ~ ~-I w s~ N
. N cn rtf 3 N N
, U7 N -a O rtf -r1 N ''d O O O N U ~ W
~ r(S N
O ~ r6 ~ ~ ~ -.-i ~ N ~ .r 'C5 >~ .R
1-~ N O
.~. r5 ~ ~ b1 N O ''~ >~ w 11 U7 O N N
(O N ~ f~' -rl ''a~ S-I -r1
N J-J ~'.,O (~ >-i r1 ~t ~ (~ ~.,' .~, '~
J-1 ,5 O ~-I O r-I fa ~-1 N Ul '~
?-~ w -rl ~-t W -rl ~ ~ r1 '-~ f3~ O -r-I
U7 r-i r1 f~ r1 (d U N ~ r1 ~-I
N 1~ w .-I ~ '-d U O N -.1 '-C3 ~
U U1 ~ ~ .-. U 1.~ U7 (~
N r-I ?-I Wd ~ O ~ -~'. U ~ +~ N N O U
'r U7 O ~'., ~-I r1
S-
(~ W N ''d S-'r i-a O 1:.,"O N U r-I N '-I
N ,t.' -~I ,fir N
O o~
U7 N r-I N r1 -rl w 1W'. ~., ~-1 S-1 (~
N ~1 U -~. ~ Sr' r"~ l~ N ~'r x r-I
U7 S~-, ~ ~ (~ f~ ~r t51 ~ ~ (a N (~ -LJ
~ -~ ->~, ~-I 1-J N b1
~
-~I ~ U o o u~ ~ >~ s~ -~I s~ r-I w ~ 3
x o ~ ~ N ~n Wo +~ s~
.u u~ R ~ z1 a~ s~ ~ .~ -.~ ~ a~ w ~I
U U o >~ ~ c~ z ~ ~
~
w ~ ~ N .~ ~ -~I >~ ~ N rtf -~I s~
N ~ N ~ o ~ ~I
3 --
b1 O 5 S-i r~ b1 (d '~ ~ w O ~ O U -r1
- ~-I U7 ~1 N ~ ~ -.-i
- N N (d -r-I ~ -~ U7 U7 ~ N N ,~ ''d
?~ U o~
~t'C~ ~-I ~ r-I r1 ~-I -r1 U7 U U ~-1 L~'
u1 ,~ ~"-, - ?yi ~-I b1 ~-I ~i V-~
O o\
~ (d -r-I ~ ~ s~ O 5r'~-i -r-I cd -~-I
~-I ,~ b1 (d S-m-I N E-~ ~ rd
O fd ''d U7 a S-W -t r-i 1~ ~ ~ .~ O i~
N ~ +.~ N ,-r-I S-i O +~
W ~--
UJ 1W-I t~ O N U f~ 1J -j',''~ ~ W-1 .Q
U7 U ~ .(', ~ '-' ~-1 ~-1
U fd O -~ .~ ra -r-I ~ (~ ~ N U7 U7
-r1 ~., ~ N .>-'. N ~ -~I ~ N S-~ r(f
~'-,
Ul '~ S-I 1.y". O N -rl (a H (d r1 -r-I
~ H U .~., 1.y-1 ~
N
~ ~ O -rl ~ r-1 O 'T1 1~ U r-i U f~ ~ O
~ '~ r1 ~ N u7 ~'.
O O >~ S-m~ r1 1-~ -rl ~ -rl N r1 u1
'~ N ~ rd N - N (d (6 U
r1 -~ rfS O b1 ~ O w W >~ rd ~ +-~ .~ +-~
~-1 'd N ~ v ~". ,~ Ol
U ~ r1 .T'. -r1 U N U7 N O W d O N ~
~.I O ~ U7 H 1-W 7 5
- -I U ~
~ N N -i
~ ~
~
~ W
" J-~ t51 I ( r
~-1 ,~ -r-I ~ r1 (a r-I -1
~ ~ r -I .
~.~U ~r~lb~w 3~noUa~
04 w>~rt-~~~3w
~ ~ U m ~ >~ o ~ .~I U s~ ~ ~ w N m
cn ~ m x cn
o ,-~ o ~ ~ ~I .~ ~ +~ ~I o ~ ~ ~ -~I >~
u~ rd .~ a~ a~ a~ .u >~ a~
>~
S-i ~ ~ O ~ 1~ ~ -rl ~ T5 ~ N >~ N U ~
5 (~ r1 ~ ~ O b1
w N N S~ i~ ~ rd ~ O U ~ w -~I ~ S~ ~
O ~., , c0 O r1 O -~I
w r1 .r'-, O >-.' ''d N ~d fd ~'-.
~r-rl U7 ~ ~y ~ 1-~ O
O F~'
U 'd b1 (d ~ ~ >~ -~ ~ ~ ~ ''~ c17 U U '~
N ,~ ~-I O ~-I O U7 v bW O U
~ f~ 'ZS '-d ~ -~ O ~ U +~ N O -r1 -rl ~
~ N N U C7 1~
11 -ri N N U fn -rl N U7 J--~ Li U7 ~
~ ~-I J.J ,~-, '-' r-I ~ O r1
'Tj
ca ~ d~ ~ w ~, ~ U1 N U ~ 3 O .~ (~ 7-t
~ N S-~ >'', -rI ~I N O ~
r-I ~ N f~ ~-I l~ (~ ri r-I S-a J-~ U
U O (a .L,' f~ ~ ~-I ~-1 ~-, (~
O ~ w U7 r1 ~ U ~ U -~I O O Ol ,Lj O ~
t~ w r1 ~ U .~
~
U7 O ~ ~ O r-I -rl ~.1 U7 r-I -rl -
'~ -r1 ~ ~ r1 t6 w , Id
O '
r-1 ~ N N -rl O >'i ~-I -r-I (~ w
'~ (d 1J (a r-I ,~-I >r TS w
~ '(~ ~
l I
~ ~ U N O O
d U N
O a ~ a~ + ~
C ~ -~.~I U7 N O S-I
U7 ~ w r~ r1 ~ ~ -~I
( ~I >.
r~ a~ ~ ~ ~ o >~
~ w a~
M 2 ,
~ l (a ~ U ~ O N f >~ ~ r~ -r1 r1
O FC U ~ u1 ~ ~
l ~
r . ~ ra N U 3 -rl
N N U U , J.-~ (l5 O N
.-I r
~ b1 +~ ~I f1-v
S-I
~ (6 O
w 'C1 U ~ -.-I b1-rl ,~
.("., 1~ N -rl
UI td ~ M ~ U ~U O
~ ''d 'LS
~1 1~ f~ U -r-I U 1-1 -ri b1 U1 -~', G~
-r1 U7 .-i U JJ f(S r-1 U1 fWU ~ '~
-r1
~.,' >_.,'.~, U1 N -r-1 .>"'.,Fi (d .-i O N
,4 ,S~ ~'., ~ U7 (~ O U (~ ~-I r1
N
-ri f~ O '~ ''d +~ -rl ri U .~', U b1
b1 O 'r O U -.-i U ~r U1 >r"., U7 (d
~ -f", "
' ''
U7 r-1 ~-i r1 (~ c~ O ~-1 ., -.1 U U
1-1 ~-I -rl +-~ -ri ~-I U7 ~ N U7 ~
~ ~ ~
r O
uJ ~ O w O ~ N N ~ ~ -~ ~ r~ ~ O -~I
N ~ ~ O P4 W
~6N~~~ I w~U~lUrl~lO~-~I UO~>~O>~,~~d
''C U 1~ ''~ ~ O N ~ O .~ 'd J-~ U Ul N
N U7 J-mrl 1~ ''d O O ~
N ~ (d N (d U f~ >~ ''d N '-d ~-~ ~ -~
''d w w 1~ N ~-I -r-1 U 1~ >~
~ f~
J--~ ca 1~ W -i r1 -r-I J.-~ r-I N -~'-n
U ~ ~', .-i S-1 r-I ,''~ O ~ 1~ U7 f~
~-I (d
U U -~ U ~, r~ J~ ''C u1 U O ri ~ r1 -~
r1 N -r1 , ~ U s~ O r1 N
''L~ U ~ I ~ rd >:'., ~ ~ 1 f~ ~ '~
'r U .f", O U1 ''L3 ~I -rl r-I
O -~
?-i '~ S-r o~ O 1-~ (0 ~-I S-I ~ x N
~ >~ H FC O ~ O -S-', ~'-, ''d U ''d
>~ U o0 ,
J-W -I J-~ lf1 .~., U7 1J (~ JJ N r-I
-r-I -r-I '~ -r-I JJ ~ (a >37 ca -r-I
r-I ((f N U7
U7 O ~ U7 N (f$ U N U7 U1 N (a J-1 -r-I
U U1 ~ ~I N ~ ''C3
.f~r y, i: ~ S-I 1~ -~I C~ 'r ~ ~-I N
?, (~ ~ 3 !~ o ~ - N ~1 S~ N rd
o, l ~ N
~ ' U1 H
~ O I N O
d '~ s
O ~-! ~I O N J r
G4 .f. c ,
U t~ O O N l .
O O -r1 ~ 1W ~ -r1 >~ U It1 ''d U ~
~ ~ ~ b1
U w ~ N
N r1 l~ ~~ M O O ~ WO H
r-I O .'W'W1 Ul S-1 ~-I
>=i ~-r ~ ~
U1 ~ O U1 ~ (d U fIS N U1 ~ 41 w ~'-,
U7 ~-I (~ ~ N U7 ~ U (~
O
fO I ,L,' f~ ,~y O ~ x La (O f0 .("., O
r1 dl 'L~ '~ --- (a N U7 .~'.,
U r-I
3 0 ~ ~ 3 ~I .u zi >, o 3 z3
U -~I ~ o -~I a~
~
L (a ~ (a U N r1 U7 ~ ~"-,w N rl U1 .(',
~ ~ -rl f.2~ N 01 N N N
(1, 1.W ~r ~ N J-~ r-I N yy O ~ (d -r-I
~ N 7.a U7 J-~ -r-I ~ ~ H ~r
-r-I o
Y.a ~ >~ ~-I O ~-I (d (~ S-I (~ ~ U7 .~G
U ~-I ~ 41 >~ c0 U r1 f~ f~ ~
'~ ~-I
(a ~ ~ (~ N U -rl U b1 (a ~'., U ~ O
rl ~-I ~ r1 v S.,' U1 -rl
O
~1 .~., ~.1 J-~ 1~ -r-I ~-I -r-I -r-I
N -r-I 11 11 r-I Q-, td Ql ~.1 ~ r-1
~', r-I N Sa J-~ J-1
~3 O ~2 ~1 '~--~ <n 'd ~ ~ c~ ~ ~r ,~
~ -~I w N >~ O w ~ FC Sa ~ ~ U1 'd
-rl
-.-i ~ -ri ~ 5r -f. ~ ~ -r-I S-~ .>~.
O ~6 ~ ~ N N -~.,. ~r N -r-I ~".,
~ U1 N ~ -r-I .T"-,
a w r-I a r-I ~ -~ , ~ H a ,~ -~I U w
W cn w ~ ~ -~ ,~ .~ ~ ~n .~ ~ ~ ~U
N H ~-i
0
~ H
J~
-I
a
o
as
~o
-~
z
O o0 0, o
Ll
N c-I v-I N
H
r1
U
Ot
~W
z~
79
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
W ~ -~>' °' ~ I o
vo .y o'"o ~ o
O ~ b O ~ y M
O w-r y y., . ~ O
b ~ '~ fn ,-i ~ v~ O "~ y p ~ U
o ~ ~O ~%~cC.O~~O
p .~ ~ O > Er w, II '-' ~ O ~
L7 N I c'~ a, ~
V7 .fl 5 w ., ,~" ~ ~ ~ >
V ~ ~ W w '~ p N
_U O ~O ~.~Cw,O~~YOO.~ ~= ~O
~ O ,~ W ~ '~' c'~C ~ m c3 :r
~_ (r ~ .~ ~ Er '~ .~ a~
N .~ 5 b~ ~ o Q W° 0~.0 ~
CL ~ W O IY '~> W Q,' ~ ~ 'w~ ~L w C~:
c~
"-' N~ .~O
. r ~ ~'~' a; ~ ,~ ,fin ~ c'~ 3 vo
M
O Ov 0-o U ~ ~ ~ G E.., M U
00 p" ~,~ ,~" ~ x N ~ ' . ~ ~'~ r7 p,
U U U P~! '~ N ~ °° °~° ~ °~ c~i ~ o ~
o ,~ o ~o °~
a.~~ O ~ ~ c . " I~ '~ 3 '.yes.. w w M i
U U U ~~'Y' ~~~30~0 ~°~Q~ °~°~f~
U ~ ° ~ M ~ c vi ~ ~ ~ ~ c U
a~
u°.. w° r°, ° ~ "~ ~--; ~ W oNO x so ~ '~ w ~ ,~
~' o
c~ ~ ~ ~ av V ~; ~ ~ >~ v~ a\
vi v~ ~ vi " v ~'' ~1 a 0 .b ~,; C'7 '" w» ~ "'' ~ o ;O ~ o°n
x~ ~ ~~~~-b
~ax , w ~ x ~~.~
fi. o ;b v~ .. E-~ ~o m ~ i U
O O V O ,_, ~ V ~ O ~ U N ~-. U ~ U
M a ~ ~ '~ a. w a b O ~ a o z ~" ~_i
O . U ~ O ..U, G ~ ~ C U
a x a~ c~ ~ a ,' ~ ,~ oo
c~ b b ~ ~ ~ ~ . N ° M ~ a
~., x a aa, a aNz a.z3 ~a xz.~w ~~ ~N.~Az
w o
b ~ $ ~ .° ~ y O ~ ~ ~; U
'b N U ~ > ~ b4 '.~ y~ c~ C/~ O V .b C
gag c'~~' ° a~ ~~C~c~d' '~'~>a'~ .~za'~c ~~°
U n ~ D . ~' U ~ b ~ '..'"1. U pn c~
n
;b G .d b ~ ~ ,r-J .r ~ OU cyC 0, ~ >; ~ ~ v~
8 O U ~ '~ ~a ~
p, coo ~ E~ .~ a ,~ ~ ~ E~ w i ~ ~ o
U .
~ <z" ~ ~ ~ Q .~ ~ ~ ~ o
w o ,? ~ U ~ ~ ~ ~ ~ 5 ~ r~ ~ c .~
a ;b ~ ~ a~ y ~ cr'a ° ~ w ~ ~ c~3 a. y ~ W..
U y p '~ U ,~ ~ 'C O U ~ ~4
a~ O 'n ~ .' y ~ ~" N G > ~ ~ ~ ~~ t-~
ay ~~ ~ 3~.~
>; ~:~ ~ ~ ~ ~.~b:~ ~~ o a ~.~x.~w ~b oa
~~ ~~ ~ ~~~~~ o~~~ ~~~,w~ ..Nw
.o. o ~e ~1 .o o .~ ~ .~ .~ ~ .~ ~ z~. v ~ O ~ ° .a°n c ~ ~
y C~ ~ L~ G o. GG ~ p ~ p" .~ ag ~ ~ ~ ~ p o ~ c'~ .
A a ~ a ~ a a ~ _~~ a ~ ~~ ~ a ~Aw
N
W
E~ U a
U ~ v~ c~
w ~ a v~ H
w
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
U
C7 ~ y
°N' w° r,;
'~ a~ w
00
o ~ '~ ~o ~ a w
° °"'
on w
a ~ v~ o o ~ ao
~d ~ c~ ~' 0 00
a~ .. ~ N C ~n
N
. ..
V N V N
it ~ C Oc~C O NCUp
p. C:~ UJ ~ N C/~ '~ N N G,
O ',~ U
rG-ii _~ ;' N G G
,b p ~ O ~ '~ -" i.y ~ v~ .~ O
Q, ~ ~ ~ ~~' Ov f~ ~_ iC O ~ ~ ~ ~ b
vp G .-. ~~ ~ OW G ~ O O V
C4 N ~00 Gp ' .
~ b a i a ~ r~ i~ ~ c~ c ;b ~ ~ ~ ° ~ Q Q
o ~ ~ ~ E~ ~ '~ ~ W a ~ A, ~ ~, U ~ c~ °
.. ~.
o ~ ~ ~~ a ~ b
~ Q, ~ ~ pq o ~ ° ~ °~ ~ ~ ~ ~ p" a
~O y, ~ b
z
au y ~
3
~ ~i oo ~ oo ~ ~ o~ a\ °Q ~ r" ~, ~ a~ a\ o
c~ N ~ 0.1 ~; v~ v ~ .~ ~ ~ ~ m ~ ° M a~ ~ _ ~ ~ ° U
c~ i~ v ~ ~ °4 ~ ~ oNO ,=, C '~ ~ ~ p; w M ~l ~ b N c~ v
a~ a~ PG P~"' ~° awb ~ op ~ c'~ c2 '~. m b p, c° W
.°~'~'° °'
~ ~ °' v° ~ W o ~ N ~ a: a i i > ~ c'~d ~ '~ ~ C '~ c~ ~
c~ N
~_ ~ ~Q ~ ~ w ~ ~ aa~3 A x~~
~ o~'o U~ E" .~"' .~., a~ ~ O O \O ~ O oo G ° ° U 'G Q W
y G ~ 00 ~ ' U ~ '~ .b v~ ~ n ~ ~ G w ~ ~ U .~
", ~ ~ U '3 ~ ~ ~ ro -d ~s o a~ '" ~ ~ t~ o a o ,~
V A4 C7 Ur °~° z W oo C ~ ~ ~r c~ vi C7 Z ° U ~ N c~
c° U C7 ~ ~
V7 b
N w ~ .° on °
c~
~ °3 _>, E-. ~ G ~ ~ ~ G
a a. °'~' ~ 3 °' ~ ° o ~, cr ~ $' o
c
b ~ '~" O by p, ~' c~. ~ '~ G U G V y
a a t3~ b y ~ '~ ,~ G ~ ~ .b ,~ ~ ~O
c~ a ~~ ,~ ~' ~ ~ ~~ 'b a ~ ~G. C1,
U G >, .~ 'V ~ A '~ ~ ~ O 7 G '~ ..G.
4. b-0 b '~ ~ N C ,=,~ ° ;b b
b ~" ~ ° _~ ~ w ""y ~'~ U c
f.. iG b-0 O '~ p b s., ~ cC v..0 ~ O
cC U G ° ~' '~~' b-0 f~ ~7 C
C ~, ~ ~ ~ "d ~ b
c~ ~ ~~' '~ ."' v, U ~ y 'b y a ~~ O
a~ ~ ~> ~ a3 3 ~ ~ '° '~ ~ U b
>, w ~ a N o4 ~~ ~ ~ ~ c~
ai Q on :~ °~ ~' ~ ~ ° . ° U
_o _ a~ a v~ ~ a ~ ~ .~
o ° ono v ~ o. ~ ° S '~ '~ ~ °' ~
.r
c ,~ r~ ~~ ~ '> a o 0
Gr ~O ~ n U ~ ~ U S'.r ~ U ~., U . ~ ~ ,_~, w, v~
04 ~ b ' ~ G ~ ~ ~ a a ~ ~ G do ~ ~ do ~ a~ °
G o w ~ ~. a, o '.3 ~, oo N 3 ~, c. ~ °? s~. ~ ~d a.
A a ~b d ~ a~ o ~ a ~ Q ~ a ~b Qb ~ a ~,
x
w
b ~ b ~ ~ ~ w
o a~ ~ ~ U o
w' a a, a, v ~ H H
81
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
<110> INCYTE GENOMICS, INC.
YUE, Henry
BANDMAN, Olga
TANG, Y. Tom
HILLMAN, Jennifer L.
LU, Dyung Aina M.
BAUGHN, Mariah R.
<120> HUMAN LYASES AND ASSOCIATED PROTEINS
<130> PF-0759 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/172,307
<151> 1999-12-16
<160> 20
<170> PERL Program
<210> 1
<211> 243
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 168714
<400> 1
Met Glu Asp Ser Phe Leu Gln Ser Phe Gly Arg Leu Ser Leu Gln
1 5 10 15
Pro Gln Gln Gln Gln Gln Arg Gln Arg Pro Pro Arg Pro Pro Pro
20 25 30
Arg Gly Thr Pro Pro Arg Arg His Ser Phe Arg Lys His Leu Tyr
35 40 45
Leu Leu Arg Gly Leu Pro Gly Ser Gly Lys Thr Thr Leu Ala Arg
50 55 60
Gln Leu Gln His Asp Phe Pro Arg Ala Leu Ile Phe Ser Thr Asp
65 70 75
Asp Phe Phe Phe Arg Glu Asp Gly Ala Tyr Glu Phe Asn Pro Asp
80 85 90
Phe Leu Glu Glu Ala His Glu Trp Asn Gln Lys Arg Ala Arg Lys
95 100 105
Ala Met Arg Asn Gly Ile Ser Pro Ile Ile Ile Asp Asn Thr Asn
110 115 120
Leu His Ala Trp Glu Met Lys Pro Tyr Ala Val Met Ala Leu Glu
125 130 135
Asn Asn Tyr Glu Val Ile Phe Arg Glu Pro Asp Thr Arg Trp Lys
140 145 150
Phe Asn Val Gln Glu Leu Ala Arg Arg Asn Ile His Gly Val Ser
155 160 165
Arg Glu Lys Ile His Arg Met Lys Glu Arg Tyr Glu His Asp Val
170 175 180
Thr Phe His Ser Val Leu His Ala Glu Lys Pro Ser Arg Met Asn
185 190 195
Arg Asn Gln Asp Arg Asn Asn Ala Leu Pro Ser Asn Asn Ala Arg
200 205 210
Tyr Trp Asn Ser Tyr Thr Glu Phe Pro Asn Arg Arg Ala His Gly
215 220 225
Gly Phe Thr Asn Glu Ser Ser Tyr His Arg Arg Gly Gly Cys His
230 235 240
His Gly Tyr
1
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
<210> 2
<211> 425
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1851727
<400> 2
Met Val Ser Lys Ala Leu Leu Arg Leu Val Ser Ala Val Asn Arg
1 5 10 15
Arg Arg Met Lys Leu Leu Leu Gly Ile Ala Leu Leu Ala Tyr Val
20 25 30
Ala Ser Val Trp Gly Asn Phe Val Asn Met Ser Phe Leu Leu Asn
35 40 45
Arg Ser Ile Gln Glu Asn Gly Glu Leu Lys Ile Glu Ser Lys Ile
50 55 60
Glu Glu Met Val Glu Pro Leu Arg Glu Lys Ile Arg Asp Leu Glu
65 70 75
Lys Ser Phe Thr Gln Lys Tyr Pro Pro Val Lys Phe Leu Ser Glu
80 85 90
Lys Asp Arg Lys Arg Ile Leu Ile Thr Gly Gly Ala Gly Phe Val
95 100 105
Gly Ser His Leu Thr Asp Lys Leu Met Met Asp Gly His Glu Val
110 115 120
Thr Val Val Asp Asn Phe Phe Thr Gly Arg Lys Arg Asn Val Glu
125 130 135
His Trp Ile Gly His Glu Asn Phe Glu Leu Ile Asn His Asp Val
140 145 150
Val Glu Pro Leu Tyr Ile Glu Val Asp Gln Ile Tyr His Leu Ala
155 160 165
Ser Pro Ala Ser Pro Pro Asn Tyr Met Tyr Asn Pro Ile Lys Thr
170 175 180
Leu Lys Thr Asn Thr Ile Gly Thr Leu Asn Met Leu Gly Leu Ala
185 190 195
Lys Arg Val Gly Ala Arg Leu Leu Leu Ala Ser Thr Ser Glu Val
200 205 210
Tyr Gly Asp Pro Glu Val His Pro Gln Ser Glu Asp Tyr Trp Gly
215 220 225
His Val Asn Pro Ile Gly Pro Arg Ala Cys Tyr Asp Glu Gly Lys
230 235 240
Arg Val Ala Glu Thr Met Cys Tyr Ala Tyr Met Lys Gln Glu Gly
245 250 255
Val Glu Val Arg Val Ala Arg Ile Phe Asn Thr Phe Gly Pro Arg
260 265 270
Met His Met Asn Asp Gly Arg Val Val Ser Asn Phe Ile Leu Gln
275 280 285
Ala Leu Gln Gly Glu Pro Leu Thr Val Tyr Gly Ser Gly Ser Gln
290 295 300
Thr Arg Ala Phe Gln Tyr Val Ser Asp Leu Val Asn Gly Leu Val
305 310 315
Ala Leu Met Asn Ser Asn Val Ser Ser Pro Val Asn Leu Gly Asn
320 325 . 330
Pro Glu Glu His Thr Ile Leu Glu Phe Ala Gln Leu Ile Lys Asn
335 340 345
Leu Val Gly Ser Gly Ser Glu Ile Gln Phe Leu Ser Glu Ala Gln
350 355 360
Asp Asp Pro Gln Lys Arg Lys Pro Asp Ile Lys Lys Ala Lys Leu
365 370 375
Met Leu Gly Trp Glu Pro Val Val Pro Leu Glu Glu Gly Leu Asn
380 385 390
Lys Ala Ile His Tyr Phe Arg Lys Glu Leu Glu Tyr Gln Ala Asn
395 400 405
Asn Gln Tyr Ile Pro Lys Pro Lys Pro Ala Arg Ile Lys Lys Gly
410 415 420
Arg Thr Arg His Ser
2
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
425
<210> 3
<211> 216
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2095185
<400> 3
Met Ser Phe Leu Phe Ser Ser Arg Ser Ser Lys Thr Phe Lys Pro
1 5 10 15
Lys Lys Asn Ile Pro Glu Gly Ser His Gln Tyr Glu Leu Leu Lys
20 25 30
His Ala Glu Ala Thr Leu Gly Ser Gly Asn Leu Arg Gln Ala Val
35 40 45
Met Leu Pro Glu Gly Glu Asp Leu Asn Glu Trp Ile Ala Val Asn
50 55 60
Thr Val Asp Phe Phe Asn Gln Ile Asn Met Leu Tyr Gly Thr Ile
65 70 75
Thr Glu Phe Cys Thr Glu Ala Ser Cys Pro Val Met Ser Ala Gly
80 85 90
Pro Arg Tyr Glu Tyr His Trp Ala Asp Gly Thr Asn Ile Lys Lys
95 100 105
Pro Ile Lys Cys Ser Ala Pro Lys Tyr Ile Asp Tyr Leu Met Thr
110 115 120
Trp Val Gln Asp Gln Leu Asp Asp Glu Thr Leu Phe Pro Ser Lys
125 130 135
Ile Gly Val Pro Phe Pro Lys Asn Phe Met Ser Val Ala Lys Thr
140 145 150
Ile Leu Lys Arg Leu Phe Arg Val Tyr Ala His Ile Tyr His Gln
155 160 165
His Phe Asp Ser Val Met Gln Leu Gln Glu Glu Ala His Leu Asn
170 175 180
Thr Ser Phe Lys His Phe Ile Phe Phe Val Gln Glu Phe Asn Leu
185 190 195
Ile Asp Arg Arg Glu Leu Ala Pro Leu Gln Glu Leu Ile Glu Lys
200 205 210
Leu Gly Ser Lys Asp Arg
215
<210> 4
<211> 343
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2342959
<400> 4
Met Asp Gly Arg Arg Val Leu Gly Arg Phe Trp Ser Gly Trp Arg
1 5 10 15
Arg Gly Leu Gly Val Arg Pro Val Pro Glu Asp Ala Gly Phe Gly
20 25 30
Thr Glu Ala Arg His Gln Arg Gln Pro Arg Gly Ser Cys Gln Arg
35 40 45
Ser Gly Pro Leu Gly Asp Gln Pro Phe Ala Gly Leu Leu Pro Lys
50 55 60
Asn Leu Ser Arg Glu Glu Leu Val Asp Ala Leu Arg Ala Ala Val
65 70 75
Val Asp Arg Lys Gly Pro Leu Val Thr Leu Asn Lys Pro Gln Gly
80 85 90
3
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
Leu Pro Val Thr Gly Lys Pro Gly Glu Leu Thr Leu Phe Ser Val
95 100 105
Leu Pro Glu Leu Ser Gln Ser Leu Gly Leu Arg Glu Gln Glu Leu
110 115 120
Gln Val Val Arg Ala Ser Gly Lys Glu Ser Ser Gly Leu Val Leu
125 130 135
Leu Ser Ser Cys Pro Gln Thr Ala Ser Arg Leu Gln Lys Tyr Phe
140 145 150
Thr His Ala Arg Arg Ala Gln Arg Pro Thr Ala Thr Tyr Cys Ala
155 160 165
Val Thr Asp Gly Ile Pro Ala Ala Ser Glu Gly Lys Ile Gln Ala
170 175 180
Ala Leu Lys Leu Glu His Ile Asp Gly Val Asn Leu Thr Val Pro
185 190 195
Val Lys Ala Pro Ser Arg Lys Asp Ile Leu Glu Gly Val Lys Lys
200 205 210
Thr Leu Ser His Phe Arg Val Val Ala Thr Gly Ser Gly Cys Ala
215 220 225
Leu Val Gln Leu Gln Pro Leu Thr Val Phe Ser Ser Gln Leu Gln
230 235 240
Val His Met Val Leu Gln Leu Cys Pro Val Leu Gly Asp His Met
245 250 255
Tyr Ser Ala Arg Val Gly Thr Val Leu Gly Gln Arg Phe Leu Leu
260 265 270
Pro Ala Glu Asn Asn Lys Pro Gln Arg Gln Val Leu Asp Glu Ala
275 280 285
Leu Leu Arg Arg Leu His Leu Thr Pro Ser Gln Ala Ala Gln Leu
290 295 300
Pro Leu His Leu His Leu His Arg Leu Leu Leu Pro Gly Thr Arg
305 310 315
Ala Arg Asp Thr Pro Val Glu Leu Leu Ala Pro Leu Pro Pro Tyr
320 325 330
Phe Ser Arg Thr Leu Gln Cys Leu Gly Leu Arg Leu Gln
335 340
<210> 5
<211> 74
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2613975
<400> 5
Met Ser Asp Thr Arg Arg Arg Val Lys Val Tyr Thr Leu Asn Glu
1 5 10 15
Asp Arg Gln Trp Asp Asp Arg Gly Thr Gly His Val Ser Ser Thr
20 25 30
Tyr Val Glu Glu Leu Lys Gly Met Ser Leu Leu Val Arg Ala Glu
35 40 45
Ser Asp Gly Ser Leu Leu Leu Glu Ser Lys Ile Asn Pro Asn Thr
50 55 60
Ala Tyr Gln Lys Gln Gln Ala Ser Ser Cys Leu Ser Leu Ile
65 70
<210> 6
<211> 176
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2683534
4
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
<400> 6
Met Ala Arg Val Leu Lys Ala Ala Ala Ala Asn Ala Val Gly Leu
1 5 10 15
Phe Ser Arg Leu Gln Ala Pro Ile Pro Thr Val Arg Ala Ser Ser
20 25 30
Thr Ser Gln Pro Leu Asp Gln Val Thr Gly Ser Val Trp Asn Leu
35 40 45
Gly Arg Leu Asn His Val, Ala Ile Ala Val Pro Asp Leu Glu Lys
50 55 60
Ala Ala Ala Phe Tyr Lys Asn Ile Leu Gly Ala Gln Val Ser Glu
65 70 75
Ala Val Pro Leu Pro Glu His Gly Val Ser Val Val Phe Val Asn
80 85 90
Leu Gly Asn Thr Lys Met Glu Leu Leu His Pro Leu Gly Arg Asp
95 100 105
Ser Pro Ile Ala Gly Phe Leu Gln Lys Asn Lys Ala Gly Gly Met
110 115 120
His His Ile Cys Ile Glu Val Asp Asn Ile Asn Ala Ala Val Met
125 130 135
Asp Leu Lys Lys Lys Lys Ile Arg Ser Leu Ser Glu Glu Val Lys
140 145 150
Ile Gly Ala His Gly Lys Pro Val Ile Phe Leu His Pro Lys Asp
155 160 165
Cys Gly Gly Val Leu Val Glu Leu Glu Gln Ala
170 175
<210> 7
<211> 374
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2801723
<400> 7
Met Val Ala Ala Glu Leu Pro Cys Ala Phe Gln Thr Ile Leu Phe
1 5 10 15
Thr Val Leu Gly Thr Ala Glu Leu Gly Asp Val Gly Gly Val Leu
20 25 30
Gly Gly Thr Val Gly Ser Ser Arg Arg Leu Cys Glu Arg Val Leu
35 40 45
Val Thr Gly Gly Ala Gly Phe Ile Ala Ser His Met Ile Val Ser
50 55 60
Leu Val Glu Asp Tyr Pro Asn Tyr Met Ile Ile Asn Leu.Asp Lys
65 70 75
Leu Asp Tyr Cys Ala Ser Leu Lys Asn Leu Glu Thr Ile Ser Asn
80 85 90
Lys Gln Asn Tyr Lys Phe Ile G1n Gly Asp Ile Cys Asp Ser His
95 100 105
Phe Val Lys Leu Leu Phe Glu Thr Glu Lys Ile Asp Ile Val Leu
110 115 120
His Phe Ala Ala Gln Thr His Val Asp Leu Ser Phe Val Arg Ala
125 130 135
Phe Glu Phe Thr Tyr Val Asn Val Tyr Gly Thr His Val Leu Val
140 145 150
Ser Ala Ala His Glu Ala Arg Val Glu Lys Phe Ile Tyr Val Ser
155 160 165
Thr Asp Glu Val Tyr Gly Gly Ser Leu Asp Lys Glu Phe Asp Glu
170 175 180
Ser Ser Pro Lys Gln Pro Thr Asn Pro Tyr Ala Ser Ser Lys Ala
185 190 195
Ala Ala Glu Cys Phe Val Gln Ser Tyr Trp Glu Gln Tyr Lys Phe
200 205 210
Pro Val Val Ile Thr Arg Ser Ser Asn Val Tyr Gly Pro His Gln
215 220 225
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
Tyr Pro Glu Lys Val Ile Pro Lys Phe Ile Ser Leu Leu Gln His
230 235 240
Asn Arg Lys Cys Cys Ile His Gly Ser Gly Leu Gln Thr Arg Asn
245 250 255
Phe Leu Tyr Ala Thr Asp Val Val Glu Ala Phe Leu Thr Val Leu
260 265 270
Lys Lys Gly Lys Pro Gly Glu Ile Tyr Asn Ile Gly Thr Asn Phe
275 280 285
Glu Met Ser Val Val Gln Leu Ala Lys Glu Leu Ile Gln Leu Ile
290 295 300
Lys Glu Thr Asn Ser Glu Ser Glu Met Glu Asn Trp Val Asp Tyr
305 310 315
Val Asn Asp Arg Pro Thr Asn Asp Met Arg Tyr Pro Met Lys Ser
320 325 330
Glu Lys Ile His Gly Leu Gly Trp Arg Pro Lys Val Pro Trp Lys
335 340 345
Glu Gly Ile Lys Lys Thr Ile Glu Trp Tyr Arg Glu Asn Phe His
350 355 360
Asn Trp Lys Asn Val Glu Lys Ala Leu Glu Pro Phe Pro Val
365 370
<210> 8
<211> 780
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3130234
<400> 8
Met Ala Pro Tyr Ser Leu Leu Val Thr Arg Leu Gln Lys Ala Leu
1 5 10 15
Gly Val Arg Gln Tyr His Val Ala Ser Val Leu Cys Gln Arg Ala
20 25 30
Lys Val Ala Met Ser His Phe Glu Pro Asn Glu Tyr Ile His Tyr
35 40 45
Asp Leu Leu Glu Lys Asn Ile Asn Ile Val Arg Lys Arg Leu Asn
50 55 60
Arg Pro Leu Thr Leu Ser Glu Lys Ile Val Tyr Gly His Leu Asp
65 70 75
Asp Pro Ala Ser Gln Glu Ile Glu Arg Gly Lys Ser Tyr Leu Arg
80 85 90
Leu Arg Pro Asp Arg Val Ala Met Gln Asp Ala Thr Ala Gln Met
95 100 105
Ala Met Leu Gln Phe Ile Ser Ser Gly Leu Ser Lys Val Ala Val
110 115 120
Pro Ser Thr Ile His Cys Asp His Leu Ile Glu Ala Gln Val Gly
125 130 135
Gly Glu Lys Asp Leu Arg Arg Ala Lys Asp Ile Asn Gln Glu Val
140 145 150
Tyr Asn Phe Leu Ala Thr Ala Gly Ala Lys Tyr Gly Val Gly Phe
155 160 165
Trp Lys Pro Gly Ser Gly Ile Ile His Gln Ile Ile Leu Glu Asn
170 175 180
Tyr Ala Tyr Pro Gly Val Leu Leu Ile Gly Thr Asp Ser His Thr
185 190 195
Pro Asn Gly Gly Gly Leu Gly Gly Ile Cys Ile Gly Val Gly Gly
200 205 210
Ala Asp Ala Val Asp Val Met Ala Gly Ile Pro Trp Glu Leu Lys
215 220 225
Cys Pro Lys Val Ile Gly Val Lys Leu Thr Gly Ser Leu Ser Gly
230 235 240
Trp Ser Ser Pro Lys Asp Val Ile Leu Lys Val Ala Gly Ile Leu
245 250 255
Thr Val Lys Gly Gly Thr Gly Ala Ile Val Glu Tyr His Gly Pro
6
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
260 265 270
Gly Val Asp Ser Ile Ser Cys Thr Gly Met Ala Thr Ile Cys Asn
275 280 285
Met Gly Ala Glu Ile Gly Ala Thr Thr Ser Val Phe Pro Tyr Asn
290 295 300
His Arg Met Lys Lys Tyr Leu Ser Lys Thr Gly Arg Glu Asp Ile
305 310 315
Ala Asn Leu Ala Asp Glu Phe Lys Asp His Leu Val Pro Asp Pro
320 325 330
Gly Cys His Tyr Asp Gln Leu Ile Glu Ile Asn Leu Ser Glu Leu
335 340 345
Lys Pro His Ile Asn Gly Pro Phe Thr Pro Asp Leu Ala His Pro
350 355 360
Val Ala Glu Val Gly Lys Val Ala Glu Lys Glu Gly Trp Pro Leu
365 370 375
Asp Ile Arg Val Gly Leu Ile Gly Ser Cys Thr Asn Ser Ser Tyr
380 385 390
Glu Asp Met Gly Arg Ser Ala Ala Val Ala Lys Gln Ala Leu Ala
395 400 405
His Gly Leu Lys Cys Lys Ser Gln Phe Thr Ile Thr Pro Gly Ser
410 415 420
Glu Gln Ile Arg Ala Thr Ile Glu Arg Asp Gly Tyr Ala Gln Ile
425 430 435
Leu Arg Asp Leu Gly Gly Ile Val Leu Ala Asn Ala Cys Gly Pro
440 445 450
Cys Ile Gly Gln Trp Asp Arg Lys Asp Ile Lys Lys Gly Glu Lys
455 460 465
Asn Thr Ile Val Thr Ser Tyr Asn Arg Asn Phe Thr Gly Arg Asn
470 475 480
Asp Ala Asn Pro Glu Thr His Ala Phe Val Thr Ser Pro Glu Ile
485 490 495
Val Thr Ala Leu Ala Ile Ala Gly Thr Leu Lys Phe Asn Pro Glu
500 505 510
Thr Asp Tyr Leu Thr Gly Thr Asp Gly Lys Lys Phe Arg Leu Glu
515 520 525
Ala Pro Asp Ala Asp Glu Leu Pro Lys Gly Glu Phe Asp Pro Gly
530 535 540
Gln Asp Thr Tyr Gln His Pro Pro Lys Asp Ser Ser Gly Gln His
545 550 555
Val Asp Val Ser Pro Thr Ser Gln Arg Leu Gln Leu Leu Glu Pro
560 565 570
Phe Asp Lys Trp Asp Gly Lys Asp Leu Glu Asp Leu Gln Ile Leu
575 580 585
Ile Lys Val Lys Gly Lys Cys Thr Thr Asp His Ile Ser Ala Ala
590 595 600
Gly Pro Trp Leu Lys Phe Arg Gly His Leu Asp Asn Ile Ser Asn
605 610 615
Asn Leu Leu Ile Gly Ala Ile Asn Ile Glu Asn Gly Lys Ala Asn
620 625 630
Ser Val Arg Asn Ala Val Thr Gln Glu Phe Gly Pro Val Pro Asp
635 640 645
Thr Ala Arg Tyr Tyr Lys Lys His Gly Ile Arg Trp Val Val Ile
650 655 660
Gly Asp Glu Asn Tyr Gly Glu Gly Ser Ser Arg Glu His Ala Ala
665 670 675
Leu Glu Pro Arg His Leu Gly Gly Arg Ala Ile Ile Thr Lys Ser
680 685 690
Phe Ala Arg Ile His Glu Thr Asn Leu Lys Lys Gln Gly Leu Leu
695 700 705
Pro Leu Thr Phe Ala Asp Pro Ala Asp Tyr Asn Lys Ile His Pro
710 715 720
Val Asp Lys Leu Thr Ile Gln Gly Leu Lys Asp Phe Thr Pro Gly
725 730 735
Lys Pro Leu Lys Cys Ile Ile Lys His Pro Asn Gly Thr Gln Glu
740 745 750
Thr Ile Leu Leu Asn His Thr Phe Asn Glu Thr Gln Ile Glu Trp
755 760 765
7
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
Phe Arg Ala Gly Ser Ala Leu Asn Arg Met Lys Glu Leu Gln Gln
770 775 780
<210> 9
<211> 594
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3256118
<400> 9
Met Gly Lys Lys Ser Arg Val Lys Thr Gln Lys Ser Gly Thr Gly
1 5 10 15
Ala Thr Ala Thr Val Ser Pro Lys Glu Ile Leu Asn Leu Thr Ser
20 25 30
Glu Leu Leu Gln Lys Cys Ser Ser Pro Ala Pro Gly Pro Gly Lys
35 40 45
Glu Trp Glu Glu Tyr Val Gln Ile Arg Thr Leu Val Glu Lys Ile
50 55 60
Arg Lys Lys Gln Lys Gly Leu Ser Val Thr Phe Asp Gly Lys Arg
65 70 75
Glu Asp Tyr Phe Pro Asp Leu Met Lys Trp Ala Ser Glu Asn Gly
80 85 90
Ala Ser Val Glu Gly Phe Glu Met Val Asn Phe Lys Glu Glu Gly
95 100 105
Phe Gly Leu Arg Ala Thr Arg Asp Ile Lys Ala Glu Glu Leu Phe
110 115 120
Leu Trp Val Pro Arg Lys Leu Leu Met Thr Val Glu Ser Ala Lys
125 130 135
Asn Ser Val Leu Gly Pro Leu Tyr Ser Gln Asp Arg Ile Leu Gln
140 145 150
Ala Met Gly Asn Ile Ala Leu Ala Phe His Leu Leu Cys Glu Arg
155 160 165
Ala Ser Pro Asn Ser Phe Trp Gln Pro Tyr Ile Gln Thr Leu Pro
170 175 180
Ser Glu Tyr Asp Thr Pro Leu Tyr Phe Glu Glu Asp Glu Val Arg
185 190 195
Tyr Leu Gln Ser Thr Gln Ala Ile His Asp Val Phe Ser Gln Tyr
200 205 210
Lys Asn Thr Ala Arg Gln Tyr Ala Tyr Phe Tyr Lys Val Ile Gln
215 220 225
Thr His Pro His Ala Asn Lys Leu Pro Leu Lys Asp Ser Phe Thr
230 235 240
Tyr Glu Asp Tyr Arg Trp Ala Val Ser Ser Val Met Thr Arg Gln
245 250 255
Asn Gln Ile Pro Thr Glu Asp Gly Ser Arg Val Thr Leu Ala Leu
260 265 270
Ile Pro Leu Trp Asp Met Cys Asn His Thr Asn Gly Leu Ile Thr
275 280 285
Thr Gly Tyr Asn Leu Glu Asp Asp Arg Cys Glu Cys Val Ala Leu
290 295 300
Gln Asp Phe Arg Ala Gly Glu Gln Ile Tyr Ile Phe Tyr Gly Thr
305 310 315
Arg Ser Asn Ala Glu Phe Val Ile His Ser Gly Phe Phe Phe Asp
320 325 330
Asn Asn Ser His Asp Arg Val Lys Ile Lys Leu Gly Val Ser Lys
335 340 345
Ser Asp Arg Leu Tyr Ala Met Lys Ala Glu Val Leu Ala Arg Ala
350 355 360
Gly Ile Pro Thr Ser Ser Val Phe Ala Leu His Phe Thr Glu Pro
365 370 375
Pro Ile Ser Ala Gln Leu Leu Ala Phe Leu Arg Val Phe Cys Met
380 385 390
Thr Glu Glu Glu Leu Lys Glu His Leu Leu Gly Asp Ser Ala Ile
8
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
395 400 405
Asp Arg Ile Phe Thr Leu Gly Asn Ser Glu Phe Pro Val Ser Trp
410 415 420
Asp Asn Glu Val Lys Leu Trp Thr Phe Leu Glu Asp Arg Ala Ser
425 430 435
Leu Leu Leu Lys Thr Tyr Lys Thr Thr Ile Glu Glu Asp Lys Ser
440 445 450
Val Leu Lys Asn His Asp Leu Ser Val Arg Ala Lys Met Ala Ile
455 460 465
Lys Leu Arg Leu Gly Glu Lys Glu Ile Leu Glu Lys Ala Val Lys
470 475 480
Ser Ala Ala Val Asn Arg Glu Tyr Tyr Arg Gln Gln Met Glu Glu
485 490 495
Lys Ala Pro Leu Pro Lys Tyr Glu Glu Ser Asn Leu Gly Leu Leu
500 505 510
Glu Ser Ser Val Gly Asp Ser Arg Leu Pro Leu Val Leu Arg Asn
515 520 525
Leu Glu Glu Glu Ala Gly Val Gln Asp Ala Leu Asn Ile Arg Glu
530 535 540
Ala Ile Ser Lys Ala Lys Ala Thr Glu Asn Gly Leu Val Asn Gly
545 550 555
Glu Asn Ser Ile Pro Asn Gly Thr Arg Ser Glu Asn Glu Ser Leu
560 565 570
Asn Gln Glu Ser Lys Arg Ala Val Glu Asp Ala Lys Gly Ser Ser
575 580 585
Ser Asp Ser Thr Ala Gly Val Lys Glu
590
<210> 10
<211> 298
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4759250
<400> 10
Met Ala Ala Arg Arg Ala Leu His Phe Val Phe Lys Val Gly Asn
1 5 10 15
Arg Phe Gln Thr Ala Arg Phe Tyr Arg Asp Val Leu Gly Met Lys
20 25 30
Val Leu Arg His Glu Glu Phe Glu Glu Gly Cys Lys Ala Ala Cys
35 40 45
Asn Gly Pro Tyr Asp Gly Lys Trp Ser Lys Thr Met Val Gly Phe
50 55 60
Gly Pro Glu Asp Asp His Phe Val Ala Glu Leu Thr Tyr Asn Tyr
65 70 75
Gly Val Gly Asp Tyr Lys Leu Gly Asn Asp Phe Met Gly Ile Thr
80 85 90
Leu Ala Ser Ser Gln Ala Val Ser Asn Ala Arg Lys Leu Glu Trp
95 100 105
Pro Leu Thr Glu Val Ala Glu Gly Val Phe Glu Thr Glu Ala Pro
110 115 120
Gly Gly Tyr Lys Phe Tyr Leu Gln Asn Arg Ser Leu Pro Gln Ser
125 130 135
Asp Pro Val Leu Lys Val Thr Leu Ala Val Ser Asp Leu Gln Lys
140 145 150
Ser Leu Asn Tyr Trp Cys Asn Leu Leu Gly Met Lys Ile Tyr Glu
155 160 165
Lys Asp Glu Glu Lys Gln Arg Ala Leu Leu Gly Tyr Ala Asp Asn
170 175 180
Gln Cys Lys Leu Glu Leu Gln Gly Val Lys Gly Gly Val Asp His
185 190 195
Ala Ala Ala Phe Gly Arg Ile Ala Phe Ser Cys Pro Gln Lys Glu
200 205 210
9
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
Leu Pro Asp Leu Glu Asp Leu Met Lys Arg Glu Asn Gln Lys Ile
215 220 225
Leu Thr Pro Leu Val Ser Leu Asp Thr Pro Gly Lys Ala Thr Val
230 235 240
Gln Val Val Ile Leu Ala Asp Pro Asp Gly His Glu Ile Cys Phe
245 250 255
Val Gly Asp Glu Ala Phe Arg Glu Leu Ser Lys Met Asp Pro Glu
260 265 270
Gly Ser Lys Leu Leu Asp Asp Ala Met Ala Ala Asp Lys Ser Asp
275 280 285
Glu Trp Phe Ala Lys His Asn Lys Pro Lys Ala Ser Gly
290 295
<210> 11
<211> 1686
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 168714
<400> 11
gggctgtgag tctcccagcg tccccagctt tccaggtagg gacgccccct cccacgcagc 60
acggttccgg cgggtggaaa ggaggggctg ggctcccagc gcccgcccct ctatccatca 120
catggccgga gagtcacaaa aacaacagct ttggccaaga ccgtgacttc agtaaaggga 180
acccggggct ctcgcagcca gccctcctgc ccatggagga cagtttcctt caatcttttg 240
ggaggctgag cctccagccc cagcagcagc agcagcggca gcggccgccc cggccgcccc 300
cgcgggggac acctcctcgc cgccacagct ttaggaaaca cctctacctc ctgcgaggcc 360
tcccgggctc cgggaaaact acactggcca gacaattgca gcatgacttt cccagggccc 420
tgattttcag cacggatgat tttttcttca gggaagatgg tgcctatgag ttcaatcctg 480
acttcctgga ggaagctcat gaatggaacc aaaaaagagc aagaaaagca atgaggaatg 540
gcatatcccc cattattatt gataatacca acctccacgc ctgggaaatg aagccctatg 600
cagtcatggc acttgaaaat aactatgaag ttatattccg agaacctgac actcgctgga 660
aattcaacgt tcaagagtta gcaagaagaa acattcatgg tgtctcaaga gaaaaaatcc 720
accgaatgaa agaacggtat gaacacgatg ttacttttca cagtgtgctt catgcagaaa 780
agccaagcag aatgaacaga aaccaggaca ggaataatgc attgccttcc aacaatgcca 840
gatactggaa ttcctacaca gagtttccaa accggagggc ccacggtgga tttacaaatg 900
agagctccta tcacagaagg ggcggttgtc accatggata ttagaggcct atcttacagc 960
caggcagaat tttcctaagt cagtttctac ttcagttttt gttatttttt gttgcatttt 1020
agtcagagct ccaattccag tgtaaatagc tgaactcaaa agtttctgag caaagtcatt 1080
atattcactt tcttcaccaa aatttgttaa agtgcttcta tatgcatggt ctgatgctgg 1140
gaattctgca gatttgagta aacagtctct ttctctaggg taagaatttg aaaccaaaac 1200
ttgagaacac acccaagaat atatttacat aggttcatag atgaaataaa gtgtttatat 1260
tatatataag cttcagtacc atttgctctg aagtgatcta tttatttttt caggaaattc 1320
atctccatcg gtaaagttgg gaaggtggag agaagtggtg gggggcagga aaagttttag 1380
tgccattgct actttgataa tctatgtatc caaaaatgtg agatgtgcga ctcttatgat 1440
actgattttc ctttaatgtt aatatgccag aaagcataca tctaagggaa cattgtcctt 1500
caaagtagac actttgggaa gttatttctt tattttaatg atgtatcatt gttaaaaatg 1560
ctgtcaaatc cttaatagct acaggagcta ctgagggaaa tcagtgtcat tatttaaagt 1620
cacgccttgt gtttttacta ctttattcag caggattaaa cctgaataac ttttggctgt 1680
tgtgct 1686
<210> 12
<211> 2053
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1851727
<400> 12
caggcgggcc cccgcgcggc agggccctgg acccgcgcgg ctcccgggga tggtgagcaa 60
ggcgctgctg cgcctcgtgt ctgccgtcaa ccgcaggagg atgaagctgc tgctgggcat 120
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
cgccttgctg gcctacgtcg cctctgtttg gggcaacttc gttaatatga gctttctact 180
caacaggtct atccaggaaa atggtgaact aaaaattgaa agcaagattg aagagatggt 240
tgaaccacta agagagaaaa tcagagattt agaaaaaagc tttacccaga aatacccacc 300
agtaaagttt ttatcagaaa aggatcggaa aagaattttg ataacaggag gcgcagggtt 360
cgtgggctcc catctaactg acaaactcat gatggacggc cacgaggtga ccgtggtgga 420
caatttcttc acgggcagga agagaaacgt ggagcactgg atcggacatg agaacttcga 480
gttgattaac cacgacgtgg tggagcccct ctacatcgag gttgaccaga tataccatct 540
ggcatctcca gcctcccctc caaactacat gtataatcct atcaagacat taaagaccaa 600
tacgattggg acattaaaca tgttggggct ggcaaaacga gtcggtgccc gtctgctcct 660
ggcctccaca tcggaggtgt atggagatcc tgaagtccac cctcaaagtg aggattactg 720
gggccacgtg aatccaatag gacctcgggc ctgctacgat gaaggcaaac gtgttgcaga 780
gaccatgtgc tatgcctaca tgaagcagga aggcgtggaa gtgcgagtgg ccagaatctt 840
caacaccttt gggccacgca tgcacatgaa cgatgggcga gtagtcagca acttcatcct 900
gcaggcgctc cagggggagc cactcacggt atacggatcc gggtctcaga caagggcgtt 960
ccagtacgtc agcgatctag tgaatggcct cgtggctctc atgaacagca acgtcagcag 1020
cccggtcaac ctggggaacc cagaagaaca cacaatccta gaatttgctc agttaattaa 1080
aaaccttgtt ggtagcggaa gtgaaattca gtttctctcc gaagcccagg atgacccaca 1140
gaaaagaaaa ccagacatca aaaaagcaaa gctgatgctg gggtgggagc ccgtggtccc 1200
gctggaggaa ggtttaaaca aagcaattca ctacttccgt aaagaactcg agtaccaggc 1260
aaataatcag tacatcccca aaccaaagcc tgccagaata aagaaaggac ggactcgcca 1320
cagctgaact cctcactttt aggacacaag actaccattg tacacttgat gggatgtatt 1380
tttggctttt ttttgttgtc gtttaaagaa agactttaac aggtgtcatg aagaacaaac 1440
tggaatttca ttctgaagct tgctttaatg aaatggatgt gcctaaaagc tcccctcaaa 1500
aaactgcaga ttttgccttg cactttttga atctctcttt ttatgtaaaa tagcgtagat 1560
gcatctctgc gtattttcaa gtttttttat cttgctgtga gagcatatgt tgtgactgtc 1620
gttgacagtt ttatttactg gtttctttgt gaagctgaaa aggaacatta agcgggacaa 1680
aaaatgccga ttttatttat aaaagtgggt acttaataaa tgagtcgtta tactatgcat 1740
aaagaaaaat cctagcagta ttgtcaggtg gtggtgcgcc ggcattgatt ttagggcaga 1800
taaaagaatt ctgtgtgaga gctttatgtt tctcttttaa ttcagagttt ttccaaggtc 1860
tacttttgag ttgcaaactt gactttgaaa tattcctgtt ggtcatgatc aaggatattt 1920
gaaatcacta ctgtgttttg ctgcgtatct ggggcggggg caggttgggg ggcacaaagt 1980
taacatattc ttggttaacc atggttaaat atgctatttt aataaaatat tgaaactcaa 2040
aaaaaaaaaa aaa 2053
<210> 13
<211> 2490
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2095185
<400> 13
gctcgaggcg aggtggggta ggcgggcaag gcgggcgccg aggtttgcaa aggctcgcag 60
cggccagaaa cccggctccg agcggcggcg gcccggcttc cgctgcccgt gagctaagga 120
cggtccgctc cctctagcca gctccgaatc ctgatccagg cgggggccag gggcccctcg 180
cctcccctct gaggaccgaa gatgagcttc ctcttcagca gccgctcttc taaaacattc 240
aaaccaaaga agaatatccc tgaaggatct catcagtatg aactcttaaa acatgcagaa 300
gcaactctag gaagtgggaa tctgagacaa gctgttatgt tgcctgaggg agaggatctc 360
aatgaatgga ttgctgtgaa cactgtggat ttctttaacc agatcaacat gttatatgga 420
actattacag aattctgcac tgaagcaagc tgtccagtca tgtctgcagg tccgagatat 480
gaatatcact gggcagatgg tactaatatt aaaaagccaa tcaaatgttc tgcaccaaaa 540
tacattgact atttgatgac ttgggttcaa gatcagcttg atgatgaaac tctttttcct 600
tctaagattg gtgtcccatt tcccaaaaac tttatgtctg tggcaaagac tattctaaag 660
cgtctgttca gggtttatgc ccatatttat caccagcact ttgattctgt gatgcagctg 720
caagaggagg cccacctcaa cacctccttt aagcacttta ttttctttgt tcaggagttt 780
aatctgattg ataggcgtga gctggcacct cttcaagaat taatagagaa acttggatca 840
aaagacagat aaatgtttct tctagaacac agttaccccc ttgcttcatc tattgctaga 900
actatctcat tgctatttgt tatagactag tgatacaaac tttaagaaaa caggataaaa 960
agatacccat tgcctgtgtc tactgataaa attatcccaa aggtaggttg gtgtgatagt 1020
ttccgagtaa gaccttaagg acacagccaa atcttaagta ctgtgtgacc actcttgttg 1080
ttatcacata gtcatacttg gttgtaatat gtgatggtta acctgtagct tataaattta 1140
cttattattc ttttactcat ttactcagtc atttctttac aagaaaatga ttgaatctgt 1200
tttaggtgac agcacaatgg acattaagaa tttccatcaa taatttatga ataagtttcc 1260
agaacaaatt tcctaataac acaatcagat tggttttatt cttttatttt acgaataaaa 1320
11
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
aatgtatttt tcagtatcct tgagatttag aacatctgtg tcacttcaga taacatttta 1380
gtttcaagtt tgtatggtag tgtttttata gataagatac gtctattttt tcaaaattca 1440
tgattgcagt ttaaatcatc atatgacgtg tgggtgggag caaccaaagt tatttttaca 1500
gggactttat tttttgatct ttatttgaga ttgttttcat atctatctaa attattagga 1560
gtgtgtgtat cagaagtaat tttttaatgt cttctaagga tggtcttcca ggcttttaaa 1620
ctgaaaagct taattcagat agtagctttt ggctgagaaa aggaatccaa aatattaata 1680
aatttagatc tcaaaaccac tatttttatt atttcattat ttttcagagg ccttaaaatt 1740
ctggataaga gaatggagga aaatactcag agtacttgat tattttattt ccttttatta 1800
aaaaattact tctatgtttt tattgtctct tgagccttag ttaagagtag tgtagaaatg 1860
catgaacttc atcctaataa ggataaaact taaggaaaac cacaataaac catgaaggtg 1920
tacacatctt ataacacaga taaagttttg gtgtgctacc tattcttgag agagtgagtg 1980
agtgtatgtg tttaaaggaa acaaaatggg agaaataagt tttaaaaaaa tcctcatttt 2040
gttaatattc aaaagatgga ctgagcttcc acttgggttt tatcttgttt taattgtttt 2100
tgtatcaaaa cttgaaattc ctctatttct attgggatat aaaagccttc cccttcagtg 2160
aagaaaacat ttatttttta tttgattcct aggatttagt aaactctagc tgtctattta 2220
aaatgtactg aggcacaaca agtattatac tggaagactt gccaaactgg caaagcttta 2280
agttcatcag cattctatgt ggttcagagc tgtgattttt gcaaagtatt ttaccaacct 2340
cctcgatggc tttgataaag gttagatttg atgttttttt ttagatttat ttttcttact 2400
ccactaaact ataaagaaaa taattactta gaaactccat tttaaataat catttcctag 2460
aaattcttaa atatatacag aattttaaag 2490
<210> 14
<211> 1230
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2342959
<400> 14
gcgcgctgtc ctggctcggg agatggacgg ccgccgtgtt ttgggccggt tctggagtgg 60
ctggcggcgg ggcctgggtg tccgcccagt gcccgaggac gcaggctttg gcaccgaagc 120
ccggcatcag aggcaacccc gcggctcctg ccaacggtcg gggcccctcg gggaccagcc 180
cttcgcgggg ctgctgccaa aaaacctcag tcgggaggag ctggttgatg cgctgcgggc 240
agccgtggtg gaccggaaag gacctctagt gacgttgaac aagccacagg gtctaccagt 300
gacaggaaaa ccaggagagc tgacgttgtt ctcagtgctg ccagagctga gccagtccct 360
agggctcagg gagcaggagc ttcaggttgt ccgagcatct gggaaagaaa gctctgggct 420
tgtactcctc tccagctgtc cccagacagc tagtcgcctc cagaagtact tcacccatgc 480
acggagagcc caaaggccca cagccaccta ctgtgctgtc actgatggga tcccagctgc 540
ttctgagggg aagatccagg ctgccctgaa actggaacac attgatgggg tcaatctcac 600
agttccagtg aaggccccat cccgaaagga catcctggaa ggtgtcaaga agactctcag 660
tcactttcgt gtggtagcca caggctctgg ctgtgccctg gtccagctgc agccactgac 720
agtgttctcc agtcaactac aggtgcacat ggtactacag ctctgccctg tgcttgggga 780
ccacatgtac tctgcccgtg tgggcactgt cctgggccag cgatttctgc tgccagctga 840
gaacaacaag ccccaaagac aggtcctgga tgaagccctc ctcagacgcc tccacctgac 900
cccctcccag gctgcccagc tgcccttgca cctccaccta catcggctcc ttctcccagg 960
caccagggcc agggacaccc ctgttgagct cctggcacca ctgccccctt atttctccag 1020
gaccctacag tgcctggggc tccgcttaca atagtcctcc ctctgttcct gaccccctca 1080
cacacactgg aaagtgaggg tgggggctct gcagtcagac aaacctaaga tcacatcctg 1140
gacaggccac ttgcttgctg tgtggcattg ggcaagtaac tttacctctc tggacttgtg 1200
ataataaaag ttcctacctc aaaaaaaaaa 1230
<210> 15
<211> 955
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2613975
<400> 15
ctcctcgcga gatgccgagc attccggcct gggaagcgcg tgcagaagcg gaggtgctgc 60
tcatgggact tgtcggccgc cgtagcccct gctaggacag cccgtgcgag cctgctggag 120
12
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
gaggaagaga aaggcagaga gagtcgggtt acaagatggc ggatctgtag tagttaccgc 180
ggcggcggga gagcaagcga gccctggggg gcaaagagac gggagagtgg gtgtatgcgc 240
gggtgaagtg agaggtaacg gggcctccgg gcggagaggc ctcagtggct cttgtcaccc 300
cttctcgcgg ctgaaccttt ggagccatgg tgaattcggg cctctccgaa gccgccgccg 360
ccgccaccgc cactactgcc tttaccgtct cctaagagtg aggagcgcgg acgaggtaag 420
cgaggaggcg gcggctagag cggtggagac agcagccacc atgtcggata cgcggcggcg 480
agtgaaggtc tataccctga acgaagaccg gcaatgggac gaccgaggca ccgggcacgt 540
ctcctccact tacgtggagg agctcaaggg gatgtcgctg ctggttcggg cagagtccga 600
cggatcacta ctcttggaat caaagataaa tccaaatact gcatatcaga aacaacaggc 660
aagtagttgt ttatctttaa tttgaaagac ttcatctgtg atcaaggaag tattaatctg 720
acaaaggtgg gaaagctttc ctgacaagaa aaaaacatgt ttggtaaaca aagatcatgt 780
gtatttctct tgcaggttaa aagtttcaga ctgaaaaaaa gtttttgtac tggtgataat 840
tatcattttt ggattgagcc actgtcggtt tattctaaga tgtatttatt agtattattt 900
aactgtagtt agccaagctc ttctatacct tgacatgaaa ccttttattc tgagt 955
<210> 16
<211> 849
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2683534
<400> 16
cgtggctagt cttgacgtgg cgggttgctt tccaaaatgg cgcgggtgct gaaggctgca 60
gccgcgaatg ccgtagggct tttttccaga cttcaagctc ccattccaac agtaagagct 120
tcttccacat cacagccctt ggatcaagtg acaggttctg tgtggaacct gggtcgactc 180
aaccatgtag ccatagcagt gccagatttg gaaaaggctg cagcatttta taagaatatt 240
ctgggggccc aggtaagtga agcggtccct cttcctgaac atggagtatc tgttgttttt 300
gtcaacctgg gaaataccaa gatggaactg cttcatccat tgggacgtga cagtccaatt 360
gcaggttttc tgcagaaaaa caaggctgga ggaatgcatc acatctgcat cgaggtggat 420
aatattaatg cagctgtgat ggatttgaaa aaaaagaaga tccgcagtct aagtgaagag 480
gtcaaaatag gagcacatgg aaaaccagtg atttttctcc atcctaaaga ctgtggtgga 540
gtccttgtgg aactggagca agcttgattt atatttgcaa gcaactaaat taattgacct 600
gaaaaagcct atcaaatact atcaaaatgt actatgacat tgagtccttc actgcttcca 660
tcatgtaaaa gttcacagtt aaagactgaa ttacagaaag attaaaatat atacatatat 720
aaatacataa atatgtatat tatttagatt aacaaacata tttgttaatt tgaatttgaa 780
gaaaatcttg attactaatt acttagggaa cattattaaa atcatataga aataaattat 840
tcctcttct 849
<210> 17
<211> 1919
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2801723
<400> 17
gctgcctctg gctgctctgt taacgtgtcc cgcgagcgag gcgcgtccga aaatggtcgc 60
ggcggaactt ccctgcgctt ttcagaccat actctttacg gtactaggca ctgctgagct 120
gggagatgtc ggcggcgtgt tgggaggaac cgtggggtct tcccggcggc tttgcgaacg 180
ggtcctggtg accggcggtg ctggtttcat tgcatcacat atgattgtct ctttagtgga 240
agattatcca aactatatga tcataaatct agacaagctg gattactgtg caagcttgaa 300
gaatcttgaa accatttcta acaaacagaa ctacaaattt atacagggtg acatatgtga 360
ttctcacttt gtgaaactgc tttttgaaac agagaaaata gatatagtac tacattttgc 420
cgcacaaaca catgtagatc tttcattcgt acgtgccttt gagtttacct atgttaatgt 480
ttatggcact cacgttttgg taagtgctgc tcatgaagcc agagtggaga agtttattta 540
tgtcagcaca gatgaagtat atggtggcag tcttgataag gaatttgatg aatcttcacc 600
caaacaacct acaaatcctt atgcatcatc taaagcagct gctgaatgtt ttgtacagtc 660
ttactgggaa caatataagt ttccagttgt catcacaaga agcagtaatg tttatggacc 720
acatcaatat ccagaaaagg ttattccaaa atttatatct ttgctacagc acaacaggaa 780
atgttgcatt catgggtcag ggcttcaaac aagaaacttc ctttatgcta ctgatgttgt 840
13
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
agaagcattt ctcactgtcc tcaaaaaagg gaaaccaggt gaaatttata acatcggaac 900
caattttgaa atgtcagttg tccagcttgc caaagaacta atacaactga tcaaagagac 960
caattcagag tctgaaatgg aaaattgggt tgattatgtt aatgatagac ccaccaatga 1020
catgagatac ccaatgaagt cagaaaaaat acatggctta ggatggagac ctaaagtgcc 1080
ttggaaagaa ggaataaaga aaacaattga atggtacaga gagaattttc acaactggaa 1140
gaatgtggaa aaggcattag aaccctttcc ggtataatca ccatttatat agtcgagaca 1200
gttgtcaaag aagaaagtta tcctacctcg ccaagtggta tgaaattaag tgaccaaatg 1260
aagtgcactc ttttcttttg gaattagatt catgactttc tgtataaaat tcaaatgcag 1320
aatgcctcaa tctttgggag agtttcagta ctggcataga atttaaatgt caaaattctt 1380
tctgaaaccc tttctcctag aaactaggaa ataataggtg tagaagactc tccctaaggg 1440
tagccaggaa gaagtctcct gattcggaca accatgaggg gtagtggtgc tagggagaag 1500
gcaaccttca ctggttttga actcagtgcc taagaaagtc tctgaaatgt tcgtttttag 1560
gcaatatagg atgtcttagg ccctaattca ccatttcttt tttaagatct gatatgctat 1620
cattgcctta ataatggaac aaaatagaag catatctaac actttttaaa ttgataattt 1680
tgtaaaattg attacgttga atgcttttta agagaagtgt gtaaagtttt tatattttca 1740
caattaacgt atgtaaaacc ttgtatcaga aatttatcat gtttactgtt taaaatgatt 1800
gtatttataa aattgtcaat atcttaatgt atttaatgta gaatattgct ttttaaaata 1860
atgtttttat tttgctgtag aaaaataaaa aaaaatttga ttataaaaaa aaaaaaaaa 1919
<210> 18
<211> 2735
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3130234
<400> 18
ctttgtcagt gcacaaaatg gcgccctaca gcctactggt gactcggctg cagaaagctc 60
tgggtgtgcg gcagtaccat gtggcctcag tcctgtgcca acgggccaag gtggcgatga 120
gccactttga gcccaacgag tacatccatt atgacctgct agagaagaac attaacattg 180
ttcgcaaacg actgaaccgg ccgctgacac tctcggagaa gattgtgtat ggacacctgg 240
atgaccccgc cagccaggaa attgagcgag gcaagtcgta cctgcggctg cggccggacc 300
gtgtggccat gcaggatgcg acggcccaga tggccatgct ccagttcatc agcagcgggc 360
tgtccaaggt ggctgtgcca tccaccatcc actgtgacca tctgattgaa gcccaggttg 420
ggggcgagaa agacctgcgc cgggccaagg acatcaacca ggaagtttat aatttcctgg 480
caactgcagg tgccaaatat ggcgtgggct tctggaagcc tggatctgga atcattcacc 540
agattattct ggaaaactat gcgtaccctg gtgttcttct gattggcact gactcccaca 600
cccccaatgg tggcggcctt gggggcatct gcattggagt tgggggtgcc gatgctgtgg 660
atgtcatggc tgggatcccc tgggagttga agtgccccaa ggtgattggc gtgaagctga 720
cgggctctct ctccggttgg tcctcaccca aagatgtgat cctgaaggtg gcaggcatcc 780
tcacggtgaa aggtggcaca ggtgcaatcg tggaatacca cgggcctggt gtagactcca 840
tctcctgcac tggcatggcg acaatctgca acatgggtgc agaaattggg gccaccactt 900
ccgtgttccc ttacaaccac aggatgaaga agtacctgag caagaccggc cgggaagaca 960
ttgccaatct agctgatgaa ttcaaggatc acttggtgcc tgaccctggc tgccattatg 1020
accaactaat tgaaattaac ctcagtgagc tgaagccaca catcaatggg cccttcaccc 1080
ctgacctggc tcaccctgtg gcagaagtgg gcaaggtggc agagaaggaa ggatggcctc 1140
tggacatccg agtgggtcta attggtagct gcaccaattc aagctatgaa gatatggggc 1200
gctcagcagc tgtggccaag caggcactgg cccatggcct caagtgcaag tcccagttca 1260
ccatcactcc aggttccgag cagatccgcg ccaccattga gcgggacggc tatgcacaga 1320
tcttgaggga tctgggtggc attgtcctgg ccaatgcttg tggcccctgc attggccagt 1380
gggacaggaa ggacatcaag aagggggaga agaacacaat cgtcacctcc tacaacagga 1440
acttcacggg ccgcaacgac gcaaaccccg agacccatgc ctttgtcacg tccccagaga 1500
ttgtcacagc cctggccatt gcgggaaccc tcaagttcaa cccagagacc gactacctga 1560
cgggcacgga tggcaagaag ttcaggctgg aggctccgga tgcagatgag cttcccaaag 1620
gggagtttga cccagggcag gacacctacc agcacccacc caaggacagc agcgggcagc 1680
atgtggacgt gagccccacc agccagcgcc tgcagctcct ggagcctttt gacaagtggg 1740
atggcaagga cctggaggac ctgcagatcc tcatcaaggt caaagggaag tgtaccactg 1800
accacatctc agctgctggc ccctggctca agttccgtgg gcacttggat aacatctcca 1860
acaacctgct cattggtgcc atcaacattg aaaacggcaa ggccaactcc gtgcgcaatg 1920
ccgtcactca ggagtttggc cccgtccctg acactgcccg ctactacaag aaacatggca 1980
tcaggtgggt ggtgatcgga gacgagaact acggcgaggg ctcgagccgg gagcatgcag 2040
ctctggagcc tcgccacctt gggggccggg ccatcatcac caagagcttt gccaggatcc 2100
acgagaccaa cctgaagaaa cagggcctgc tgcctctgac cttcgctgac ccggctgact 2160
acaacaagat tcaccctgtg gacaagctga ccattcaggg cctgaaggac ttcacccctg 2220
14
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
gcaagcccct gaagtgcatc atcaagcacc ccaacgggac ccaggagacc atcctcctga 2280
accacacctt caacgagacg cagattgagt ggttccgcgc tggcagtgcc ctcaacagaa 2340
tgaaggaact gcaacagtga gggcagtgcc tccccgcccc gccgctggcg tcaagttcag 2400
ctccacgtgt gccatcagtg gatccgatcc gtccagccat ggcttcctat tccaagatgg 2460
tgtgaccaga catgcttcct gctccccgct tagcccacgg agtgactgtg gttgtggtgg 2520
gggggttctt aaaataactt tttagccccc gtcttcctat tttgagtttg gttcagatct 2580
taagcagctc catgcaactg tatttatttt tgatgacaag actcccatct aaagtttttc 2640
tcctgcctga tcatttcatt ggtggctgaa ggattctaga gaaccttttg ttcttgcaag 2700
gaaaacaaga atccaaaacc aaaaaaaaaa aaaaa 2735
<210> 19
<211> 2822
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3256118
<400> 19
cccgtccggc cgccgccgcc gccaccgccg ccaccgcctg ggggttggtt gaggcggacg 60
gcggggtccg ggccggagta cgtcgttccc gctgcgctag gggaagcggg cagtcagaaa 120
aatgggtaag aagagtcgag taaaaactca gaaatctggc actggtgcta cagcaactgt 180
gtcaccaaag gaaatcttga acctgaccag tgagctgctg cagaaatgca gcagtccggc 240
gcctggccca ggaaaagagt gggaagagta tgtgcagatc cggactctgg ttgagaaaat 300
acggaaaaag caaaaaggtc tgtccgttac ttttgatgga aaaagagaag attactttcc 360
tgatctaatg aaatgggcct ctgaaaatgg ggcttctgtc gagggttttg aaatggttaa 420
cttcaaagaa gagggctttg gtttgagagc aacaagagat atcaaggcag aagaattgtt 480
tttatgggtt ccacgaaaat tgctaatgac tgttgaatct gctaaaaatt cagtgttggg 540
gcccttatat tctcaagacc gaatccttca agccatggga aacatcgcac tggcctttca 600
tttgctgtgt gagcgagcca gccctaactc cttctggcag ccctatattc aaaccctccc 660
cagtgaatat gacactcctc tctactttga agaagatgaa gttcggtatc ttcagtccac 720
acaagctata catgatgtct tcagccagta taaaaacaca gctcgacagt acgcctactt 780
ctataaagtc atccagaccc atcctcatgc caacaaacta cccttgaagg attctttcac 840
ttacgaggac tacaggtggg cagtctcttc tgttatgacg aggcaaaacc aaattcccac 900
agaggatggt tcccgcgtga ccctggctct gattccttta tgggatatgt gtaaccacac 960
caacggcctg atcactactg gttacaacct ggaagatgac cgctgtgagt gtgtggctct 1020
gcaggatttt cgggctggag agcagattta cattttttat ggcactcgat ccaacgcaga 1080
gtttgtgatc cacagtggtt ttttctttga caataactca cacgacagag tgaaaataaa 1140
gcttggagtg agtaaaagtg acagactcta cgccatgaag gccgaggtct tggctcgtgc 1200
cggcatcccc acttccagtg tttttgcatt gcattttacc gagccgccca tctctgctca 1260
gcttttggct tttctccgag tattctgtat gactgaagaa gaactgaaag aacacttgct 1320
gggagacagc gctattgata gaatcttcac cttggggaac tcggaatttc ctgttagctg 1380
ggacaacgag gtcaaacttt ggacatttct tgaagataga gcctcacttc ttttaaaaac 1440
atataaaaca actattgagg aagataaatc cgtcttgaaa aaccacgatc tttctgttcg 1500
tgcaaaaatg gccatcaaat tgcgcttagg tgagaaagag attttggaaa aagcagtaaa 1560
gagtgcagct gtcaaccggg aatactatcg ccaacagatg gaggaaaagg ctccgcttcc 1620
caaatatgaa gagagtaacc ttgggctgtt ggagagcagc gtgggggact cgaggctccc 1680
cctggtcttg agaaacctcg aggaggaggc tggagtgcag gatgccttga acatcagaga 1740
ggcaatcagc aaagcaaagg ccacagaaaa cgggcttgta aacggtgaaa actctatccc 1800
taatgggacc aggtccgaaa atgaaagtct caatcaagaa agtaaaagag cagttgaaga 1860
cgccaaagga tcttcttcag acagcactgc tggagttaag gagtagctcg aggtgaagct 1920
ggatggggga tccagtggag caggagttga cggacagtcc gttcacatcg ctgtgtttcc 1980
ttgttaacat ttttctttct gcagagagga agatatgttt ttgctgcttt atataaaaat 2040
ggttttttta agttatttta aaaatctagc ttcccttttt gattaagatt gccatcttgc 2100
ttttaggcaa aacaaaccaa ttaacaaaca accacaagaa agggagaaga ggtgcctgtg 2160
ggagattttg cagacctatt gtgggtatag gtattttctt cctggggaag aattcagttc 2220
ccgtctcagc tgtacttttg tgggcctgtc atcttgatga ccagaatgaa agcttgctct 2280
gcctcctgcc agccagaatt ggtggcggga cttggggata cagcgtgaag gtggggaagt 2340
tgcacagcag aaaacagaat tgaagttggg aaactctaga gtctgggcaa aatgtttggt 2400
tttttctctt aaaaaaaata acaccccatt accaaaagaa aaggtaaggt ggcaacctta 2460
tttttaatag tttgaaatga tgataatcct aattatataa aaatatatat ataaacacac 2520
atatatatag tgatttctaa agatttgttt acttttgtgt tttgttttac tgtactaaga 2580
acttgtcctt tctccttgaa tcaaagtagg acatgcatca tcctcctaat tttaaatgtt 2640
ggctctgatt ttaaagtggt gcatttgatt ccagccttgg taatggagag tttgcaaaca 2700
cacagcggcc cacagcttca cgtggtggtg tgcagtgtga ggcagctcct tggctttcct, 2760
CA 02390689 2002-06-10
WO 01/44445 PCT/US00/33815
ggttttcaca acaagctaga gattttcaaa gctacacttt tgagtaaaaa cccttattaa 2820
2822
as
<210> 20
<211> 1774
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4759250
<400> 20
gtgacggctg cgtgcggcgg gaatcatggc tgctcgcaga gctctgcact tcgtattcaa 60
agtgggaaac cgcttccaga cggcgcgttt ctatcgggac gtcctgggga tgaaggttct 120
gcggcatgag gaatttgaag aaggctgcaa agctgcctgt aatgggcctt atgatgggaa 180
atggagtaaa acaatggtgg gatttgggcc tgaggatgat cattttgtcg cagaactgac 240
ttacaattat ggcgtcggag actacaagct tggcaatgac tttatgggaa tcacgctcgc 300
ttctagccag gctgtcagca acgccaggaa gctggagtgg ccactgacgg aagttgcaga 360
aggtgttttt gaaaccgagg ccccgggagg atataagttc tatttgcaga atcgcagtct 420
gcctcagtca gatcctgtat taaaagtaac tctagcagtg tctgatcttc aaaagtcctt 480
gaactactgg tgtaatctac tgggaatgaa aatttatgaa aaagatgaag aaaagcaaag 540
ggctttgctg ggctatgctg ataaccagtg taagctggag ctacagggcg tcaagggtgg 600
ggtggaccat gcagcagctt ttggaagaat tgccttctct tgcccccaga aagagttgcc 660
agacttagaa gacttgatga aaagggagaa ccagaagatt ctgactcccc tggtgagcct 720
ggacacccca gggaaagcaa cagtacaggt ggtcattctg gccgaccctg acggacatga 780
aatttgcttt gtcggggatg aagcatttcg agaactttct aagatggatc cagagggaag 840
caaattgttg gatgatgcaa tggcagcaga taaaagtgac gagtggtttg ccaaacacaa 900
taaacccaaa gcttcaggtt aacggaagac atgatgcaga gcaagcctct gtgattcctg 960
cccagcacct gtgaggcctg acgtgtcagt tcccaataaa tgctcttctg atttgtttcc 1020
cgtacaggca aggaggcttg ggtagtgcag atttgtgtat ttcaatcttt gaaagctctg 1080
atgtaattta gaaatgaaat ccaatcatga gtccaggtag agaacgcctg ctgtaatcta 1140
cactgttgct gggactgcgc attctgtata taactgtgtt ggatgagtga cagatgattg 1200
tccagactag gacagcggca tgaacatgac tttggttggg attgcggata gttagggtta 1260
cctctgaatc gtgtagcttt tatgagagca gctgtgcaag tgaatccaca ttaatgcctt 1320
gtcgtggtgc cattcccagc gcctgacgat acgctcttct attgtcttat tctggcaggt 1380
tttgacgttt taaatttttt aaagaaattt tattccttgg accaaaaggt ttggttaacc 1440
acccccctct tacttgcttt cacattttga gtgtccagag gaaacagaaa ggaatgagtg 1500
tgtgacgttg ctgcacgcct gactctgtgc gagcttcttt ctgtgtatat attttgtttt 1560
atttttttcc gtgtatattt ttaatcccga cagaacatca tgtgagattt ctttaaaatg 1620
gattaaacga tttcttcagc ctgaaaaaaa aggttttgaa aatgttttct tgtagttttg 1680
tttggttcta aacaacaaat aggttttaat cactcgaaat ggaattatat tgtgtattca 1740
ttgaataaat tttttttgaa agtaaaaaaa aaaa 1774
16
