Note: Descriptions are shown in the official language in which they were submitted.
CA 02349586 2001-05-02
WO OO/Z6350 PCT/US99/25820
COENZYME A-UTILIZING ENZYMES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of coenzyme A-
utilizing
enzymes and to the use of these sequences in the diagnosis, treatment, and
prevention of neoplastic,
immunological, neurological, vesicle trafficking, and muscle disorders.
BACKGROUND OF THE INVENTION
A large number of cellular biosynthetic intermediary metabolism processes
involve
intermolecular transfer of carbon atom-containing substrates (carbon
substrates). Examples of such
reactions include the tricarboxylic acid cycle, synthesis of fatty acids and
long-chain phospholipids,
synthesis of alcohols and aldehydes, synthesis of intermediary metabolites,
and reactions involved in
the amino acid degradation pathways. Some of these reactions require input of
energy, usually in the
form of conversion of ATP to either ADP or AMP and pyrophosphate.
In many cases, a carbon substrate is derived from a small molecule containing
at least two
carbon atoms. The carbon substrate is often covalently bound to a larger
molecule which acts as a
carbon substrate carrier molecule within the cell. In the biosynthetic
mechanisms described above,
the corner molecule is coenzyme A. Coenzyme A (CoA) is structurally related to
derivatives of the
nucleotide ADP and consists of 4'-phosphopantetheine linked via a
phosphodiester bond to the alpha
phosphate group of adenosine 3',5'-bisphosphate. The terminal thiol group of
4'-phosphopantetheine
acts as the site for carbon substrate bond formation. The predominant carbon
substrates which utilize
CoA as a carrier molecule during biosynthesis and intermediary metabolism in
the cell are acetyl,
succinyl, and propionyl moieties, all of which are examples of acyl groups.
Other carbon substrates
include enoyl lipid, which acts as a fatty acid oxidation intermediate, and
carnitine, which acts as an
acetyl-CoA flux regulator/mitochondrial acyl group transfer protein. Acyl-CoA
and acetyl-CoA are
synthesized in the cell by acyl-CoA synthetase and acetyl-CoA synthetase,
respectively.
Activation of fatty acids is mediated by at least three forms of acyl-CoA
synthetase activity:
i) acetyl-CoA synthetase, which activates acetate and several other low
molecular weight carboxylic
acids and is found in muscle mitochondria and the cytosol of other tissues;
ii) medium-chain acyl-
CoA synthetase, which activates fatty acids containing between four and eleven
carbon atoms
(predominantly from dietary sources), and is present only in liver
mitochondria; and iii) acyl CoA
synthetase, which is specific for long chain fatty acids with between six and
twenty carbon atoms, and
is found in microsomes and the mitochondria. Proteins associated with aryl-CoA
synthetase activity
have been identified from many sources including bacteria, yeast, plants,
mouse, and man. The
activity of aryl-CoA synthetase may be modulated by phosphorylation of the
enzyme by cAMP-
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
dependent protein kinase.
Lysophosphatidic acid (LPA) and phosphatidic acid (PA) are two phospholipids
involved in
signal transduction and in lipid biosynthesis in cells. LPA acyltransferase
(LPAAT), also known as 1-
acyl sn-glycerol-3-phosphate acetyltransferase (EC 2.3.1.51), catalyzes the
conversion of LPA to PA,
utilizing acyl-CoA as the acyl donor. This enzyme is found in microsomes and
the plasma membrane
and may be regulated by phosphorylation in response to interleukin-1. LPA
serves as an intermediate
in membrane phospholipid metabolism and is also produced by activated
platelets. West, J. et al.
(1997, DNA Cell Biol. 16:691-701) identified messenger RNA encoding two
proteins, LPAAT-a and
LPAAT-(3, which contain extensive sequence similarities to microbial or plant
LPAAT sequences.
LPAAT-a mRNA was detected in all tissues, with the highest expression in
skeletal muscle, whereas
LPAAT-[3 was expressed predominantly in heart and liver tissues.
Overexpression of the two cDNAs
in mammalian cells led to increased LPAAT activity in cell-free extracts using
an in vitro assay. The
assay measured the conversion of fluorescently labeled LPA to PA. This
increase in LPAAT activity
correlated with enhancement of transcription and synthesis of tumor necrosis
factor-alpha and
interleukin-6 in cells upon stimulation with interleukin-lei, suggesting that
LPAAT overexpression
may amplify cellular signaling responses from cytokines (West, supra.).
In separate experiments, recombinant protein produced in COS 7 cells exhibited
LPAAT
activity, with a preference for LPA as the phosphoglycerol acceptor and
arachidonyl coenzyme A as
the aryl donor. Northern blot analysis demonstrated that the mRNA was
expressed in most human
tissues including a panel of brain subregions. Expression was highest in liver
and pancreas and
lowest in placenta (Eberhardt, C. et al. (1997) J. Biol. Chem. 272:20299-
20305). LPA may also
participate in the pathophysioiogy of neurodegenerative processes by causing
vasoconstriction as well
as impairment of glutamate and glucose uptake by astrocytes. In addition, LPA
is a potent promoter
of tumor cell growth and invasion through activation of Ras and the
Raf/mitogen-activated protein
kinase pathway, stimulation of phospholipases C and D, inhibition of adenylyl
cyclase, and tyrosine
phosphorylation of focal adhesion proteins which accompanies actin
cytoskeleton remodeling
(Eberhardt, supra).
Another human homolog of bacterial, yeast, and plant LPAAT was found in human
U937
cells. Northern blot analysis indicated high levels of expression in immune
cells and in epithelium.
3o Rapid amplification of cDNA ends revealed differentially expressed splice
variants (Stamps, A.C. et
al. (1997) Biochem. J. 326:455-461).
The discovery of new coenzyme A-utilizing enzymes and the polynucleotides
encoding them
satisfies a need in the art by providing new compositions which are useful in
the diagnosis,
prevention, and treatment of neoplastic, immunological, neurological, vesicle
trafficking, and muscle
disorders.
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, coenzyme A-
utilizing enzymes,
referred to collectively as "CoAEN" and individually as "CoAEN-1," "CoAEN-2,"
"CoAEN-3,"
"CoAEN-4," and "CoAEN-5." 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-
5 and fragments thereof. The invention also includes a polypeptide comprising
an amino acid
sequence that differs by one or more conservative amino acid substitutions
from an amino acid
sequence selected from the group consisting of SEQ ID NO:1-5.
The invention fiuther provides a substantially purified variant having at
least 90% amino acid
identity to at least one of the amino acid sequences selected from the group
consisting of SEQ ID
NO:1-5 and fiugments thereof. The invention also provides an isolated and
purified polynucleotide
encoding the polypeptide comprising an amino acid sequence selected from the
group consisting of
SEQ ID NO:1-5 and fragments thereof. The invention also includes an isolated
and purified
polynucleotide variant having at least 70% polynucleotide sequence identity to
the polynucleotide
encoding the polypeptide comprising an amino acid sequence selected from the
group consisting of
SEQ ID NO:1-5 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:1-5 and fi~agments
thereof. The invention
also provides 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:1-5 and fi~agments thereof.
The invention also provides a method for detecting a polynucleotide in a
sample containing
nucleic acids, the method comprising the steps of (a) hybridizing the
complement of the
polynucleotide sequence to at least one of the polynucleotides of the 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 in the
sample. In one aspect,
the method further comprises amplifying the polynucleotide prior to
hybridization.
The invention also provides an isolated and purified polynucleotide comprising
a
polynucleotide sequence selected from the group consisting of SEQ ID N0:6-10
and fragments
thereof. The invention further provides an isolated and purified
polynucleotide variant having at least
70% polynucleotide sequence identity to the polynucleotide sequence selected
from the group
consisting of SEQ ID N0:6-10 and fi~agments thereof. The invention also
provides 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 N0:6-10
and fragments
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
thereof.
The invention fiuther provides an expression vector containing at least a
fragment of the
polynucleotide encoding the polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-5. In another aspect, the expression vector is
contained within a host cell.
The invention also provides a method for producing a polypeptide, the method
comprising the
steps of (a) culturing the host cell containing an expression vector
containing a polynucleotide of the
invention 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:1-5
and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a
polypeptide selected from
the group consisting of SEQ ID NO:1-5 and fragments thereof. The invention
also provides a purified
agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder
associated with
decreased expression or activity of CoAEN, the method comprising administering
to a subject in need
of such treatment an effective amount of a pharmaceutical composition
comprising a substantially
purified polypeptide having the amino acid sequence selected from the group
consisting of SEQ ID
NO:1-5 and fragments thereof, in conjunction with a suitable pharmaceutical
carrier.
The invention also provides a method for treating or preventing a disorder
associated with
increased expression or activity of CoAEN, the method comprising administering
to a subject in need
of such treatment an effective amount of an antagonist of a polypeptide having
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-5 and fragments
thereof.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs),
clone identification numbers (clone IDs), cDNA libraries, and cDNA fi~agments
used to assemble full-
length sequences encoding CoAEN.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of CoAEN.
Table 3 shows the tissue-specific expression patterns of each nucleic acid
sequence as
determined by northern analysis; diseases, disorders, or conditions associated
with these tissues; and
the vector into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA clones
encoding CoAEN were isolated.
4
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
Table 5 shows the tools, programs, and algorithms used to analyze CoAEN, along
with
applicable descriptions, .references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"CoAEN" refers to the amino acid sequences of substantially purified CoAEN
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, marine, equine, and
human, and from any source, whether nat~ual, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
CoAEN. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of CoAEN either by
directly interacting with
CoAEN or by acting on components of the biological pathway in which CoAEN
participates.
An "allelic variant" is an alternative form of the gene encoding CoAEN.
Allelic variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
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 CoAEN include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as CoAEN or a
polypeptide with at least one functional characteristic of CoAEN. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding CoAEN, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
CoAEN. 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 CoAEN. Deliberate amino acid substitutions may be made on the basis
of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the
residues, as long as the biological or immunological activity of CoAEN is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to 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" 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.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of CoAEN. Amagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of CoAEN either by
directly interacting with CoAEN or by acting on components of the biological
pathway in which
CoAEN participates.
The tenor "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind CoAEN polypeptides can be prepared using intact
polypeptides or using
6
CA 02349586 2001-05-02
WO 00/Z6350 PCT/US99125820
fragments containing small peptides of interest as the immunizing antigen. The
polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLI~. The coupled peptide is
then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition 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"
or "minus" can refer to the
antisense strand, and the designation "positive" or "plus" can refer to the
sense strand.
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 CoAEN, or of any
oligopeptide thereof, to induce a
specific immune response in appropriate animals or cells and to bind with
specific antibodies.
The temts "complementary" and "complementarily" refer to the natural binding
of
polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the
complementary sequence "3' T-C-A 5'." 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
amplification reactions, which
depend upon binding between nucleic acid strands, and in the design and use of
peptide nucleic acid
(PNA) molecules.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding CoAEN or fragments
of CoAEN may
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
be employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
resequenced to
resolve uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk
CT) in the 5' and/or
the 3' direction, and resequenced, or which has been assembled from the
overlapping sequences of
one or more Incyte Clones and, in some cases, one or more public domain ESTs,
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI). Some sequences have been both extended and assembled to produce the
consensus sequence.
"Conservative amino acid substitutions" are those substitutions that, when
made, least
interfere with the properties of the original protein, i.e., the sriucture and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Residue Conservative Substitution
_ __ ~." .",.
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
3o Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, a.s a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
absence of one or more amino acid residues or nucleotides.
The term "derivative" 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, aryl, hydroxyl, or amino group.
A derivative
polynucleotide encodes a polypeptide which retains at least one biological or
immunological function
of the natural molecule. A derivative polypeptide is one modified by
glycosylation, pegylation, or any
similar process that retains at least one biological or immunological function
of the polypeptide from
which it was derived.
A "fragment" is a unique portion of CoAEN or the polynucleotide encoding CoAEN
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fingment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10, 15,
20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid residues
in length. Fragments may be preferentially selected from certain regions of a
molecule. For example,
a polypeptide fragment may comprise a certain length of contiguous amino acids
selected from the
first 250 or 500 amino acids (or first 25% or 50% of a polypeptide) as shown
in a certain defined
sequence. Clearly these lengths are exemplary, and any length that is
supported by the specification,
including the Sequence Listing, tables, and figures, may be encompassed by the
present embodiments.
A fragment of SEQ ID N0:6-10 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:6-10, for example, as distinct from any
other sequence in the same
genome. A fragment of SEQ ID N0:6-10 is useful, for example, in hybridization
and amplification
technologies and in analogous methods that distinguish SEQ ID N0:6-10 from
related polynucleotide
sequences. The precise length of a fragment of SEQ ID N0:6-10 and the region
of SEQ ID N0:6-10
to which the fragment corresponds are routinely determinable by one of
ordinary skill in the art based
on the intended purpose for the fiagment.
A fragment of SEQ ID NO:1-5 is encoded by a fragment of SEQ ID N0:6-10. A
fiagment of
SEQ ID NO:1-5 comprises a region of unique amino acid sequence that
specifically identifies SEQ ID
NO:1-5. For example, a fragment of SEQ ID NO:1-5 is useful as an immunogenic
peptide for the
development of amibodies that specifically recognize SEQ ID NO:1-5. The
precise length of a
fragment of SEQ ID NO:1-5 and the region of SEQ ID NO:1-5 to which the
fragment corresponds are
routinely determinable by one of ordinary skill in the art based on the
intended purpose for the
fragment.
The term "similarity" refers to a degree of complementarity. There may be
partial similarity
or complete similarity. The word "identity" may substitute for the word
"similarity." A partially
9
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
complementary sequence that at least partially inhibits an identical sequence
from hybridizing to a
target nucleic acid is referred to as "substantially similar." The 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 complementarity
(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" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences rnay be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window--4, and "diagonals saved"=4. The
'freighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequence pairs.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available from
several sources, including the NCBI, Bethesda, MD, and on the Internet at
http:/hwvw.ncbi.nlm.nih.eovBLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
Sequences" can be accessed and used interactively at
http:hfwww.ncbi.nlm.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blasts and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blasts with the "BLAST 2
Sequences" tool Version
2Ø9 (May-07-1999) set at default parameters. Such default parameters may be,
for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: S and Extension Gap: 2 penalties
Gap x drop-off 50
Expect: 10
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the
hydrophobicity arid acidity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
11
CA 02349586 2001-05-02
WO 00/26350 PGTNS99/25820
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø9
(May-07-1999) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
Open Gap: Il and Extension Gap: 1 penalties
Gap x drop-o,~': SD
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for
stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" 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" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of identity. Specific
hybridization complexes form under permissive annealing conditions and remain
hybridized after the
"washing" step(s). The washing steps) is particularly important in determining
the stringency of the
hybridization process, with more stringent conditions allowing less non-
specific binding, i.e., binding
between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for
annealing of nucleic acid sequences are routinely determinable by one of
ordinary skill in the art and
may be consistent among hybridization experiments, whereas wash conditions may
be varied among
experiments to achieve the desired stringency, and therefore hybridization
specificity. Permissive
annealing conditions occur, for example, at 68°C in the presence of
about 6 x SSC, about 1 % (w/v)
12
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
SDS, and about 100 ~g/ml denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Generally, such wash temperatures
are selected to be about
5°C to 20°C lower than the thermal melting point (Tin) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook et al., 1989,
Molecular Cloning: A Laboratory Manual, 2"° ed., vol. 1-3, Cold Spring
Harbor Press, Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1 % SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration
may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
denatured salmon sperm DNA at about 100-200 pg/ml. Organic solvent, such as
formamide at a
concentration of about 35-50% v/v, may also be used under particular
circumstances, such as for
RNA:DNA hybridizations. Useful variations on these wash conditions will be
readily apparent to
those of ordinary skill in the art. Hybridization, particularly under high
stringency conditions, may be
suggestive of evolutionary similarity between the nucleotides. Such similarity
is strongly indicative
of a similar role for the nucleotides and their encoded polypeptides.
The term 'hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support {e.g., paper, membranes, filters; chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on
a substrate.
The terms "element" and "array element" in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
13
CA 02349586 2001-05-02
WO 00/26350 PCTNS99125820
The term "modulate" refers to a change in the activity of CoAEN. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of CoAEN.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucieotide, 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.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with the second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Generally, operably linked DNA sequences may be in close proximity
or contiguous and,
where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation,
and may be pegylated to extend their lifespan in the cell.
"Probe" refers to nucleic acid sequences encoding CoAEN, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence; e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
3o may be considerably longer than these examples, and it is understood that
any length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2'~
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel et al.,1987, Current Protocols in
Molecular Biolostv,
Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis et al., 1990.
PCR Protocols. A
14
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
Guide to Methods and Applications, Academic Press, San Diego CA. PCR primer
pairs can be
derived from a known sequence, for example, by using computer programs
intended for that purpose
such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas T~ is capable of choosing specific
primers from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead Institute/MIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for
example, as PCR or sequencing primers, microarray elements, or specific probes
to identify filly or
partially complementary polynucleotides in a sample of nucleic acids. Methods
of oligonucleotide
selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding CoAEN, or fragments thereof, or CoAEN itself, may comprise a
bodily fluid; an
extract from a cell, chromosome, organelle, or membrane isolated from a cell;
a cell; genomic DNA,
RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print;
etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least about 60% free,
preferably about 75% free, and most preferably about 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or
nucleotides by
different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
"Transformation" describes a process by which exogenous DNA enters and changes
a
recipient cell. Transformation may occuI under natural or artificial
conditions according to various
methods well known in the art, and may rely on any known method for the
insertion of foreign nucleic
acid sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected
based on the type of host cell being transformed and may include, but is not
limited to, 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 a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
16
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
95% or at least 98% or
greater sequence identity over a certain defined length. A variant may be
described as, for example,
an "allelic" (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may
have significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides generally will have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucieotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least
98% or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human coenzyme A-utilizing
enzymes
(CoAEN), the polynucleotides encoding CoAEN, and the use of these compositions
for the diagnosis,
treatment, or prevention of neoplastic, immunological, neurological, vesicle
trafficking, and muscle
disorders.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
CoAEN. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs)
of the
polypeptide and nucleotide sequences, respectively. Column 3 shows the clone
IDs of the Incyte
clones in which nucleic acids encoding each CoAEN were identified, and column
4 shows the cDNA
libraries from which these clones were isolated. Column 5 shows Incyte clones
and their
corresponding cDNA libraries. Clones for which cDNA libraries are not
indicated were derived from
pooled cDNA libraries. The Incyte clones in column 5 were used to assemble the
consensus
nucleotide sequence of each CoAEN and are usefi~l as fiagments in
hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column I references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
17
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
shows the identity of each protein; and column 7 shows analytical methods and
in some cases,
searchable databases to which the analytical methods were applied. The methods
of column 7 were
used to characterize each polypeptide through sequence homology and protein
motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding CoAEN. The first column of Table
3 lists the
nucleotide SEQ ID NOs. Column 2 lists tissue categories which express CoAEN as
a fraction of total
tissues expressing CoAEN. Column 3 lists diseases, disorders, or conditions
associated with those
tissues expressing CoAEN as a fraction of total tissues expressing CoAEN.
Column 4 lists the vectors
used to subclone each cDNA library.
The following fragments of the nucleotide sequences encoding CoAEN are useful
in
hybridization or amplification technologies to identify SEQ ID N0:6-10 and to
distinguish between
SEQ ID N0:6-10 and related polynucleotide sequences. The encoded polypeptide
sequences are
useful, for example, in generating antibodies. The useful fragments are the
fragment of SEQ ID N0:6
from about nucleotide 515 to about nucleotide 607; the fragments of SEQ ID
N0:7 from about
nucleotide 1229 to about nucleotide 1264 and from about nucleotide 1619 to
about nucleotide 1642;
the fragment of SEQ ID N0:8 from about nucleotide 1258 to about nucleotide
1437; the fragment of
SEQ ID N0:9 from about nucleotide 1191 to about nucleotide 1229; and the
fragment of SEQ ID
NO:10 from about nucleotide 77 to about nucleotide 139.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
finm which cDNA clones encoding CoAEN were isolated. Column 1 references the
nucleotide SEQ
ID NOs, column 2 shows the cDNA libraries from which these clones were
isolated, and column 3
shows the tissue origins and other descriptive information relevant to the
cDNA libraries in column 2.
The invention also encompasses CoAEN variants. A preferred CoAEN variant is
one which
has at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the CoAEN amino acid sequence, and which contains at
least one functional or
structural characteristic of CoAEN.
The invention also encompasses polynucleotides which encode CoAEN. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:6-10, which encodes CoAEN.
The invention also encompasses a variant of a polynucleotide sequence encoding
CoAEN. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at
least about 85%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding CoAEN. A particular aspect of the invention encompasses a
variant of a
18
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:6-
which has at least about 70%, or alternatively at least about 85%, or even at
least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the
group consisting of
SEQ ID N0:6-10. Any one ofthe polynucleotide variants described above can
encode an amino acid
5 sequence which contains at least one functional or structural characteristic
of CoAEN.
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 CoAEN, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
10 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 CoAEN, and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode CoAEN and its variants are
generally capable
of hybridizing to the nucleotide sequence of the naturally occurnng CoAEN
under appropriately
selected conditions of stringency, it may be advantageous to produce
nucleotide sequences encoding
CoAEN 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 CoAEN 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 invemion also encompasses production of DNA sequences which encode CoAEN
and
CoAEN 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 CoAEN or any fragnent 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
N0:6-10 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
19
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Perkin-
Elmer), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway
NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Perkin-Elmer). Sequencing is then carried out using either the ABI 373 or 377
DNA sequencing
system (Perkin-Elmer), the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics,
Sunnyvale CA), or other systems known in the art. The resulting sequences are
analyzed using a
variety of algorithms which are well known in the art. (See, e.g., Ausubel,
F.M. ( 1997 Soho-rt
Protocols in Molecular Biolosy, John Wiley & Sons, New York NY, unit 7.7;
Meyers, R.A. (1995)
Molecular Biology and Biotechnolo~v, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding CoAEN may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. ( 1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al.
(1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction
enzyme digestions and
ligations may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 Primer Analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
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. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
l0 software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, 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 CoAEN may be cloned in recombinant DNA molecules that direct
expression of
CoAEN, or fragments or functional equivalents thereof, in appropriate host
cells. Due to the inherent
degeneracy of the genetic code, other DNA sequences which encode substantially
the same or a
functionally equivalent amino acid sequence may be produced and used to
express CoAEN.
The nucleotide sequences ofthe present invention can be engineered using
methods generally
known in the art in order to alter CoAEN~ncoding 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 CoAEN may be synthesized, in whole
or in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.)
Alternatively, CoAEN itself or a fragment thereof may be synthesized using
chemical methods. For
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-Elmer). Additionally, the amino acid
sequence of CoAEN, 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
21
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
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, New
York NY.)
In order to express a biologically active CoAEN, the nucleotide sequences
encoding CoAEN
or derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which
contains the necessary elements for transcriptional and translational control
of the inserted coding
sequence in a suitable host. These elements include regulatory sequences, such
as enhancers,
constitutive and inducible promoters, and 5' and 3' untranslated regions in
the vector and in
polynucleotide sequences encoding CoAEN. Such elements may vary in their
strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding CoAEN. Such signals include the ATG initiation codon and
adjacent sequences,
e.g. the Kozak sequence. In cases where sequences encoding CoAEN and its
initiation codon and
upstream regulatory sequences are inserted into the appropriate expression
vector, no additional
transcriptional or translational control signals may be needed. However, in
cases where only coding
sequence, or a fragment thereof, is inserted, exogenous translational control
signals including an in-
frame ATG initiation codon should be provided by the vector. Exogenous
translational elements and
initiation codons may be of various origins, both natural and synthetic. The
efficiency of expression
may be enhanced by the inclusion of enhancers appropriate for the particular
host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding CoAEN 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 Clonine. A
Laboratory Manual, Cold Spring Harboi Press, Plainview NY, ch. 4, 8, and 16-
17; Ausubel, F.M. et
al. (1995) 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 CoAEN. 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
22
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
upon the use intended for polynucleotide sequences encoding CoAEN. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding CoAEN can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORTI
plasmid (Life Technologies). Ligation of sequences encoding CoAEN 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 CoAEN are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of CoAEN may be used.
For example, vectors
containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of CoAEN. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
Qastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, G.A. et at. ( 1987) Methods Enzymol. 153:516-544; and
Scorer, C.A. et al. ( 1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of CoAEN. Transcription of
sequences
encoding CoAEN may be driven viral promoters, e.g., the 35S and 19S promoters
of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(198?) 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; Brogue, 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. The McGraw Hill Yearbook of Science
and Technolo~r
( 1992) McGraw Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding CoAEN
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E 1 or E3 region of the viral genome
may be used to obtain
infective virus which expresses CoAEN in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
23
CA 02349586 2001-05-02
WO 00/26350 PGTNS99/25820
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
CoAEN in cell lines is preferred. For example, sequences encoding CoAEN 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 1 to 2 days
in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer resistance
to a selective agent, and its presence allows growth and recovery of cells
which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. ( 1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
~ Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins,
green fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate 1i-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding CoAEN is inserted within a marker gene sequence, transformed
cells containing
sequences encoding CoAEN can be identified by the absence of marker gene
function. Alternatively,
a marker gene can be placed in tandem with a sequence encoding CoAEN under the
control of a
single promoter. Expression of the marker gene in response to induction or
selection usually indicates
24
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding CoAEN
and that express
CoAEN 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 CoAEN
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
monoclonal antibodies reactive to two non-interfering epitopes on CoAEN is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. ( 1990) Serological Methods. a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunoloav, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding CoAEN
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding CoAEN, 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 polymerise
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding CoAEN may be
cultured under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
containing polynucleotides which encode CoAEN may be designed to contain
signal sequences which
direct secretion of CoAEN through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
CA 02349586 2001-05-02
WO 00/Z6350 PCT/US99/25820
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Dit~erent host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding CoAEN may be ligated to a heterologous sequence resulting
in translation of a
fission protein in any of the aforementioned host systems. For example, a
chimeric CoAEN protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of CoAEN
activity. Heterologous protein
and peptide moieties may also facilitate purification of fusion proteins using
commercially available
IS amity matrices. Such moieties include, but are not limited to, glutathione
S transferase (GST),
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their cognate
fusion proteins on immobilized glutathione, maltose, phenylarsine oxide,
calmodulin, and metal-
chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the CoAEN encoding sequence and the
heterologous protein
sequence, so that CoAEN may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel (1995 s. s. upra, ch. 10).
A variety of commercially available kits may also be used to facilitate
expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled CoAEN may
be achieved
in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 'SS-methionine.
Fragments of CoAEN may be produced not only by recombinant means, 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 ABI 431A peptide synthesizer (Perkin-Elmer).
Various fragments of
26
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
CoAEN may be synthesized separately and then combined to produce the full
length molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists between
regions of CoAEN and coenzyme A-utilizing enzymes. In addition, the expression
of CoAEN is
closely associated with cancer, immune response, inflammation, and
development; and in brain and
muscle tissues. Therefore, CoAEN appears to play a role in neoplastic,
immunological, neurological,
vesicle trafficking, and muscle disorders. In the treatment of disorders
associated with increased
CoAEN expression or activity, it is desirable to decrease the expression or
activity of CoAEN. In the
treatment of disorders associated with decreased CoAEN expression or activity,
it is desirable to
increase the expression or activity of CoAEN.
Therefore, in one embodiment, CoAEN or a fragment or derivative thereof may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or activity
of CoAEN. Examples of such disorders include, but are not limited to, an
immunological disorder,
such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult
respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis,
cholecystitis, contact dermatitis,
Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
poiymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections, and trauma; a
neurological disorder, such as
epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms,
Alzheimer's disease, Pick's
disease, Huntington's disease, dementia, Parkinson's disease and other
extrapyramidal disorders,
amyotrophic lateral sclerosis and other motor neuron disorders, progressive
neural muscular atrophy,
retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other
demyelinating diseases, bacterial
and viral meningitis, brain abscess, subdural empyema, epidural abscess,
suppurative intracranial
thrombophlebitis, myelitis and radiculitis, viral central nervous system
disease; prion diseases
including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker
syndrome; fatal
familial insomnia, nutritional and metabolic diseases of the nervous system,
neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central nervous system,
cerebral palsy,
27
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
neuroskeletal disorders, autonomic nervous system disorders, cranial nerve
disorders, spinal cord
diseases, muscular dystrophy and other neuromuscular disorders, peripheral
nervous system
disorders, dermatomyositis and polymyositis; inherited, metabolic, endocrine,
and toxic myopathies;
myasthenia gravis, periodic paralysis; mental disorders including mood,
anxiety, and schizophrenic
disorders; akathesia, amnesia, catatonia, diabetic neuropathy, tardive
dyskinesia, dystonias, paranoid
psychoses, postherpedc neuralgia, and Tourette's disorder; a vesicle
trafficking disorder, such as
cystic fibrosis, glucose-galactose malabsorption syndrome,
hypercholesterolemia, diabetes mellitus,
diabetes insipidus, hyper- and hypoglycemia, Grave's disease, goiter,
Cushing's disease, and
Addison's disease; gastrointestinal disorders including ulcerative colitis,
gastric and duodenal ulcers;
other conditions associated with abnormal vesicle trafficking, including
acquired immunodeficiency
syndrome (AIDS); allergies including hay fever, asthma, and urticaria (hives):
autoimmune hemolytic
anemia; proliferative glomerulonephritis; inflammatory bowel disease; multiple
sclerosis; myasthenia
gravis; rheumatoid and osteoarthritis; scleroderma; Chediak-Higashi and
Sjogren's syndromes;
systemic lupus erythematosus; toxic shock syndrome; traumatic tissue damage;
and viral, bacterial,
fungal, helminthic, and protozoa) infections; and a muscle disorder, such as
cardiomyopathy,
myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy,
myotonic dystrophy,
central core disease, nemaline myopathy, centronuclear myopathy, Iipid
myopathy, mitochondria)
myopathy, infectious myositis, polymyositis, dermatomyositis, inclusion body
myositis, thyrotoxic
myopathy, ethanol myopathy, angina, anaphylactic shock, arrhythmias, asthma,
cardiovascular shock,
Cushing's syndrome, hypertension, hypoglycemia, myocardial infarction,
migraine, and
pheochromocytoma, and myopathies including cardiomyopathy, encephalopathy,
epilepsy,
Kearns-Sayre syndrome, lactic acidosis, myoclonic disorder, and
ophthalmoplegia. A muscle disorder
is defined as any impairment or alteration in the normal action of muscle and
includes, but is not
limited to, disorders of the blood vessels, gastrointestinal tract, heart,
uterus, bladder, and skeletal
muscle.
In another embodiment, a vector capable of expressing CoAEN or a fragment or
derivative
thereof may be administered to a subject to treat or prevent a disorder
associated with decreased
expression or activity of CoAEN including, but not limited to, those described
above.
In a fiuther embodiment, a pharmaceutical composition comprising a
substantially purified
CoAEN in conjunction with a suitable pharmaceutical carrier may be
administered to a subject to treat
or prevent a disorder associated with decreased expression or activity of
CoAEN including, but not
limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of CoAEN
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or activity
of CoAEN including, but not limited to, those listed above.
28
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
In a further embodiment, an antagonist of CoAEN may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of CoAEN.
Examples of such
disorders include, but are not limited to, a neoplastic disorder, such as,
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; and disorders such as those
described above. In one
aspect, an antibody which specifically binds CoAEN may be used directly as an
antagonist or
indirectly as a targeting or delivery mechanism for bringing a pharmaceutical
agent to cells or tissues
which express CoAEN.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding CoAEN may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of CoAEN including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of CoAEN may be produced using methods which are generally known
in the
art. In particular, purified CoAEN may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind CoAEN.
Antibodies to CoAEN 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
fiagments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with CoAEN or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, platonic 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
29
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/258Z0
CoAEN have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein and contain the
entire amino acid sequence of a small, naturally occurring molecule. Short
stretches of CoAEN
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 CoAEN may be prepared using any technique which
provides for
the production of antibody molecules by continuous cell lines in culture.
These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
CoAEN-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g.,
Burton, D.R (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for CoAEN may also be
generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
CoAEN and its
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering CoAEN epitopes is generally used, but a
competitive binding assay
may also be employed {Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for CoAEN.
Affinity is expressed as an
association constant, I~, which is defined as the molar concentration of CoAEN-
antibody complex
divided by the molar concentrations of flee antigen and free antibody under
equilibrium conditions.
The Kg determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple CoAEN epitopes, represents the average affinity, or
avidity, of the antibodies
for CoAEN. The K, determined for a preparation of monoclonal antibodies, which
are monospecific
for a particular CoAEN epitope, represents a true measure of affinity. High-
affinity antibody
preparations with K, ranging from about 109 to 10'2 L/mole are preferred for
use in immunoassays in
which the CoAEN-antibody complex must withstand rigorous manipulations. Low-
affinity antibody
preparations with K, ranging from about 106 to 10' L/mole are preferred for
use in
immunopurification and similar procedures which ultimately require
dissociation of CoAEN,
preferably in active form, from the antibody (Catty, D. (1988) Antibodies.
Volume I~ A Practical
Approach, IRL Press, Washington, DC; Liddell, J.E. and Cryer, A. (1991) A
Practical Guide to
Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably
5-10 mg specific antibody/mI, is generally employed in procedures requiring
precipitation of CoAEN-
antibody complexes. Procedures for evaluating antibody specificity, titer, and
avidity, and guidelines
for antibody quality and usage in various applications, are generally
available. (See, e.g., Catty,
supra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding CoAEN, or
any
fingment or complement thereof, may be used for therapeutic purposes. In one
aspect, the
complement of the polynucleotide encoding CoAEN may be used in situations in
which it would be
desirable to block the transcription of the mRNA. In particular, cells may be
transformed with
sequences complementary to polynucleotides encoding CoAEN. Thus, complementary
molecules or
fragments may be used to modulate CoAEN activity, or to achieve regulation of
gene function. Such
technology is now well known in the art, and sense or antisense
oligonucleotides or larger fragments
can be designed from various locations along the coding or control regions of
sequences encoding
CoAEN.
Expression vectors derived from retroviruses, adenoviruses, or herpes or
vaccinia viruses, or
31
CA 02349586 2001-05-02
wo oons~so Pcrius99nsg2o
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 can be used
to construct vectors to express nucleic acid sequences complementary to the
polynucleotides encoding
CoAEN. (See, e.g., Sambrook, supra; Ausubel, 1995, suDla.)
Genes encoding CoAEN can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding CoAEN. 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 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 CoAEN. Oligonucleotides derived from
the transcription
initiation site, e.g., between about positions -10 and +10 from the start
site, may be employed.
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 Immunologic Approaches, Futura Publishing,
Mt. Kisco NY, pp.
163-177.) A complementary sequence or antisense molecule may also be designed
to block
translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding CoAEN.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides.
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
32
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding CoAEN. Such DNA sequences may be incorporated into a wide
variety of
vectors with suitable RNA polymerase promoters such as T7 or SP6.
Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can
be introduced into
cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
l0 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.
15 Many methods for introducing vectors into cells or tissues are available
and equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
20 Biotechno1.15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
pharmaceutical
25 or sterile composition, in conjunction with a pharmaceutically acceptable
carrier, for any of the
therapeutic effects discussed above. Such pharmaceutical compositions may
consist of CoAEN,
antibodies to CoAEN, and mimetics, agonists, antagonists, or inhibitors of
CoAEN. 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
30 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,
35 enteral, topical, sublingual, or rectal means.
33
CA 02349586 2001-05-02
wo oon6~so Pcnus99nsszo
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 Reminstton's
Pharmaceutical Sciences (Maack Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically acceptable carriers well known in the art in dosages suitable
for oral 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
compounds with solid excipient and processing the resultant mixture of
granules (optionally, after
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' solution, Ringer's
solution, or physiologically buffered saline. Aqueous injection suspensions
may contain substances
which increase the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or
dextran. Additionally, suspensions of the active compounds may be prepared as
appropriate oily
injection suspensions. Suitable iipophilic solvents or vehicles include fatty
oils, such as sesame oil, or
34
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/258Z0
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
burner to be
permeated are used in the formulation. Such penetrants are generally known in
the art.
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
acids. Salts tend to be more soluble in aqueous or other protonic solvents
than are the corresponding
free base forms. In other cases, the preparation may be a lyophilized powder
which may contain any
or all of the following: 1 mM to 50 mM histidine, 0. I % to 2% sucrose, and 2%
to 7% mannitol, at a
pH range of 4.5 to 5.5, 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 CoAEN, such
labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein
the active ingredients are contained in an effective amount to achieve the
intended purpose. The
determination of an effective dose is well within the capability of those
skilled in the art.
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 of
administration. Such information can then be used to determine useful doses
and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
CoAEN or fragments thereof, antibodies of CoAEN, and agonists, antagonists or
inhibitors of
CoAEN, which ameliorates the symptoms or condition. Therapeutic efficacy and
toxicity may be
determined by standard pharmaceutical procedures in cell cultures or with
experimental animals, such
as by calculating the EDso (the dose therapeutically effective in 50% of the
population) or LDP (the
dose lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LD~/ED~ 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
CA 02349586 2001-05-02
wo oon635o PcTnrs99nsszo
compositions is preferably within a range of circulating concentrations that
includes the EDT with
little or no toxicity. The dosage varies within this range depending upon the
dosage form employed,
the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting 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 ~cg 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
methods of delivery is provided in the literature and generally available to
practitioners in the art.
I S 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.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind CoAEN may be used
for the
diagnosis of disorders characterized by expression of CoAEN, or in assays to
monitor patients being
treated with CoAEN or agonists, antagonists, or inhibitors of CoAEN.
Antibodies useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for CoAEN include methods which utilize the antibody and a
label to detect
CoAEN in human body fluids or in extracts of cells or tissues. The antibodies
may be used with or
without modification, and may be labeled by covalent or non-covalent
attachment of a reporter
molecule. A wide variety of reporter molecules, several of which are described
above, are known in
the art and may be used.
A variety of protocols for measuring CoAEN, including ELISAs, RIAs, and FACS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of CoAEN expression.
Normal or standard values for CoAEN expression are established by combining
body fluids or cell
extracts taken from normal mammalian subjects, for example, human subjects,
with antibody to
CoAEN under conditions suitable for complex formation. The amount of standard
complex formation
may be quantitated by various methods, such as photometric means. Quantities
of CoAEN 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.
36
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
In another embodiment of the invention, the polynucleofides encoding CoAEN may
be used
for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of CoAEN
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of CoAEN, and to monitor regulation of CoAEN levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding CoAEN or closely related
molecules may be used
to identify nucleic acid sequences which encode CoAEN. The specificity of the
probe, whether it is
made from a highly specific region, e.g., the 5' regulatory region, or from a
less specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding CoAEN, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the CoAEN encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:6-10 or from
genomic sequences including promoters, enhancers, and introns of the CoAEN
gene.
Means for producing specific hybridization probes for DNAs encoding CoAEN
include the
cloning of polynucleotide sequences encoding CoAEN or CoAEN derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as ~P or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidinlbiotin coupling
systems, and the like.
Polynucleotide sequences encoding CoAEN may be used for the diagnosis of
disorders
associated with expression of CoAEN. Examples of such disorders include, but
are not limited to, a
neoplastic disorder, such as 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;
an immunological disorder, such as acquired immunodeficiency syndrome (AIDS),
Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis,
dermatomyositis, diabetes
mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis, erythema
37
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/Z5820
nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout,
Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irntable bowel syndrome, multiple
sclerosis, myasthenia
gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis,
psoriasis, Reiter's syndrome, rheumatoid arthritis, sclerodenna, Sjogren's
syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoa), and helminthic
infections, and trauma; a
neurological disorder, such as epilepsy, ischemic cerebrovascular disease,
stroke, cerebral neoplasms,
Alzheimer's disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and other
extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron
disorders, progressive
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple
sclerosis and other
demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis,
viral central nervous
system disease; prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders,
peripheral nervous system disorders, dermatomyosids and polymyositis;
inherited, metabolic,
endocrine, and toxic myopathies; myasthenia gravis, periodic paralysis; mental
disorders including
mood, anxiety, and schizophrenic disorders; akathesia, amnesia, catatonia,
diabetic neuropathy,
tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and
Tourette's disorder; a
vesicle trafficking disorder, such as cystic fibrosis, glucose-galactose
malabsorption syndrome,
hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper- and
hypoglycemia, Grave's
disease, goiter, Cushing's disease, and Addison's disease; gastrointestinal
disorders including
ulcerative colitis, gastric and duodenal ulcers; other conditions associated
with abnormal vesicle
trafficking, including acquired immunodeficiency syndrome (AIDS); allergies
including hay fever,
asthma, and urticaria (hives); autoimmune hemolytic anemia; proliferative
glomerulonephritis;
inflammatory bowel disease; multiple sclerosis; myasthenia gravis; rheumatoid
and osteoarthritis;
scleroderma; Chediak-Higashi and Sjogren's syndromes; systemic lupus
erythematosus; toxic shock
syndrome; traumatic tissue damage; and viral, bacterial, fungal, helminthic,
and protozoa) infections;
and a muscle disorder, such as cardiomyopathy, myocarditis, Duchenne's
muscular dystrophy,
Becker's muscular dystrophy, myotonic dystrophy, central core disease,
nemaline myopathy,
centronuclear myopathy, lipid myopathy, mitochondria) myopathy, infectious
myositis, polymyositis,
38
CA 02349586 2001-05-02
WO 00/26350 PCTNS99l25820
dermatomyositis, inclusion body myositis, thyrotoxic myopathy, ethanol
myopathy, angina,
anaphylactic shock, arrhythmias, asthma, cardiovascular shock, Cushing's
syndrome, hypertension,
hypoglycemia, myocardial infarction, migraine, and pheochromocytoma, and
myopathies including
cardiomyopathy, encephalopathy, epilepsy, Kearns-Sayre syndrome, lactic
acidosis, myoclonic
disorder, and ophthalmoplegia. The polynucleotide sequences encoding CoAEN may
be used in
Southern or northern analysis, dot blot, or other membrane-based technologies;
in PCR technologies;
in dipstick, pin, and multiformat ELISA-like assays; and in microarrays
utilizing fluids or tissues from
patients to detect altered CoAEN expression. Such qualitative or quantitative
methods are well known
in the art.
In a particular aspect, the nucleotide sequences encoding CoAEN may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding CoAEN may be labeled by standard methods and added to a
fluid or tissue
sample from a patient under conditions suitable for the formation of
hybridization complexes. After a
suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to a
control sample then the presence of altered levels of nucleotide sequences
encoding CoAEN 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
CoAEN, 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 CoAEN, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from
normal subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
39
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
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 fiuther progression of the cancer.
s Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
CoAEN 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 CoAEN, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
CoAEN, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
Methods which may also be used to quantify the expression of CoAEN include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et aI. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples rnay be
accelerated by running the assay in a high-throughput format where the
oligomer of interest is
presented in various dilutions and a spectrophotometric or colorimetric
response gives rapid
quantitation.
In fiuther 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 identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, 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.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalon, D. et al.
( 1995) PCT application W095/35505; Heller, RA. et al. ( 1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
In another embodiment of the invention, nucleic acid sequences encoding CoAEN
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 (PACs), bacterial artificial chromosomes (BACs), bacterial P1
constructions, or single
chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price,
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/Z5820
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, suura,
pp. 965-968.) Examples of genetic map data can be found in various scientific
journals or at the
Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation
between the
location of the gene encoding CoAEN 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 ofthe 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 placement 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 cnidely
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 336:577-580.) The nucleotide sequence ofthe 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, CoAEN, 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 CoAEN and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding a.~nity to the protein of interest. (See, e.g.,
Geyser, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with CoAEN,
or fragments thereof,
and washed. Bound CoAEN is then detected by methods well known in the art.
Purified CoAEN can
also be coated directly onto plates 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
41
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
antibodies capable of binding CoAEN specifically compete with a test compound
for binding CoAEN.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with CoAEN.
In additional embodiments, the nucleotide sequences which encode CoAEN may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. No. [Attorney Docket No. PF-0622 P, filed November 3,
1998], are hereby
expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some
tissues were homogenized and lysed in guanidinium isothiocyanate, while others
were homogenized
and lysed in phenol or in a suitable mixture of denaturants, such as TR,IZOL
(Life Technologies), a
monophasic solution of phenol and guanidine isothiocyanate. The resulting
lysates were centrifuged
over CsCI cushions or extracted with chloroform. RNA was precipitated from the
lysates with either
isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) IZNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit {Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997s supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
42
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORTI plasmid (Life Technologies), or
pINCY (Incyte
Pharmaceuticals, Palo Alto CA). Recombinant plasmids were transformed into
competent E. coli
cells including XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH10B,
or
ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids were recovered from host cells by in vivo excision using the UNIZAP
vector system
(Stratagene) or by cell lysis. Plasmids were purified using at least one of
the following: a Magic or
WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep
purification kit (Edge
Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,
QIAWELL 8
Ultra Plasmid purification systems or the RE.A.L. PREP 96 plasmid purification
kit from QIAGEN.
Following precipitation, plasmids were resuspended in 0.1 ml of distilled
water and stored, with or
without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
cDNA sequencing reactions were processed using standard methods or high
throughput
instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) thermal cycler or
the PTC-200
thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser
(Robbins Scientific) or
the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-
Elmer).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Perkin-Elmer) in conjunction with
standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
43
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the techniques
disclosed in Example V.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 5 summarizes the tools, programs, aid algorithms used and provides
applicable
descriptions, references, and threshold parameters. The first column of Table
5 shows the tools,
programs, and algorithms used, the second column provides brief descriptions
thereof, the third
column presents appropriate references, all of which are incorporated by
reference herein in their
entirety, and the fourth column presents, where applicable, the scores,
probability values, and other
parameters used to evaluate the strength of a match between two sequences (the
higher the score, the
greater the homology between two sequences). Sequences were analyzed using
MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software
(DNASTAR). Polynucleotide and polypeptide sequence alignments were generated
using the default
parameters specified by the clustal algorithm as incorporated into the
MEGALIGN multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned
sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then queried
against a selection of public databases such as the GenBank primate, rodent,
mammalian, vertebrate,
and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences
were assembled
into full length polynucleotide sequences using programs based on Phred,
Phrap, and Conned, and
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences 'were translated to derive the
corresponding full length
amino acid sequences, and these full length sequences were subsequently
analyzed by querying
against databases such as the GenBank databases (described above), SwissProt,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family
databases such
as PFAM. HMM is a probabilistic approach which analyzes consensus primary
structures of gene
families. (See, e.g., Eddy, S.R (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide and
amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:6-10. Fragments from about 20 to about 4000 nucleotides which are useful in
hybridization and
amplification technologies were described in The Invention section above.
IV. Northern Analysis
44
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook..
supra, ch. 7; Ausubel,
1995, supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in nucleotide databases such as GenBank or LIFESEQ (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:
% se4uence 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 I % 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 analyses are reported as a percentage distribution of
libraries in which
the transcript encoding CoAEN occurred. Analysis involved the categorization
of cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeietal, nervous,
reproductive, and urologic. The disease/condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
sequence of interest was counted and divided by the total number of libraries
across all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in
Table 3.
V. Extension of CoAEN Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:6-10 were produced by
extension of an
appropriate fragment of the full length molecule using oligonucleotide primers
designed from this
fragment. One primer was synthesized to initiate 5' extension of the known
fragment, and the other
primer, to initiate 3' extension of the known fragment. The initial primers
were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68 °C to about 72 °C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
lnc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mgz ; (NH4)ZS04,
and ~3-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerise (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94 ° C, 3 min; Step 2: 94 ° C, 15
sec; Step 3: 60 ° C, 1 min; Step 4: 68 °C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 ° C, 5
min; Step 7: storage at 4 ° C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68 ° C, 5 min; Step 7: storage at 4 °C.
The concentration of DNA in each well was determined by dispensing 100 Ell
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 pl of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
IS Acton MA), allowing the DNA to bind to the reagent. The plate was scanned
in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 to 10 ~l aliquot of the reaction mixture was
analyzed by
electrophoresis on a I % agarose mini-gel to determine which reactions were
successful in extending
the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, individual colonies were picked and cultured
overnight at 37°C in 384-
well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham Pharmacia Biotech) and Pfu DNA polymerise (Stratagene) with the
following
parameters: Step 1: 94 ° C, 3 min; Step 2: 94 ° C, 15 sec; Step
3: 60 ° C, 1 min; Step 4: 72 °C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72 ° C, 5 min;
Step 7: storage at 4 ° C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with
46
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID N0:6-10 are used to obtain
5' regulatory
sequences using the procedure above, oligonucleotides designed for such
extension, and an
appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:6-10 are employed to screen cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~cCi of
[,~ 31P) adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at mom temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VII. Microarrays
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,
W, 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 complementarily 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
47
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
the elements of the microarray. Fragments suitable for hybridization can be
selected using software
well known in the art such as LASERGENE software (DNASTAR). 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; Shalon, 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 CoAEN-encoding sequences, or any parts thereof,
are used
to detect, decrease, or inhibit expression of naturally occurring CoAEN.
Although use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
I S designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of CoAEN. To
inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5' sequence
and used to prevent promoter binding to the coding sequence. To inhibit
translation, a complementary
oligonucleotide is designed to prevent ribosomal binding to the CoAEN-encoding
transcript.
IX. Expression of CoAEN
Expression and purification of CoAEN is achieved using bacterial or virus-
based expression
systems. For expression of CoAEN in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21 (DE3).
Antibiotic resistant bacteria express CoAEN upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of CoAEN in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autoscraphica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding CoAEN by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect SQodoutera frusiperda (S~) 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.
48
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
7:1937-1945.)
In most expression systems, CoAEN is synthesized as a fusion protein with,
e.g., glutathione
S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His,
petrnitting rapid, single-step,
amity-based purification of recombinant fusion protein finm crude cell
lysates. GST, a 26-
kilodalton enzyme from Schistosomayanonicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
CoAEN at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity
purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues, enables
purification on metal-chelate
resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel ( 1995,
supra. ch. 10 and 16). Purified CoAEN obtained by these methods can be used
directly in the
following activity assay.
X. Demonstration of CoAEN Activity
CoAEN activity is determined by measuring the transfer of malonyl groups from
malonyl-
CoA to [3HJ-labeled carnitine to yield [~H]-labeled palmitoyl carnitine
according to the method of
Bremer, J. (1981, Biochim. Biophys. Acta 665:628-631). Mitochondria are
prepared from
mammalian tissue using methods well-known in the art. The mitochondria)
preparations (35-40 fig)
are preincubated with various concentrations of CoAEN and malonyl-CoA (0-1 ~M)
in an incubation
mixture containing 75 mM KCI, 50 mM mannitol, 25 mM HEPES (pH 7.3), 2 mM NaCN,
0.2 mM
EGTA, 1 mM dithiothreitol, and 1 % bovine serum albumin (essentially fatty
acid free) at 30°C for 3
minutes. Following this incubation, 0.1 ~cCi of L-[methyl-3H]carnitine is
added to give a final L-
carnitine concentration of 200 ~M, and the incubation is continued for a
fiuther 6 minutes. The
reaction is stopped by adding 100 ~cl of 10 N HCI. [3H]palmitoyl camitine is
extracted with butanol
and counted using standard liquid scintillation procedures. The amount of
[3H]palmitoyl camitine
recovered,is proportional to the amount of CoAEN in the sample.
Alternatively, CoAEN activity is determined according to the method of Aguado
and
Campbell ( 1998, J. Biol. Chem. 273:4096-4105). CoAEN activity is determined
by measuring the
reaction of the thiol group of the released CoA with 5,5'-dithiobis(2-
nitrobenzoic acid) (DTNB)
3o spectrophotometrically which results in an increase in absorbance at 413
nm. A suitable mammalian
cell line, such as COS?, is transiently transfected with a vector containing
CoAEN. After a suitable
period of incubation with cell culture media to allow transient expression of
CoAEN, the cells are
collected and homogenized using methods well-known in the art. Cell
homogenates are then
incubated with buffered substrates. A typical incubation mixture consists of
100 mM Tris-HCI, pH
7.4, 1 mM DTNB, 50 ~M LPA (oleoyl-sn-glycerol 3-phosphate), 10-45 ~M acyl-CoA,
and 50-150 ~cg
49
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
of cell homogenate in a total volume of 1 ml. DTNB is added as a 0.01 M
solution in 0.1 M
potassium phosphate buffer, pH 7Ø The reaction is initiated by the addition
of acyl-CoA after
preincubation of the sample with all of the other components for 2 minutes. A
molar absorbance of
13,600 units is used to calculate the coupling of released CoA with DTNB. The
increase in
absorbance at 413 nm is proportional to the amount of CoAEN in the sample.
XI. Functional Assays
CoAEN function is assessed by expressing the sequences encoding CoAEN at
physiologically elevated levels in mammalian cell culture systems. cDNA is
subcloned into a
mammalian expression vector containing a strong promoter that drives high
levels of cDNA
expression. Vectors of choice include pCMV SPORT (Life Technologies) and pCR3.
l (Invitrogen,
Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ~g of
recombinant vector
are transiently transfected into a human cell line, for example, an
endothelial or hematopoietic cell
line, using either liposome formulations or electroporation. 1-2 ~cg of an
additional plasmid
containing sequences encoding a marker protein are co-transfected. Expression
of a marker protein
provides a means to distinguish transfected cells from nontransfected cells
and is a reliable predictor
of cDNA expression from the recombinant vector. Marker proteins of choice
include, e.g., Green
Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow
cytometry (FCM),
an automated, laser optics-based technique, is used to identify transfected
cells expressing GFP or
CD64-GFP and to evaluate the apoptotic state of the cells and other cellular
properties. FCM detects
and quantifies the uptake of fluorescent molecules that diagnose events
preceding or coincident with
cell death. These events include changes in nuclear DNA content as measured by
staining of DNA
with propidium iodide; changes in cell size and granularity as measured by
forward light scatter and
90 degree side light scatter; down-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 C~rtometrv, Oxford,
New York NY.
The influence of CoAEN on gene expression can be assessed using highly
purified
populations of cells transfected with sequences encoding CoAEN and either CD64
or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind
to conserved regions
of human immunoglobulin G (IgG). Transfected cells are efficiently separated
from nontransfected
cells using magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake
Success NY). mRNA can be purified from the cells using methods well known by
those of skill in the
art. Expression of mRNA encoding CoAEN and other genes of interest can be
analyzed by northern
analysis or microarray techniques.
CA 02349586 2001-05-02
WO 00/26350 . PGT/US99/25820
XII. Production of CoAEN Specific Antibodies
CoAEN substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harnngton, M.G. ( 1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
s Alternatively, the CoAEN amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995 s, unra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Perkin-Elmer) using finoc-chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995,~ra.) Rabbits are immunized with the
oligopeptide-
KLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-
CoAEN activity by, for example, binding the peptide or CoAEN to a substrate,
blocking with 1
BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated
goat anti-rabbit IgG.
XIII. Purification of Naturally Occurring CoAEN Using Specific Antibodies
Naturally occurring or recombinant CoAEN is substantially purified by
immunoaffinity
chromatography using antibodies specific for CoAEN. An immunoaffinity column
is constructed by
covalently coupling anti-CoAEN antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing CoAEN are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of CoAEN (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/CoAEN 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 CoAEN is collected.
XIV. Identification of Molecules Which Interact with CoAEN
CoAEN, or biologically active fragments thereof, are labeled with "~I Bolton-
Hunter
reagent. (See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-
539.) Candidate
molecules previously arrayed in the wells of a mufti-well plate are incubated
with the labeled CoAEN,
washed, and any wells with labeled CoAEN complex are assayed. Data obtained
using different
concentrations of CoAEN are used to calculate values for the number, affinity,
and association of
CoAEN with the candidate molecules.
51
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the invention.
Although the invention has been described in connection with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are
obvious to those skilled in molecular biology or related fields are intended
to be within the scope of
the following claims.
52
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
. .
. x
M ~f'I H
r-i v~
.-r O O O - N cr M
O
O H H H O M O
H
z w ~ o
o rw z
o
z c~ z H ~. oo z
w
~ ~ ate'" x
d x
~
a x cn o x . H
ao
_ GL
U M ~
.-- -- u7
M M
l0 N 19 ~f7 O
~-i O
.-i Cu .-~ ~ o H r
x H H
x o x m cx .-a C~.
r o D
co ~n M w o~ .fl ~,
in Z H
~o v~ o~ N .-i .-i
M oo U D
CD O1 V' N D L~ N
N ill
r ~ o~ ~o . a~
o~ ~ z a
M dD N O1 tI7 ~ N
.-i w fx1
ri tD ~r lD M
v~ .-a ~-
M .--a M
O
H t0
.-i
. .-. . a w
x
~ .-~ -- H
o ~a o ov
-i o o o ~ z ~n c~
~ N
o H H H o a ~ N
H r
oo
~ Hz z H
m
z z ~
~ W N M
O O t~ W .-i ~
U
D U W C7 W
CL
o .~ ~ ~ ~ x ., w
,~
~- M ~--
ill C1
O
.mo ~ ,~ in o v~
,-,
z ,-a x c.. .~ in
cz x H H
x o~ w oo H o ~ C..,
N o c
U r-i CO l4 M..-i u~
v~ Oo H Z u)
tf7 00 .-~ 01 lO OD
OD OD D (!) O
r r N ~!'7M rl O
01 ~ 0.' F
O N tl7 N a H
l0 l0
O l0 C' M . (n CO
01 01 ~ CO
tf7 '-1 .-1 C~ .-. N
M H v U7
ri r-i M N
O l0
i-i
. . E,
c~.,
x
.-i rl .-1 ~- O .-.
ep M ~ O M
.-1 O rl N z r rl
O O O l0 O
o H H H o L1 o
H H ~ ~ H
H O U O CO ~C 04
H O o ~ O
o z H a a o o
a z ~ ~n z
N ~ C9 w pp Z
r.~ fY. r r ?~
x x z ~ ~ -- ~
a o .~ M x
a w ~ x w a
m U H
C7 U a H ~
~
H M H U -~ H
-- I
f~ -- r I
r-i N .-I ~o ~r ri
lD .-1 ri O I
C r-~
x ~ x x ~ x H ~
H N H a~
i
x x o~ u: .-~ rx
o~ r o~ D O ~r
o
-i m mr ,mo H c
o ,- m z ~
r
O~ M 4D O .-i N
r r N Z H N
O
c r ~r ~o o~ o
r M r, a cl~ c~
r1 a0 V' tf7 O1 ~-I
N ~D C' O fl.' N
o~ M r ~r umn U v~
r cn W f~7 M
M 01 N r-I .-I .-~ r-1
V' N C>) '-' M
.-1 H ~ c~ M
?i O O O H O
I
H H H H H H
Z ~ 2 2 2
17 ~ 2 H H ?i
a ~ o ~ ~ H
D U PO CL1 L1
o~ oo ,--m r
u~ oo co ~r .-i
G r CO OD M N
A
O O r w ~ v~
r-1 OD N l0 ~-I N
H
U w ~ a~ co M
.-i .-i ~--~ N M
v
.
'v
o
~.~
z
,o r ao o,
Ua
~w
z
cn
~o
I
v
11 r-1 N M V' u7
~
O
~'
a
a~
~
53
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
v~ vi cn cn w vi
w
ro
N
U
U +~ U U U +~ Z U
-~ O ~ O O O O
U
~ ~
00 ~ oo ro o0
~n
.w w
ro ,
~ U ~
+~ .1 ~ a~ H H a~ H r
H +~ o
4.
FC m z ~n cn m Z . rn z U
-rl Ga
ro ro ro ro H ~ ro H cn
H +~ o
-I .-~ .-~ ~-I cx .~ x a~
cx o E rx
aA oo m ao w x w w ~n
w ~ w
H
N '
!n N -r-I
O ro v7 ~ U
-'i N l..r r.~ N ro
i-~ .~ N O +~ U
ro .u w U N -rl N
U G U I ,.~ -b tO
-~ ~, s~ ~I +~ -~I ro
w fO ro ~ C, 1J f_I
-~ sa o - >, ro v
a-' r.C +~ ~ G v~ ~ w
o a~ G a~ .
.-I a~ a ~n
a~
' ~
o i -~ b a o 0
o m
H rl a.J C. U .~ S-I
J-1 -rl
J-~
1~ C'-. .p r-I O r-I
~-. l~
S-1
N i-~ I O J, tn ~,
rl '>7
~ ~
va. n. .~ am
Ca.~
v ~
u7 I~ o~
1.~ M 01 Op
~
v~ c N H N N
OD
~D
~ Ov W D u7 ~ c' a 1~ N
M ~
~ ~ C7 ~7 W I I tn 1 M M
C1~ U'
V' .-I
'
I I I t-l 1 O O I N U'
I U
t~ I r1 rl 00 00 N I F
I~ I O
M
M OD N l0 v~ N rl r-I .1
U7 M r-1 01
A A m ~n x a o H .2. 5.r'
~ c~ C/7
G
r-i
ri
ro
+~
-rl I~ N
ro
V7
1~
.-i
N
G ~ M M
+~
N U7
-ri
+~
O
Cn
O v~ N
H ~ N N M
c~ z z z z
tn l0 O N
01 l0 M l0 OD ~f7
01 M
o ~m n o M H cn H N ~
~n
-r-I r-I
~ ~ H
t~ H H cn M ~r ~
H to
ro M c. c~ M o 0
ro .~
-rl rl 1~ N N l0 M M O
r-I O tn '-1 tn
UJ
i.~ M O .-~ tD [-y., fn N '-1
?, r-1 v0 ?~
N
N +~ .-~ H ~-I cl1 M H v1
r-I
N O In H N l0 U7 O M
r1 (n OW
JJ t~ I~ r-1 O ~ CO
~. cT u--i l0
(n
O C1~ O l0 ~ I'~ cr N ~(7 M N ~
~-i N tl~ M
a" b' ~ ~ ~ ~ ~ H H H rl H
N ~n H cn
O ?~ H v1 H c!~ H
o m oo m ~ M r
v~
W M tn N ~O l0 l0 M !~ M
O OD rl u7
01
c O1 .-1 M .-I .-a ao
01 40 ~r .-~ cr .-i
c~
H cn cn cn E-~ cn cn
v1 H E-~ H H cn
H
QI
C ~ O 01 ~ O Cfl
~
o w ,-a N m
H H N r c
N
pG
0
H
.-i N M v~
W
54
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
o
~,
" z z
o z
U
z
'
~. n, (n a, H
7
' (~ ~a
C
.,.j
b
(,'
~
O
H
U
ro
+~
H
O
O _
H
. O N N M
W
N O N 01 a
O
d N N ,1
'b
C
O O O O
..r ~ _ _ _
v
f~ .-a O ,n
.i-~
.-I N N N
U
D ~ ~ ~ N ,.'~N
ro
ya r1 r1
L.a J-~ 1~ O O J~ O .N
v V
O S-I M S-1 u7 N O
O N ~ N Y.1
O N M N N OD O~ 01
O C C C
v' N N
lO M W N ~ O a O C O
W O W W
N
. .,1 . .,.~
.
ri O rl O ri O O +~ O +~ O iJ
O r-I r-1
o ~ o ~ o .r .., .- --
_. ro ro ro
p o
s
a w a w ro a ~ s a
ro ~ a ~
~ s~
a~ a~ ~ a~ . o~
~ ro a~ a~
.~ ro ro
U ~ U rl ~ U .-I U .-i U .-1
~ ro .-i rl
C I-I C .-1 C W C W C 4-1
ro ro .t~ .-i r-1
ro ro a~ ro ro ro
a~ a a C C
s~ o~ v a~
U U U U H U H U H U H
H Cu U U
C
.-1
o
ro
~n
o
~ M
H
N cr
~
w
_
'
O
x O ~ .-. ~ O M O
W O ~ -I .-i ~ O v~
C
p to .~ ~ .-a .-i .- W-a
O
N O ~ N .-. , N
-r-I ,-~
.N O N N O r-1 aD M O t0
O O O
C
U7 "~ N ~ ~-i ~ Y M ~- O v
U O O
v) O N rl -- M M M
ro
.,i r-1 O ,-i . ,--~. ,..i. ,-i
S..I
[-a ~0 --. ~- O ro O -- O ro
fc, O ro O -- ro H
N
C Sa ~r C ~- -- ~- C "w
-- r-I C O
N C
-.-1 v~ r-I wl ~ rl .i
ro -1-I ~ U
U
~ ro +~ w .u v ,.-,~ ,~
a~ rn ,~ +~ ...i
~n .N m > ~ m > ~ v~
~ ~ . ~n a
>
fU O C N rl .-1 r1
U rl ~ N O Ul
rl O N N
.y-i - N J.~ t.i +m-- +~
U +~ y~ +~ ~
.i-m ~-- rl
C ro E C U U C U C U C
U .-i O
.i fn f1, ~ .~I ~ tn ~ .i
,~ rl ~ t~ r1 (1,
~ t0
O O ~ O O 'p 'Cf T3
T3 Cf J-~ O ~ O
C O O
~ rl O .-I O f.-iO O O S.I
O S-1 O O 1-1
N
+~ ~ a~ .N s~ s~ w +~
o s~ .-~ .~ 5 ro
~ ~ +~
m s-~ N > ~n Q. f1 Q,
O. L1 N ~ H U
H m
ro a~ a~ o~ a~ m ro
ro ro a~ ro w a~
a~ x a~ ro
~ c.~ z o ~ x c~ rx rx
x x ~n z z ~
c~ x
H
r o0 0,
w '-'
CA 02349586 2001-05-02
wo oon~so rcrius99nss2o
I I I W
O N S-1 S-I 1-I O o~ ro I
-I ' ~ ro O a.~ ro -- O ~, Ts o,
> >, -~
, ~ W ~ ~
c H ro ~ - >. ~, O M -a~
~ O ro zf ro
o ro a I >, -.~ I W -o .-i a ro
+~ a > a~
ro N a~ w ,-a O
.u O .-I Q
v7 O~ .L7 ~ O ro .~ tD >, T3 m -.i W ro
S.I rl U
H ~ ~ -c N tr ro -.'~ x O U
O c
O .C W N ro ro O > ro O H sa ro .rl
U -rl H
H ~ ~ ?~ -.1 .--i tT Cl. O N "O
W >, v >~ .~ ~ +~ ro
~ ,-a ,-I o
v ro m o ro ~ -~I a H o ~ o~ s~
' a~ ~
o ..~ > ro 'a H w o ~ H +~ ro U r,
v .~r o ro
N ~ O Z1 ~ W s~ ro W ro N G ~ U -rl
~ >~ ?~
~ ro ~ ~ G -rl G-i W r-i W N (n .1-~
.--I ro p~
-rl N +~ -rl Z3 ?~ U 'p U N O O
Cu U L7 -.-1 G
ro H ro >r v ~ >'I , -n
v a a -- ~ o ~-I
.u U ~. -.~ > o ro ro > >, c >, ro
ro
L~ N .~ .-i O 1-~ U O ~ .4.1 Q1 ~, O
Jr r-I -~-I O >~
~
O O ~ N
U
v) N N rl a O +~ .-. O ?,
ro N O N -.-I ro
b
N +~ tn .i tl t~ ro I-1 U v~ TS 'Lf ~ f1
U O .4~ 'a7 ~ -~-1
a W ro rl N N C a ro ~
+.i
O
v +.r >, N ro U N +-~ W ~ ~ ~-I ro
t N v) f1 ts. O a
v) p~ .-I a ~ ~ N a v1 O O U -.-I S-W,
~ .-~ >,
~ ~ ~ v U
ro ~ ~
.u .-I cn N ~n ~ C1
T1 s-I v . O
~ ?, U +.I
O rl O ~ -~I .-I -~I M . -~I .u
~ M o v
-1 O .C +~ t~ p. C +~ N a W ?~ ~ +~
a ~ C1. ro U
~ sa
N ro ? ml N +.~ .i S-a W N
C O ~ N 3 v) W ro
~ ~
.i.-~ -r1 +.~ fl. 'rJ -.-IN 'a7
T1 .~ .~.~ O N G C~
.
f!l ~ LL ~ v7 v7 ro v1
U ~ '~
r-I ~ -
' -rl
C3 O TJ ro ro s-I ro ro . O U3
-ri i s-a N ~
C1 O
O .u r-I N ~ 't7 O~ ~ rn ~ WO .-I ~ f-I
O w -rl N .-1 p,
a ro O >, .t~ l-a N ~ 1.-I -.-I -rt ~ ?~
UJ v) .~ T7 ro ?i
'
O CJ . .O 'O v ,L7 T1 N G .-I
N U ~ ro N ro y.~
a~ ~
~
s-I O .-1 C > 'O UJ N D ro v) ~ ro .
v) 'Cf N .
U G ~ i~ U ~ N -.1 ~ 'I] 1 ~ ro ~ ro
ro .-I v7 f~ f-W7 ~,
Q O t~ O U O O +~ N , O G m O 3 N . O H
-~ ~ .~ ro ro 1.-I
U i-I f.I N l.1 x t(1 H N ro C -rl 1-I
ro tn O .W(] ro U N ro
.~G ro
W L1 W rl V) W N ' r-i W .J~ ~ -.i ro W Cl.
v1 ~ 1~ -.1 O v)
O ro N ~ N s-I x t~ U ~ ro ~ ..-i
' ?r -~-i
H Z3 0 U Tt rl .i p N .-~ f-I O -d
.-i C7 N ro W .G $ -O ~-I
tm s.a
ro a~ a~ u~ a~ ro s~. a~ ro ~ +~ a~
ro ro r-I ro
a Q.
H .u +-~ .-I u H a >, .u ~ a U -.~ -v
U ro a >, H .~
ro
ro ro ro ro a~ ro ro ro ro U >~ ro
.I 3 v~ .~ U -c ai ro +.~
~ ~
-
~ .~ .N rl +~ ~n .~ H .-, H o .
a o~ a H ~n .-., .-,
H
,~
o ro o o a~ o ro o ro o a~ -a ro ro o ro
-~, o -~I v~ ro a -.~
v7 v~ 1~ v1 ~--I vW .-r N +-m v)
s-I a ~ N ~ ~ U v) ?, v U C1.
~ 3
.-I ,..i v) -..i -.-I '-I ro +~ U O .-i
ro r-I +.~ O O ~ 'O v) v)
ro ro ~n s~ ro -r-I ~-I ro a a a~ b -~
d' E .a .'-I -.~ ro
~
r.C -I U D ~ r.C
z z a r.~ U 'O a N
+ a ~ a
o, rr - z ,; -~ u x ro z rr
v o +~
a a x a .o c~ ro ~ x a -n ro a~ rx
a~ ro ~I >. s..l a
a
_ a ~ ~ 0
ro U
f-1 CT S-a T b~ . O~ ro . ~ N
-I-i -1 1-I -i L1 U .-1 ~ v
J N N 1
-W
a a c a .~ a a a~ v ~ >, -,~ ro ro ~ a
a ' +~ H a~
'
'-I >~ .1.~ f.l .-1 ..I CT N ~ +~ -ri
C1 O N Gl, S~ 'O U H N ~
O -~-I 1-I
v) v~ .--1 v) N a ?~ v) ~ a ro O ro u~
-~I ro U p,
a a~ a a~ a a m ~ a ,~ v~ .-I .u a a~
v) a, a
i
rl -~ c o v~ s-I s-~ ~n ro ~n ~
a , --t .I ro >, -o aJ
~ ro zs ro b -r.l a Ts a -~ aJ ro .
a~ a ,~ ro >.I --I >.-I Ts
a ro
a
a~ a~ ~ .~ v a~ w v~ w o +.~ U +~ a~
~ .-~ .-i a~ ~ ~o ro ~
+~ a~
+~ w a~ o +~ .-I -~I +~ a v Q. v a +~
a~ a .-I a ar
H -~
U W U W , U ro S-1 U ~ ~1 TJ yLy U W
N ~ ~ C U O +~
a a O 3 a ~ O N a .-~1 O O .-I a ro
~ v1 >,-I 1-i
H c H a v H a~ ~ -q H ro ~ . a a a G
-o o ro o a~ a,
+~ w ro a +.~ w a ,.,
ro s~ .c~ ~ v '
.~ U
~n v~ ..~ m +~ ~ o v~ ~
.r-I a~ ro ~ E ~ .~ ri .~
~ a~
G m ~ u) v) s; >~ N ~ W O ro +> H sr
~ m is U ro v)
C
O ro O ro p. O ro 't7 O 'Cf ~ ~ ,-i .
b~ ro -C 'tJ 1~ ~ -rl U O i0
E-~
U U U U ~ U -~-i N U ~ tv .~ 3 C1 U U
-r-i +~ a N +~
a ~ ~ ?i O T3 +i .-1 ro a~ U s-I >,
f-I O b~
v) v) ro v1 s~ ro v) -,~ ro H o ~n
ro H C w U v v~ ~ H c ro
N ro
ro ro U o ro H .,~ ro u, .,~ ro ro
U -~I o ~ U ~ ~ o a v
.c~ -~
3 3 3 3
ro ~ o ~ ~
o ~ zJ .u v o o ~ o ~ v ~ ro
~ ~
>, ~ ~I ro >, m cn >, a ~n ~ a v~ >,
.-a v~ ~ a~ rn .-I
a~ ~ ~-I
H o H o H ro ~n H ro ~n a~ ~ H o
~ U ro H 2s H ro o s~ a
ro ro I ro ro w ro ~ .-i H ~ ro
i o .-i o a a~ ro U ro v .~ I
a H
f-1 1-I N >'I +~ ~-I , f-I
1.1 r-i !~ U !-I ro J-~ i1 S-I
.-i L1 C1 N U N
.Q .C7 ro .Q T3 N -C1 U N ~ O .~ >~
ro W N O v) U C C ro
U i1
-.~ ~ a~ a -~ ~ .>~ .~I .~ .~ -, -
a~ v v ~I ,~ ro w ~ v .u ro ..~
a a~
>,
a >, a >, -.~ a o .u ,~ a o +~ M o .-, a >,
-~ ro ~ -~ U ~ ro U .a
r-i f-1 ~ d' M
O O
O ,1 O
o
z w z z
z
o a H ~'
c cH x
n
rx H
ca a w a
o ,
o, .1 c
a~
l~ 00 OD
M N
c~
N ,....,
U m t ~ N
D O M
-i ,-I ,1 N M
C~
H
r ao a, o
56
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
0
_ a
W 'e~
p, o .d ~ ~ . ~~ ~ ~ r~°, 04
':° o ~ ~' ~; ea ~ o ~ aT 4: ~ , ~ 0 0.w,
~b ~ ~ ~ ~ ~ .a °° r ~ 'r a ~ ~~
O~ p N .-r C ~ ~ :~ .C~"
~,a ~ .... W ~ ~ II ~ ~ O o a ~ e'~ c °
~7 v ~ fi~ ,--i ~ ~ ~ _~ ~ fi I~" S !/a ~ ~'~' O op
Id i~~~'~O~'W~~ ~°1:~
~ e'~ mn ~
~ ~~C ~~ 'w~ w v~~ ~ v~bw
Q U x ~b v~
o c_°'. ~ 0 00 ~ ~ ~ N '~ .. ...a ~"
W r N ~ M v U ,X~ ,~ ~ ~'~ M
cd G t~ ~ ~O ~ ~ _Ov W N
V ~ ~ M ~ ~ 00 ~ x ~ v ~ O ~Y vp
v1 ~ ~ ~ " Vii, pip f~' ~ ~' ,d_, o ~ ~ e~ N ~'" ~ N
O O ~ O ~~ Uj M a N .--i cd ~-; ~..~ Y
Ov M ~ o . U
V N A N ~ ~ ~ x ~ ~ E"~
O' R" A. '~ ~3 ~ ~ ~n ~ N
~a ~'a a~'a ~~x °~.~b~ ~~~~ gv ~a
V b V b~ Gz;c~ W'.,~ o c~N
ab'~w
w~ w~ ~M ~ ~'~~r~~ ~~,~b ~' ~oz
o ~ ° ~ V ~ o
a.w ww ww aNz ~,z~~~' x~x°~
a~
on y,,., ~ " p
H
Nb wg~~ ~~ o
b ~
'Cy ~ U ~~ ~ ~~" 'Cf '° p N v c~
Nw
°° b ~ ~ ~ ~ ~ ~ -8
o:~ ° ~ ~ ~ d w ~~D'i
8 ~ ~ ~ ~ ~ ~"w
a~ U .5 ° v~ o ~d ~ ~, ai c' 3, °" '~ ~ en
s
rn ~ w E~ ,~ ~ ~ e~" '$ ~ '~ ~ ~ .ty $"
.cy paq ~~." '~ ~ ~ ~ ~ ~ '~ ~ w
'' ~r o
o ~ ~ .d ~ ~ , ~ e~ a o o a~
U .
a ~~ ~ '~ ~ .~ ~c w ~' ~ a w ~ w a ~ ~ .~
..
a ~ ~ ~ ~ x
w ~ Q v~ E~"
w
57
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
"' .
N
p U C~ ~ 'd'
.C ~ '~ ,..i ~ O
.,° a~ ~., ~ g o a
V
O
N
a. z ~ ~ c~
d
'° ~~ ~~a°;
.9 °'
,~, o,
w~ v i v Ii, ~~ tt., C' W.dr
~ c~N Ubn.~r ~~ ~ O U ~'~ .C~' O
n1 p o0 ~ ~ p
U ~ ~ d
3~'~yd
_ .. ... ~ 3 0o r'
G~ ~ ~ N00~~ v "~_~ .~r~(7
'~.' ~ ~ ~i "~ tn LJ 'H '~ v ~ ~ N ~ ~ M ~ ~ pv .r,
_ 00 ~ C~ N r p ~1'
~ U U ~ ~ ifr U ~i ~ ~ N
o o vo V
per'
r~; .a .3~ ~ ~ .~ ~5 0. 3 ~ ''~ oo ~ ~ O '~3 ~ a, ~
x ~~°~z wx5°; ~~'~~3 u~x~ z°~
0
,p ~ a, ~ 3 ~~ ~
~"i W° °° ~ ~ ~ ~ c. a H
8 a. :~ ,~' .~ ,~
.o ~3 ~ .~ ~ ~ A o~u ~, N 8'
.~ on ~ ~ ~° b :3
., ., ..,
b' ~ ~ ~~~ b ~ ~ HQ
0 0 .~ ~ ~ ~ ~ o c~
3 °' °~
y
v ~~ ~ ~,a ~ g ~~ '~~
w ~ a~ ~ b ~~ ~.~ ,
f/1 U ~ ~ U
p.~,, ~ ,~ ~ a~ ~ o
A ~ ~b d ~ dU o ~ d d ~ d~
0.~ . ~ per" o
58
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
TANG, Y. Tom
CORLEY, Neil C.
GUEGLER, Karl J.
GORGONE, Gina A.
AZIMZAI, Yalda
KASER, Matthew R.
YUE, Henry
<120> COENZYME A-UTILIZING ENZYMES
<130> PF-0622 PCT
<140> To Be Assigned
<141> Herewith
<150> 09/185,217; unassigned
<151> 1998-11-03; 1998-11-03
<160> 10
<170> PERL Program
<2I0> 1
<211> 100
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1580751CD1
<400> 1
Met Lys Gly Ala Thr Thr Asn Ile Cys Tyr Asn Val Leu Asp Arg
1 5 10 15
Asn Val His Glu Lys Lys Leu Gly Asp Lys Val Ala Phe Tyr Trp
20 25 30
Pro Cys Gln Arg Asp Gln Asp Gly Tyr Tyr Trp Ile Thr Gly Arg
35 40 45
Ile Asp Asp Met Leu Asn Val Ser Gly Glu Gly Gln Gly Pro Pro
50 55 60
Ser His Leu Ile Asn Ser Ala Pro Leu Thr Thr Pro Ser Arg Ser
65 70 75
Leu Pro Gln Glu Pro Arg Ser Val Leu Trp Pro Asp His Val Leu
80 85 90
Ser Val Ala Phe Ser Ser Gly Pro Arg Phe
95 100
<210> 2
<211> 159
1/1~
CA 02349586 2001-05-02
wo oon~so Pcrms99ns~zo
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 16278~89CD1
<400> 2
Met Ser Gly Gln Gly Val Asp Arg His Leu Phe Ala Leu Tyr Ile
1 5 10 15
Val Ser Arg Phe Leu His Leu Gln Ser Pro Phe Leu Thr Gln Val
20 25 30
His Ser Glu Gln Trp Gln Leu Ser Thr Ser Gln Ile Pro Val Gln
35 40 45
Gln Met His Leu Phe Asp Val His Asn Tyr Pro Asp Tyr Val Ser
50 55 60
Ser Gly G1y Gly Phe Gly Pro Ala Asp Asp His Gly Tyr Gly Val
65 70 75
Ser Tyr Ile Phe Met Gly Asp Gly Met Ile Thr Phe His Ile Ser
80 85 90
Ser Lys Lys Ser Ser Thr Lys Thr Asp Ser His Arg Leu Gly Gln
95 100 105
His Ile Glu Asp Ala Leu Leu Asp Val Ala Ser Leu Phe Gln Ala
110 115 120
Gly Gln His Phe Lys Arg Arg Phe Arg Gly Ser Gly Lys Glu Asn
125 130 135
Ser Arg His Arg Cys Gly Phe Leu Ser Arg Gin Thr Gly Ala Ser
140 145 150
Lys Ala Ser Met Thr Ser Thr Asp Phe
155
<210> 3
<211> 215
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1965888CD1
<400> 3
Met Asn Arg Pro Ser Ala Arg Asn Ala Leu Gly Asn Val Phe Val
1 5 10 15
Ser Glu Leu Leu Glu Thr Leu Ala Gl.n Leu Arg Glu Asp Arg Gln
20 25 30
Val Arg Val Leu Leu Phe Arg Ser Gly Val Lys Gly Val Phe Cys
35 40 45
Ala Gly Ala Asp Leu Lys Glu Arg Glu Gln Met Ser Glu Ala Glu
50 55 60
Val Gly Val Phe Val Gln Arg Leu Arg Gly Leu Met Asn Asp Ile
65 70 75
Ala Ser Ser Ala Val Met Gly Leu Ile Glu Thr Thr Arg Gly Leu
80 85 90
Leu Pro Gly Ala Gly Gly Thr Gln Arg Leu Pro Arg Cys Leu Gly
2/10
CA 02349586 2001-05-02
wo oons~so rcrius99nss~o
95 100 105
Val Ala Leu Ala Lys Glu Leu Ile Phe Thr Gly Arg Arg Leu Ser
110 115 120
Gly Thr Glu Ala His Val Leu Gly Leu Val Asn His Ala Val Ala
125 130 135
Gln Asn Glu Glu Gly Asp Ala Ala Tyr Gln Arg Ala Arg Ala Leu
140 145 150
Ala Gln Glu Ile Leu Pro Gln Ala Pro Ile Ala Val Arg Leu Gly
155 160 165
Lys Val Ala Ile Asp Arg Gly Thr Glu Val Asp Ile Ala Ser Gly
170 175 180
Met Ala Ile Glu Giy Met Cys Tyr Ala Gln Asn Ile Pro Thr Arg
185 190 195
Asp Arg Leu Glu Gly Met Ala Ala Phe Arg Glu Lys Arg Thr Pro
200 205 210
Lys Phe Val Gly Lys
215
<210> 4
<211> 720
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2816341CD1
<400> 4
Met Asn Asn His Val Ser Ser Lys Pro Ser Thr Met Lys Leu Lys
1 5 10 15
His Thr Ile Asn Pro Ile Leu Leu Tyr Phe Ile His Phe Leu Ile
20 25 30
Ser Leu Tyr Thr Ile Leu Thr Tyr Ile Pro Phe Tyr Phe Phe Ser
35 40 45
Glu Ser Arg Gln Glu Lys Sex Asn Arg Ile Lys Ala Lys Pro Val
50 55 60
Asn Ser Lys Pro Asp Ser Ala Tyr Arg Ser Val Asn Ser Leu Asp
65 70 75
Gly Leu Ala Ser Val Leu Tyr Pro Gly Cys Asp Thr Leu Asp Lys
80 85 90
Val Phe Thr Tyr Ala Lys Asn Lys Phe Lys Asn Lys Arg Leu Leu
95 100 105
Gly Thr Arg Glu Val Leu Asn Glu Glu Asp Glu Val Gln Pro Asn
110 115 120
Gly Lys Ile Phe Lys Lys Val Ile Leu Gly Gln Tyr Asn Trp Leu
125 130 135
Ser Tyr Glu Asp Val Phe Val Arg Ala Phe Asn Phe Gly Asn Gly
140 145 150
Leu Gln Met Leu Gly Gln Lys Pro Lys Thr Asn Ile Ala Ile Phe
155 160 165
Cys Glu Thr Arg Ala Glu Trp Met Ile Ala Ala Gln Ala Cys Phe
170 175 180
Met Tyr Asn Phe Gln Leu Val Thr Leu Tyr Ala Thr Leu Gly Gly
185 190 195
3/10
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/25820
Pro Ala Ile Val His Ala Leu Asn Glu Thr Glu Val Thr Asn Ile
200 205 210
Ile Thr Ser Lys Glu Leu Leu Gln Thr Lys Leu Lys Asp Ile Val
215 220 225
Ser Leu Val Pro Arg Leu Arg His Ile Ile Thr Val Asp Gly Lys
230 235 240
Pro Pro Thr Trp Ser Glu Phe Pro Lys Gly Ile Ile Val His Thr
245 250 255
Met Ala Ala Val Glu Ala Leu Gly Ala Lys Ala Ser Met Glu Asn
260 265 270
Gln Pro His Ser.Lys Pro Leu Pro Ser Asp Ile Ala Val Ile Met
275 280 285
Tyr Thr Ser Gly Ser Thr Gly Leu Pro Lys Gly Val Met Ile Ser
290 295 300
His Ser Asn Ile Ile Ala Gly Ile Thr Gly Met Ala Glu Arg Ile
305 310 315
Pro Glu Leu Gly Glu Glu Asp Val Tyr Ile Gly Tyr Leu Pro Leu
320 325 330
Ala His Val Leu Glu Leu Ser Ala Glu Leu Val Cys Leu Ser His
335 340 345
Gly Cys Arg Ile Gly Tyr Ser Ser Pro Gln Thr Leu Ala Asp Gln
350 355 360
Ser Ser Lys Ile Lys Lys Gly Ser Lys Gly Asp Thr Ser Met Leu
365 370 375
Lys Pro Thr Leu Met Ala Ala Val Pro Glu Ile Met Asp Arg Ile
380 385 390
Tyr Lys Asn Val Met Asn Lys Val Ser Glu Met Ser Ser Phe Gln
395 400 405
Arg Asn Leu Phe Ile Leu Ala Tyr Asn Tyr Lys Met Glu Gln Ile
410 415 420
Ser Lys Gly Arg Asn Thr Pro Leu Cys Asp Ser Phe Val Phe Arg
425 430 435
Lys Val Arg Ser Leu Leu Gly Gly Asn Ile Arg Leu Leu Leu Cys
440 445 450
Gly Gly Ala Pro Leu Ser Ala Thr Thr Gln Arg Phe Met Asn Ile
455 460 465
Cys Phe Cys Cys Pro Val Gly Gln Gly Tyr Gly Leu Thr Glu Ser
470 475 480
Ala Gly Ala Gly Thr Ile Ser Glu Val Trp Asp Tyr Asn Thr Gly
485 490 495
Arg Val Gly Ala Pro Leu Val Cys Cys Glu Ile Lys Leu Lys Asn
500 505 510
Trp Glu Glu Gly Gly Tyr Phe Asn Thr Asp Lys Pro His Pro Arg
515 520 525
Gly Glu Ile Leu Ile Gly Gly Gln Ser Val Thr Met Gly Tyr Tyr
530 535 540
Lys Asn Glu Ala Lys Thr Lys Ala Asp Phe Phe Glu Asp Glu Asn
545 550 555
Gly Gln Arg Trp Leu Cys Thr Gly Asp Ile Gly Glu Phe Glu Pro
560 565 570
Asp Gly Cys Leu Lys Ile Ile Asp Arg Lys Lys Asp Leu Val Lys
575 580 585
Leu Gln Ala Gly Glu Tyr Val Ser Leu Gly Lys Val Glu Ala Ala
590 595 600
Leu Lys Asn Leu Pro Leu Val Asp Asn Ile Cys Ala Tyr Ala Asn
4/10
CA 02349586 2001-05-02
WO OO/Z6350 PCf/US99I25820
605 610 615
Ser Tyr His Ser Tyr Val Ile Gly Phe Val Val Pro Asn Gln Lys
620 625 630
Glu Leu Thr Glu Leu Ala Arg Lys Lys Gly Leu Lys Gly Thr Trp
635 640 645
Glu Glu Leu Cys Asn Ser Cys Glu Met Glu Asn Glu Leu Leu Lys
650 655 660
Val Leu Ser Glu Ala Ala Ile Ser Ala Ser Leu Glu Lys Phe Glu
665 670 675
Ile Leu Val Lys Ile Arg Leu Ser Pro Glu Pro Trp Thr Pro Glu
680 685 690
Thr Gly Leu Val Thr Asp Ala Phe Lys Leu Lys Arg Lys Glu Leu
695 700 705
Lys Thr His Tyr Gln Ala Asp Ile Glu Arg Met Tyr Gly Arg Lys
710 715 720
<210> 5
<211> 456
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3324214CD1
<400> 5
Met Phe Leu Leu Leu Thr Phe Asp Ser Leu Ile Val Asn Leu Leu
1 5 10 15
Gly Ile Ser Leu Thr Val Leu Phe Thr Leu Leu Leu Val Phe Ile
20 25 30
Ile Val Pro Ala Ile Phe Gly Val Ser Phe Gly Ile Arg Lys Leu
35 40 45
Tyr Met Lys Ser Leu Leu Lys Ile Phe Ala Trp Ala Thr Leu Arg
50 55 60
Met Glu Arg Gly Ala Lys Glu Lys Asn His Gln Leu Tyr Lys Pro
65 70 75
Tyr Thr Asn Gly Ile Ile Ala Lys Asp Pro Thr Ser Leu Glu Glu
80 85 90
Glu Ile Lys Glu Ile Arg Arg Ser Gly Ser Ser Lys Ala Leu Asp
95 100 105
Asn Thr Pro Glu Phe Glu Leu Ser Asp Ile Phe Tyr Phe Cys Arg
110 115 120
Lys Gly Met Glu Thr Ile Met Asp Asp Glu Val Thr Lys Arg Phe
125 130 135
Ser Ala Glu Glu Leu Glu Ser Trp Asn Leu Leu Ser Arg Thr Asn
140 145 150
Tyr Asn Phe Gln Tyr Ile Ser Leu Arg Leu Thr Val Leu Trp Gly
155 160 165
Leu Gly Val Leu Ile Arg Tyr Cys Phe Leu Leu Pro Leu Arg Ile
170 175 180
Ala Leu Ala Phe Thr Gly Ile Ser Leu Leu Val Val Gly Thr Thr
185 190 195
Val Val Gly Tyr Leu Pro Asn Gly Arg Phe Lys Glu Phe Met Ser
200 205 210
$~1~
CA 02349586 2001-05-02
WO 00126350 PCTNS99/25820
Lys His Val His Leu Met Cys Tyr Arg Ile Cys Val Arg Ala Leu
215 220 225
Thr Ala Ile Ile Thr Tyr His Asp Arg Glu Asn Arg Pro Arg Asn
230 235 240
Gly Gly Ile Cys Val Ala Asn His Thr Ser Pro Ile Asp Val Ile
245 250 255
Ile Leu Ala Ser Asp Gly Tyr Tyr Ala Met Val Gly Gln Val His
260 265 270
Gly Gly Leu Met Gly Val Ile Gln Arg Ala Met Val Lys Ala Cys
275 280 285
Pro His Val Trp Phe Glu Arg Ser Glu Val Lys Asp Arg His Leu
290 295 300
Val Ala Lys Arg Leu Thr Glu His Val Gln Asp Lys Ser Lys Leu
305 310 315
Pro Ile Leu Ile Phe Pro Glu Gly Thr Cys Ile Asn Asn Thr Ser
320 325 330
Val Met Met Phe Lys Lys Gly Ser Phe Glu Ile Gly Ala Thr Val
335 340 345
Tyr Pro Val Ala Ile Lys Tyr Asp Pro Gln Phe Gly Asp Ala Phe
350 355 360
Trp Asn Ser Ser Lys Tyr Gly Met Val Thr Tyr Leu Leu Arg Met
365 370 375
Met Thr Ser Trp Ala Ile Val Cys Ser Val Trp Tyr Leu Pro Pro
380 385 390
Met Thr Arg Glu Ala Asp Glu Asp Ala Val Gln Phe Ala Asn Arg
395 400 405
Val Lys Ser Ala Ile Ala Arg Gln Gly Gly Leu Val Asp Leu Leu
410 415 420
Trp Asp Gly Gly Leu Lys Arg Glu Lys Val Lys Asp Thr Phe Lys
425 430 435
Glu Glu Gln Gln Lys Leu Tyr Ser Lys Met Ile Val Gly Asn His
440 445 450
Lys Asp Arg Ser Arg Ser
455
<210> 6
<211> 687
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 63
<223> a or g or c or t, un3cnown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 1580751CB1
<400> 6
cggttctagg aacttgacgt gatggggctt cctgaggagc gggtccggag cggcagcggg 60
ganccggggc caggaggaag ctggagccgg aggccgggcg cggagttggt ctccgccgcc 120
cgaggtcagc cgctccgcgc acgtcccctc gctgcagcgc taccgcgagc tgcaccggcg 180
ctccgtggag gagccgcggg aattctgggg agacattgcc aaggaatttt actggaagac 240
6/10
CA 02349586 2001-05-02
WO 00/26350 PCTNS99/25820
tccatgccct ggcccattcc ttcggtacaa ctttgatgtg actaaaggga aaatcttcat 300
tgagtggatg aaaggagcaa ctaccaacat ctgctacaat gtactggatc gaaatgtcca 360
tgagaaaaag cttggagata aagttgcttt ttactggcct tgccagcggg accaggatgg 420
ctattactgg atcactggca ggattgatga catgctcaat gtatctggtg agggccaggg 480
gccaccttcc catcttatta actctgctcc tctgacaaca cccagccgaa gccttccgca 540
agagcccagg agtgtccttt ggccagacca tgtactaagt gtagcattca gttctgggcc 600
caggttttag aggagtaatg aacactggaa tgtgcctaga gtaggggaac ttggatggtg 660
gggtgactga aagcatgtca tctgagg 687
<210> 7
<211> 1803
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1627889CB1
<400> 7
caaattttca tttttaatag agatgggttc accaaggtgg caaggctggt tttgattccc 60
gatcccagaa ccacctgccc tgggcccccc aaagtgcggg atctacaggc atgagccccc 120
gtgctcagac taaagctttt ctttatacgg gggttcatgt tggtaaaaat ccattgaatt 180
atatacgtag gatttttgaa cttatttcta taaagtttta tatttcaata aaaagcttaa 240
agatatatat atattatttt ccatacatga caagtattgt atcatatata ctatttttga 300
acttattcct ataaaatgtt atatttcaat aaaaactgac agatatatta cattattttc 360
catccatgac aagtattatt atatcataca tgctattttt tttttttttt ttttttttga 420
gatggagttt cgcttgttgc ccaggctgga gtgcaatggc gccatctcgg cccaccgcaa 480
cctctgcctc ccaggttcaa gcgattctcc tgcctcagcc tcctgagtag ctgggactac 540
agcatgcgcc accataccca gctaattttg tgtttttagt agagatgggg tttctccata 600
ctggtcaggc tggtctcaaa ctcctgacct caggtgatcc gcctgcctca gcctcccaaa 660
gtgctgggat tacaggcgtg accaatgcac ctggctggaa ctccattttt acaacgtaac 720
tctgcccatt taacctcttt gtgacttgtg accttccttt gcacccctgt accctctctg 780
cccagatccc acctgccctg cacctcttct cccttgccct acacctagcc ccgcacctac 840
aaggtattgt ggttccatgg aaggagtttg gactctgggg ccagacacac ctaggaaccc 900
gcctggctct ccctggctgt gtgaacctgg ccaaatgatt tcccctctct aagcctcagt 960
tccccatctg taaaatgggg ttgatattcc caccttgcag ggatgtggca aactcagttg 1020
aggccagatg tgccgcagcc agccctaagt cgacttcctg tctttccatg acctgtgacc 1080
tccctgggga ctgcaggacc cacagtgcct cgccctgttc cgcgtggcag tggacaagca 1140
ccaggctctg ctgaaggcag ccatgagcgg.gcagggagtt gaccgccacc tgtttgcgct 1200
gtacatcgtg tcccgattcc tccacctgca gtcgcccttc ctgacccagg tccattcgga 1260
gcagtggcag ctgtccacca gccagatccc tgttcagcaa atgcatctgt ttgacgtcca 1320
caattacccg gactatgttt cctcaggcgg tggattcggg cctgctgatg accatggtta 1380
tggtgtttct tatatcttca tgggggatgg catgatcacc ttccacatct ccagcaaaaa 1440
atcaagcaca aaaacggatt cccacaggct ggggcagcac attgaggacg cactgctgga 1500
tgtggcctcc ctgttccagg cgggacagca ttttaagcgc cggttcagag ggtcagggaa 1560
ggagaactcc aggcacaggt gtggatttct ctcccgccag actggggcct ccaaggcctc 1620
aatgacatcc accgacttct gactccttcc agcaggcagc tggcctctcc aaggaataag 1680
ggtgaaattg ccacagctgg ctgacacagg acaggggcaa ctggtttggc aaccccacat 1740
ccaggccaat aaagatgtgt gagctggaca cttataaaaa aaacaaatat aaaaaaaaaa 1800
aaa 1803
<210> 8
<211> 1340
7/10
CA 02349586 2001-05-02
wo oon~so Pc~rius99nssZo
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1965888CB1
<400> 8
ccagcctgcg ccccgatgct gcgcgttctg tgcctcctgc gcccctggag gccccttcgg 60
gcccgcggct gcgcttccga cggggcggcc gggggctcag agatccaagt gcgcgccctg 120
gcgggtccgg accaagttta caagtccttc cactttctct ctgaggcaga aagagcaagg 180
gtttttctct ccattttatg gttgggaaaa ttgaggcctg cctgagtgtg tgacttgtgg 240
caagtcactc tggtcatcta gggcagaggc tccccagatc ccaggcctcc tgcctccagt 300
ccccagcccg cagcccagga ttaggcagag ccagctgctt tcccgtggct gccctgactc 360
cttacaggga tcactgagat tctgatgaac agaccttctg cccgcaatgc cttggggaat 420
gtcttcgtca gtgagctgct ggaaactctg gcccagctgc gggaggaccg gcaagtgcgt 480
gtcctgctct tcagaagtgg agtgaagggc gtgttctgtg caggtgcaga cctgaaggag 540
cgggaacaga tgagtgaagc agaggtgggg gtgtttgtcc agcgactccg gggcctgatg 600
aatgacatcg cttcctcggc agtcatggga ctgattgaga ccacgcgagg gctcctcccg 660
ggggcaggag ggactcagag gctgccccgt tgtctggggg tggccctggc gaaggagctc 720
atcttcacgg gccgacgact gagtggaact gaggcccacg tactggggct ggtgaatcac 780
gctgtggccc agaacgagga gggggacgcc gcctaccagc gggcacgagc actggcccag 840
gagatcctgc cccaggcccc cattgccgtg cggctgggca aagtagccat tgaccgagga 900
acggaggtgg acattgcatc tgggatggcc attgaaggga tgtgctatgc ccagaatatt 960
ccaacccggg accggctaga gggcatggca gccttcaggg agaagcggac tcccaaattt 1020
gttggcaaat gacccccatt ttaaccttca gcatgggaga tgcatgccct gaagagcagg 1080
atccagaagg aagatttgtg gccagattgc cttcatcatt tcacctctcc agacttccat 1140
ttcttcacaa ggatgatgat ggaaataaaa tgactggcgt gatgcctgga accaaggtgc 1200
tgatcctacc acctactgct accttcctta gcttcaccct ggctagaaat aatcacgagg 1260
gttgggtttg ctttggaaaa tgcctgtctc tctacttgaa tgataaagaa.ttaaattaga 1320
tctctctgaa aaaaaaaaaa 1340
<210> 9
<211> 4027
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2816341CB1
<400> 9
gcgccgggct gcgacactgc agttgtctac gcggccgggg ccgggacgag gaggcgttgg 60
acggggtcgc atacgttcgt cccctcgcat tgcggccccg acagctgcgc caggatcccc 120
gggcggcggc gcggggcgtg aacgctctgg ggctcagcca ggcctgcgcg ggcccgatgc 180
cggaggaacc cggactccgg cgtagcggtt ttgacacaag ggcgcatatc ttcaaagcac 240
ctagtacctc ctaccattgt caactgatac agaattcgtt gttgggaagg actggggaaa 300
cagctgtaac atttgccacc ctcagaagct gctggtcctg tgtcacacca ccttagcctc 360
ttgatcgagg aagattctcg ctgaagtctg ttaattctac tttttgagta cttatgaata 420
accacgtgtc ttcaaaacca tctaccatga agctaaaaca taccatcaac cctattcttt 480
tatattttat acattttcta atatcacttt atactatttt aacatacatt ccgttttatt 540
ttttctccga gtcaagacaa gaaaaatcaa accgaattaa agcaaagcct gtaaattcaa 600
aacctgattc tgcatacaga tctgttaata gtttggatgg tttggcttca gtattatacc 660
8/10
CA 02349586 2001-05-02
WO 00/26350 PCT/US99/Z5820
ctggatgtga tactttagat aaagttttta catatgcaaa aaacaaattt aagaacaaaa 720
gactcttggg aacacgtgaa gttttaaatg aggaagatga agtacaacca aatggaaaaa 780
tttttaaaaa ggttattctt ggacagtata attggctttc ctatgaagat gtctttgttc 840
gagcctttaa ttttggaaat ggattacaga tgttgggtca gaaaccaaag accaacatcg 900
ccatcttctg tgagaccagg gccgagtgga tgatagctgc acaggcgtgt tttatgtata 960
attttcagct tgttacatta tatgccactc taggaggtcc agccattgtt catgcattaa 1020
atgaaacaga ggtgaccaac atcattacta gtaaagaact cttacaaaca aagttgaagg 1080
atatagtttc tttggtccca cgcctgcggc acatcatcac tgttgatgga aagccaccga 1140
cctggtccga gttccccaag ggcatcattg tgcataccat ggctgcagtg gaggccctgg 1200
gagccaaggc cagcatggaa aaccaacctc atagcaaacc attgccctca gatattgcag 1260
taatcatgta cacaagtgga tccacaggac ttccaaaggg agtcatgatc tcacatagta 1320
acattattgc tggtataact gggatggcag aaaggattcc agaactagga gaggaagatg 1380
tctacattgg atatttgcct ctggcccatg ttctagaatt aagtgctgag cttgtctgtc 1440
tttctcacgg atgccgcatt ggttactctt caccacagac tttagcagat cagtcttcaa 1500
aaattaaaaa aggaagcaaa ggggatacat ccatgttgaa accaacactg atggcagcag 1560
ttccggaaat catggatcgg atctacaaaa atgtcatgaa taaagtcagt gaaatgagta 1620
gttttcaacg taatctgttt attctggcct ataattacaa aatggaacag atttcaaaag 1680
gacgtaatac tccactgtgc gacagctttg ttttccggaa agttcgaagc ttgctagggg 1740
gaaatattcg tctcctgttg tgtggtggcg ctccactttc tgcaaccacg cagcgattca 1800
tgaacatctg tttctgctgt cctgttggtc agggatacgg gctcactgaa tctgctgggg 1860
ctggaacaat ttccgaagtg tgggactaca atactggcag agtgggagca ccattagttt 1920
gctgtgaaat caaattaaaa aactgggagg aaggtggata ctttaatact gataagccac 1980
accccagggg tgaaattctt attgggggcc aaagtgtgac aatggggtac tacaaaaatg 2040
aagcaaaaac aaaagctgat ttctttgaag atgaaaatgg acaaaggtgg ctctgtactg 2100
gggatattgg agagtttgaa cccgatggat gcttaaagat tattgatcgt aaaaaggacc 2160
ttgtaaaact acaggcaggg gaatatgttt~ctcttgggaa agtagaggca gctttgaaga 2220
atcttccact agtagataac atttgtgcat atgcaaacag ttatcattct tatgtcattg 2280
gatttgttgt gccaaatcaa aaggaactaa ctgaactagc tcgaaagaaa ggacttaaag 2340
ggacttggga ggagctgtgt aacagttgtg aaatggaaaa tgagctactt aaagtgcttt 2400
ccgaagctgc tatttcagca agtctggaaa agtttgaaat tctagtaaaa attcgtttga 2460
gtcctgaacc gtggacccct gaaactggtc tagtgacaga tgccttcaag ctgaaacgca 2520
aagagcttaa aacacattac caggcggaca ttgagcgaat gtatggaaga aaataattat 2580
tctcttctgg catcagtttg ctacagtgag ctcagatcaa ataggaaaat acttgaaatg 2640
catgtctcaa gctgcaaggc aaactccatt cctcatatta aactattact tctcatgacg 2700
tcaccatttt taactgacag gattagtaaa acattaagac agcaaacttg tgtctgtctc 2760
ttctttcatt ttccccgcca ccaacttact ttaccaccta tgactgtact tgtcagtatg 2820
agaatttttc tgaatcatat tggggaagca gtgattttaa aacctcaagt ttttaaacat 2880
gatttatatg ttctgtataa tgttcagttt gtaacttttt aaaagtttgg atgtatagag 2940
ggataaatag gaaatataag aattggttat ttgggggctt ttttacttac tgtatttaaa 3000
aatacaaggg tattgatatg aaattatgta aatttcaaat gcttatgaat caaatcattg 3060
ttgaacaaaa gatttgttgc tgtgtaatta ttgtcttgta tgcatttgag agaaataaat 3120
atacccatac ttatgtttta agaagttgag atcttgtgaa tatatgcctg tcagtgtctt 3180
ctttatatat ttatttttta ttagaaaaaa tgaagtttgg ttggtgatgc atgaaacaaa 3240
atagcaagag agggttatag tttaatagta agggagataa cacagcatgt gtagcaccag 3300
ttgataattg gtctctagta gcttactgtc aaaatgttca atgaagtctt ctgttcatct 3360
gttgaaacta ggaaaatacc caaacttaaa tggaagaatt ctgaaagaga ggatagaatt 3420
taaagaacaa gagtatataa agttattctt tgaatatttc attgactata tgtacattga 3480
gttatctata tttgtaaaca aattagtcat ggaaaattat tctatcccaa agtctccttt 3540
tagtctagat aatcattatt tcattttaaa attagtgttt ttcatagttt gcactgatgc 3600
gtgtatggat gtgtgtgagt cagtggtagc ttatttaaaa agcaccttat cctttctccc 3660
ataacctttg tacactaaaa aatgaaagaa tttagaatgt atttgatgat agcattctca 3720
ctaagacaca tgagaattta actttataac cgcgtgagtt aagatttaat tcataggttt 3780
tgatgtcatt gttgaagtta tttgtaattc agaaaccttg cttgtgtgat acatagtaag 3840
tctcttcatt tattactgct tgtctgttgt tatatctgga ttatcaaaag caatagtgca 3900
ccaattaaga tgtgctcaaa tcaggactta aatcataggc accacatttt tcatgtcaga 3960
9/ 10
CA 02349586 2001-05-02
wo oon63so pcTius99nsa~o
ctagttactt tgttgattct cagttactgt aggcatcaaa aggcaaaaat caaaaaaaaa 4020
aaaaagg 4027
<210> 10
<211> 1808
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 1597, 1650, 1656, 1713, 1719, 1754, 1778, 1791, 1794, 1796,
<221> unsure
<222> 1800
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 3324214CB1
<400> 10
ctgtcaggaa ggaccatctg aaggctgcaa tttgttctta gggaggcagg tgctggcctg 60
gcctggatct tccaccatgt tcctgttgct gacttttgat agcctgattg tcaaccttct 120
gggcatctcc ctgactgtcc tcttcaccct ccttctcgtt ttcatcatag tgccagccat 180
ttttggagtc tcctttggta tccgcaaact ctacatgaaa agtctgttaa aaatctttgc 240
gtgggctacc ttgagaatgg agcgaggagc caaggagaag aaccaccagc tttacaagcc 300
ctacaccaac ggaatcattg caaaggatcc cacttcacta gaagaagaga tcaaagagat 360
tcgtcgaagt ggtagtagta aggctctgga caacactcca gagttcgagc tctctgacat 420
tttctacttt tgccggaaag gaatggagac cattatggat gatgaggtga caaagagatt 480
ctcagcagaa gaactggagt cctggaacct gctgagcaga accaattata acttccagta 540
catcagcctt cggctcacgg tcctgtgggg gttaggagtg ctgattcggt actgctttct 600
gctgccgctc aggatagcac tggctttcac agggattagc cttctggtgg tgggcacaac 660
tgtggtggga tacttgccaa atgggaggtt taaggagttc atgagtaaac atgttcactt 720
aatgtgttac cggatctgcg tgcgagcgct gacagccatc atcacctacc atgacaggga 780
aaacagacca agaaatggtg gcatctgtgt ggccaatcat acctcaccga tcgatgtgat 840
catcttggcc agcgatggct attatgccat ggtgggtcaa gtgcacgggg gactcatggg 900
tgtgattcag agagccatgg tgaaggcctg cccacacgtc tggtttgagc gctcggaagt 960
gaaggatcgc cacctggtgg ctaagagact gactgaacat gtgcaagata aaagcaagct 1020
gcctatcctc atcttcccag aaggaacctg catcaataat acatcggtga tgatgttcaa 1080
aaagggaagt tttgaaattg gagccacagt_ttaccctgtt gctatcaagt atgaccctca 1140
atttggcgat gccttctgga acagcagcaa atacgggatg gtgacgtacc tgctgcgaat 1200
gatgaccagc tgggccattg tctgcagcgt gtggtacctg cctcccatga ctagagaggc 1260
agatgaagat gctgtccagt ttgcgaatag ggtgaaatct gccattgcca ggcagggagg 1320
acttgtggac ctgctgtggg atgggggcct gaagagggag aaggtgaagg acacgttcaa 1380
ggaggagcag cagaagctgt acagcaagat gatcgtgggg aaccacaagg acaggagccg 1440
ctcctgagcc tgcctccagc tggctggggc caccgtgcgg ggtgccaacg ggctcagagc 1500
tggagttgcc gccgccgccc ccactgctgt gtcctttcca gactccaggg ctcccagggc 1560
tgctctggat cccagggctt cggctttggc gagccgnagg ggatcctgtg gacccggcgc 1620
acttaccttg gtggtttaaa cggatgtgtn ggtttngacc aggagggatg cttgtttttt 1680
taaatagtgt ttgaggatgc attaagtgaa ttnaatttna agttttgggt tatgttggga 1740
attggcatgt tttnttggtg ggtaaggttt tttaagcntt tcaggttttt nggngnaatn 1800
tttaggat 1808
10/10
