Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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HUMAN HYDROLASE HOMOLOGS: N-TERMINAL ASPARAGINE AMIDOHYDROLASE,
GLYCOSYL HYDROLASE, GLUCOHYDROLASE, BIOTINIDASE, AND N
ACETYLGLUCOSAMINE 6-P DEACETYLASE
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human
hydrolase homologs and to the use of these sequences in the diagnosis,
treatment. and
prevention of reproductive. carbohydrate metabolism. and cell proliferation
disorders.
BACKGROUND OF THE INVENTION
to Hydrolysis is a common enzymatic mechanism. There are numerous enzymes
whose catalytic mechanism involves breaking a covalent bond in a substrate by
the
addition of a molecule of water across the bond. The reaction involves a
nucleophilic
attack by the water molecule's oxygen atom on a target bond within the
substrate. which
results in a splitting of the water molecule across the target bond, thereby
breaking the
15 bond and generating two product molecules. This general mechanism applies
to a wide
variety of enzymes. including phosphatases, glycosyl hydrolases,
lysophospholipases,
peptidases and amidohydrolases.
The protein phosphorylation/dephosphorylation cycle is one of the major
regulatory mechanisms employed by eukaryotic cells to control cellular
activities. During
~0 protein phosphorylation, phosphate groups are transferred from adenosine
triphosphate
molecules to a protein by protein kinases. During protein dephosphoryiation.
phosphate
groups are removed from a protein by protein phosphatases. using a hydrolytic
mechanism. In this manner. phosphatases are involved in the control of many
cellular
signaling events that regulate cell growth and differentiation. cell-to-cell
contact, the cell
25 cycle, and oncogenesis. (Cohen P. (1989) Annu. Rev. Biochem. 58:453-508.)
Glycosyl hydrolases are a large group of hydrolase enzymes that cleave the
glycosidic bond between two carbohydrates. or between a carbohydrate and a non-
carbohydrate moiety. Based on sequence similarity. these enzymes have been
classified
into at least ~4 families that are named according to their substrate
specificities. Examples
30 of glycosyl hy~drolases include glucosidases. glucanases. xylanases.
amylases.
galactosidases. sialidases. and mannosidases. Disorders of carbohydrate
metabolism are
frequently associated with glycosyl hydrolases. For example. mutations in a-1.-
1-
-1-
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2
glucosidase allow the accumulation of glycogen resulting in Pompe's disease,
while
Krabbe disease results from the accumulation of galactocerebroside due to non-
functional
galactosylceramidase. The buildup of mannose-containing oligosaccharides, or
mannosidosis, results from a-mannosidase insufficiency. Thus, the glycosyl
hydrolases
are involved in disorders of carbohydrate metabolism. (Davies G. and Henrissat
B. (1995)
Structure 3:853-859; PROSITE documents PDOC 00621 and PDOC 00511.)
Lysophospholipases (LPLs) are widely distributed enzymes that metabolize
intracellular
lipids, and occur in numerous isoforms. These isoforms vary in molecular mass,
the substrate
metabolized, and the optimum pH required for activity. Small isoforms.
approximately 15-30 kD,
function as hydrolases; large isoforms, those exceeding 60 kD. function both
as hydrolases and
transacylases. A particular substrate for LPLs, lysophosphatidylcholine,
causes lysis of cell
membranes when it is formed or imported into a cell. LPLs are regulated by
lipid factors
including acylcarnitine. arachidonic acid, and phosphatidic acid. These lipid
factors are signaling
molecules important in numerous pathways, including the inflammatory response.
(Anderson, R.
et al. (1994) Toxicol. Appl. Pharmacol. 125:176-183; Selle, H. et al. (1993);
Eur. J. Biochem.
212:411-416.)
Hydrolases are involved in many protein degradation pathways. Peptidases, such
as
pyroglutamyl peptidase and N-terminal asparagine amidohydrolase. hydrolyze
peptide bonds and
therefore participate in protein degradation. For example, N-terminal
asparagine amidohydrolase
cleaves the N-terminal asparagine from target proteins, thereby conferring
metabolic instability by
tagging the target for further degradation. Other hydrolases participate in
scavenging non-protein
cofactors during protein degradation. Biotin is an essential water-soluble
vitamin that acts as a
cofactor for various carboxvlases involved in normal metabolism. Biotinidase,
or biotin-amide
amidohydrolase, cleaves the biotin-lysine bond, allowing the recycling of
biotin during protein
35 degradation. Biotinidase deficiency, or multiple carboxylase deficiency,
can result from
insufficient recycling of biotin. (Rawlings N.D. and Barrett A.J. ( 1993)
Biochem. J. 290:205-218;
Barrett A.J. and Rawlings N.D. ( 1995) Arch. Biochem. Biophys. 318:247-250:
Pomponio R.J. et
al. (1997) Hum. Molec. Genet. 6:?39-745.)
The discovery of new human hydrolase homologs and the polynucleotides encoding
them
satisfies a need in the art by providing new compositions which are useful in
the diagnosis,
treatment, and prevention of reproductive, carbohydrate metabolism. and cell
proliferation
disorders.
SUMMARY OF THE INVENTION
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The invention features substantially purifed polypeptides, human hydrolase
homologs,
referred to collectively as "NHH" and individually as ''HHH-1", "HHH-2", "HHH-
3". "HHH-4",
"HHH-S". "HHH-6" and "HHH-7." In one aspect. the invention provides a
substantially purified
polypeptide comprising an amino acid sequence selected from the group
consisting of SEQ ID
NO:1. SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4. SEQ ID NO:S. SEQ ID N0:6. and SEQ
ID
N0:7 (SEQ ID NO:1 through SEQ ID N0:7) and fragments thereof.
The invention further provides a substantially purified variant having at
least 90% amino
acid identity to the amino acid sequences of SEQ ID NO:1 through SEQ ID N0:7.
or to a fragment
of any of these sequences. The invention also provides an isolated and
purified polynucleotide
encoding the poiypeptide comprising an amino acid sequence selected from the
croup consisting
of SEQ ID NO: l through SEQ ID N0:7 and fragments thereof. The invention also
includes an
isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity
to the polynucleotide encoding the polypeptide comprising an amino acid
sequence selected from
the group consisting of SEQ ID NO:1 through SEQ ID N0:7 and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide
which
hybridizes under stringent conditions to the polynucleotide encoding the
polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID NO:1
through SEQ ID
N0:7 and fragments thereof, as well as an isolated and purified polynucleotide
having a sequence
which is complementary to the polynucleotide encoding the polypeptide
comprising the amino
acid sequence selected from the group consisting of SEQ ID NO:I through SEQ ID
N0:7 and
fragments thereof.
The invention also provides an isolated and purified polynucleotide comprising
a
polynucleotide sequence selected from the group consisting of SEQ ID N0:8, SEQ
ID N0:9, SEQ
ID NO:10, S~Q ID NO:11, SEQ iD N0:12, SEQ ID N0:13, and SEQ ID N0:14 (SEQ ID
N0:8
through SEQ ID N0:14) and fragments thereof. The invention further provides an
isolated and
purified polynucleotide variant having at least 90% polynucleotide sequence
identity to the
polynucleotide sequence comprising a polynucleotide sequence selected from the
group consisting
of SEQ ID N0:8 through SEQ ID N0:14 and fragments there of, as well as an
isolated and
purified polynucleotide having a sequence which is complementary to the
polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:8
through SEQ ID N0:14 and fragments thereof.
The invention further provides an expression vector containing at least a
fragment of the
polynucieotide encoding the polypeptide comprising an amino acid sequence
selected from the
group consisting of SEQ ID NO:I through SEQ ID N0:7 and fragments thereof. In
another
aspect. the expression vector is contained within a host cell.
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4
The invention also provides a method for producing a polypeptide comprising
the amino
acid sequence selected from the group consisting of SEQ ID NO: I through SEQ
ID N0:7 and
fragments thereof. the method comprising the steps of: (a) culturing the host
cell containing an
expression vector containing at least a fragment of a polynucleotide encoding
the polypeptide
under conditions suitable for the expression of the polypeptide: and (b)
recovering the polypeptide
from the host cell culture.
The invention also provides a pharmaceutical composition comprising a
substantially
purified polypeptide having the amino acid sequence selected from the group
consisting of SEQ
ID NO:1 through SEQ ID N0:7 and fragments thereof in conjunction with a
suitable
pharmaceutical carrier.
The invention further includes a purified antibody which binds to a
polypeptide
comprising the amino acid sequence selected from the group consisting of SEQ
ID NO:1 through
SEQ ID N0:7 and fragments thereof. as well as a purified agonist and a
purified antagonist to the
polypeptide. The invention also provides a method for treating or preventing a
reproductive
disorder, the method comprising administering to a subject in need of such
treatment an effective
amount of an antagonist of the polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1 through SEQ ID N0:7 and fragments thereof. The
invention
further provides a method for treating or preventing an carbohydrate
metabolism disorder, the
method comprising administering to a subject in need of such treatment an
effective amount of an
antagonist of the polypeptide having an amino acid sequence selected from the
group consisting of
SEQ ID NO:1 through SEQ ID N0:7 and fragments thereof. The invention also
provides a
method for treating or preventing a cell proliferation disorder. the method
comprising
administering to a subject in need of such treatment an effective amount of an
antagonist of the
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1
through SEQ ID N0:7 and fragments thereof.
The invention also provides a method for detecting a polynucleotide encoding
the
polypeptide comprising the amino acid sequence selected from the group
consisting of SEQ ID
NO:1 through SEQ ID N0:7 and fragments thereof, in a biological sample
containing nucleic
acids, the method comprising the steps of: (a) hybridizing the complement of
the polynucleotide
sequence encoding the polypeptide comprising the amino acid sequence selected
from the group
consisting of SEQ ID NO:1 through SEQ ID N0:7 and fragments thereof. to at
least one of the
nucleic acids of the biological sample. thereby forming a hybridization
complex: and (b) detecting
the hybridization complex, wherein the presence of the hybridization complex
correlates with the
presence of a polynucleotide encoding the polypeptide in the biological
sample. In one aspect. the
nucleic acids of the biological sample are amplified by the polyrnerase chain
reaction prior to the
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hybridizing step.
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 methodology,
protocols. cell lines,
vectors, and reagents 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.
It must be noted that as used herein and in the appended claims, the singular
forms "a,"
l0 "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 methods and materials similar or equivalent to those
described herein can
be used in the practice or testing of the present invention, the preferred
methods, devices, and
materials are now described. All publications mentioned herein are cited for
the purpose of
describing and disclosing the cell lines, vectors, and methodologies 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
pnor mvent~on.
DEFINITIONS
''HHH," as used herein, refers to the amino acid sequences of substantially
purified HHH
obtained from any species, particularly a mammalian species, including bovine,
ovine, porcine,
murine, equine. and preferably the human species, from any source, whether
natural, synthetic,
semi-synthetic, or recombinant.
The term "agonist," as used herein. refers to a molecule which, when bound to
HHH,
increases or prolongs the duration of the effect of HHH. Aeonists may include
proteins, nucleic
acids, carbohydrates. or any other molecules which bind to and modulate the
effect of HHH.
An "allelic variant," as this term is used herein. is an alternative form of
the gene encoding
HHH. 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.
Anv given natural or recombinant eene may have none. one. or many allelic
forms. Common
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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 HHH. as described herein, include
those
sequences with deletions, insertions. or substitutions of different
nucleotides, resulting in a
polynucleotide the same as HHH or a polypeptide with at least one functional
characteristic of
HHH. Included within this definition are polymorphisms which may or may not be
readily
detectable using a particular oligonucleotide probe of the polynucleotide
encoding HHH, and
improper or unexpected hybridization to allelic variants, with a locus other
than the normal
chromosomal locus for the poiynucleotide sequence encoding HHH. 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 HHH.
Deliberate amino acid
substitutions may be made on the basis of similarity in polarity, charge,
solubility, hydrophobicity,
hydrophilicity, andlor the amphipathic nature of the residues, as long as the
biological or
immunological activity of HHH is retained. For example, negatively charged
amino acids may
include aspartic acid and glutamic acid, positively charged amino acids may
include lysine and
arginine, and amino acids with uncharged polar head groups having similar
hydrophilicity values
may include leucine, isoleucine, and valine; glycine and alanine; asparagine
and glutamine: serine
and threonine; and phenylalanine and tyrosine.
The terms "amino acid" or ''amino acid sequence," as used herein, refer to an
oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any
of these, and to
naturally occurring or synthetic molecules. In this context, ''fragments,"
"immunogenic
fragments," or "antigenic fragments'' refer to fragments of HHH which are
preferably at least 5 to
about 1~ amino acids in length, most preferably at least 14 amino acids, and
which retain some
biological activity or immunologicai activity of HHH. Where "amino acid
sequence" is recited
herein to refer to an amino acid 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," as used herein, relates to the production of additional
copies of a nucleic
acid sequence. Amplification is generally carried out using polymerise chain
reaction (PCR)
technologies well known in the art. (See, e.g.. Dieffenbach, C.W. and G.S.
Dveksier ( 1995) PCR
Primer. a Laboratory Manual, Cold Spring Harbor Press. Plainview, NY, pp.i-5.)
The term "antagonist." as it is used herein. refers to a molecule which, when
bound to
HHH, decreases the amount or the duration of the effect of the biological or
immunological
activity of HHH. Antagonists may include proteins. nucleic acids.
carbohydrates. antibodies, or
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any other molecules which decrease the effect of HHH.
As used herein, the term "antibody" refers to intact molecules as well as to
fragments
thereof, such as Fab, F(ab'),. and Fv fragments, which are capable of binding
the epitopic
determinant. Antibodies that bind HHH 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 conjueated to a
carrier protein if
desired. Commonly used carriers that are chemically coupled to peptides
include bovine serum
albumin, thyroglobulin, and keyhole limpet hemocyanin (1CLH). The coupled
peptide is then used
l0 to immunize the animal.
The term "antigenic determinant," as used herein, refers to that fragment 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 bind specifically to antigenic determinants
(given 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." as used herein, refers to any composition containing a
nucleic acid
sequence which is complementary to the ''sense" strand of a specific nucleic
acid sequence.
Antisense molecules may be produced by any method including synthesis or
transcription. Once
introduced into a cell, the complementary nucleotides combine with natural
sequences produced
by the cell to form duplexes and to block either transcription or translation.
The designation
"negative" can refer to the antisense strand, and the designation "positive''
can refer to the sense
strand.
As used herein, the term "biologically active," refers to a protein having
structural,
regulatory, or biochemical functions of a naturally occurring molecule.
Likewise,
"immunologically active" refers to the capability of the natural, recombinant.
or synthetic HHH, or
of any oligopeptide thereof, to induce a specific immune response in
appropriate animals or cells
and to bind with specific antibodies.
The terms ''complementary" or ''complementarily," as used herein, refer to the
natural
binding of polynucleotides by base pairing. For example, the sequence "A-G-T"
binds to the
complementary sequence "T-C-A." Complementarily between two single-stranded
molecules
may be "partial," such that only some of the nucleic acids bind, or it may be
"complete,'' such that
total complementarily exists between the single stranded molecules. The degree
of
complementarily between nucleic acid strands has significant effects on the
efficiency and strength
of the hybridization between the nucleic acid strands. This is of particular
importance in
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amplification reactions, which depend upon binding between nucleic acids
strands, and in the
design and use of peptide nucleic acid (PNA) molecules.
A "composition comprising a given polynucleotide sequence" or a "composition
comprising a given amino acid sequence." as these terms are used herein, 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 HHH or fragments of HHH 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," as used herein. refers to a nucleic acid sequence which
has been
resequenced to resolve uncalled bases, extended using XL-PCRTM (Perkin Elmer,
Norwalk, CT) in
the 5' and/or the 3' direction, and resequenced. or which has been assembled
from the overlapping
t5 sequences of more than one Incyte Clone using a computer program for
fragment assembly, such
as the GELVIEWTM Fragment Assembly system (GCG, Madison, WI). Some sequences
have
been both extended and assembled to produce the consensus sequence.
As used herein, the term "correlates with expression of a polynucleotide"
indicates that the
detection of the presence of nucleic acids. the same or related to a nucleic
acid sequence encoding
HHH. by Northern analysis is indicative of the presence of nucleic acids
encoding HHH in a
sample, and thereby correlates with expression of the transcript from the
polynucleotide encoding
HHH.
A "deletion," as the term is used herein. 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," as used herein. refers to the chemical modification of
a polypeptide
sequence, or a polynucleotide sequence. Chemical modifications of a
polynucleotide sequence
can include, for example, replacement of hydrogen by an alkyl, acyl, 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.
The term "similarity," as used herein, refers to a degree of complementariy.
There may
be partial similarity or complete similarity. The word "identity" may
substitute for the word
"similarity." A partially complementary sequence that at least partially
inhibits an identical
sequence trom hybridizing to a target nucleic acid is referred to as
''substantially similar." The
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inhibition of hybridization of the completely complementary sequence to the
target sequence may
be examined using a hybridization assay (Southern or Northern blot. solution
hybridization, and
the like) under conditions of reduced stringency. A substantially similar
sequence or hybridization
probe will compete for and inhibit the binding of a completely similar
(identical) sequence to the
target sequence under conditions of reduced stringency. This is not to say
that conditions of
reduced stringency are such that non-specific binding is permitted, as reduced
stringency
conditions require that the binding of two sequences to one another be a
specific (i.e., a selective)
interaction. The absence of non-specific binding may be tested by the use of a
second target
sequence which lacks even a partial degree of complementarily (e.g., less than
about 30%
similarity or identity). In the absence of non-specific binding, the
substantially similar sequence
or probe will not hybridize to the second non-complementary target sequence.
The phrases "percent identity" or "% identity" refer to the percentage of
sequence
similarity found in a comparison of two or more amino acid or nucleic acid
sequences. Percent
identity can be determined electronically, e.g., by using the MegAlignT"'
program (DNASTAR,
Inc., Madison WI). The MegAlignT'" program can create alignments between two
or more
sequences according to different methods, e.g., the clustal method. (See,
e.g., Higgins, D.G. and
P.M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups sequences
into clusters by
examining the distances between all pairs. The clusters are aligned pairwise
and then in groups.
The percentage similarity between two amino acid sequences, e.g., sequence A
and sequence B, is
calculated by dividing the length of sequence A, minus the number of gap
residues in sequence A,
minus the number of gap residues in sequence B, into the sum of the residue
matches between
sequence A and sequence B, times one hundred. Gaps of low or of no similarity
between the two
amino acid sequences are not included in determining percentage similarity.
Percent identity
between nucleic acid sequences can also be counted or calculated by other
methods known in the
art, e.g., the Jotun Hein method. (See, e.g., Hein, J. ( 1990) Methods
Enzymol. 183:626-645.)
Identity between sequences can also be determined by other methods known in
the art, e.g., by
varying hybridization conditions.
"Human artificial chromosomes'" (HACs), as described herein. 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 stable mitotic chromosome segregation
and maintenance.
(See, e.g., Harrington, J.J. et al. ( 1997) Nat Genet. 15:345-3~~.)
The term "humanized antibody." as used herein, refers to antibody molecules 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." as the term is used herein. refers to any process by which a
strand of
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nucleic acid binds with a complementary strand through base pairing.
As used herein. the term ''hybridization complex" refers to a complex formed
between two
nucleic acid sequences by virtue of the formation of hydrogen bonds bet,veen
complementary
bases. A hybridization complex may be formed in solution (e.g., Cot or R"t
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" or "addition," as used herein, refer to changes in an
amino acid or
nucleotide sequence resulting in the addition of one or more amino acid
residues or nucleotides,
10 respectively, to the sequence found in the naturally occurring molecule.
"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.
IS The term "microarray," as used herein, refers to an arrangement of distinct
polynucleotides arrayed on a substrate, e.g., paper, nylon or any other type
of membrane, filter,
chip, glass slide, or any other suitable solid support.
The terms ''element" or "array element" as used herein in a microarray
context. refer to
hybridizable polynucleotides arranged on the surface of a substrate.
The term "modulate," as it appears herein, refers to a change in the activity
of HHH. For
example, modulation may cause an increase or a decrease in protein activity,
binding
characteristics. or any other biological, functional, or immunological
properties of HHH.
The phrases "nucleic acid" or "nucleic acid sequence," as used herein. 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 antisense strand, to peptide nucleic acid
(PNA), or to any
DNA-like or RNA-like material. In this context, ''fragments" refers to those
nucleic acid
sequences which, when translated, would produce polypeptides retaining some
functional
characteristic, e.g., antigenicity, or structural domain characteristic, e.g.,
ATP-binding site, of the
full-length polypeptide.
The terms "operably associated' or "operably linked." as used herein, refer to
functionally
related nucleic acid sequences. A promoter is operably associated or operable
linked with a
coding sequence if the promoter controls the translation of the encoded
polypeptide. While
operably associated or operably linked nucleic acid sequences can be
contiguous and in the same
reading frame. certain genetic elements, e.g., repressor genes. are not
contiQUOUSIy linked to the
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11
sequence encoding the polypeptide but still bind to operator sequences that
control expression of
the polypeptide.
The term "oligonucleotide." as used herein. refers to a nucleic acid sequence
of at least
about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides.
and most preferably
about 20 to 25 nucleotides. which can be used in PCR amplification or in a
hybridization assay or
microarray. As used herein. the term "oligonucleotide" is substantially
equivalent to the terms
"amplimer," "primer," "oligomer," and "probe." as these terms are commonly
defined in the art.
"Peptide nucleic acid" (PNA), as used herein. refers to an antisense molecule
or anti-gene
agent which comprises an oligonucleotide of at least about ~ nucleotides in
length linked to a
l0 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. (See, e.g.,
Nielsen. P.E. et al. {1993) Anticancer Drug Des. 8:53-63.)
The term "sample," as used herein. is used in its broadest sense. A biological
sample
I S suspected of containing nucleic acids encoding HHH, or fragments thereof,
or HHH 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 solid
support; a tissue: a
tissue print; etc.
As used herein, the terms "specific binding" or "specifically binding'' refer
to that
20 interaction between a protein or peptide and an agonist. an antibody, or an
antagonist. 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 containing the
epitope A. or the presence
of free unlabeled A, in a reaction containing free labeled A and the antibody
will reduce the
25 amount of labeled A that binds to the antibody.
As used herein, the term "stringent conditions" refers to conditions which
permit
hybridization between polynucleotides and the claimed polynucleotides.
Stringent conditions can
be defined by salt concentration, the concentration of organic solvent. e.g..
formamide,
temperature. and other conditions well known in the art. In particular.
stringency can be increased
30 by reducing the concentration of salt. increasing the concentration of
forcnamide. or raising the
hybridization temperature.
The term "substantially purified," as used herein. refers to nucleic acid or
amino acid
sequences that are removed from their natural environment and are isolated or
separated, and are
at least about 60% free. preferably about 7~% free, and most preferably about
90% free from other
35 components with which they are naturally associated.
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12
A "substitution," as used herein. refers to the replacement of one or more
amino acids or
nucleotides by different amino acids or nucleotides, respectively.
"Transformation." as defined herein, describes a process by which exogenous
DNA enters
and changes 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 eukarvotic
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, 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 "variant" of HHH polypeptides. as used herein. refers to an amino acid
sequence that is
altered by one or more amino acid residues. The variant may have
"conservative" changes,
wherein a substituted amino acid has similar structural or chemical properties
(e.g., replacement of
(eucine with isoleucine). More rarely, a variant may have "nonconservative"
changes (e.g.,
replacement of glycine with tryptophan). Analogous minor variations may also
include amino
acid deletions or insertions. or both. Guidance in determining which amino
acid residues may be
substituted, inserted, or deleted without abolishing biological or
immunological activity may be
found using computer programs well known in the art, for example. LASERGENET"'
software.
The term "variant," when used in the context of a polynucleotide sequence, may
encompass a polynucleotide sequence related to HHH. This definition may also
include, for
example, "allelic" (as defined above). ''splice," "species.'' or "polymorphic"
variants. A splice
variant may have significant identity to a reference molecule, but will
generally have a greater or
lesser number of polynucleotides due to alternate splicing of exons during
mRNA processing. The
corresponding polypeptide may possess additional functional domains or an
absence of domains.
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
base. The presence
of SNPs may be indicative of. for example, a certain population. a disease
state. or a propensity for
a disease state.
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13
THE INVENTION
The invention is based on the discovery of new human hydrolase homologs (HHH),
the
polynucleotides encoding HHH, and the use of these compositions for the
diagnosis, treatment, or
prevention of reproductive, carbohydrate metabolism. and cell proliferation
disorders. In Table l,
columns 1 and 2 show the sequence identification numbers (SEQ ID NO:) of the
amino acid and
nucleic acid sequence. respectively. Column 3 shows the Clone ID of the Incyte
Clone in which
nucleic acids encoding each HHH .were first identified. and column 4, the cDNA
library of this
clone. Column ~ describes fragments, and shows the Incyte clones (and
libraries) and shotgun
sequences useful as fragments, e.g., in hybridization technologies, and which
are part of the
consensus nucleotide sequence of each HHH.
The columns of Table 2 show various properties of the polypeptides of the
invention:
column l references the SEQ ID NO; column 2 shows the number of amino acid
residues: column
3, potential phosphorylation sites; column ~, potential glycosylation sites:
column ~, the identity of
the protein; and column 6; analytical methods used to identify the protein
through sequence
homologies and protein motifs.
The columns of Table 3 show the tissue expression of each nucleic acid
sequence by
northern analysis, diseases or disorders associated with this tissue
expression, and the vector into
which each cDNA was cloned.
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14
TABLE 1
Protein NucleotideClone Library Fragments
lD
SEQ 1D SEQ ID
NO: NO:
321510H1 (EOSIHET02)
2593359H1 (LUNGNOT22)
1 8 321510 EOSIHET03 290233776 (DRGCNOTO1
)
3016872Ei1 (MUSCNOT07)
3513795F6 (ENDINOT02)
56227786 (NEUTLPTO1
)
634343H1 (NEUTGMTO1)
634343X12 (NEUTGMTOI)
2 9 634343 NEUTGMT01 634343X 14 (NEUTGMT01
)
634343X 17 (NEUTGMT01
)
121775671 (7VEUTGMT01
)
406420H1 (EOS1HET02)
75566986 (BRAITUT02)
75566976 (BRAITUT02)
3 10 2017918 THP1NOT01 915932H1 (BRSTNOT04)
2017918H1 (THPINOT01)
201791876 (THP1NOT01
)
3t 19919F6 (LUNGTUT13)
121549271 (BRSTTUT01)
1269841F1 (BRAINOT09)
133996971 (COLNTUT03)
1513403F1 (PANCTUT01)
1528049F1 (UCMCLSTO1)
165234976 (PROSTUT08)
4 I l 2175072 ENDCNOT03 1657538F6 (URETTUT01
)
1812758F6 (PROSTUT12)
1856319F6 (PROSNOT18)
2175072H1 (ENDCNOT03)
2613966H 1 (THYRNOT09)
3030372H1 (HEARFET02)
3773551H1 (BRSTNOT25)
121:148681 ( BRSTTUT01
)
! 517312 F6 ( PANCTUT01
)
2197153H1 (SPLNFET02)
12 2403107 ENDANOT01 '_'_6276376 (UTRSNOT02)
2403107H1 (ENDANOT01)
SANA00 E 53F 1 SANA00458F
1
SANA01878F1 SANA02367F1
1284408F6 (COLNNOT16)
i362518F6 (LUNGNOT12)
1514414F6 (PANCTUT01
)
6 13 3069540 UTRSNOR01 1578848F6 (DUODNOTO1
)
3069540H I (UTRSNOR01
)
3257058HI (OVARTUNO1)
030600H 1 (THP INOBOI
)
2016607H 1 (UTRSNOT08)
7 14 4182350 BRAUNOTO? ?445385F6 (THP1NOT03)
2812186F6 (OVARNOT10)
-1182350H 1 (BRAUNOT02)
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TABLE 2
Amino Potential Potential Analytical
Protein Acid PhosphorylationGlycosylationIdentificationMethod
SEQ 1D ResiduesSites Sites
NO:
S15 S58 N-terminal
T91 T98
5111 5145 asparaeine BLAST
S 193
1 310 5216 S58 N252 amidohydrolaseGI 1373365
5108
S136
S46 T78
T210
T246 S302 N39 N273 Vanin-1 BLAST
T424 N347
5_0 -X26 T336 N357 N41 GI 4138229
S370 l N468
S423
T326 S74 N133 Glycosyl hydrolaseBLOCKS
T135
3 346 T232 5269 (BL00592)
S303 T135
S179
S202 T236
S308 GIYcosyl hydrolase
4 401 S363 S17 N12 N88 BLOCKS
S61 (BL00775)
S308 S346
5367
5392
SS S93 T7
T 18
T105 5132
5134
5166 T241 GlucohydrolaseBLAST
S303
5 506 S344 S374 N386 GI 728850
T397
T441 S35
S 109
T352 S494
386 S113 S'l2 acetylglucosamineB
5329
6 G
53 618856
T201 S
_ 6-P deacetvlase
7 206 T128 S 132 Glycosyl hydrolasep~TS
C203
(PR00746)
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16
TABLE 3
Tissue Expression DiseaselClass
Seq ID Vector
NO:
(Fraction of Total)(Fraction of Total)
Reproductive (0.205)Cancer (0.432)
1 Gastrointestinal Inflammation (0.364)pBluescript
(0.182)
Cardiovascular (0.136)Fetal (0.136)
Hematopoietic/lmmuneInflammation (0.474)
(0.421 }
2 Gastrointestinal Cancer (0.421 ) pSPORTI
(0.316)
Reproductive (0.158)
Hematopoietic/ImmuneCancer (0.500)
(0.333)
3 Cardiovascular (0.250)Inflammation (0.500)pINCY
Nervous (0.250) Fetal (0.167)
Reproductive (0.340)Cancer (0.538)
4 Hematopoietic/lmmuneInflammation (0.292)pINCY
(0.142)
Gastrointestinal Fetal (0.179)
(0.132)
Reproductive (0.242)Fetal (0.333)
Cardiovascular (0.212)Inflammation (0.242)pINCY
Developmental (0.182)Cancer (0.212)
Reproductive (0.290)Cancer (0.677)
6 Gastrointestinal Inflammation (0.226)pINCY
(0.226)
Cardiovascular (0.129)Fetal (0.129)
Reproductive (0.308)Cancer (0.500)
7 Hematopoietic/ImmuneFetal (0.192} pINCY
(0.192)
Nervous (0.192) Inflammation (0.192)
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17
The invention also encompasses HHH variants. A preferred HHH variant is one
which
has at least about 80%, more preferably at least about 90%, and most
preferably at least about 95%
amino acid sequence identity to the HHH amino acid sequence, and which
contains at least one
functional or structural characteristic of HHH.
The invention also encompasses polynucleotides which encode HHH. In a
particular
embodiment, the invention encompasses polynucleotide sequences comprising the
sequences of
SEQ ID N0:8, SEQ ID N0:9. SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID
N0:13,
and SEQ ID N0:14 which encode HHH-1, HHH-2, HHH-3, HHH-4. HHH-5, HHH-b, and
HHH-
7, respectively.
The invention also encompasses a variant of a polynucleotide sequence encoding
HHH.
In particular, such a variant polynucleotide sequence will have at least about
80%, more preferably
at least about 90%, and most preferably at least about 95% polynucleotide
sequence identity to the
poiynucleotide sequence encoding HHH. A particular aspect of the invention
encompasses a
variant of SEQ ID N0:8 which has at least about 80%, more preferably at least
about 90%, and
most preferably at least about 95% polynucleotide sequence identity to SEQ ID
N0:8. The
invention further encompasses a polynucleotide variant of SEQ ID N0:9 having
at least about
80%, more preferably at least about 90%, and most preferably at least about
95% polynucleotide
sequence identity to SEQ ID N0:9. The invention further encompasses a
polynucleotide variant
of SEQ ID NO:10 having at least about 80%, more preferably at least about 90%,
and most
preferably at least about 95% polynucleotide sequence identity to SEQ ID
NO:10. The invention
further encompasses a polynucleotide variant of SEQ ID NO:11 having at least
about 80%, more
preferably at least about 90%. and most preferably at least about 95%
polynucleotide sequence
identity to SEQ ID NO:11. The invention further encompasses a polynucleotide
variant of SEQ
ID N0:12 having at least about 80%. more preferably at least about 90%. and
most preferably at
least about 95% polynucleotide sequence identity to SEQ ID N0:12. The
invention further
encompasses a polynucleotide variant of SEQ ID N0:13 having at least about
80%, more
preferably at least about 90%, and most preferably at least about 95%
polynucleotide sequence
identity to SEQ ID N0:13. The invention further encompasses a polynucleotide
variant of SEQ
ID N0:14 having at least about 80%. more preferably at least about 90%. and
most preferably at
least about 95% polynucieotide sequence identity to SEQ ID N0:14. Any one of
the
poiynucieotide variants described above can encode an amino acid sequence
which contains at
least one functional or structural characteristic of HHH.
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 HHH. some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene. may be
CA 02329076 2000-11-28
WO 99161626 PCT/US99/12021
18
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 HHH. and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode HHH and its variants are preferably
capable
of hybridizing to the nucleotide sequence of the naturally occurring HHH under
appropriately
selected conditions of stringency, it may be advantageous to produce
nucleotide sequences
encoding HHH 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 HHH 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 HHH
and
HHH 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 HHH 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
N0:8 through SEQ ID N0:14 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.) For example, stringent salt
concentration will ordinarily
be less than about 750 mM NaCI and 75 mM trisodium citrate, preferably less
than about 500 mM
NaCI and ~0 mM trisodium citrate, and most preferably less than about 250 mM
NaCI and 25 mM
trisodium citrate. Low stringency hybridization can be obtained in the absence
of organic solvent,
e.g., formamide. while high stringency hybridization can be obtained in the
presence of at least
about 35% formamide. and most preferably at least about 50% formamide.
Stringent temperature
conditions will ordinarily include temperatures of at least about 30°C,
more preferably of at least
about 37°C, and most preferably of at least about 42°C. Varying
additional parameters, such as
hybridization time. the concentration of detergent. e.g., sodium dodecyl
sulfate (SDS). and the
inclusion or exclusion of carrier DNA, are well known to those skilled in the
art. Various levels of
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I9
stringency are accomplished by combining these various conditions as needed.
In a preferred
embodiment, hybridization will occur at 30°C in 750 mM NaCI. 7~ mM
trisodium citrate, and I%
SDS. In a more preferred embodiment. hybridization will occur at 37°C
in X00 mM NaCI, 50 mM
trisodium citrate, 1% SDS, 3~% formamide, and 100 ~cg/ml denatured salmon
sperm DNA
(ssDNA). In a most preferred embodiment, hybridization will occur at
42°C in 250 mM NaCI, 25
mM trisodium citrate, i% SDS, 50 % formamide, and 200 ~g/ml ssDNA. Useful
variations on
these conditions will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash
stringency conditions can be defined by sail concentration and by temperature.
As above, wash
stringency can be increased by decreasing salt concentration or by increasing
temperature. For
example, stringent salt concentration for the wash steps will preferably be
less than about 30 mM
NaCI and 3 mM trisodium citrate. and most preferably less than about 15 mM
NaCI and 1.5 mM
trisodium citrate. Stringent temperature conditions for the wash steps will
ordinarily include
temperature of at least about 25°C, more preferably of at least about
42°C. and most preferably of
at least about 68°C. In a preferred embodiment, wash steps will occur
at 25°C in 30 mM NaCI, 3
mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps
will occur at
42°C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS. In a most
preferred embodiment,
wash steps will occur at 68°C in 1 ~ mM NaCI, 1.5 mM trisodium citrate,
and 0.1 % SDS.
Additional variations on these conditions will be readily apparent to those
skilled in the art.
Methods for DNA sequencing and analysis are well known and generally available
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 poiymerase I, SEQUENASE~
(Amersham
Pharmacia Biotech, Piscataway NJ), Taq polymerase (Perkin Elmer). thetmostable
T7 polymerase
(Amersham Phatmacia Biotech), or combinations of polymerases and proofreading
exonucleases
such as those found in the ELONGASE Amplification System (Life Technologies,
Gaithersburg
MD). Preferably, sequence preparation is automated with machines such as the
ABI Catalyst 800
(Perkin Elmer) or a Hamilton Micro Lab 2200 (Hamilton. Reno NV) in combination
with thenmal
cyclers (for PCR). Sequencing is then carried out using either ABI 373 or 377
DNA sequencers
(Perkin Elmer) or the MEGABASE capillary electrophoresis (Molecular Dynamics),
and the
sequences are analyzed using tools (computer programs and algorithms) which
are well known in
the art. (Ausubel (1997, unit 7.7); Meyers, R.A. (1995; Molecular Bioiogv and
Biotechnolosy,
Wiley VCH, lnc, New York NY. p 856-853).
The nucleic acid sequences encoding HHH 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 e~campie. one method
which may be
CA 02329076 2000-11-28
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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
5 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 LNIA fragments adjacent to known sequences in human and yeast
artificial
chromosome DNA. (See, e.g., Lagerstrom. M. et al. (1991) PCR Methods Appiic.
1:111-119.) In
this method, multiple restriction enzyme digestions and ligations may be used
to insert an
l0 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. 19:3055-306). Additionally,
one may use PCR,
nested primers, and PromoterFinderT'" libraries to walk genomic DNA (Clontech,
Palo Alto. CA).
This procedure avoids the need to screen libraries and is useful in finding
intron/exon junctions.
15 For all PCR-based methods, primers may be designed using commercially
available software, such
as OLIGOT"' 4.06 Primer Analysis software (National Biosciences Inc.,
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
20 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.
Capillary electrophoresis systems which are commercially available may be used
to
analyze the size or confirm the nucleotide sequence of sequencing or PCR
products. tn 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., GenotyperTM and Sequence NavigatorTM, Perkin
Elmer), 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 HHN may be cloned in recombinant DNA molecules that direct
expression of HHH,
or fragments or functional equivalents thereof. in appropriate host cells. Due
to the inherent
CA 02329076 2000-11-28
WO 99161626 PCT/US99/12021
21
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 HHH.
The nucleotide sequences of the present invention can be engineered using
methods
generally known in the art in order to alter HHH-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.
In another embodiment, sequences encoding HHH 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) Nucl.
Acids Res. Symp. Ser. 21 S-223, and Horn, T. et al. ( 1980) Nucl. Acids Res.
Symp. Ser. 225-232.)
Alternatively, HHH itself or a fragment thereof may be synthesized using
chemical methods. For
l5 example, peptide synthesis can be performed using various solid-phase
techniques. (See, e.g.,
Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be
achieved using
the ABI 431A Peptide Synthesizer (Perkin Eimer). Additionally, the amino acid
sequence of
HHH, 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.
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, T. ( 1984) Proteins, Structures and
Molecular Properties, WH
Freeman and Co., New York, NY.)
In order to express a biologically active NHH, the nucleotide sequences
encoding HHH 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' untransiated regions in
the vector and in
polynucleotide sequences encoding HHH. Such elements may vary in their
strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding HHH. Such signals include the ATG initiation codon and
adjacent sequences,
e.g. the Kozak sequence. (n cases where sequences encoding HHH and its
initiation codon and
upstream regulatory sequences are inserted into the appropriate expression
vector, no additional
transcriptional or transiational control signals may be needed. However, in
cases where only
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22
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-I 62.)
Methods which are well known to those skilled in the art may be used to
construct
expression vectors containing sequences encoding HHH 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; and Ausubel. F.M. et al. ( 1995, and periodic supplements) Current
Protocols in Molecular
BioloQV, 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 HHH. 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. The invention
is not limited 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 HHH. For
example,
routine cloning, subcloning, and propagation of polynucleotide sequences
encoding HHH can be
achieved using a multifunctional E, coli vector such as Bluescript~
(Stratagene) or pSportlT'"
plasmid (GIBCO BRL). Ligation of sequences encoding HHH 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 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 HHH are needed. e.g. for
the production
of antibodies, vectors which direct high level expression of HHH 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 HHH. A number of
vectors
containing constitutive or inducible promoters. such as alpha factor. alcohol
oxidase. and PGH,
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23
may be used in the yeast Saccharomyces cerevisiae or Pichiapastoris. 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, supra; and
Grant et al. { 1987) Methods Enzymol. 153:516-54: Scorer. C. A. et al. (1994)
Biol1"echnology
12:1$1-184.)
Plant systems may also be used for expression of HHH. Transcription of
sequences
encoding HHH may be driven viral promoters. e.g., the 35S and 195 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. G. et al. (1984) EMBO
J. 3:1671-1680;
Broglie, R. et al. ( 1984) Science 224:838-843; and Winter, J. et al. ( 1991 )
Results Probl. Cell
Differ. 17:85-105.) These constructs can be introduced into plant cells by
direct DNA
transformation or pathogen-mediated transfection. (See, e.g., Hobbs, S. or
Murry, L.E. in
McGraw Hill Yearbook of Science and Technolosy ( 1992) McGraw Hill, New York,
NY; pp.
191-196.)
1n 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 HHH
may be ligated
into an adenovirus transcription/translation complex consisting of the late
promoter and tripartite
leader sequence. Insertion in a non-essential Ei or E3 region of the viral
genome may be used to
obtain infective virus which expresses HHH in host cells. (See, e.g., Logan,
J. and T. Shenk
( 1984) Proc. Natl. Acad. Sci. 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.
For long term production of recombinant proteins in mammalian systems, stable
expression of HHH in cell lines is preferred. For example, sequences encoding
HHH can be
transformed into cell lines using expression vectors which may contain viral
origins of replication
and/or endogenous 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 i 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 aeent. and its presence allows
growth and recovery of
cells which successfully express the introduced sequences. Resistant clones of
stably transformed
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24
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 or api cells. respectively.
(See, e.g., Wigler, M. et
S al. ( 1977) Cell 11:223-232: and 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 or pat confer resistance to chlorsulfuron and phosphinotricin
acetyltransferase,
respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
77:3567-3570;
Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14; and Murry, supra.)
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.
85:8047-8051.) Visible
markers, e.g., anthocyanins, green fluorescent proteins (GFP) (Clontech, Palo
Alto, CA), B
glucuronidase and its substrate (3-D-glucuronoside, or luciferase and its
substrate fuciferin 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. et al. ( 1995) Methods Mol. Biol. SS: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 HHH is inserted within a marker gene
sequence, transformed
cells containing sequences encoding HHH can be identified by the absence of
marker gene
function. Alternatively, a marker gene can be placed in tandem with a sequence
encoding HHH
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.
2S In general, host cells that contain the nucleic acid sequence encoding HHH
and that
express HHH 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 HHH using
either
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
3S monoclonal antibodies reactive to two non-interfering epitopes on HHH is
preferred. but a
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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. Section IV; Coligan, J. E. et al. ( 1997 and periodic supplements)
Current Protocols in
lmmunoloay, Greene Pub. Associates and Wilev-interscience, New York, NY; and
Maddox. D.E.
~ et al. ( 1983) J. Exp. Med. 158:1211-1216).
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 polynucieotides
encoding HHH
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled
10 nucleotide. Alternatively, the sequences encoding HHH. 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
IS Pharmacia & Upjohn (Kalamazoo, MI), Promega (Madison, WI), and U.S.
Biochemical Corp.
(Cleveland, OH). Suitable reporter molecules or labels which may be used for
ease of detection
include radionuciides, enrymes, fluorescent, chemiluminescent, or chromogenic
agents. as well as
substrates. cofactors, inhibitors, magnetic particles. and the like.
Host cells transformed with nucleotide sequences encoding HHH may be cultured
under
20 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 HHH may be designed to contain
signal
sequences which direct secretion of HHH through a prokaryotic or eukaryotic
cell membrane.
25 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-translationai processing
which cleaves a "prepro"
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. Bethesda. MD) and may be chosen to
ensure the
correct modification and processing of the foreign protein.
In another embodiment of the invention. natural. modified. or recombinant
nucleic acid
sequences encoding HHH may be ligated to a heterologous sequence resulting in
translation of a
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26
fusion protein in any of the aforementioned host systems. For example. a
chimeric HHH protein
containing a heterologous moiety that can be recognized by a commercially
available antibody
may facilitate the screening of peptide libraries for inhibitors of HHH
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, Ttx. 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-mvc, and
hemagglutinin (HA) enable
l0 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 proteolvtic cleavage site located between the HHH
encoding sequence and
the heterologous protein sequence, so that HHH may be cleaved away from the
heterologous
moiety following purification. Methods for fusion protein expression and
purification are
discussed in Ausubel, F. M. et at. ( 1995 and periodic supplements) Current
Protocols in Molecular
Biolosv, John Wiley & Sons, New York, NY, 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 radiolabeied HHH may be
achieved
in vitro using the TNTT'" rabbit reticulocyte iysate or wheat germ extract
systems (Promega,
Madison, WI). 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. preferably '3S-methionine.
Fragments of HHH may be produced not only by recombinant production, but also
by
direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton.
supra pp. 55-60.)
Protein synthesis may be performed by manual techniques or by automation.
Automated synthesis
may be achieved, for example. using the Applied Biosystems 431 A Peptide
Synthesizer (Perkin
Elmer). Various fragments of HHH may be synthesized separately and then
combined to produce
the full length molecule.
THERAPEUTICS
Chemical and structural similarity exists between HHH-1 and N-terminal
asparagine
amidohydrolase from mouse (GI 1373365). In addition. HHH-I is expressed in
libraries from
reproductive and gastrointestinal tissues. Chemical and structural similarity
exists between HHH-
2 and vanin-1 from mouse (GI 4138229). In addition. HHH-2 is expressed in
libraries from
hematopoietic/immune and gastrointestinal tissues. Protein sequence analysis
identifies HHH-3 as
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27
a glycosyl hydrolase (BL00592). In addition. HHH-3 is expressed in libraries
from
hematopoietic/immune and cardiovascular tissues. Protein sequence analysis
identifies HHH-4 as
a glycosyl hydrolase (BL007775). In addition. HHH-4 is expressed in libraries
from reproductive
and hematopoietic/immune tissues. Chemical and structural similarity exists
between HHH-S and
glucohydrolase from yeast (G1 728850). In addition. HHH-5 is expressed in
libraries from
reproductive and cardiovascular tissues. Chemical and structural similarity
exists between HHH-6
and N-acetylglucosamine 6-P deacetylase from Bacillus subtiiis (GI 2618856),
and HHH-6
contains a signal peptidases signature sequence. In addition, HHH-6 is
expressed in libraries from
reproductive and gastrointestinal tissues. Protein sequence analysis
identifies HHH-7 as a
glycosyl hydrolase (PR00746). In addition, HHH-7 is expressed in libraries
from reproductive
and hematopoietic/immune tissues. Finally, HHH is associated with libraries
made from
cancerous or proliferative tissues. Therefore. HHH appear to play roles in
reproductive.
carbohydrate metabolism, and cell proliferation disorders.
Therefore, in one embodiment, an antagonist of HHH may be administered to a
subject to
treat or prevent a reproductive disorder. Such a disorder may include, but is
not limited to,
disorders of prolactin production; infertility, including tubal disease,
ovulatory defects, and
endometriosis; disruptions of the estrous cycle. disruptions of the menstrual
cycle, polycystic
ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian
tumors, uterine
fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis; cancer
of the breast,
fibrocystic breast disease, and galactorrhea; disruptions of spermatogenesis,
abnormal sperm
physiology, cancer of the testis, cancer of the prostate, benign prostatic
hyperplasia. prostatitis,
Peyronie's disease. carcinoma of the male breast, and gynecomastia. In one
aspect, an antibody
which specifically binds HHH may be used directly as an antagonist or
indirectly as a targeting or
delivery mechanism for bringing a pharmaceutical agent to cells or tissue
which express HHH.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding HHH may be administered to a subject to treat or prevent a
reproductive disorder
including, but not limited to, those described above.
In another embodiment, HHH or a fragment or derivative thereof may be
administered to a
subject to treat or prevent a disorder of carbohydrate metabolism. Such
disorders can include, but
are not limited to, diabetes, insulin-dependent diabetes mellitus, non-insulin-
dependent diabetes
mellitus, hypoglycemia, glucagonoma, galactosemia, hereditary fructose
intolerance,
fructose-l.6-diphosphatase deficiency, obesity. congenital type II
dyserythropoietic anemia.
mannosidosis, neuraminidase deficiency. galactose epimerase deficiency,
glycogen storage
diseases, lysosomal storage diseases. fructosuria. pentosuria. and inherited
abnormalities of
pyruvate metabolism.
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28
In yet another embodiment, a vector capable of expressing HHH or a fragment or
derivative thereof may be administered to a subject to treat or prevent a
disorder of carbohydrate
metabolism including, but not limited to, those described above.
In a further embodiment, a pharmaceutical composition comprising a
substantially
purified HHH in conjunction with a suitable pharmaceutical carrier may be
administered to a
subject to treat or prevent a disorder of carbohydrate metabolism including,
but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of HHH
may be
administered to a subject to treat or prevent a disorder of carbohydrate
metabolism including, but
not limited to, those listed above.
In yet another embodiment, an antagonist of HHH may be administered to a
subject to
treat or prevent a cell proliferation disorder. Such a disorder may include,
but is not limited to,
actinic keratosis, arteriosclerosis, atherosclerosis. bursitis, cirrhosis,
hepatitis, mixed connective
tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera,
t5 psoriasis, primary thrombocythemia, and cancers including adenocarcinoma,
leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and. in particular,
cancers 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 one aspect, an
antibody which specifically binds HHH may be used directly as an antagonist or
indirectly as a
targeting or delivery mechanism for bringing a pharmaceutical agent to cells
or tissue which
express HHH.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding HHH may be administered to a subject to treat or prevent a cell
proliferation disorder
including cancer such as 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 treatmem
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 HHH may be produced using methods which are generally known
in the
art. In particular. purified HHH may be used to produce antibodies or to
screen libraries of
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29
pharmaceutical agents to identify those which specifically bind HHH.
Antibodies to HHH may
also be generated using methods that are well known in the art. Such
antibodies may include, but
are 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 especially 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 HHH or with any fragment
or
oligopeptide thereof which has immunogenic properties. Dependine 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
Corvnebacterium parvum
are especially preferable.
It is preferred that the oligopeptides. peptides, or fragments used to induce
antibodies to
5 HHH have an amino acid sequence consisting of at least about 5 amino acids,
and, more
preferably, 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 and
contain the entire amino acid sequence of a small, naturally occurring
molecule. Short stretches of
HHH 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 HHH 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 26:495-497;
Kozbor. D. et al.
( 1985) J. Immunol. Methods 81:31-42: Cote, R.J. et al. ( 1983) Proc. Natl.
Acad. Sci.
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. 81:6851-6855; Neuberger. M.S. et al. (1984) Nature
312:604-608; and
Takeda, S. et al. ( 1985) Nature 314:452-4~4.) Alternatively, techniques
described for the
production of single chain antibodies may be adapted. using methods known in
the art, to produce
HHH-specific single chain antibodies. Antibodies with related specificity, but
of distinct idiotypic
composition. may be generated by chain shuffling from random combinatorial
immunoglobulin
3.~ libraries. (See. e.g., Burton D.R. ( 1991 ) Proc. Natl. Acad. Sci.
88:1013.1-10137.)
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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. 86:
3833-3837: and Winter, G. et al. (1991) Nature 319:293-299.)
Antibody fragments which contain specific binding sites for HHH may also be
venerated.
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.
10 (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
15 HHH and its specific antibody. A two-site, monoclonal-based immunoassay
utilizing monoclonal
antibodies reactive to two non-interfering HHH epitopes is preferred, but a
competitive binding
assay may also be employed. (Maddox, supra.)
In another embodiment of the invention. the polynucleotides encoding HHH, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect. the
20 complement of the polynucleotide encoding HHH may be used in situations in
which it would be
desirable to block the transcription of the mRNA. In particular, cells may be
transfotlrted with
sequences complementary to polynucleotides encoding HHH. Thus, complementary
molecules or
fragments may be used to modulate HHH activity. or to achieve regulation of
gene function. Such
technology is now well known in the art, and sense or antisense
oligonucleotides or larger
25 fragments can be designed from various locations along the coding or
control regions of sequences
encoding HHH.
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. Methods which are well known to
those skilled in the art
30 can be used to construct vectors to express nucleic acid sequences
complementary to the
polynucieotides encoding HHH. (See, e.g., Sambrook. supra; and Ausubel,
supra.)
Genes encoding HHH can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof.
encoding HHH. Such
constructs may be used to introduce untranslatable sense or antisense
sequences into a cell. Even
in the absence of integration into the DNA, such vectors may continue to
transcribe RNA
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31
molecules until they are disabled by endogenous nucleases. Transient
expression may last for a
month or more with a non-replicating vector. and may last even longer if
appropriate replication
elements are part of the vector system.
As mentioned above, modifications of gene expression can be obtained by
designing
complementary sequences or antisense molecules (DNA, RNA. or PNA) to the
control. 5', or
regulatory regions of the gene encoding HHH. Oligonucleotides derived from the
transcription
initiation site, e.g., between about positions -10 and +l0 from the start
site. are preferred.
Similarly, inhibition can 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 Immunolo ig c Approaches,
Futura Publishing
Co., 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 HHH.
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 riborymes 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 oligonucieotides such as solid
phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules may be
generated by
in vitro and in vivo transcription of DNA sequences encoding HHH. Such DNA
sequences may
be incorporated into a wide varien~ 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
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32
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.
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) Nature Biotechnology 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 dogs, cats, cows,
horses, rabbits,
monkeys, and most preferably, humans.
An additional embodiment of the invention relates to the administration of a
pharmaceutical or sterile composition, in conjunction with a pharmaceutically
acceptable carrier,
for any of the therapeutic effects discussed above. Such pharmaceutical
compositions may consist
of HHH, antibodies to HHH, and mimetics, agonists, antagonists, or inhibitors
of HHH. The
compositions may be administered alone or in combination with at least one
other agent, such as a
stabilizing compound. which may be administered in any sterile, biocompatible
pharmaceutical
carrier including, but not limited to, saline, buffered saline, dextrose, and
water. The compositions
may be administered to a patient alone, or in combination with other agents,
drugs, or hormones.
The pharmaceutical 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. transdermal. subcutaneous,
intraperitoneal, intranasal,
enteral, topical. sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain
suitable pharmaceutically-acceptable carriers comprising excipients and
auxiliaries which
facilitate processing of the active compounds into preparations which can be
used
pharmaceutically. Further details on techniques for formulation and
administration may be found
in the latest edition of Remin~eton's Pharmaceutical Sciences (Maack
Publishing Co.. Easton, PA).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically acceptable carriers well known in the art in dosages suitable
for oral
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
33
administration. Such carriers enable the pharmaceutical compositions to be
formulated as tablets,
pills, dragees, capsules, liquids, gels. syrups, slurries, suspensions, and
the like, for ingestion by
the patient.
Pharmaceutical preparations for oral use can be obtained through combining
active
S compounds with solid excipient and processing the resultant mixture of
granules (optionally, afrer
grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be
added, if desired. Suitable
excipients include carbohydrate or protein fillers, such as sugars, including
lactose, sucrose,
mannitol, and sorbitol: starch from corn, wheat, rice, potato. or other
plants: cellulose, such as
methyl cellulose, hydroxypropylmethyl-cellulose, or sodium
carboxymethylcellulose; gums,
including arabic and tragacanth: and proteins. such as gelatin and collagen.
If desired,
disintegrating or solubilizing agents may be added, such as the cross-linked
polyvinyl pyrrolidone,
agar, and alginic acid or a salt thereof, such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as
concentrated
sugar solutions, which may also contain gum arabic, talc.
polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable
organic solvents or
solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee
coatings for
product identification or to characterize the quantity of active compound,
i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as well as soft, sealed capsules made of gelatin and a coating, such
as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or
binders, such as lactose or
starches, lubricants, such as talc or magnesium stearate, and. optionally,
stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in suitable
liquids. such as fatty
oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hanks's solution,
Ringer's solution, or physiologically buffered saline. Aqueous injection
suspensions may contain
substances which increase the viscosity of the suspension, such as sodium
carboxymethy)
cellulose, sorbitol, or dextran. Additionally, suspensions of the active
compounds may be
prepared as appropriate oily injection suspensions. Suitable lipophilic
solvents or vehicles include
fatty oils, such as sesame oil, or synthetic fatty acid esters. such as ethyl
oleate, triglycerides, or
liposomes. Non-lipid polycationic amino polymers may also be used for
delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to increase the
solubility of the
compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art.
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34
The pharmaceutical compositions of the present invention may be manufactured
in a
manner that is known in the art, e.g., by means of conventional mixing.
dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping, or
lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many
acids. including but not limited to. hydrochloric, sulfuric. acetic. lactic.
tartaric. malic. and
succinic acid. Salts tend to be more soluble in aqueous or other protonic
solvents than are the
corresponding free base forms. 1n other cases, the preferred preparation may
be a lyophilized
powder which may contain any or all of the following: 1 mM to SO mM histidine,
0.1 % to 2%
sucrose. and 2% to 7% mannitoh at a pH range of 4.S to S.S, that is combined
with buffer prior to
use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate container and labeled for treatment of an indicated condition. For
administration of
HHH, such labeling would include amount, frequency, and method of
administration.
Pharmaceutical compositions suitable for use in the invention include
compositions
1S 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.
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, or
pigs. An animal model may also be used to determine the appropriate
concentration range and
route ofadministration. 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
HHH or fragments thereof, antibodies of HHH, and agonists, antagonists or
inhibitors of HHH,
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 ED5° (the dose therapeutically effective in
SO% of the population) or
LD,o (the dose lethal to SO% of the population) statistics. The dose ratio of
therapeutic to toxic
effects is the therapeutic index. and it can be expressed as the
ED<°/LDa° ratio. Pharmaceutical
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 EDS° 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.
3S The exact dosage will be determined by the practitioner, in light of
factors related to the
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
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
5 sensitivities. and response to therapy. Long-acting pharmaceutical
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 ~g to 100,000 fig, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
10 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. conditions, locations, etc.
15 DIAGNOSTICS
In another embodiment, antibodies which specifically bind HHH may be used for
the
diagnosis of disorders characterized by expression of HHH, or in assays to
monitor patients being
treated with HHH or agonists, antagonists, or inhibitors of HHH. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic
20 assays for HHH include methods which utilize the antibody and a label to
detect HHH 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.
25 A variety of protocols for measuring HHH. including ELISAs, RIAs, and FACS.
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of HHH expression.
Normal or standard values for HHH expression are established by combining body
fluids or cell
extracts taken from normal mammalian subjects. preferably human, with antibody
to HHH under
conditions suitable for complex formation The amount of standard complex
formation may be
30 quantitated by various methods, preferably by photometric means. Quantities
of HHH 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 HHH may
be used
35 for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide
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WO 99/61626 PCT/US99/12021
36
sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides
may be
used to detect and quantitate gene expression in biopsied tissues in which
expression of HHH may
be correlated with disease. The diagnostic assay may be used to determine
absence. presence, and
excess expression of HHH, and to monitor regulation of HHH levels during
therapeutic
S intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide sequences. including genomic sequences, encoding HHH or closely
related
molecules may be used to identify nucleic acid sequences which encode HHH. The
specificity of
the probe, whether it is made from a highly specific region, e.g., the ~'
regulatory region, or from a
less specific region, e.g., a conserved motif, and the stringency of the
hybridization or
amplification (maximal, high, intermediate, or low), will determine whether
the probe identifies
only naturally occurring sequences encoding HHH, allelic variants, or related
sequences.
Probes may also be used for the detection of related sequences. and should
preferably
have at least SO% sequence identity to any of the HHH encoding sequences. The
hybridization
I 5 probes of the subject invention may be DNA or RNA and may be derived from
the sequence of
SEQ ID N0:8 through SEQ ID N0:14 or from genomic sequences including
promoters,
enhancers, and introns of the HHH gene.
Means for producing specific hybridization probes for DNAs encoding HHH
include the
cloning of polynucleotide sequences encoding HHH or HHH 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
poiymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as'=P or''S. or
by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding HHH may be used for the diagnosis of a
disorder
associated with expression of HHH. Examples of such a disorder include. but
are not limited to,
reproductive disorder such as disorders of prolactin production; infertility,
including tubal disease,
ovulatory defects, and endometriosis: disruptions of the estrous cycle,
disruptions of the menstrual
cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome.
endometrial and ovarian
tumors, uterine fibroids. autoimmune disorders, ectopic pregnancies. and
teratogenesis; cancer of
the breast, fibrocystic breast disease. and galactorrhea: disruptions of
spermatogenesis, abnormal
sperm physiology, cancer of the testis. cancer of the prostate. benign
prostatic hyperplasia,
prostatitis. Peyronie's disease. carcinoma of the male breast. and
gynecomastia. carbohydrate
metabolism disorders such as diabetes, insulin-dependent diabetes mellitus,
non-insulin-dependent
s diabetes mellitus, hypoglycemia. ~:lucagonoma. galactosemia. hereditaw tW
ctose intolerance,
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WO 99/61626 PCT/US99/12021
37
fructose-1.6-diphosphatase deficiency, obesity, congenital type II
dyserythropoietic anemia,
mannosidosis. neuraminidase deficiency, galactose epimerase deficiency,
glycogen storage
diseases, lysosomal storage diseases, fructosuria. pentosuria, and inherited
abnormalities of
pyruvate metabolism, and cell proliferation disorders, such as actinic
keratosis. arteriosclerosis,
atherosclerosis. bursitis, cirrhosis, hepatitis. mined connective tissue
disease (MCTD),
myelofibrosis. paroxysmal nocturnal hemoglobinuria. polycythemia vera,
psoriasis, primary
thrombocvthemia, and cancers including adenocarcinoma, leukemia. lymphoma,
melanoma,
myeloma. sarcoma, teratocarcinoma, and, in particular, cancers ofthe 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 HHH
may be used in
Southern or Northern analysis, dot blot, or other membrane-based technologies;
in PCR
technologies: in dipstick, pin, and >rLISA assays: and in microarrays
utilizing fluids or tissues
from patients to detect altered HHH expression. Such qualitative or
quantitative methods are well
known in the art.
In a particular aspect, the nucleotide sequences encoding HHH may be useful in
assays
that detect the presence of associated disorders, particularly those mentioned
above. The
nucleotide sequences encoding HHH 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 quantitated
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 HHH 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.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
HHH. 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 HHH. 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.
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WO 99/61626 PCT/US99/12021
38
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 a relatively high amount of transcript
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
HHH 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 HHH, or a fragment of a polynucleotide complementary
to the
IS polynucleotide encoding HHH, 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 quantitation of closely related DNA or RNA sequences.
Methods which may also be used to quantitate the expression of HHH 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: and Duplaa, C. et al. (1993) Anal. Biochem. 229-236.) The
speed of
quantitation of multiple samples may be accelerated by running the assay in an
ELISA format
where the oligomer 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 targets in a
microarray. The
microarray can be used to monitor the expression level of large numbers of
genes simultaneously
and to idemify 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, and
to develop and monitor the activities of therapeutic agents.
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. 93:10614-10619: Baldeschweiler et al. (1995) PCT application
W095/2~ 1116; Shalon.
D. et al. ( 1995) PCT application W095/35505: Heller. R.A. et al. ( 1997)
Proc. Natl. Acad. Sci.
94:2150-215: and Heller. M.J. et al. ( 1997) U.S. Patent No. ~.60~.662.)
CA 02329076 2000-11-28
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39
In another embodiment of the invention, nucleic acid sequences encoding HHH
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic
sequence. 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
PI constructions, or single chromosome cDNA libraries. (See, e.g., Price, C.M.
(1993) Blood
Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7:149-154.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich,
et al. (1995) in
Meyers, R.A. (ed.) Molecular Biology and Biotechnoloey, VCH Publishers New
York, NY, pp.
965-968.) Examples of genetic map data can be found in various scientific
journals or at the
Online Mendelian Inheritance in Man (OMIM) site. Correlation between the
location of the gene
encoding HHH on a physical chromosomal map and a specific disorder, or a
predisposition to a
specific disorder, may help define the region of DNA associated with that
disorder. The
nucleotide sequences of the invention may be used to detect differences in
gene sequences among
normal, carrier, and affected individuals.
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 piacement of a gene on the chromosome of another mammalian
species, such as
mouse, may reveal associated markers even if the number or arm of a particular
human
chromosome is not known. New sequences can be assigned to chromosomal arms by
physical
mapping. This provides valuable information to investigators searching for
disease genes using
positional cloning or other gene discovery techniques. Once the disease or
syndrome has been
crudely localized by genetic linkage to a particular genomic region, e.g.,
ataxia-telangiectasia to
l 1q22-23, 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 336:77-
580.) The nucleotide
sequence of the subject 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. HHH, 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 HHH 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.. Geysen, et al.
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WO 99/61626 PCT/US99/12021
( 1984) PCT application W084/03564.) In this method. large numbers of
different small test
compounds are synthesized on a solid substrate, such as plastic pins or some
other surface. The
test compounds are reacted with HHH, or fragments thereof. and washed. Bound
HHH is then
detected by methods well known in the art. Purified HHH can also be coated
directly onto plates
5 for use in the aforementioned drug screening techniques. 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 HHH specifically compete with a
test compound for
binding HHH. In this manner, antibodies can be used to detect the presence of
any peptide which
10 shares one or more antigenic determinants with HHH.
In additional embodiments, the nucleotide sequences which encode HHH 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.
IS 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 entire disclosure of all applications, patents, and publications, cited
above and below,
20 and of US provisional application 60/087,236 (filed May 29, 1998), is
hereby incorporated by
reference.
EXAMPLES
I. cDNA Library Construction
25 RNA was purchased from Clontech (Palo Alto. CA) or isolated at Incyte from
tissues
described in Table 4. The tissue was homogenized and lysed in guanidinium
isothiocyanate, and
the lysate was centrifuged over a CsCI cushion. Alternatively, the tissue was
homogenized and
lysed in phenol or a suitable mixture of denaturants such as TRIZOL reagent
(Life Technologies),
a monophasic solution of phenol and guanidine isothiocyanate, and the lysate
was extracted with
30 chloroform ( 1:~ v/v). RNA was precipitated from lysates with either
isopropanol or sodium
acetate and ethanol. Alternatively, RNA was purified from lysates by
preparative agarose gel
electrophoresis and recovered from Whatman P81 paper (Whatman. Lexington. MA).
Phenol
extraction and precipitation of RNA were repeated as necessary to increase RNA
purity, and RNA
was maintained in RNase-free solutions. In some cases. RNA was treated with
DNase. For most
35 libraries. polv.~+) RNA was isolated using oligo d(T)-coupled paramagnetic
particles (Promega.
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41
Madison, WI), Oligotex resin. or the Oligotex kit (QIAGEN Inc, Chatsworth,
CA). Alternatively,
RNA was isolated directly from tissue lysates using the RNA Isolation kit
(Stratagene) or the
Ambion PolyA Quick kit (Ambion. Austin. TX).
RNA was used for cDNA synthesis and construction of the cDNA libraries
according to
procedures recommended in the UNIZAP vector (Stratagene. La Jolla. CA) or
Superscript
plasmid system (Life Technologies, Inc), both of which are based on methods
well known in the
art (Ausubel, 1997, units S.1-6.6). Alternatively, cDNA libraries were
constructed by Stratagene
using RNA provided by Incyte. Reverse transcription was initiated using oligo
d(T) or random
primers. Synthetic oligonucleotide adapters were ligated to double stranded
cDNA, and cDNA
was digested with an appropriate restriction enzyme(s). For most libraries,
cDNA was size-
selected (300-1000 bp) using Sephacryl S 1000 or Sepharose CL2B or 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 (Stratagene), pSPORT 1 (Life Technologies), pINCY
(Incyte
Pharmaceuticals Inc, Palo Aito. CA). pINCY was amplified in JM 109 cells and
purified using the
QiaQuick column (QIAGEN Inc). Recombinant plasmids were transformed into
competent E. coli
cells, e.g., XL1-Blue, XL1-Blue MRF, or SOLR (Stratagene) or DHSn, DHI OB, or
ElectroMAX
DH l OB (Life Technologies).
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42
TABLE -t
Nucleotide
SEQ ID Library Library Description
NO:
The EOSIHET02 library was constructed
using
8 EOSIHET02 ~'' isolated from peripheral
blood cells
apheresed from a 48-year-old
Caucasian male.
Patient history included hypereosinophilia.
The NEUTGMTOI library was constructed
using
RNA isolated from peripheral
blood granulocvtes
collected by density gradient
centrifugation
9 NEUTGMTO1 through Ficoll-Hypaque. The cells
were isolated
from huffy coat units obtained
from 20 unrelated
male and female donors. Cells
were cultured in 10
nM GM-CSF for 1 hour before washing
and
harvesting for RNA preparation.
The THP1NOT01 library was constructed
using
RNA isolated from THP-1 cells.
THP-1 (ATCC
THPINOTOI TIB 202) is a human promonocyte
line derived
from the peripheral blood of
a 1-year-old
Caucasian male with acute monocytic
leukemia.
The ENDCNOT03 library was constructed
using
1 I ENDCNOT03 ~A isolated from dermal microvascular
endothelial cells removed from
a neonatal
Caucasian male.
The ENDANOTOI library was constructed
using
12 ENDANOTO1 RNA isolated from aortic endothelial
cell tissue
from an e~cplanted heart removed
from a male
during a heart transplant.
The UTRSNORO 1 library was constructed
using
RNA isolated from uterine endometrium
tissue
13 UTRSNORO1 removed from a 29-year-old Caucasian
female
during a vaginal hysterectomy
and cystocele
repair.
The BRAUNOT02 library was constructed
using
14 BRAUNOT02 ~'4 isolated from globus pallidus/substantia
innominata tissue removed from
the brain of a 35-
year-old Caucasian male.
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43
II. Isolation of cDNA Clones
Plasmids were recovered from host cells by in vivo excision ( UniZAP vector
system,
Stratagene) or by cell lysis. Plasmids v~ere purified using the MAGIC
MINIPREPS DNA
purification system (Promeea, Madison. WI); Miniprep kit (Advanced Genetic
Technologies
Corporation, Gaithersburg. MD): QIAwell-8 Plasmid, QIAwell PLUS DNA, or
QIAwell ULTRA
DNA purification systems: or REAL Prep 96 plasmid kit (QIAGEN Inc) using the
recommended
protocol. Following precipitation, plasmids were resuspended in 0. I 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
(Rao. V.B. (1994) Anal. Biochem. 216:1-14) in a high-throughput format. Host
cell lysis and
thermal cycling steps were carried out in a single reaction mixture. Samples
were processed and
stored in 384-well plates (Genetix Ltd, Christchurch UK) and concentration of
amplified plasmid
DNA was quantified fluorometrically using Pico Green Dye (Molecular Probes,
Eugene OR) and a
Fluoroscan II fluorescence scanner (Labsystems Oy, Helsinki. Finland).
III. Sequencing and Analysis
The cDNAs were prepared for sequencing using either an AB1 Catalyst 800
(Perkin
Elmer) or a Hamilton Micro Lab 2200 (Hamilton, Reno, NV) in combination with
Pettier Thermal
Cyclers (PTC200; MJ Research. Watertown MA). The cDNAs were sequenced on the
ABI 373
or 377 DNA Sequencing systems (Perkin Elmer) by the method of Sanger F. and
A.R. Coulson
(1975; J. Mol. Biol. 94:441-448) using standard ABI protocols, base calling
software, and kits.
Alternatively, cDNAs were sequenced using solutions and dyes from Amersham
Pharmacia
IS Biotech. Reading frame was determined using standard methods (Ausubel,
supra).
The cDNA sequences presented in Table 1 and the full length nucleotide and
amino acid
sequences disclosed in the Sequence Listing were Queried against databases
such as GenBank
primate (pri), rodent (rod), mammalian (mamp), vertebrate (vrtp), and
eukaryote (eukp) databases,
SwissProt, BLOCKS, and other databases which contain previously identified and
annotated
motifs and sequences. Algorithms such as Smith Waterman which deal with
primary sequence
patterns and secondary structure gap penalties (Smith, T. et al. ( 1992)
Protein Engineering 5:35-
51 ) and programs and algorithms such as BLAST (Basic Local Alignment Search
Tool; Altschul,
S.F. ( 1993) J. Mol. Evol 36:290-300: and Altschul et al. ( 1990) J. Mol.
Biol. 215:403-410), and
HMM (Hidden Markov Models: Eddy. S.R. (1996) Cur. Opin. Str. Biol. 6:361-36~,
and
Sonnhammer. E.L.L. et al. ( 1997) Proteins 28:405-420) were used to assemble
and analyze
nucleotide and amino acid sequences. The databases, programs. algorithms,
methods and tools are
available, well known in the art. and described in Ausubel (supra. unit 7.7),
in Meyers. R.A.
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
44
(1995; Molecular BioloQV and BiotechnoloiP.~y, Wiley VCH, Inc, New York NY, p
856-853), in
documentation provided with software (Genetics Computer Group (GCG). Madison
WI), and on
the world wide web (www). Two comprehensive websites which list, describe,
and/or link many
of the databases and tools are: 1 ) the www resource in practical sequence
analysis
(http://genome.wustl.edu/eddy/bio5495/online-resources.html), and 2) the
bibliography of
computational gene recognition (http://linkage.rockefeller.edu/wli/gene/
programs.html). For
example, the first website links PFAM as a database (http://genome.wustl.
edu/Pfam/) and as an
HMM search tool (http://genome.wustl.edu/eddy/cgi-bin/hmm_page.cgi).
TABLE 5 summarizes the databases and toots used herein. The first column of
TABLE 5
shows the tool, program, or algorithm: the second column, the database; the
third column, a brief
description: and the fourth column (where applicable), scores for determining
the strength of a
match between two sequences (the higher the value, the more homologous).
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
-- o
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L v: < ~ 'CL L ... C " ~ ~~- < ~ < C ~ J
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GL
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
46
IV. Northern Analysis
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;
and Ausubel, supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST are used to search for identical
or
related molecules in nucleotide databases such as GenBank or LIFESEQT~"
database (Incyte
Pharmaceuticals). 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:
sequence identity x % maximum BLAST score
100
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. For example, with a product score of 40, the
match will be exact
within a 1% to 2% error, and, with a product score of 70, the match will be
exact. Similar
molecules are usually identified by selecting those which show product scores
between 15 and 40,
although lower scores may identify related molecules.
The results of Northern analysis are reported as a list of libraries in which
the transcript
encoding HHH occurs. Abundance and percent abundance are also reported.
Abundance directly
reflects the number of times a particular transcript is represented in a cDNA
library, and percent
abundance is abundance divided by the total number of sequences examined in
the cDNA library.
V. Extension of HHH Encoding Polynucleotides
The nucleic acid sequences of Incyte Clones 321510, 634343. ?017918, 2175072,
2403107, 3069540, and 4182350 were used to design oligonucleotide primers for
extending partial
nucleotide sequences to full length. For each nucleic acid sequence. one
primer was synthesized
to initiate extension of an antisense polynucleotide, and the other was
synthesized to initiate
extension of a sense pofynucleotide.] Primers were used to facilitate the
ewension of the known
sequence "outward" generating amplicons containing new unknown nucleotide
sequence for the
region of interest. The initial primers were designed from the cDNA using
OLIGOTM 4.06
(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 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.
CA 02329076 2000-11-28
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47
Selected human cDNA libraries (GIBCO BR1.) were used to extend the sequence.
If more
than one extension is necessary or desired, additional sets of primers are
designed to further
extend the known region.
High fidelity amplification was obtained by following the instructions for the
XL-PCRT'"
kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix. PCR was
performed
using the Pettier Thermal Cycler (PTC200; M.J. Research, Watertown, MA),
beginning with 40
pmol of each primer and the recommended concentrations of all other components
of the kit, with
the following parameters:
Step 1 94 C for I min (initial denaturation)
l0 Step 2 65 C for 1 min
Step 3 68 C for 6 min
Step 4 94 C for 15 sec
Step ~ 65 C for 1 m i n
Step 6 68 C for 7 min
Step 7 Repeat steps 4 through 6 for an additional
15 cycles
Step 8 94 C for 15 sec
Step 9 65 C for 1 min
Step 10 68 C for 7:1 S min
Step I 1 Repeat steps 8 through 10 for an
additional 12 cycles
Step 12 72 C for 8 min
Step 13 4 C (and holding)
A 5 ~1 to 10 ul aliquot of the reaction mixture was analyzed by
electrophoresis on a low
concentration (about 0.6% to 0.8%) agarose mini-gel to determine which
reactions were successful
in extending the sequence. Bands thought to contain the largest products were
excised from the
gel, purified using QIAQUICKT"' (QIAGEN Inc.). and trimmed of overhangs using
Klenow
enzyme to facilitate religation and cloning.
After ethanol precipitation, the products were redissolved in 13 ul of
ligation buffer, lul
T4-DNA lipase ( 15 units) and lul T4 poiynucleotide kinase were added, and the
mixture was
incubated at room temperature for 2 to 3 hours. or overnight at l6° C.
Competent E. colt cells (in
~I of appropriate media) were transformed with 3 ul of ligation mixture and
cultured in 80 ~cl
of S(~C medium. (See, e.g., Sambrook, supra. Appendix A, p. 2.) After
incubation for one hour at
37°C, the E. colt mixture was plated on Luria Bertani (LB) agar (See,
e.g., Sambrook. sUDra,
Appendix A. p. 1 ) containing carbenicillin (2t carb). The following day,
several colonies were
35 randomly picked from each plate and cultured in 1 ~0 ul of liquid LBI2x
carb medium placed in an
individual welt of an appropriate commercially-available sterile 96-well
microtiter plate. The
following day. ~ ul of each overnight culture was transferred into a non-
sterile 96-well plate and,
after dilution 1:10 with water, 5 ~cl from each sample was transferred into a
PCR array.
For PCR amplification. 18 ul of concentrated PCR reaction mix (3.3x)
containing 4 units
40 of rTth DNA polymerase. a vector primer, and one or both of the gene
specific primers used for
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48
the extension reaction were added to each well. Amplification was performed
using the following
conditions:
Step 94 C for 60 sec
1
Step 94 C for 20 sec
2
Step >j C for 30 sec
3
Step 7'_' C for 90 sec
4
Step Repeat steps 2 through 4 for an additional
~ 29 cycles
Step 72 C for 180 sec
6
Step 4 C (and holding)
7
Aliquots of the PCR reactions were run on agarose gels together with molecular
weight
markers. The sizes of the PCR products were compared to the original partial
cDNAs. and
appropriate clones were selected. ligated into plasmid, and sequenced.
In like manner. the nucleotide sequence of SEQ ID N0:8 through SEQ ID N0:14
are
I S used to obtain 5' regulatory sequences using the procedure above.
oligonucleotides designed for 5'
extension, and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:8 through SEQ ID N0:14 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 OLIGOT"' 4.06 software (National Biosciences) and labeled by combining
SO pmol of
each oligomer, 250 uCi of {y-'=P] adenosine triphosphate (Amersham, Chicago,
IL), and T4
polynucleotide kinase (DuPont NEN~', Boston, MA). The labeled oligonucleotides
are
substantially purified using a Sephade~cT"' G-25 superfine size exclusion
dextran bead column
(Pharmacia & Upjohn, Kalamazoo. MI). 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
I. Xbal, or Pvu II
(DuPont NEN, Boston, MA).
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 increasingly stringent conditions up to 0.1 x saline sodium citrate and
0.5% sodium dodecyl
sulfate. After XOMAT ARTS' film (Kodak. Rochester. NY) is exposed to the blots
to film for
several hours, hybridization patterns are compared visually.
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49
VII. rlicroarrays
A chemical coupling procedure and an ink jet device can be used to synthesize
array
elements on the surface of a substrate. (See. e.g., Baldeschweiler, supra.) An
array 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 by
hand or using available methods and machines and contain any appropriate
number of elements.
After hybridization, nonhybridized probes are removed and a scanner used to
determine the levels
and patterns of fluorescence. The degree of complementarity and the relative
abundance of each
probe which hybridizes to an element on the microarray may be assessed through
analysis of the
scanned images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may
comprise the elements of the microarray. Fragments suitable for hybridization
can be selected
using software well known in the art such as LASERGENET~'. Full-length cDNAs.
ESTs, or
fragments thereof corresponding to one of the nucleotide sequences of the
present invention, or
selected at random from a cDNA library relevant to the present invention, are
arranged on an
appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide
using, e.g., UV cross-
linking followed by thermal and chemical treatments and subsequent drying.
(See. e.g., Schena,
M. et al. ( 1995) Science 270:467-470; and Shalom D. et al. ( 1996) Genome
Res. 6:639-645.)
Fluorescent probes are prepared and used for hybridization to the elements on
the substrate. The
substrate is analyzed by procedures described above.
VIII. Complementary Polynucleotides
Sequences complementary to the HHH-encoding sequences, or any parts thereof,
are
used to detect, decrease, or inhibit expression of naturally occurring HHH.
Although use of
oligonucleotides comprising from about t S to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides
are designed using OLIGOT"' 4.06 software and the coding sequence of HHH. To
inhibit
transcription, a complementary oligonucleotide is designed from the most
unique ~' 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 HHH-encoding
transcript.
IX. Expression of HHH
Expression and purification of HHH is achieved using bacterial or virus-based
expression
systems. For expression of HHH in bacteria. cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of
CA 02329076 2000-11-28
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cDNA tr~attscription. 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 HHH upon induction with
isopropyl beta-D-
5 thiogaiactopyranoside (IPTG). Expression of HHN in eukaryotic cells is
achieved by infecting
insect or mammalian celi lines with recombinant Autographica californica
nuclear polyhedrosis
virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin
gene of
baculovirus is replaced with cDNA encoding HHH by either homologous
recombination or
bacterial-mediated transposition involving transfer plasmid intermediates.
Viral infectivity is
10 maintained and the strong polyhedrin promoter drives high levels of cDNA
transcription.
Recombinant baculovirus is used to infect Spodoptera frugiperda (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.)
15 In most expression systems, HHH is synthesized as a fusion protein with,
e.g.,
glutathione S-transferase (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
20 antigenicity (Pharmacia, Piscataway, NJ). Following purification, the GST
moiety can be
proteolytically cleaved from HHH at specifically engineered sites. FLAG, an 8-
amino acid
peptide. enables immunoaffinity purification using commercially available
monoclonal and
polyclonal anti-FLAG antibodies (Eastman Kodak, Rochester. NY). 6-His, a
stretch of six
consecutive histidine residues, enables purification on metal-chelate resins
(QIAGEN Inc,
25 Chatsworth, CA). Methods for protein expression and purification are
discussed in Ausubel, F. M.
et al. (1995 and periodic supplements) Current Protocols in Molecular Bioloev,
John Wiley &
Sons, New York, NY, ch 10, 16. Purified HHH obtained by these methods can be
used directly in
the following activity assay.
30 X. Demonstration of HHH Activity
For purposes of example, an assay measuring the ~i-glucosidase activity of an
HHH
molecule is described. Varying amounts of HHH are incubated with 1 mM 4-
nitrophenyl (i-D-
glycopyranoside (a substrate) in ~0 mM sodium acetate buffer, pH 5.0, for
various times (typically
1-5 minutes) at 37°C. The reaction is halted by heating to 100°C
for 2 minutes. The absorbance
35 is measured spectrophotometrically at 410 nm. and is proportional to the
activity of HHH in the
CA 02329076 2000-11-28
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51
sample. (Htmova, M. et al. (1998) J. Biol. Chem. 273:11134-l 1143.)
XI. Functional Assays
HHH function is assessed by expressing the sequences encodine HHH 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 SPORTT"~ (Life Technologies, Gaithersburg, MD)
and pCRT"s
3.1 (Invitrogen, Carlsbad, CA, both of which contain the cytomegalovirus
promoter. 5-10 ug of
recombinant vector are transiently transfected into a human cell line,
preferably of endothelial or
hematopoietic origin, using either liposome formulations or electroporation. 1-
2 ~g 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, Palo Alto,
CA), CD64, or a
I S 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
properties, for
example, their apoptotic state. 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-
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 Cvtometry, Oxford, New York, NY.
The influence of HHH on gene expression can be assessed using highly purified
populations of cells transfected with sequences encoding HHH 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 (1gG). 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 HHH and other genes
of interest can be
analyzed by Northern analysis or microarray techniques.
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52
XII. Production of HHH Specific Antibodies
HHH substantially purified using polyacrvlamide 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 urine standard protocols.
Alternatively. the HHH amino acid sequence is analyzed using LASERGENET'"
software
(DNASTAR Inc.) to determine regions of high immunogenicity, and a
corresponding ofigopeptide
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 supra, ch. I 1.)
Typically. oligopeptides I S residues in length are synthesized using an
Applied
Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry and coupled to
KLH (Sigma,
St. Louis, MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS) to
increase immunogenicity. (See, e.g., Ausubel suyra.) Rabbits are immunized
with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide activity by, for example, binding the peptide to plastic, blocking
with 1 % BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
XIII. Purification of Naturally Occurring HHH Using Specific Antibodies
Naturally occurring or recombinant HHH is substantially purified by
immunoaffinity
chromatography using antibodies specific for HHH. An immunoaffinity column is
constructed by
covaiently coupling anti-HHH antibody to an activated chromatographic resin,
such as
CNBr-activated Sepharose (Pharmacia & Upjohn). After the coupling, the resin
is blocked and
washed according to the manufacturer's instructions.
Media containing HHH are passed over the immunoaffinity column, and the column
is
washed under conditions that allow the preferential absorbance of HHH (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/HHH 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 HHH is collected.
XIV. Identification of Molecules Which Interact with HHH
HHH. or biologically active fragments thereof: are labeled with'=51 Bolton-
Hunter
reagent. (See, e.e., Bolton et al. ( 1973) Biochem. J. 133:~?9.) Candidate
molecules previously
arrayed in the wells of a multi-well plate are incubated with the labeled HHH.
washed, and anv
wells with labeled HHH complex are assayed. Data obtained using different
concentrations of
HHH are used to calculate values for the number. affinity. and association of
HHH with the
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53
candidate molecules.
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
specific preferred
embodiments, it should be understood that the invention as claimed should not
be unduly limited
to such specific embodiments. Indeed, various modifications of the 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.
CA 02329076 2000-11-28
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SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
BANDMAN, Olga
HILLMAN, Jennifer L.
YUE, Henry
t_nr., Preeti
CORLEY, Neil C.
GUEGLER, Karl J.
PATTERSON, Chandra
BAUGHN, Mariah R.
<120> HUMAN HYDROLASE HOMOLOGS
<130> PF-0534 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/087,236
<151> 1998-05-29
<160> 54
<170> PERL Program
<210> 1
<211> 310
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 321510
<400> 1
Met Pro Leu Leu Val Glu Gly Arg Arg Val Arg Leu Pro Gln Ser
1 5 10 15
Ala Gly Asp Leu Val Arg Ala His Pro Pro Leu Glu Glu Arg Ala
20 25 30
Arg Leu Leu Arg Gly Gln Ser Val Gln Gln Val Gly Pro Gln Gly
35 40 45
Leu Leu Tyr Val Gln Gln Arg Glu Leu Ala Val Thr Ser Pro Lys
50 55 60
Asp Gly Ser Ile Ser Ile Leu Gly Ser Asp Asp Ala Thr Thr Cys
65 70 75
His Ile Val Val Leu Arg His Thr Gly Asn Gly Ala Thr Cys Leu
80 85 90
Thr His Cys Asp Gly Thr Asp Thr Lys Ala Glu Val Pro Leu Ile
95 100 105
Met Asn Ser Ile Lys Ser Phe Ser Asp His Ala Gln Cys Gly Arg
110 115 120
Leu Glu Val His Leu Val Gly Gly Phe Ser Asp Asp Arg Gln Leu
125 130 135
Ser Gln Lys Leu Thr His Gln Leu Leu Ser Glu Phe Asp Arg Gln
1/33
CA 02329076 2000-11-28
WO 99/61626 PCTNS99/12021
140 145 150
Glu Asp Asp Ile His Leu Val Thr Leu Cys Val Thr Glu Leu Asn
155 160 165
Asp Arg Glu Glu Asn Glu Asn His Phe Pro Val Ile Tyr Gly Ile
I70 175 I80
Ala Val Asn Ile Lys Thr Ala Glu Ile Tyr Arg Ala Ser Phe Gln
185 190 195
Asp Arg Gly Pro Glu Glu Gln Leu Arg Ala Ala Arg Thr Leu Ala
200 205 210
Gly Gly Pro Met Ile Ser Ile Tyr Asp Ala Glu Thr Glu Gln Leu
215 2'20 225
Arg Ile Gly Pro Tyr Ser Trp Thr Pro Phe Pro His Val Asp Phe
230 235 240
Trp Leu His Gln Asp Asp Lys Gln Ile Leu Glu Asn Leu Ser Thr
245 250 255
Ser Pro Leu Ala Glu Pro Pro His Phe Val Glu His Ile Arg Ser
260 265 270
Thr Leu Met Phe Leu Lys Lys His Pro Ser Pro Ala His Thr Leu
275 280 285
Phe Ser Gly Asn Lys Ala Leu Leu Tyr Lys Lys Asn Glu Asp Gly
290 295 300
Leu Trp Glu Lys Ile Ser Ser Pro Gly Ser
305 310
<210> 2
<211> 520
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 634343
<400> 2
Met Val Thr Ser Ser Phe Pro Ile Ser Val Ala Val Phe Ala Leu
1 5 10 15
Ile Thr Leu Gln Val Gly Thr Gln Asp Ser Phe Ile Ala Ala Val
20 25 30
Tyr Glu His Ala Val Ile Leu Pro Asn Lys Thr Glu Thr Pro Val
35 40 45
Ser Gln Glu Asp Ala Leu Asn Leu Met Asn Glu Asn Ile Asp Ile
50 55 60
Leu Glu Thr Ala Ile Lys Gln Ala Ala Glu Gln Gly Ala Arg Ile
65 70 75
Ile Val Thr Pro Glu Asp Ala Leu Tyr Gly Trp Lys Phe Thr Arg
80 85 90
Glu Thr Val Phe Pro Tyr Leu Glu Asp Ile Pro Asp Pro Gln Val
95 100 105
Asn Trp Ile Pro Cys Gln Asp Pro His Arg Phe Gly His Thr Pro
110 115 120
Val Gln Ala Arg Leu Ser Cys Leu Ala Lys Asp Asn Ser Ile Tyr
125 130 135
Val Leu Ala Asn Leu Gly Asp Lys Lys Pro Cys Asn Ser Arg Asp
140 145 150
Ser Thr Cys Pro Pro Asn Gly Tyr Phe Gln Tyr Asn Thr Asn Val
2/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
155 160 165
Val Tyr Asn Thr Glu Gly Lys Leu Val Ala Arg Tyr His Lys Tyr
170 175 180
His Leu Tyr Ser Glu Pro Gln Phe Asn VaI Pro Glu Lys Pro Glu
185 190 195
Leu val Thr Phe Asn Thr Ala Phe Gly Arg Phe Gly Ile Phe Thr
200 205 210
Cys Phe Asp Ile Phe Phe Tyr Asp Pro Gly Val Thr Leu Val Lys
2i5 220 225
Asp Phe His Val Asp Thr Ile Leu Phe Pro Thr Ala Trp Met Asn
230 235 240
Val Leu Pro Leu Leu Thr Ala Ile Glu Phe His Ser Ala Trp Ala
245 250 255
Met Gly Met Gly Val Asn Leu Leu Val Ala Asn Thr His His Val
260 265 270
Ser Leu Asn Met Thr Gly Ser Gly Ile Tyr Ala Pro Asn Gly Pro
275 280 285
Lys Val Tyr His Tyr Asp Met Lys Thr Glu Leu Gly Lys Leu Leu
290 295 300
Leu Ser Glu Val Asp Ser His Pro Leu Ser Ser Leu Ala Tyr Pro
305 310 315
Thr Ala Val Asn Trp Asn Ala Tyr Ala Thr Thr Ile Lys Pro Phe
320 325 330
Pro Val Gln Lys Asn Thr Phe Arg Gly Phe Ile Ser Arg Asp Gly
335 340 345
Phe Asn Phe Thr Glu Leu Phe Glu Asn Ala Gly Asn Leu Thr Val
350 355 360
Cys Gln Lys Glu Leu Cys Cys His Leu Ser Tyr Arg Met Leu Gln
365 370 375
Lys Glu Glu Asn Glu Val Tyr Val Leu Gly Ala Phe Thr Gly Leu
380 385 390
His Gly Arg Arg Arg Arg Glu Tyr Trp Gln Val Cys Thr Met Leu
395 400 405
Lys Cys Lys Thr Thr Asn Leu Thr Thr Cys Gly Arg Pro Val Glu
410 415 420
Thr Ala Ser Thr Arg Phe Glu Met Phe Ser Leu Ser Gly Thr Phe
425 430 435
Gly Thr Glu Tyr Val Phe Pro Glu Val Leu Leu Thr Glu Ile His
440 445 450
Leu Ser Pro Gly Lys Phe Glu Val Leu Lys Asp Gly Arg Leu Val
455 460 465
Asn Lys Asn Gly Ser Ser Gly Pro Ile Leu Thr Val Ser Leu Phe
474 475 480
Gly Arg Trp Tyr Thr Lys Asp Ser Leu Tyr ser Ser Cys Gly Thr
485 490 495
Ser Asn Ser Ala Ile Thr Tyr Leu Leu Ile Phe Ile Leu Leu Met
500 505 510
Ile Ile Ala Leu Gln Asn Ile Val Met Leu
515 520
<210> 3
<211> 346
<212> PRT
<213> Homo Sapiens
3/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
<220>
<221> misc_feature
<223> Incyte Clone No: 2017918
<400> 3
Met Gln Ala His Val Ser Ile Ile Leu Pro Val His Asn Ala Glu
1 5 10 15
Pro Trp Leu Asp Glu Cys Leu Arg Ser Val Leu Gln Gln Asp Phe
20 25 30
Glu Gly Thr Met Glu Leu Ser Val Phe Asn Asp Ala Ser Lys Asp
35 40 45
Lys Ser Gly Ala Ile Ile Glu Lys Trp Arg Val Lys Leu Glu Asp
50 55 60
Ser Gly Val His Val Ile Ile Gly Gly His Asp Ser Pro Ser Pro
65 70 75
Arg Gly Val Gly Tyr Ala Lys Asn Gln Ala Val Ala Gln Ser Ser
80 85 90
Gly Ser Tyr Leu Cys Phe Leu Asp Ser Asp Asp Val Met Met Pro
95 100 105
Gln Arg Val Arg Leu Gln His Glu Ala Ala Val Gln His Pro Ser
110 115 120
Ser Ile Ile Gly Cys Arg Val Arg Arg Asp Pro Pro Asn Ser Thr
125 130 135
Glu Arg Tyr Thr Arg Trp Ile Asn Gln Leu Thr Pro Glu Gln Leu
140 145 150
Leu Thr Gln Val Phe Thr Ser Asn Gly Pro Thr Val Ile Met Pro
155 160 165
Thr Trp Phe Cys Ser Arg Ala Trp Phe Ser His Val Gly Pro Phe
170 175 180
Asn Glu Gly Gly Gln Gly Val Pro Glu Asp Leu Leu Phe Phe Tyr
185 190 195
Glu His Leu Arg Lys Gly Gly Gly Val Ile Arg Val Asp Gln Ser
200 205 210
Leu Leu Leu Tyr Arg His His Pro Gln Ala Ala Thr His Cys Val
215 220 225
Leu Glu Thr Thr Ile Trp Thr His Arg Val Arg Phe Leu Glu Glu
230 235 240
Gln Ala Leu Pro Arg Trp Ala Ala Phe Thr Ile Trp Asn Ala Gly
245 250 255
Lys Gln Gly Arg Arg Leu Tyr Arg Ser Leu Thr Ala Gly Ser Gln
260 265 270
Arg Lys Val Val Ala Phe Cys Asp Val Asp Glu Asn Lys Ile Arg
275 280 285
Lys Gly Phe Tyr Cys His Glu Asp Ser Gln Glu Arg Pro Lys Pro
290 295 300
Arg Ile Pro Ile Leu His Phe Arg Ala Ala Arg Pro Pro Phe Val
305 310 315
Ile Cys Val Lys Leu Asp Leu Thr Gly Gly Thr Phe Glu Asp Asn
320 325 330
Leu Arg Ser Leu His Leu Gln Glu Gly Gln Asp Phe Leu His Phe
335 340 345
Ser
4/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
<210> 4
<211> 401
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2175072
<400> 4
Met Pro Val Gln Leu Ser Glu His Pro Glu Trp Asn Glu Ser Met
1 5 10 15
His Ser Leu Arg Ile Ser Val Gly Gly Leu Pro Val Leu Ala Ser
20 25 30
Met Thr Lys Ala Ala Asp Pro Arg Phe Arg Pro Arg Trp Lys Val
35 40 45
Ile Leu Thr Phe Phe Val Gly Ala Ala Ile Leu Trp Leu Leu Cys
50 55 60
Ser His Arg Pro Ala Pro Gly Arg Pro Pro Thr His Asn Ala His
65 70 75
Asn Trp Arg Leu Gly Gln Ala Pro Ala Asn Trp Tyr Asn Asp Thr
80 85 9p
Tyr Pro Leu Ser Pro Pro Gln Arg Thr Pro Ala Gly Ile Arg Tyr
95 100 105
Arg Ile Ala Val Ile Ala Asp Leu Asp Thr Glu Ser Arg Ala Gln
110 115 120
Glu Glu Asn Thr Trp Phe Ser Tyr Leu Lys Lys Gly Tyr Leu Thr
125 130 135
Leu Ser Asp Ser Gly Asp Lys Val Ala Val Glu Trp Asp Lys Asp
140 145 150
His Gly Val Leu Glu Ser His Leu Ala Glu Lys Gly Arg Gly Met
155 160 165
Glu Leu Ser Asp Leu ile Val Phe Asn Gly Lys Leu Tyr Ser Val
170 175 180
Asp Asp Arg Thr Gly Val Val Tyr Gln Ile Glu Gly Ser Lys Ala
185 I90 195
Val Pro Trp Val Ile Leu Ser Asp Gly Asp Gly Thr Val Glu Lys
200 205 210
Gly Phe Lys Ala Glu Trp Leu Ala Val Lys Asp Glu Arg Leu Tyr
215 220 225
Val Gly Gly Leu Gly Lys Glu Trp Thr Thr Thr Thr Gly Asp Val
230 235 240
Val Asn Glu Asn Pro Glu Trp Val Lys Val Val Gly Tyr Lys Gly
245 250 255
Ser Val Asp His Glu Asn Trp Val Ser Asn Tyr Asn Ala Leu Arg
260 265 270
Ala Ala Ala Gly Ile Gln Pro Pro Gly Tyr Leu Ile His Glu Ser
275 280 285
Ala Cys Trp Ser Asp Thr Leu Gln Arg Trp Phe Phe Leu Pro Arg
290 295 300
Arg Ala Ser Gln Glu Arg Tyr Ser Glu Lys Asp Asp Glu Arg Lys
305 310 315
Gly Ala Asn Leu Leu Leu Ser Ala Ser Pro Asp Phe Gly Asp Ile
320 325 330
Ala Val Ser His Val Gly Ala Val Val Pro Thr His Gly Phe Ser
335 340 345
~/~.i
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
Ser Phe Lys Phe Ile Pro Asn Thr Asp Asp Gln Ile Ile Val Ala
350 355 360
Leu Lys Ser Glu Glu Asp Ser Gly Arg Val Ala Ser Tyr Ile Met
365 370 375
Ala Phe Thr Leu Asp Gly Arg Phe Leu Leu Pro Glu Thr Lys Ile
380 385 390
Gly Ser Val Lys Tyr Glu Gly Ile Glu Phe Ile
395 400
<210> 5
<211> 506
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2403107
<400> 5
Met Lys Arg Arg Ser Val Thr Met Thr Asp Gly Leu Thr Ala Asp
1 5 10 15
Lys Val Thr Arg Ser Asp Gly Cys Pro Thr Ser Thr Ser Leu Pro
20 25 30
Arg Pro Arg Asp Ser Ile Arg Ser Cys Ala Leu Ser Met Asp Gln
35 40 45
Ile Pro Asp Leu His Ser Pro Met Ser Pro Ile Ser Glu Ser Pro
50 55 60
Ser Ser Pro Ala Tyr Ser Thr Val Thr Arg Val His Ala Ala Pro
65 70 75
Ala Ala Pro Ser Ala Thr Ala Leu Pro Ala Sex Pro Val Ala Arg
80 85 90
Arg Ser Ser Glu Pro Gln Leu Cys Pro Gly Ser Ala Pro Lys Thr
95 100 105
His Gly Glu Ser Asp Lys Gly Pro His Thr Ser Pro Ser His Thr
110 115 120
Leu Gly Lys Ala Ser Pro Ser Pro Ser Leu Ser Ser Tyr Ser Asp
125 130 135
Pro Asp Ser Gly His Tyr Cys Gln Leu Gln Pro Pro Val Arg Gly
140 145 150
Ser Arg Glu Trp Ala Ala Thr Glu Thr Ser Ser Gln Gln Ala Arg
155 160 165
Ser Tyr Gly Glu Arg Leu Lys Glu Leu Ser Glu Asn Gly Ala Pro
170 175 180
Glu Gly Asp Trp Gly Lys Thr Phe Thr Val Pro Ile Val Glu Val
185 190 195
Thr Ser Ser Phe Asn Pro Ala Thr Phe Gln Ser Leu Leu Ile Pro
200 205 210
Arg Asp Asn Arg Pro Leu Glu Val Gly Leu Leu Arg Lys Val Lys
215 220 225
Glu Leu Leu Ala Glu Val Asp Ala Arg Thr Leu Ala Arg His Val
230 235 240
Thr Lys Val Asp Cys Leu Val Ala Arg Ile Leu Gly Val Thr Lys
245 250 255
Glu Met Gln Thr Leu Met Gly Val Arg Trp Gly Met Glu Leu Leu
260 265 270
6133
CA 02329076 2000-11-28
WO 99/61626 PCTNS99/12021
Thr Leu Pro His Gly Arg Gln Leu Arg Leu Asp Leu Leu Glu Arg
275 280 285
Phe His Thr Met Ser Ile Met Leu Ala Val Asp Ile Leu Gly Cys
290 295 300
Thr Gly Ser Ala Glu Glu Arg Ala Ala Leu Leu His Lys Thr Ile
305 310 315
Gln Leu Ala Ala Glu Leu Arg Gly Thr Met Gly Asn Met Phe Ser
320 325 330
Phe Ala Ala Val Met Gly Ala Leu Asp Met Ala Gln Ile Ser Arg
335 340 345
Leu Glu Gln Thr Trp Val Thr Leu Arg Gln Arg His Thr Glu Gly
350 355 360
Ala Ile Leu Tyr Glu Lys Lys Leu Lys Pro Phe Leu Lys Ser Leu
365 370 375
Asn Glu Gly Lys Glu Gly Pro Pro Leu Ser Asn Thr Thr Phe Pro
380 385 390
His Val Leu Pro Leu Ile Thr Leu Leu Glu Cys Asp Ser Ala Pro
395 400 405
Pro Glu Gly Pro Glu Pro Trp Gly Ser Thr Glu His Gly Val Glu
410 415 420
Val Val Leu Ala His Leu Glu Ala Ala Arg Thr Val Ala His His
425 430 435
Gly Gly Leu Tyr His Thr Asn Ala Glu Val Lys Leu Gln Gly Phe
440 445 450
Gln Ala Arg Pro Glu Leu Leu Glu Val Phe Ser Thr Glu Phe Gln
455 460 465
Met Arg Leu Leu Trp Gly Ser Gln Gly Ala Ser Ser Ser Gln Ala
470 475 480
Arg Arg Tyr Glu Lys Phe Asp Lys Val Leu Thr Ala Leu Ser His
485 490 495
Lys Leu Glu Pro Ala Val Arg Ser Ser Glu Leu
500 505
<210> 6
<211> 386
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 3069540
<400> 6
Met Arg Gly Glu Gln Gly Ala Ala Gly Ala Arg Val Leu Gln Phe
10 15
Thr Asn Cys Arg Ile Leu Arg Gly Gly Lys Leu Leu Arg Glu Asp
20 25 30
Leu Trp Val Arg Gly Gly Arg Ile Leu Asp Pro Glu Lys Leu Phe
35 40 45
Phe Glu Glu Arg Arg Val Ala Asp Glu Arg Arg Asp Cys Gly Gly
50 55 60
Arg Ile Leu Ala Pro Gly Phe Ile Asp Val Gln Ile Asn Gly Gly
65 70 75
Phe Gly Val Asp Phe Ser Gln Ala Thr Glu Asp Val Gly Ser Gly
80 85 90
7/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
Val Ala Leu Val Ala Arg Arg Ile Leu Ser His Gly Val Thr Ser
95 100 105
Phe Cys Pro Thr Leu Val Thr Ser Pro Pro Glu Val Tyr His Lys
110 115 120
Val Val Pro Gln Ile Pro Val Lys Ser Gly Gly Pro His Gly Ala
125 130 135
Gly Val Leu Gly Leu His Leu Glu Gly Pro Phe Ile Ser Arg Glu
140 195 150
Lys Arg Gly Ala His Pro Glu Ala His Leu Arg Ser Phe Glu Ala
155 160 165
Asp Ala Phe Gln Asp Leu Leu Ala Thr Tyr Gly Pro Leu Asp Asn
170 175 180
Val Arg Ile Val Thr Leu Ala Pro Glu Leu Gly Arg Ser His Glu
185 190 195
Val Ile Arg Ala Leu Thr Ala Arg Gly Ile Cys Val Ser Leu Gly
200 205 210
His Ser Val Ala Asp Leu Arg Ala Ala Glu Asp Ala Val Trp Ser
215 220 225
Gly Ala Thr Phe Ile Thr His Leu Phe Asn Ala Met Leu Pro Phe
230 235 240
His His Arg Asp Pro Gly Ile Val Gly Leu Leu Thr Ser Asp Arg
245 250 255
Leu Pro Ala Gly Arg Cys Ile Phe Tyr Gly Met Ile Ala Asp Gly
260 265 270
Thr His Thr Asn Pro Ala Ala Leu Arg Ile Ala His Arg Ala His
275 280 285
Pro Gln Gly Leu Val Leu Val Thr Asp Ala Ile Pro Ala Leu Gly
290 295 300
Leu Gly Asn Gly Arg His Thr Leu Gly Gln Gln Glu Val Glu Val
305 310 315
Asp Gly Leu Thr Ala Tyr Val Ala Gly Cys Ser Met Glu Ser Ala
320 325 330
Leu Glu Ala Ala Ser Leu His Pro Ala Gln Leu Leu Gly Leu Glu
335 340 345
Lys Ser Lys Gly Thr Leu Asp Phe Gly Ala Asp Ala Asp Phe Val
350 355 360
Val Leu Asp Asp Ser Leu His Val Gln Ala Thr Tyr Ile Ser Gly
365 370 375
Glu Leu Val Trp Gln Ala Asp Ala Ala Arg Gln
380 385
<210> 7
<211> 206
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 4182350
<400> 7
Met Ala Arg Gly Gly Trp Arg Arg Leu Arg Arg Leu Leu Ser Ala
1 5 10 15
Gly Gln Leu Leu Phe Gln Gly Arg Ala Leu Leu Val Thr Asn Thr
20 25 30
8/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
Leu Gly Cys Gly Ala Leu Met Ala Ala Gly Asp Gly Val Arg Gln
35 40 45
Ser Trp Glu IIe Arg Ala Arg Pro Gly Gln Val Phe Asp Pro Arg
50 55 60
Arg Ser Ala Ser Met Phe Ala Val Gly Cys Ser Met Gly Pro Phe
65 70 75
Leu His Tyr Trp Tyr Leu Ser Leu Asp Arg Leu Phe Pro Ala Ser
80 85 90
Gly Leu Arg Gly Phe Pro Asn Val Leu Lys Lys Val Leu Val Asp
95 100 105
Gln Leu Val Ala Ser Pro Leu Leu Gly Val Trp Tyr Phe Leu Gly
110 115 I20
Leu Gly Cys Leu Glu Gly Gln Thr Val Gly Glu Ser Cys Gln Glu
125 130 135
Leu Arg Glu Lys Phe Trp Glu Phe Tyr Lys Ala Asp Trp Cys Val
140 145 150
Trp Pro Ala Ala Gln Phe Val Asn Phe Leu Phe Val Pro Pro Gln
155 160 165
Phe Arg Val Thr Tyr Ile Asn Gly Leu Thr Leu Gly Trp Asp Thr
170 175 180
Tyr Leu Ser Tyr Leu Lys Tyr Arg Ser Pro Val Pro Leu Thr Pro
185 190 195
Pro Gly Cys Val Ala Leu Asp Thr Arg Ala Asp
200 205
<210> 8
<211> 1126
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 321510
<400> 8
cgcctccgcg atgccgctgc tcgtcgaggg gcggcgagtg cggctgccgc agtcagccgg 60
ggacctcgtc cgagcccacc cgcctttgga ggaaagagcc agacttctca gaggtcagtc 120
tgttcaacaa gtgggacccc agggccttct gtatgttcag caaagagagc ttgcagtgac 180
ctccccaaag gatggctcca tctccattct gggttctgat gatgccacta cttgtcacat 240
tgtggtcctg aggcacacag gtaatggggc cacctgcttg acacattgtg acggaaccga 300
caccaaagct gaggtcccct tgatcatgaa ctccataaaa tccttttctg accacgctca 360
atgtggaagg ctggaagtac accttgttgg aggcttcagt gacgacaggc agttgtcaca 420
aaaactcact catcaacttc ttagtgaatt tgacaggcaa gaagatgaca ttcacttagt 480
gacattatgt gtgacagaat taaatgaccg ggaagaaaac gaaaaccact ttccagtaat 540
atatggcatt gctgtcaaca ttaagactgc agagatttac agagcatcct ttcaagatcg 600
gggtccggag gagcagcttc gtgctgcgcg aactttagca ggaggaccaa tgattagcat 66o
ttatgatgca gagacagaac aacttcgtat aggaccgtac tcctggacac catttccaca 720
tgtggatttc tggttgcacc aagatgacaa gcaaatacta gagaatcttt ccacttcgcc 780
tctggctgag ccaccccact ttgttgaaca tattagatct accttgatgt ttttaaaaaa 840
acacccatct ccagctcaca cactgttttc tggaaataaa gccctactct acaaaaaaaa 900
tgaagatggc ttgtgggaaa agatctcttc tccaggaagt taaaaaacat gaattaccaa 960
agaaagcacc ttcttggcct gacagaccat tggtggggct ggcacgaatc cagatctgga 1020
tcctacatct gttgggtctt aggcctcctt ccctcctcag tgtctttcaa atgactttca 1080
tcaaatgact ttcaaaataa aaccttattt tggcaaaaaa aaaaaa 1126
9/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
<210> 9
<211> 2260
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 634343
<400> 9
cccccttgac taaagctcca aggacagaga aaaacatcca gatttgggaa cacaataaca 60
gatatgattg tccccacttc tactgccaaa attataaaac tgttaactcc tcctcatcag 220
cttacctgac tacttaaaag caaaagagtt aattaagtat tactaattgg tgatactaga 180
tcaatgaaga gaaagcagct gagatccaga ggagtggaag gtcccccttg actaaagcta 240
aatcactaaa ccttggccat ggtcacttcc tcttttccaa tctctgtggc agtttttgcc 300
ctaataaccc tgcaggttgg tactcaggac agttttatag ctgcagtgta tgaacatgct 360
gtcattttgc caaataaaac agaaacacca gtttctcagg aggatgcctt gaatctcatg 420
aacgagaata tagacattct ggagacagcg atcaagcagg cagctgagca gggtgctcga 480
atcattgtga ctccagaaga tgcactttat ggatggaaat ttaccaggga aactgttttc 540
ccttatctgg aggatatccc agaccctcag gtgaactgga ttccgtgtca agacccccac 600
agatttggtc acacaccagt acaagcaaga ctcagctgcc tggccaagga caactctatc 660
tatgtcttgg caaatttggg ggacaaaaag ccatgtaatt cccgtgactc cacatgtcct 720
cctaatggct actttcaata caataccaat gtggtgtata atacagaagg aaaactcgtg 780
gcacgttacc ataagtacca cctgtactct gagcctcagt ttaatgtccc tgaaaagccg 840
gagttggtga ctttcaacac cgcatttgga aggtttggca ttttcacgtg ctttgatata 900
ttcttctatg atcctggtgt taccctggtg aaagatttcc atgtggacac catactgttt 960
cccacagctt ggatgaacgt tttgcccctt ttgacagcta ttgaattcca ttcagcttgg 1020
gcaatgggaa tgggagttaa tcttcttgtg gccaacacac atcatgtcag cctaaatatg 1080
acaggaagtg gcatttatgc accaaatggt cccaaagtgt atcattatga catgaagaca 1140
gagttgggaa aacttctcct ttcagaggtg gattcacatc ccctatcctc gcttgcctac 1200
ccaacagctg ttaattggaa tgcctacgcc accaccatca aaccatttcc agtacagaaa 1260
aacactttca ggggatttat ttccagggat gggttcaact tcacagaact ttttgaaaat 1320
gccggaaacc ttacagtctg tcaaaaggag ctttgctgtc atttaagcta cagaatgtta 1380
caaaaagaag agaatgaagt atacgttcta ggagctttta caggattaca tggccgaagg 1440
agaagagagt actggcaggt ctgcacaatg ctgaagtgca aaactactaa tttgacaact 1500
tgtggacggc cagtagaaac tgcttctaca agatttgaaa tgttctccct cagtggcaca 1560
tttggaacag agtatgtttt tcctgaagtg ctacttaccg aaattcatct gtcacctgga 1620
aaatttgagg tgctgaaaga tgggcgtttg gtaaacaaga atggatcatc tgggcctata 1680
ctaacagtgt cactctttgg gaggtggtac acaaaggact cactttacag ctcatgtggg 1740
accagcaatt cagcaataac ttacctgcta atattcatat tattaatgat catagctttg 1800
caaaatattg taatgttata gggcgtctct ttatcactca gcttctgcat catatgcttg 1860
gctgaatgtg. tttatcggct tcccaagttt actaagaaac tttgaagggc tatttcagta 1920
gtatagacca gtgagtccta aatatttttt ctcatcaata attatttttt aagtattatg 1980
ataatgttgt ccattttttt ggctactctg aaatgttgca gtgtggaaca atggaaagag 2040
cctgggtgtt tgggtcagat aaatgaagat caaactccag ctccagcctc atttgcttga 2100
gactttgtgt gtatggggga cttgtatgta tgggagtgag gagtttcagg gccattgcaa 2160
acatagctgt gcccttgaag agaatagtaa tgatgggaat ttagaggttt atgactgaat 2220
tccctttgac attaaagact atttgaattc aaaaaaaaaa 2260
<210> 10
<211> 1675
<212> DNA
<213> Homo Sapiens
10/3 ~
CA 02329076 2000-11-28
WO 99/b1b26 PCT/US99/12021
<220>
<221> misc_feature
<223> Incyte Clone No: 2017918
<400> 10
ccaggccatg caggcccacg tgtctattat cctcccagtc cacaacgctg aaccgtggct 60
ggacgaatgt ttgaggtctg ttttgcaaca ggactttgaa ggtaccatgg agctgtctgt 120
tttcaatgat gccagtaagg acaagtctgg ggctatcatt gaaaaatgga gagtgaagct 180
ggaagattct ggtgtccacg tgatcattgg ggggcacgat tctccctctc ctagaggcgt 240
cggatacgct aaaaatcaag cagttgccca gagctcaggg tcttaccttt gctttttgga 300
ttcggatgac gtcatgatgc cccagcgggt gaggctgcaa cacgaggctg ccgttcagca 360
cccgtcgagc atcattggtt gcagagtgag gagagatccc cctaactcca ccgaacgata 420
cacacgttgg atcaaccagc tgacgccgga gcagctccta acccaggttt tcacctcaaa 480
tggccccacg gtgatcatgc ccacctggtt ctgctcgcga gcgtggttct cccacgtggg 540
cccctttaac gagggaggtc agggcgtccc ggaggacctg ctgttcttct acgagcacct 600
caggaagggc ggcggcgtca tccgcgtgga ccagagtctc ctgctgtatc gccaccaccc 660
acaggcggcc acgcactgcg tcctcgagac gaccatctgg acccaccgcg tccgcttcct 720
ggaagagcag gccctgcccc gctgggcggc cttcaccatc tggaacgctg gcaagcaggg 780
gcgccggctg taccgcagct tgactgccgg cagccagcgc aaggtggtgg cattctgtga 840
cgtggacgag aacaagatca ggaaaggctt ctattgccac gaggactctc aggaaagacc 900
caagccccga atccccatcc tgcacttccg agccgcccgg ccacccttcg tcatctgcgt 960
gaagctggac ctcacagggg gcacctttga ggncaacctg aggtcactgc acttgcagga 1020
gggccaggac ttccttcact tcagctgacg gacccatggc agctgctccc agtgcatccc 1080
aggccagcag gagctttgtg agctgcaggc atggcgatgg tgcgcctgtt ccacacccag 1140
caggcgcaac cagagtctcg tgtgtgccga ccacaggagc caagcctttt ccactgtgtg 1200
gactcatgtg gccaaggcta ggcctggtca cccaggaccc tcaccacgtg accccagcca 1260
atcgggacag ttcaaggagg aggagacccc tattacacag gttggaataa aatatttaaa 1320
tctcgtagaa taaaggacta ggggtgatag ggggagtatg ggataggagg aatgggtggg 1380
cgggaaaaaa gaaagggcgc ccgcttctgg ggggtccgag tttatccgtc cgcttggatg 1440
cggaactgca gagcccttct aatggggggc acctaaattt tcaatctcct gggcccggcg 1500
gttttacaga gctgttggct ggggaaaaca cctggggggt tacgccaagt tgaaatcgcc 1560
ttgggaggag aagtccccct ttgtgggcaa cttggggggt aaatggcgag aagggccccg 1620
gacgcgattg ggcctttccc aaagtgtttg cgccggcctt gattgggggg gaggg 1675
<210> 11
<211> 3203
<212> DNA
<2I3> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2175072
<400> 11
gagatggagt aattttgctg tggaaagact tcacgtcttg ccgaatgaaa tcagcctcca 60
gcgcctgcag cttccggaac taagatgtga ctgggctgta attttgctgt ggaaagactt 120
cacgtcttgc cgaatgaaag tcccgcctgt ctgtcacgct gatgcccgtg cagctgtctg 180
agcacccgga atggaatgag tctatgcact ccctccggat cagtgtgggg ggccttcctg 240
tgctggcgtc catgaccaag gccgcggacc cccgcttccg cccccgctgg aaggtgatcc 300
tgacgttctt tgtgggtgct gccatcctct ggctgctctg ctcccaccgc ccggcccccg 360
gcaggccccc cacccacaat gcacacaact ggaggctcgg ccaggcgccc gccaactggt 420
acaatgacac ctaccccctg tctcccccac aaaggacacc ggctgggatt cggtatcgaa 480
tcgcagttat cgcagacctg gacacagagt caagggccca agaggaaaac acctggttca 540
gttacctgaa aaagggctac ctgaccctgt cagacagtgg ggacaaggtg gccgtggaat 600
11/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
gggacaaaga ccatggggtc ctggagtccc acctggcgga gaaggggaga ggcatggagc 660
tatccgacct gattgttttc aatgggaaac tctactccgt ggatgaccgg acgggggtcg 720
tctaccagat cgaaggcagc aaagccgtgc cctgggtgat tctgtccgac ggcgacggca 780
ccgtggagaa aggcttcaag gccgaatggc tggcagtgaa ggacgagcgt ctgtacgtgg 840
gcggcctggg caaggagtgg acgaccacta cgggtgatgt ggtgaacgag aacccggagt 900
gggtgaaggt ggtgggctac aagggcagcg tggaccacga gaactgggtg tccaactaca 960
acgccctgcg ggctgctgcc ggcatccagc cgccaggcta cctcatccat gagtctgcct 1020
gctggagtga cacgctgcag cgctggttct tcctgccgcg ccgcgccagc caggagcgct 1080
acagcgagaa ggacgacgag cgcaagggcg ccaacctgct gctgagcgcc tcccctgact 1140
tcggcgacat cgctgtgagc cacgtcgggg cggtggtccc cactcacggc ttctcgtcct 1200
tcaagttcat ccccaacacc gacgaccaga tcattgtggc cctcaaatcc gaggaggaca 1260
gcggcagagt cgcctcctac atcatggcct tcacgctgga cgggcgcttc ctgttgccgg 1320
agaccaagat cggaagcgtg aaatacgaag gcatcgagtt catttaactc aaaacggaaa 1380
cactgagcaa ggccatcagg actcagcttt tataaaaaca agaggagtgc acttttgttt 1440
tgttttgttc tttttggaac tgtgcctggg ttggaggtct ggacagggag cccagtcccg 1500
ggccccatag tggtgcgggg cactgggacc cccgggcccc acggaggccg cggtctgaac 1560
tgctttccat gctgccatct ggtggtgatt tcggtcactt caggcattga ctcaaggcct 1620
gcctaactgg ctgggtcgtt tcttccatcc gacctcgttt cttttctttc ctatgttctt 1680
ttgttcagtg aatatcccta gagctcctac catatgtcag gccctatgcc tcaccctgag 1740
aacgcagtat gcatgaggtg gacctgtttg ctgggaaccc caggtcaccc ccttttcttc 1800
ctactctgtg cctggagcat catgtccacc cctgcagatc cttggaaaag aaaatgttta 1860
tgttgcaggg tattgcatgg tcacgagtga gggcaggccc ctgggggaca catctgccca 1920
cagctgcaca ggccagggcg caggcacatc tgttggttct caggcctcag ataaaaccat 1980
ctccgcatca tatggccagt gaccgctttc tcccttcaag aaaattctgt ggctgtgcag 2040
tactttgaag ttttaattat taacctgctt taattaaagc agtttccttt cttataaagt 2100
ggaatcacca aatcttatca cacagagcac agtcctgtag ttacccagcc cgctccagca 2160
gtgcgggaga ttgtaaggaa gcggtggcgg ctggtgaagc aagtctcaca tgtcggtgtt 2220
cttggccaat ggatacaaag ataaagaaaa tgttgccttt ttctaggaac tgtcagaaat 2280
cctcatgcct ttcaagactt ctgtgaatga cttgaatttt ttattccctg cctagggtct 2340
gtgaacgagg cctgtctctt ccctggggtt tctttccatg gcctttattt ctcctcttcc 2400
agtgggagtt ttgcaggctc ttctctgtgg aaacttcacg agcgttggct gggcctcggc 2460
ttcgctggag tgtactccag ggtgaaggca gagtgggatt tgagacccag gttaggcacg 2520
acccaggtct gagaagggac gtttccatca ttcacagtgc cctccccaca gcactacctc 2580
accccgaccc ccaccctcac tcctacccca ccccgcgatc gtcaggggtg ccacggtggg 2640
ccggagggtg ccggctctgg ctgtccctgt gccggtccct cacaaacctc tccccctttg 2700
aaactcaagc acagctgcga ggagggcagc gaggagggac ccctctctca tggttgtctc 2760
tttcccccgc tatgtcatag gtagtggagg aagcgaagga agtgaacgct gaatgtgacg 2820
catttctgaa gagctcagct gtcaccgggc atagcctgga agccccaagt ctgttctgac 2880
tttgcctggc tgtctccttg acccgcctcc tagatcattg tccttgatgt ccaggctggg 2940
tcatttaaaa tagagatgca atcaggaagg ttgggggact tgggactgtg gctgaattga 3000
gaccttgctg atgtattcat gtcagcacct gagtcacagc ccaggtgccc ggaagcagcc 3060
tcttcgcata ggcagtgatt tgcgattact ttaaagctca ccttttttct tcccctctct 3120
gttcgctgct gtcagcataa tgattgtgtt ccttccctat gggatccatc tgttttgtaa 3180
acaataaagc gtctgaggga tgt 3203
<210> 12
<211> 2648
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2403107
12/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
<400> 12
cggtggccct caggaggaag gaaggaaaaa cagccgtctc tggcctctgg cctctccagg 60
ctctccaccc tgggggcaga atcaattctg tagccctcta gcaccggcag tggctccgca 120
gtcagctggg cagtggccgt gactgggtca ctctacccta ggaagtgagt gcaggggctg 180
ctggagagcc agaggctggc agcgactatg tgaagttctc caaggagaag tacatcctgg 240
actcatcgcc agagaaactc cacaaggaat tggaggagga gctcaaactc agcagcacgg 300
atctccgcag ccatgcctgg taccatggcc gcatcccccg agaggtctcg gagaccttgg 360
tacaacgcaa cggcgacttc ctcatccggg actcactcac cagcctgggc gactatgtgc 420
tcacgtgctg ctggcgcaac caggccttgc acttcaagat caacaaggtg gtggtgaagg 480
caggcgagag ctacacacac atccagtacc tgtttgagca ggagagcttt gaccacgtgc 540
ccgccctcgt gcgctatcat gtgggcagcc gcaaggctgt gtcagagcag agtggtgcca 600
tcatctactg cccggtgaac cgcaccttcc cactgcgcta cctcgaggcc agctatggcc 660
tgggacaggg gagtagcaag cctgctagcc ccgtcagccc ctcaggcccc aagggcagcc 720
acatgaagcg gcgcagcgtc accatgaccg atgggctcac tgctgacaag gtcacccgca 780
gcgatggctg ccccaccagt acgtcgctgc cccgccctcg ggactccatc cgcagctgtg 840
ccctcagcat ggaccagatc ccagacctgc actcacccat gtcgcccatc tccgagagcc 900
ctagctcccc tgcctacagc actgtaaccc gtgtccatgc cgcccctgca gccccttctg 960
ccacagcatt gcctgcctcc cctgtcgccc gccgttccag tgagccccag ctgtgtcccg 1020
gaagtgcccc aaagacccat ggggagtcag acaagggccc ccacaccagc ccctcccaca 1080
cccttggcaa ggcctccccg tcaccatcac tcagcagcta cagtgacccg gactctggcc 1140
actactgcca gctccagcct cccgtgcgtg gcagccgaga gtgggcagcg actgagacct 1200
ccagccagca ggccaggagc tatggggaga ggctaaagga actgtcagaa aatggggccc 1260
ctgaagggga ctggggcaag accttcacag tccccatcgt ggaagtcact tcttccttca 1320
acccggccac cttccagtca ctactgatcc ccagggataa ccggccactg gaggtgggcc 1380
ttctgcgcaa ggtcaaggag ctgctggcag aagtggatgc ccggacgctg gcccggcatg 1440
tcaccaaggt ggactgcctg gttgctagga tactgggcgt taccaaggag atgcagaccc 1500
taatgggagt ccgctggggc atggaactgc tcaccctccc ccatggccgg cagctacgcc 1560
tagacctgct ggaaaggttc cacaccatgt ccatcatgct ggccgtggac atcctgggct 1620
gcaccggctc tgcggaggag cgggcagcgc tgctgcacaa gaccattcag ctggcggccg 1680
agctgcgggg gactatgggc aacatgttca gcttcgcggc ggtcatgggt gccctggaca 1740
tggctcagat ttctcggctg gagcagacat gggtgaccct gcggcagcga cacacagagg 1800
gtgccatcct gtacgagaag aagctcaagc cttttctcaa gagcctcaac gagggcaaag 1860
aaggcccgcc gctgagcaac accacgtttc ctcatgtgct gcccctcatc accctgctgg 1920
agtgtgactc ggccccacca gagggccctg agccctgggg cagcacggag cacggcgtgg 1980
aggtggtgct ggctcacctg gaggccgccc gcacagtggc acaccacgga ggcctgtacc 2040
acaccaatgc tgaagtcaag ctgcaggggt tccaggcccg gccggagctc ctggaggtgt 2100
tcagcacgga gttccagatg cgccttctct ggggcagtca gggtgccagc agcagccagg 2160
cccggcgcta tgagaagttc gacaaggtcc tcactgccct gtcccacaag ctggaacctg 2220
ctgtccgctc cagcgagctg tgaccccagg gacatttccc ctctgcagct gcggacagcg 2280
tcaggggcag aggggcacac aactttcccc agagcacccc aaggacactg tgatcaaccc 2340
gagaatgttc tgggttcaac tcaagcatct cccttgcacc tccagggtcc tgcgtggacc 2400
ctgggttcca tcccaactgc tacaagctca acaggtctcc attgatggag cacaggaacg 2460
gcggttcccc caccagcctt tgctgcttcc cttcctgctg tgggttcctg gtttcggacc 2520
ctcggaagcc aggtgcttca ggctccctca atctctgtgc tggacctcta caaagatcaa 2580
agtctccatt ttaaatgtga aataagtctc tttgtctgga aaattgatgt tcagaaaggg 2640
ggaccccg 2648
<210> 13
<211> 1361
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 1321, 1333, 1354, 1355
<223> a or g or c or t, unknown, or other
13/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
<220>
<221> misc_feature
<223> Incyte Clone No: 3069540
<400> 13
ggggctccgg agccgctcgc tcccgacacg gctcacgatg cgcggcgagc agggcgcggc 60
gggggcccgc gtgctccagt tcactaactg ccggatcctg cgcggaggga aactgctcag 120
ggaggatctg tgggtgcgcg gaggccgcat cttggaccca gagaagctgt tctttgagga 180
gcggcgcgtg gccgacgagc ggcgggactg cgggggccgc atcttggctc ccggattcat 240
cgacgtgcag atcaacggtg gatttggtgt tgacttctct caagccacgg aggacgtggg 300
ttcgggggtt gccctcgtgg cccggaggat cctgtcgcac ggcgtcacct ccttctgccc 360
caccctggtc acttccccac cggaggttta tcacaaggtt gttcctcaga tccctgtgaa 420
gagtggtggt ccccatgggg caggggtcct cgggctgcac ctggagggcc ccttcatcag 480
ccgggagaag cggggcgcgc accccgaggc ccacctccgc tccttcgagg ccgatgcctt 540
ccaggacttg ctggccacct acgggcccct ggacaatgtc cgcatcgtga cgctggcccc 600
agagttgggc cgtagccacg aagtgatccg ggcgctgacg gcccgtggca tctgcgtgtc 660
cctagggcac tcagtggctg acctgcgggc ggcagaggat gctgtgtgga gcggagccac 720
cttcatcacc cacctcttca acgccatgct gcctttccac caccgcgacc caggcatcgt 780
ggggctcctg accagcgacc ggctgcccgc aggccgctgc atcttctatg ggatgattgc 840
agatggcacg cacaccaacc ccgccgccct gcggatcgcc caccgtgccc atccccaggg 900
gctggtgctg gtcaccgatg ccatccctgc cttgggcctg ggcaacggcc ggcacacgct 960
gggacagcag gaagtggaag tggacggtct gacggcctac gtggcaggct gcagcatgga 1020
gtcggccctg gaggctgcat ccctgcaccc cgcccagttg ctggggctgg agaagagtaa 1080
ggggaccctg gactttggtg ctgacgcaga cttcgtggtg ctcgacgact cccttcacgt 1140
ccaggccacc tacatctcgg gtgagctggt gtggcaggcg gacgcagcta ggcagtgaca 1200
aggacctcgg ctgagaggac acctggccgc agcgggatgc catcagggcc gggtggttgg 1260
ggagctggtc tccagggagt gagtcgggag ccctgctgga ttgatgccca tggcctgtgc 1320
ngtgccctgg agncggtggc tgggataaac gtgnncccag c 1361
<210> 14
<211> 1401
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 4182350
<400> 14
gcaatttggg atcgacagtg actcgcgact ggtcggcgcg gcgaaagcag agcggcgcgc 60
cggttccttg gttcctgagg gcgatggcgc ggggtggctg gcgccggcta cgccgcctgt 120
tatccgcggg gcagcttcta ttccagggcc gcgcgctgct cgtcactaac acgctgggct 180
gcggcgcgct catggcggcc ggtgatggcg tgcgccagtc ctgggagatc cgcgcccggc 240
ccggccaggt tttcgaccca cggcgctccg cgagcatgtt tgcggtgggc tgcagcatgg 300
gtcccttcct gcactactgg tacttgtcgc tggaccgcct attccctgcg tctggcctcc 360
gaggcttccc aaatgtcctc aagaaggtcc tcgtggatca gctggtagcc tctccattgc 420
tgggcgtctg gtacttcttg ggccttggct gcctggaggg tcagacagtg ggtgagagct 480
gccaggagct gcgggagaag ttctgggaat tctacaaggc agactggtgc gtgtggcctg 540
ctgcgcagtt cgtgaacttc ctcttcgtgc ccccccaatt tcgagtcacc tacatcaacg 600
gcctgacgct gggctgggac acgtacctgt cctacttgaa gtaccggagc ccagttcctc 660
tgacaccccc aggctgtgtg gccctggaca cccgagcaga ctgaactgtc tgcttcctgg 720
accagatgca agactgtctc ctggcggacc accccctctg acagaagggg aatgggctcc 780
tgcagcaagc tcgggtcttg agccacgtcc cagcaccact tcagctccgg agcattgggc 840
tgagccgccc tttccaagct cacttctggg actgagtttc ctcaaccgga acacacccat 900
14/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
gaagatggat gatcatcccc tagcccttct cagcaggaac ctcatgcgac ctgtgaccaa 960
gatgtcccat cctcagcaca gggcccactc tgccaaccag tctcaagcac cagcccctca 1020
acactgccat ccacctggct ctgggccaag ccaccaatcc agagctccct caggtcctgg 1080
gactaaggcg gggacatgac tgatcccctc agagcaggct caggcctgga gtcggccccc 1140
aaaagtttca catagggcca ggcagcctct gtgtttcttt ccctggtcct gaactgtgga 1200
aatgccatta aactctctct ataatgtaac tgaaactgct ggctgggcgc agtggctccc 1260
acctgtaatc tcagcccttc ccgaggctga ggtggaagga ttgctcgagg ccaggtgttc 1320
gagaccagcc tgggcaacat agcaagaccc tgtctctatt tatattgaaa aataaaaata 1380
acaccagagg aataaaaaaa a 1401
<210> 15
<211> 223
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 95, 182
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 030600H1
<400> 15
ccggagccca gttcctctga cacccccagg ctgtgtggcc ctggacaccc gagcagactg 60
aactgtctgc ttcctggacc agatgcaaga ctgtntcctg gcggaccacc ccctctgaca 120
gaaggggaat gggctcctgc agcaagctcg ggtcttgagc cacgtcccag caccacttca 180
gntccggagc attgggctga gccgcccttt ccaagctcac ttt 223
<210> 16
<211> 530
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 94, 414, 437, 527
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 121448681
<400> 16
gcggcagcga cacacagagg gtgccatcct gtacgagaag aagctcaagc cttttctcaa 60
gagcctcaac gagggcaaag aaggcccgcc gcanagcaac accacgtttc ctcatgtgct 120
gcccctcatc accctgctgg agtgtgactc ggccccacca gagggccctg agccctgggg 180
cagcacggag cacggcgtgg aggtggtgct ggctcacctg gaggccgccc gcacagtggc 240
acaccacgga ggcctgtacc acaccaatgc tgaagtcaag ctgcaggggt tccaggcccg 300
gccggagctc ctggaggtgt tcagcacgga gttccagatg cgccttctct ggggcagtca 360
gggtgccagc agcagccagg cccggcgcta tgagaagttc gacaaggtcc tcantgccct 420
gtcccacaag ctggaanctg ctgtccgctc cagcgagctg tgaacccagg gacatttccc 480
15/33
ctgcgcagtt cgtgaacttc ctcttcgtgc ccccccaatt tcg
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
ctctgcagct gggacaggtc aggggcaaag ggcaacaatt tcccagngcc 530
<210> 17
<211> 691
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 531, 540, 588, 612, 665
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 121549271
<400> 17
gctttattgt ttacaaaaca gatggatccc atagggaagg aacacaatca ttatgctgac 60
agcagcgaac agagagggga agaaaaaagg tgagctttaa agtaatcgca aatcactgcc 120
tatgcgaaga ggctgcttcc gggcacctgg gctgtgactc aggtgctgac atgaatacat 180
cagcaaggtc tcaattcagc cacagtccca agtcccccaa ccttcctgat tgcatctcta 240
ttttaaatga cccagcctgg acatcaagga caatgatcta ggaggcgggt caaggagaca 300
gccaggcaaa gtcagaacag acttggggct tccaggctat gcccggtgac agctgagctc 360
ttcagaaatg cgtcacattc agcgttcact tccttcgctt cctccactac ctatgacata 420
gcgggggaaa gagacaacca tgagagaggg gtccctcctc gctgccctcc tcgcagtgtg 480
cttgagtttc aaagggggag aggtttgtga gggaccggca cagggacagc nagagccggn 540
accctccggc ccaccgtggc acccctgacg attcccgggg tggggtanga attaagggtg 600
ggggtcgggg tnaaggaagt cctttgggga gggcacttgt taattattgg aaaacgtccc 660
ttttnagctt gggtcgtgcc taaccttggg t 691
<210> 18
<211> 615
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 568, 571
<223> a or g or c or t, unknor~m, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 121775671
<400> 18
atagtcttta atgtcaaagg gaattcagtc ataaacctct aaattcccat cattactatt 60
ctcttcaagg gcacagctat gtttgcaatg gccctgaaac tcctcactcc catacataca 120
agtcccccat acacacaaag tctcaagcaa atgaggctgg agctggagtt tgatcttcat 180
ttatctgacc caaacaccca ggctctttcc attgttccac actgcaacat ttcagagtag 240
ccaaaaaaat ggacaacatt atcataatac ttaaaaaata attattgatg agaaaaaata 300
tttaggactc actggtctat actactgaaa tagcccttca aagtttctta gtaaacttgg 360
gaagccgata aacacattca gccaagcata tgatgcagaa gctgagtgat aaagagacgc 420
cctataacat tacaatattt tgcaaagcta tgatcattaa taatatgaat attagcaggt 480
16/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
aagttattgc tgaattgctg gtcccacatg agctgtaaag tgagtccttt gtgtaccacc 540
tcccaaagag tgacactggt tagtatangg ncccagatgg ttccattcct ggtttaccaa 600
acggcccatt ttttc 615
<210> 19
<211> 547
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 22, 40, 460, 468, 488, 541
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 1269841F1
<400> 19
ggcccctggg gacacatctg cncacagctg cacaggccan ggcgcaggca catctgttgg 60
ttctcaggcc tcagataaaa ccatctccgc atcatatggc cagtgaccgc tttctccctt 120
caagaaaatt ctgtggctgt gcagtacttt gaagttttaa ttattaacct gctttaatta 180
aagcagtttc ctttcttata aagtggaatc accaaatctt atcacacaga gcacagtcct 240
gtagttaccc agcccgctcc agcagtgcgg gagattgtaa ggaagcggtg gcggctggtg 300
aagcaagtct cacatgtcgg cgttcttggc caatggatac aaagataaag aaaatgttgc 360
ctttttctag gaactgtcag aaatcctcga tgcctttcaa gacttctgtg aatgactgaa 420
ttttttattc cctgcctagg gtctgtgaac gaagcctgtn tcttcccngg ggggtttctt 480
tccatggnct ttattctcct cttccagtgg gaatttttgc aggcgccttc gcgtgggaac 540
ntcacga 547
<210> 20
<211> 502
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 89, 460
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 1284408F6
<400> 20
gcggcgcgtg gccgacgagc ggcgggactg cgggggccgc atcttggctc ccggattcat 60
cgacgtgcag atcaacggtg gatttggtnt tgacttctct caagccacgg aggacgtggg 120
ttcgggggtt gccctcgtgg cccggaggat cctgtcgcac ggcgtcacct ccttctgccc 180
caccctggtc acttccccac cggaggttta tcacaaggtt gttcctcaga tccctgtgaa 240
gagtggtggt ccccatgggg caggggtcct cgggctgcac ctggagggcc ccttcatcag 300
ccgggagaag cggggcgcgc accccgaggc ccacctccgc tccttcgagg ccgatgcctt 360
ccaggattgc tggccaccta cgggcccctg gacaatgtcc gcatcgtgac gctggcccag 420
atttgggcgt aaccacgaag taattccggg cgctgacggn ccgtggcatc tgcgtgttcc 480
17/3 3
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
taaggaatca attggttgac tt
502
<210> 21
<211> 707
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 3, 7, 21, 25, 34, 36, 53, 454, 495, 499, 549, 552, 596, 617, 623,
654, 656, 661, 678, 681, 683
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 133996971
<400> 21
atnaatntat agatggatcc natanggaag gaananaatc attatgctga cancagcgaa 60
cagagagggg aagaaaaaag gtgagcttta aagtaatcgc aaatcactgc ctatgcgaag 120
aggctgcttc cgggcacctg ggctgtgact caggtgctga catgaataca tcagcaaggt 180
ctcaattcag ccacagtccc aagtccccca accttcctga ttgcatctct attttaaatg 240
acccagcctg gacatcaagg acaatgatct aggaggcggg tcaaggagac agccaggcaa 300
agtcagaaca gacttggggc ttccaggcta tgcccggtga cagctgagct cttcagaaat 360
gcgtcacatt cagcgttcac ttccttcgct tcctccacta cctgggtctc aaatcccact 420
ctgccttcac cctggagtac actccagcga agcngaggcc cagccaacgc tcgtgaagtt 480
tccacagaga agagnctgna aaactcccac tggaagagga gaaataaagg ccatggaaag 540
aaaccccang gnagagacag gcctcgttca cagaccctaa ggcagggaat aaaaantcca 600
gtcattcaca gaagtcntga aangcatgag ggatttctga cattcctaga aaangncaca 660
nttccttaac cttggaanca ntngccaaga agccgacatg tgagact 707
<210> 22
<211> 577
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 324, 412, 459, 468, 486, 514, 526, 530, 543, 544, 568, 576
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 1362518F6
<400> 22
ccgcgaccca ggcatcgtgg ggctcctgac cagcgaccgg ctgcccgcaa gccgctgcat 60
cttctatggg atgattgcag atggcacgca caccaacccc gccgccctgc ggatcgccca 120
ccgtgcccat ccccaggggc tggtgctggt caccgatgcc atccctgcct tgggcctggg 180
caacggccgg cacacgctgg gacagcagga agtggaagtg gacggtctga cggcctacgt 240
ggcaggctgc agcatggagt cggccctgga ggctgcatcc ctgcaccccg cccagttgct 300
ggggctggag aagagtaagg ggancctggg atttggtgct gacgcagatt cgtggtgctc 360
gacgactccc ttcaacgtcc aaggccaact acatctcggg tgaactggtg tnggcaaggc 420
18/33
CA 02329076 2000-11-28
WO 99/61626 PCTNS99/12021
ggacgaagct aggcagtgac aaggacttcg ggttgagang acaacttngg ccgcaacggg 480
ggattncatt aagggcccgg ttggttttgg gaanttggtc ctccangggn ttgagttccg 540
gannccctgc ttggatttga atgcccangg ggcctnt 577
<210> 23
<211> 424
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 92
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 1513403F1
<400> 23
agcttataaa gtggaatcac caaatcttat cacacagagc acagtcctgt agttacccag 60
cccgctccag cagtgcggga gattgtaagg angcggtggc ggctggtgaa gcaagtctca 120
catgtcggcg ttcttggcca atggatacaa agataaagaa aatgttgcct ttttctagga 180
actgtcagaa atcctcatgc ctttcaagac ttctgtgaat gacttgaatt ttttattccc 240
tgcctagggt ctgtgaacga ggcctgtctc ttccctgggg tttctttcca tggcctttat 300
ttctcctctt ccagtgggag ttttgcaggc tcttctctgt ggaaacttca cgagcgttgg 360
ctgggcctcg gcttcgctgg agtgtactcc agggtgaagc agaatgggat ttgagacccc 420
aggt 424
<210> 24
<211> 524
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 71, 319, 351, 400, 409, 415, 430, 453, 466, 490, 507, 519
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 1514414F6
<400> 24
tggcatctgc gtgtccctag ggcactcagt ggctgacctg cgggcggcag aggatgctgt 60
gtggagcgga ncaccttcat cacccacctc ttcaacgcca tgctgccttt ccaccaccgc 120
gacccaggca tcgtggggct cctgaccagc gaccggctgc ccgcaggccg ctgcatcttc 180
tatgggatga ttgcagatgg cacgcacacc aaccccgccg ccctgcggat cgcccaccgt 240
gcccatcccc aggggctggt gctggtcacc gatgccatcc ctggccttgg gcctgggcaa 300
cggccggcac acgctgggna aacaggaagt ggaagtggac ggtctgacgg ntacgtggca 360
ggcaccaaga cgctgagtgg cagccatagc cccaatggan gtctgtgtnc gggantttct 420
gcaaggccan aaggttgcag gaatggagtc ggncctggag ggctgnattc ctgcaacccg 480
gcccaaattn ctgggggttg gagaagngta aaggggganc cctt 524
19/33
CA 02329076 2000-11-28
WO 99161626 PCT/US99/12021
<210> 25
<211> 684
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 44, 173, 314, 318, 350, 381, 383, 385, 389, 398, 403, 418, 423, 424,
427, 434, 445, 455, 459, 462, 481, 498, 500, 506, 516, 518, 535, 541, 544,
556, 564, 566, 579, 586, 590, 598, 606, 611, 612, 625, 629, 639, 642, 644,
648, 671, 677
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 1517312F6
<400> 25
gccgctgggt ttaggaggtc cccgggttgc cggcggcgac agcnggggaa gcatgactgc 60
tgtgggccga agtgccccgc gctggggtcc cgaggggctg ctggagagcc agaggtggca 120
gcgactatgt gaagttctcc aaggagaagt acatcctgga ctcatcgcca ganaaactcc 180
acaaggaatt ggaggaggag ctcaaactca gcagcacgga tctccgcagc catgcctggt 240
accatggccg catcccccga gaggtctcgg agaccttggt acaacgcaac gggcgattct 300
catccgggac tcantcanca gcctggggga ctatgtgctc aagtgccggn tgggggcaac 360
caaggccttt gcactttcaa ngntnaacna aggtggtngt ttnaagggca aggcgaanaa 420
gtnncancac cacnttccaa gttancttgt ttttnaaanc anggaaaaaa gctttttgaa 480
nccaaaggtt ggcccggncn cctttnggtg gggggntnaa ttcaattttt ggggnaaaaa 540
nccnggcaaa agggtnttgt gttntnaagg aaggccaana aatttnggtn tgccccantg 600
aaattnttaa nntttgcccc cgggnttgna aaacggggna ancntttncc cccaaattgg 660
ggggtttaaa ntttcgngag gggg 684
<210> 26
<211> 490
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 125, 484
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 1528049F1
<400> 26
gctgcgagga gggcagcgag gagggacccc tctctcatgg ttgtctcttt cccccgctat 60
gtcataggta gtggaggaag cgaaggaagt gaacgctaaa tgtgacgcat ttctgaagag 120
ctcanctgtc accgggcata gcctggaagc cccaagtctg ttctgacttt gcctggctgt 180
ctccttgacc cgcctcctag atcattgtcc ttgatgtcca ggctgggtca tttaaaatag 240
agatgcaatc aggaaggttg ggggacttgg gactgtggct gaattgagac cttgctgatg 300
tattcatgtc agcacctgag tcacagccca ggtgcccgga agcagcctct tcgcataggc 360
agtgatttgc gattacttta aagctcacct tttttcttcc cctctctgtt cgctgctgtc 420
agcataatga ttgtgttcct tccctatggg atccatctgt tttgtaaaca ataaagcgtc 480
20/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
tganggatgt 490
<210> 27
<211> 556
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 444, 471, 479, 496, 518, 544
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 1578848F6
<400> 27
ggggctccgg agccgctcgc tcccgacacg gctcacgatg cgcggcgagc agggcgcggc 60
gggggcccgc gtgctccagt tcactaactg ccggatcctg cgcggaggga aactgctcag 120
ggaggatctg tgggtgcgcg gaggccgcat cttggaccca gagaagctgt tctttgagga 180
gcggcgcgtg gccgacgagc ggcgggactg cgggggccgc atcttggctc ccggattcat 240
cgacgtgcag atcaacggtg gatttggtgt tgattctctc aagccacgga ggacgtgggt 300
tcgggggttg ccctcgtggc ccggaagatc ctgtcgcaag gcgtcaactc cttctgcccc 360
aacctggtca tttcccaccg gagtttatca caaagttgtt cctcagattc ctgtgaagaa 420
tggtggtccc atggggcaag ggtnctcggg ctgcactgga ggcccttata nccggaaanc 480
gggcggcacc cgaggncact tcgtcctcga gcgatgcntc aaacttctgc actaagggcc 540
tganaattcg atgtaa 5S6
<210> 28
<211> 594
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 232, 376, 419, 521, 523, 533, 541, 555, 561, 571, 577
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 165234976
<400> 28
tctttgtttt tataaaagct gagtcctgat ggccttgctc agtgtttccg ttttgagtta 60
aatgaactcg atgccttcgt atttcacgct tccgatcttg gtctccggca acaggaagcg 120
cccgtccagc gtgaaggcca tgatgtagga ggcgactctg ccgctgtcct cctcggattt 180
gagggccaca atgatctggt cgtcggtgtt ggggatgaac ttgaaggacg anaagccgtg 240
agtggggacc accgccccga cgtggctcac agcgatgtcg ccgaagtcag gggaggcgct 300
cagcagcagg ttggcgccct tgcgctcgtc gtccttctcg ctgtagcgct cctggctggc 360
gcggcgcggc aggaanaacc agcgctgcag cgtgtcactc cagcaggcag actcatggnt 420
gaggtagcct ggcggctgga tgccggcagc aacccgcagg tgtgtagttg gacacccagt 480
tctcgtggtc cacgctgcct tgtagcccac cacttcacca ntncggttcc gtnacacata 540
nccgtagtgt gtcantcttg ncaggcgcca ntaaacntgt cttaattcag catc 594
21/33
CA 02329076 2000-11-28
WO 99/G1626 PCT/US99/12021
<210> 29
<211> 514
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 19, 361, 409, 439, 450, 482, 484, 493, 496, 497, 513
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 1657538F6
<400> 29
ctgaaaaagg gctacctgna ccctgtcaga cagtggggac aaggtggccg tggaatggga 60
caaagaccat ggggtcctac gagtcccacc tggcggagaa ggggagaggc atggagctat 120
ccgacctgat tgttttcaat gggaaactct actccgtgga tgaccggacg ggggtcgtct 180
accagatcga aggcagcaaa gccgtgccct gggtgattct gtccgacggc gacggcaccg 240
tggagaaagg cttcaaggcc gaatggtggc agtgaaggac gagcgtctgt acgtgggcgg 300
cctgggcaag gagtggacga ccactacggg tgatgtggtg aacgagaacc cggagtgggt 360
naaggtggtg ggctacaagg gcagcgtgga ccacgagaac tgggtgtcna actacaacgc 420
cctgcgggtt gctgccggna tccagccgcn agttaccttc atccatgagt tgcctgctgg 480
antnaaacct tgnagnntgg ttcttcttgc cgnc 514
<210> 30
<211> 524
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 361, 369, 488
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 1812758F6
<400> 30
gagcaaggcc atcaggactc agcttttata aaaacaagag gagtgcactt ttgttttgtt 60
ttgttctttt tggaactgtg cctgggttgg aggtctggac agggagccca gtcccgggcc 120
ccatagtggt gcgggcactg gacccccggg ccccacggag gccgcggtct gaactgcttt 180
ccatgctgcc atctggtggt gatttcggtc acttcaggca ttgactcaag gcctgcctaa 240
ctggctgggt cgtttcttcc atccgacctc gtttcttttc tttcctatgt tcttttgttc 300
agtgaatatc cctagagctc ctaccatatg tcaggcccta tgctcaccct gagaacgcat 360
nagcatgang tggactgttt gctgggaacc caggtcaccc cttttcttcc tatctgtgct 420
ggagatcatg tcaaccctgc agatcctgga aaagaaatgt tatgttgcaa ggtattgcat 480
gtcacgantg aaggcaggcc ctggggaaca tctgcccaca gctg 524
<210> 31
<211> 568
22/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 41, 169, 170, 182, 253, 256. 257, 301, 340, 359, 374, 386, 394, 415,
445, 473, 474, 481, 509, 519, 533, 542, 557, 559
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 1856319F6
<400> 31
gtaattttgc tgtggaaaga cttcacgtct tgccgaatga nagtcccgcc tgtctgtcac 60
gctgatgccc gtgcagtgtc tgagcacccg gaatggaatg agtctatgca ctccctccgg 120
atcagtgtgg ggggccttcc tgtgctggcg tccatgacca aggccgcgnn cccccgcttc 180
cngccccgct ggaaggtgat cctgacgttc tttgtgggtg ctgccatcct ctggctgctc 240
tgctcccacc gcncgnnccc cggcaggccc cccacccaca atgcacacaa ctggaggctc 300
ngccaggcgc ccgccaactg gtacaatgac acctaccccn tgtctccccc acaaaggana 360
ccggctggga ttcngtatcg aatcgngtta tcgnagacct ggacacagag tcaanggccc 420
aagaggaaaa cacctggtta gttanctgaa aagggtactg accctgtaag aanntgggga 480
naaggtggcc gtggaatgga caaaacatng ggtctgganc caactggcga aanggggaag 540
gntggagctt cgactgntnt ttaatgga 568
<210> 32
<211> 282
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 64, 85, 126, 134, 165, 178, 182, 191, 226, 246, 249, 269, 270, 279
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 2016607H1
<400> 32
gtgactcgcg actggtcggc gcggcgaaag cagagcggcg cgccggttcc ttggttcctg 60
aggncgatgg cgccgggtgg ctggngcggc tacgccgcct gttatccgcg gggcagcttc 120
tattcnaggg ccgngcgctg ctcgtcacta acacgctgag ctgcngcgcg ctcatggngg 180
cnggtgatag ngtgcgccag tcctggcgag atccgcggcc cggcancggc caggttttcg 240
acccanggng ggtccacgaa catgtttgnn gtgggctgna gc 282
<210> 33
<211> 650
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
23/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
<222> 6, 11, 21, 22, 26, 31, 44, 46, 55, 56, 64, 72, 74, 77, 79, 83, 85,
86, 102, 160, 172, 178, 210. 280, 307, 319, 322, 344. 388, 394, 444, 462,
503, 521, 526, 549, 561, 567. 570, 599, 603, 609. 618, 626, 635, 648
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 201791876
<400> 33
atactnctac nccaacctgt nnaaanggag nctcctcctc cttnancttt cccgnnaggg 60
tggngtcacg tngngangnt ccngnntgac caggcctagc cntggccaca tgagtccaca 120
cagtggaaaa ggcttggctc ctgtggtcgg cacacacgan actctggttg cncctgcngg 180
gtgtggaaca ggcgcaccat cgccatgccn gcagctcaca aagctcctgc tggcctggga 240
tgcactggga gcagctgcca tgggtccgtc agctgaagtn aaggaagtcc tggccctcct 300
gcaagtncag tgacctcang tngtcctcaa aggcgccccc tgtnaggtcc agcttcacgc 360
agatgacgaa gggtggccgg gcggctcnga agtncaggat ggggattcgg ggcttgggtc 420
tttcctgaga gtcctcgtgg caanagaagc ctttcctgat cntggttctc gtccacggtc 480
acagaatgcc accaccttgc gcngggctgc cggcagtcaa nctgcngtac agccggcgcc 540
cctgctttnc cagcgttcca natggtnaan ggccgcccca gcggggcaag ggccttgcnc 600
ttncaaggna atcggacncc gttggntccc aaatngtcgt ctcccaanga 650
<210> 34
<211> 247
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2197153H1
<400> 34
cggtggccct caggaggaag gaaggaaaaa cagccgtctc tggcctctgg cctctccagg 60
ctctccaccc tgggggcaga atcaattctg tagccctcta gcaccggcag tggctccgca 120
gtcagctggg cagtggccgt gactgggtca ctctacccta ggaagtgagt gcaggggctg 180
ctggagagcc agaggctggc agcgactatg tgaagttctc caaggagaag tacatcctgg 240
actcatc 247
<210> 35
<211> 581
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 564
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 226276376
24/3 3
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
<400> 35
gagatattta ttaacagatg ggggtgctgg tgtgggggcc cttgtctgac tccccatggg 60
tctttggggc acttccggga cacagctggg gctcactgga acggcgggcg acaggggagg 120
caggcaatgc tgtggcagaa ggggctgcag gggcggcatg gacacgggtt acagtgctgt 180
aggcagggga gctagggctc tcggagatgg gcgacatggg tgagtgcagg tctgggatct 240
ggtccatgct gagggcacag ctgcggatgg agtcccgagg gcggggcagc gacgtactgg 300
tggggcagcc atcgctgcgg gtgaccttgt cagcagtgag cccatcggtc atggtgacgc 360
tgcgccgctt catgtggctg cccttggggc ctgaggggct gacggggcta gcaggcttgc 420
tactcccctg tcccaggcca tagctggcct cgaggtagcg cagtgggaag gtgcggttca 480
ccgggcagta gatgatggca ccactctgct ctgacacagc ttgcggctgc ccacatatag 540
cgcacgaggc ggcacgtggt caanctctct gctcaaacag g 581
<210> 36
<211> 553
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 24, 63, 334, 377, 409, 415, 435, 439, 444, 456, 459, 466, 473, 498,
504, 511, 521, 527, 535, 549, 552
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 2445385F6
<400> 36
cggcgctccg cgagcatgtt tgcngtgggc tgcagcatgg gtcccttcct gcactactgg 60
tanttgtcgc tggaccgcct attccctgcg tctggcctcc gaggcttccc aaatgtcctc 120
aagaaggtcc tcgtggatca gctggtagcc tctccattgc tgggcgtctg gtacttcttg 180
ggccttggct gcctggaggg tcagacagtg ggtgagagct gccaggagct gcgggagaag 240
ttctggggaa ttctacaagg cagactggtg cgtgtggcct gctgcgcagt tcgtgaactt 300
cctcttcgtg cccccccaat ttcgagtcac ctanatcaac ggcctgacgc tgggctgggg 360
acacgtacct gtcctanttg aagtaccgga gcccagttcc tctgacacnc cccangctgt 420
gtggccctgg acacncganc agantgaact gtctgnttnc tggacnagat ggnagactgt 480
ctcctggggg acaccccntc tggncagaaa ngggatgggg ntcctgnagc aaagntcggg 540
gtcttgagnc ang
553
<210> 37
<211> 269
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2593359H1
<400> 37
ccagctcaca cactgttttc tggaaataaa gccctactct acaaaaaaaa tgaagatggc 60
ttgtgggaaa agatctcttc tccaggaagt taaaaaacat gaattaccaa agaaagcacc 120
ttcttggcct gacagaccat tggtggggct ggcacgaatc cagatctgga tcctacatct 180
gttgggtctt aggcctcctt ccctcctcag tgtctttcaa atgactttca tcaaatgact 240
25/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
ttcaaaataa aaccttattt tggcaaaag 269
<210> 38
<211> 234
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 101, 233
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 2613966H1
<400> 38
gagatggagt aattttgctg tggaaagact tcacgtcttg ccgaatgaaa tcagcctcca 60
gcgcctgcag cttccggaac taagatgtga ctgggcttgc ngaggcgctg actcctctcc 120
tccctccctg gctgtgcagg tcccgcctgt ctgtcacgct gatgcccgtg cagctgtctg 180
agcacccgga atggaatgag tctatgcact ccctccggat cagtgtgggg ggnt 234
<210> 39
<211> 604
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 306, 335, 377, 383, 388, 400, 402, 409, 434, 463, 467, 471, 473, 476,
477, 479, 480, 482, 490, 495, 506, 516, 520, 523, 525, 532, 539, 541, 543,
545, 552, 553, 556, 557, 558, 559, 564, 566, 573, 575, 579, 584, 585, 587,
589, 590, 593, 598
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 2812186F6
<400> 39
gtttcctcaa ccggaacaca cccatgaaga tggatgatca tcccctagcc cttctcagca 60
ggaacctcat gtgacctgtg accaagatgt cccatcctca gcacagggcc cactctgcca 120
accagtctca agcaccagcc cctcaacact gccatccacc tggctctggg ccaagccacc 180
aatccagagc tccctcaggt cctgggacta aggcggggac atgactgatc ccctcagagc 240
aggctcaggc ctggagtcgg cccccaaaag tttcacatag ggccaggcag cctctgtgtt 300
tctttncctg gtctgaactg tggaaatgcc attanactct ctctaatgta actgaaactt 360
gctggctggg ggcgcantgg gcntcccnac cttgtaattn tngggcccnt ttccccggag 420
ggctttaaag gttngaaaag gggaattggc ctccaaaggg gcncaanggg ntngtnntnn 480
gnaaaaaacn aagcnccttg gggggngcca atacanttgn ggnanaagaa gnccccccnt 540
ngntnccccc annatnnnnt aaanantttt gtngngaana atannantnn aanattgnta 600
gctt 604
26/33
CA 02329076 2000-11-28
WO 99/61626 PCTNS99/12021
<210> 40
<211> 599
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 21, 58, 60, 117, 489, 504, 510, 512
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 290233776
<400> 40
ccaaaataag gttttatttt naaagtcatt tgatgaaagt catttgaaag acactgangn 60
gggaaggagg cctaagaccc aacagatgta ggatccagat ctggattcgt gccagcncca 120
ccaatggtct gtcaggccaa gaaggtgctt tctttggtaa ttcatgtttt ttaacttcct 180
ggagaagaga tcttttccca caagccatct tcattttttt tgtagagtag ggctttattt 240
ccagaaaaca gtgtgtgagc tggagatggg tgttttttta aaaacatcaa ggtagatcta 300
atatgttcaa caaagtgggg tggctcagcc agaggcgaag tggaaagatt ctctagtatt 360
tgcttgtcat cttggtgcaa ccagaaatcc acatgtggaa atggtgtcca ggagtacggt 420
cctatacgaa gtgttctgtc tctgcatcat aaatgctaat cattggtcct cctgctaaag 480
ttcgcgcana cgaagctgct cctncggacn cngatcttga aaggatgctc tgtaaatctc 540
tgcagtctta atgttgacag caatgccata tattactgga aagtgggttt cgttttctt 599
<210> 41
<211> 227
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 3016872H1
<400> 41
gccaaaataa ggttttattt tgaaagtcat ttgatgaaag tcatttgaaa gacactgagg 60
agggaaggag gcctaagacc caacagatgt aggatccaga tctggattcg tgccagcccc 120
accaatggtc tgtcaggcca agaaggtgct ttctttggta attcatgttt tttaacttcc 180
tggagaagag atcttttccc acaagccatc ttcatttttt ttgtaga 227
<210> 42
<211> 309
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 115
<223> a or g or c or t, unknown, or other
<220>
<221> misc feature
27/33
CA 02329076 2000-11-28
WO 99/61626 PCTNS99/12021
<223> Incyte Clone No: 3030372H1
<400> 42
gtcgtttctt ccatccgacc tcgtttcttt tctttcctat gttcttttgt tcagtgaata 60
tccctagagc tcctaccata tgtcaggccc tatgcctcac cctgagaacg cagtncgcat 120
gaggtggacc tgtttgctgg gaaccccagg tcaccccctt ttcttcctac tctgtgcctg 180
gagcatcatg tccacccctg cagatccttg gaaaagaaaa tgtttatgtt gcagggtatt 240
gcatggtcac gagtgagggc aggcccctgg ggacacatct gcccacagct gcacaggcca 300
gggcgcagg 309
<210> 43
<211> 574
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 209, 414, 471, 476, 506, 563
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 3119919F6
<400> 43
aggactttga aggtaccatg gagctgtctg ttttcaatga tgccagtaag gacaagtctg 60
gggctatcat tgaaaaatgg agagtgaagc tggaagattc tggtgtccac gtgatcattg 120
gggggcacga ttctccctct cctagaggcg tcggatacgc taaaaatcaa gcagttgccc 180
agagctcagg gtcttacctt tgctttttng attcggatga cgtcatgatg ccccagcggg 240
tgaggctgca acacgaggct gccgttcagc acccgtcgag catcattggt tgcagagtga 300
ggagagatcc ccctaactcc accgaacgat acacacgttg gatcaaccag ctgacgccgg 360
agcagctcct aacccagcac gttccaagag ccaggctgct gcatgcgatg ttgnacaaca 420
aggtgtcctc ggcaatggac tgtgttctgt gagactggac acgggggccg nccgcngttg 480
ccgccttcgg ggtatgactt ggctgncgtc ttgaagggaa ggggactttg cccgttgcca 540
aattgggttg gcttgtccct tgncatccct cttg 574
<210> 44
<211> 240
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 237, 239
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 3257058H1
28/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
<400> 44
ctcagctctc ggctggggtt cgtcactggg cgcgggattt ggccgccgcg gggctccgga 60
gccgctcgct cccgacacgg ctcacgatgc gcggcgagca gggcgcggcg ggggcccgcg 120
tgctccagtt cactaactgc cggatcctgc gcggagggaa actgctcagg gaggatctgt 180
gggtgcgcgg aagccgcatc ttggacccag agaagctgtt ctttgaggag cggcgcntng 240
<210> 45
<211> 591
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 40, 46, 150, 159, 217, 295, 362, 372, 401, 405, 409, 415, 422, 450,
457, 458, 473, 496, 498, 502, 521, 534, 563, 565, 583, 584
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 3513795F6
<400> 45
cgcctccgcg atgccgctgc tcgtcgaggg gcggcgagtn cggctnccgc agtcagccgg 60
ggacctcgtc cgagcccacc cgcctttgga ggaaagagcc agacttctca gaggtcagtc 120
tgttcaacaa gtgggacccc agggccttcn gtatgttcng caaagagagc ttgcagtgac 180
ctccccaaag gatggctcca tctccattct gggttcngat gatgccacta cttgtcacat 240
tgtggtcctg aggcacacag gtaatggggc cacctgcttg acacattgtg acggnaccga 300
caccaaagct gaagtcccct tgatcatgaa ctccataaaa tcttttctga ccacgctcaa 360
tntggaaagc tngaagtaca ccttgttgga agctcagtga nggcnagcnt tgttncaaaa 420
antccctcat caaattctta gtggatttgn caaggcnnga aggtggccat tcnccttagt 480
gggcattatg tgtggncnga anttaaattg accggggaag naaacgaaaa accncttttc 540
cagggaaaat attggcattt gcntngtcca aaattttaaa acnnggccga g 591
<210> 46
<211> 304
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 3773551H1
<300>
<400> 46
cgaatcgcag ttatcgcaga cctggacaca gagtcaaggg cccaagagga aaacacctgg 60
ttcagttacc tgaaaaaggg ctacctgacc ctgtcagaca gtggggacaa ggtggccgtg 120
gaatgggaca aagaccatgg ggtcctggag tcccacctgg cggagaaggg gagaggcatg 180
gagctatccg acctgattgt tttcaatggg aaactctact ccgtggatga ccggacgggg 240
gtcgtctacc agatcgaagg cagcaaagcc gtgccctggg tgattctgtc cgacggcgac 300
ggca 304
29/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
<210> 47
<211> 227
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 406420H1
<400> 47
gatcaaccag ctgacgccgg agcagctcct aacccaggtt ttcacctcaa atggccccac 60
ggtgatcatg cccacctggt tctgctcgcg agcgtggttc tcccacgtgg gcccctttaa 120
cgaaggaggt cagggcgtcc cggaggacct gctgttcttc tacgagcacc tcaggaaggg 180
cggcggcgtc atccgcgtgg accagagtct cctgctgtat cgccacc 227
<210> 48
<211> 289
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 18
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 56227786
<400> 48
ttatttttta agtattanga taatgttgtc catttttttg gctactctga aatgttgcag 60
tgtggaacaa tggaaagagc ctgggtgttt gggtcagata aatgaagatc aaactccagc 120
tccagcctca tttgcttgag actttgtgtg tatgggggac ttgtatgtat gggagtgagg 180
agtttcaggg ccattgcaaa catagctgtg cccttgaaga gaatagtaat gatgggaatt 240
tagaggttta tgactgaatt ccctttgaca ttaaagacta tttgaattc 289
<210> 49
<211> 756
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 192, 459, 650, 657, 683, 689, 696, 699, 744, 749, 755
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 634343X12
30/33
CA 02329076 2000-11-28
WO 99/61626 PCTNS99/12021
<400> 49
gggaatggga gttaatcttc ttgtggccaa cacacatcat gtcagcctaa atatgacagg 60
aagtggtatt tatgcaccaa atggtcccaa agtgtatcat tatgacatga agacagagtt 120
gggaaaactt ctcctttcag aggtgggttc acatccccta tcctcgcttg cctacccaac 180
agctgttaat tngaatgcct acgccaccac catcaaacca tttccagtac agaaaaacac 240
tttcagggga tttatttcca gggatgggtt caacttcaca gaactttttg aaaatgcagg 300
aaaccttaca gtctgtcaaa aggagctttg ctgtcattta agctacagaa tgttacaaaa 360
agaagagaat gaagtatacg ttctaggagc ttttacagga ttacatggaa ggagaagaga 420
gtactggcag gtctgcacaa tgctgaagtg caaactacna atttgacaac ttgtggacgg 480
ccagtagaaa ctgcttctac aagatttgaa atgttctccc tcagtggcac atttggacac 540
agagtatgtt tttcctgaag tgctacttac cgaagattca tctgtcacct ggaaaatttg 600
aggtgctgaa agatggggcg tttggtaaac aagaatggac tcatctgggn ctatacnaac 660
cagtgtcact ccttggggag gtngtacana aagggntcnc tttacagctc atgtggggac 720
cagcaattcc gcccttaacc ttancccgng ggtant 756
<210> 50
<211> 740
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 24, 533, 6I2, 615, 617, 639, 655, 660, 668, 679, 687, 692, 710, 719,
722, 734
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 634343X14
<400> 50
ctcgtggcac gttaccataa gtancacctg tactctgagc ctcagtttaa tgtccctgaa 60
aagccggagt tggtgacttt caacaccgca tttggaaggt ttggcatttt cacgtgcttt 120
gatatattct tctatgatcc tggtgttacc ctggtgaaag atttccatgt ggacaccata 180
ctgtttccca cagcttggat gaacgttttg ccccttttga cagctattga attccattca 240
gcttgggcaa tgggaatggg agttaatctt cttgtggcca acacacatca tgtcagccta 300
aatatgacag gaagtggtat ttatgcacca aatggtccca aagtgtatca ttatgacatg 360
aagacagagt tgggaaaact tctcctttca gaggtggatt cacatcccct atcctcgctt 420
gcctacccaa cagctgttaa ttggaatgcc tacgccacca ccatcaaacc atttccagta 480
cagaaaaaca ctttcagggg atttatttcc agggatgggt tcaacttcac agnacttttt 540
gaaaatgcag gaaaccttac agtctgtcaa aaggggcttt gctgtcattt aagctacaga 600
atgttaccaa angangngaa tgaagtatac gttctaggng ctttacaggg ttacngggan 660
ggggaagngg tacctggcng tctgcanatg cnccagtgca aaaccaccan ttggccacnt 720
gnggagggcc ctanaaactg 740
<210> 51
<211> 889
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 622, 701, 771, 773, 794, 810, 831. 841, 853, 858, 882, 886, 887, 888
<223> a or g or c or t, unknown, or other
31/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
<220>
<221> misc_feature
<223> Incyte Clone No: 634343X17
<400> 51
cccccttgac taaagctcca aggacagaga aaaacatcca gatttgggaa cacaataaca 60
gatatgattg tccccacttc tactgccaaa attataaaac tgttaactcc tcctcatcag 120
cttacctgac tacttaaaag caaaagagtt aattaagtat tactaattgg tgatactaga 180
tcaatgaaga aatcactaaa ccttggccat ggtcacttcc tcttttccaa tctctgtgtc 240
agtttttgcc ctaataaccc tgcaggttgg tactcaggac agttttatag ctgcagtgta 300
tgaacatgct gtcattttgc caaataaaac agaaacacca gtttctcagg aggatgcctt 360
gaatctcatg aacgagtgga gacagcgatc aagcaggcag ctgagcaggg tgctcgaatc 420
attgtgactc cagaagatgc actttatgga tggaaattta ccagggaaac tgttttccct 480
tatctggagg atatcccaga ccctcaggtg aactggattc cgtgtcaaga cccccacaga 540
tttggtcaca caccagtaca agcaagactc agctgcctgg ccaaggacaa ctctatctat 600
gtcttggcaa atttggggga cnaaaagcca tgtaattccc gtgactccac gtgtcctcct 660
aatgggctac tttcaataca taccatgtgg tgtataatac ngaaggaaaa ctcgtggcac 720
gttacccata agtacccacc tgtaactctg agccctcagt ttaatgtccc ngnaaaagcc 780
ggagtttggt gacnttcaac accgcatttn ggaaggttgg gcatttcacg ngcttggaaa 840
nattctccag atnccggngt accccggtga aagatttcct gngggnnnt 889
<210> 52
<211> 496
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 63, 65, 88, 274, 278, 279. 280, 281, 283, 284, 288, 292, 294, 298,
300, 301, 302, 306, 309, 311, 314, 317, 322, 323, 324, 329, 335, 342, 344,
345, 347, 351. 353, 363, 371, 374, 376, 382, 389, 390, 398, 400, 402, 406,
416, 417, 418, 430, 435. 446, 449, 464, 465, 466, 470, 482, 485, 486, 487
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 75566986
<400> 52
ggcagctgct cccagtgcat cccaggccag caggagcttt gtgagctgca ggcatggcga 60
tgntncgcct gttccacacc cagcaggngc aaccagagtc tcgtgtgtgc cgaccacagg 120
agccaagcct tttccactgt gtggactcat gtggccaagg ctaggcctgg tcacccagga 180
ccctcaccac gtgaccccag ccaatcggga cagttcaagg aggaggagac ccctattaca 240
caggttggaa taaaatattt aaatctcgta aaanaaannn nanntggnga angngggnan 390
nngtgnatng nggnaanaga tnnnaagcna aaaanaaggg gngnncngct ntnagggttc 360
cgngttttgt ntancngttg cntgcggann tccagagncn cnttcnaaag ggggannncc 420
taaagttttn aattnccacg gggccngtng gttttgaaaa aggnnnttgn cctggggaaa 480
anccnnnggg ggttta 496
<210> 53
<211> 252
<2I2> DNA
<213> Homo Sapiens
32/33
CA 02329076 2000-11-28
WO 99/61626 PCT/US99/12021
<220>
<221> unsure
<222> 58, 71, 125, 166, 172, 175, 183, 185, 187, 196, 198, 208, 226, 234,
244, 249
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 755669T6
<400> 53
ttattccaac ctgtgtaata ggtgtctcct cctccttgaa ctgtcccgat tggctggngt 60
cacgtggtga nggtcctggg tgaccaggcc tagccttggc cacatgagtc cacacagtgg 120
aaaangcttg gctcctgtgg tcggcacaca cgagactctg gttgcncctg cnggntgtgg 180
aanangngca ccatcnanat tccctgtngc tcacaaagct cctgcnggcc tggnatgcac 240
tggnagcanc tg
252
<210> 54
<211> 297
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 6, 24, 31, 34, 117, 141, 189, 195, 205, 206, 2I6, 228, 231, 239
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 915932H1
<400> 54
ccaggncatg caggcccacg tgtntattat nctnccagtc cacaacgctg aaccgtggct 60
ggacgaatgt ttgaggtctg ttttgcaaca ggactttgaa ggtaccatgg agctgtntgt 120
tttcaatgat gccagtaagg ncaagtctgg ggctatcatt gaaaaatgga gagtgaagct 180
gggggattnt ggtgnccacg gtggnnattt gggggnacgg ttttgccngt nctagaggng 240
tcggatacgg taaaaatcaa gcagttgccc agagctcagg gtcttacctt tgctttt 297
33/33
