Note: Descriptions are shown in the official language in which they were submitted.
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CARBOHYDRATE-MODIFYING E~,TZYMES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of
carbohydrate-modifying
enzymes and to the use of these sequences in the diagnosis, treatment, and
prevention of carbohydrate
metabolism disorders, autoimmune/inflammatory disorders, and cancer.
BACKGROUND OF THE INVENTION
Carbohydrates, including sugars or saccharides, starch, and cellulose, are
aldehyde or ketone
compounds with multiple hydroxyl groups. The importance of carbohydrate
metabolism is
demonstrated by the sensitive regulatory system in place for maintenance of
blood glucose levels.
Two pancreatic hormones, insulin and glucagon, promote increased glucose
uptake and storage by
cells and increased glucose release from cells, respectively. Carbohydrates
have three important roles
in mammalian cells. First, carbohydrates are used as energy stores, fuels, and
metabolic
intermediates. Carbohydrates are broken down to form energy in glycolysis and
are stored as
glycogen for later use. Second, the sugars deoxyribose and ribose form part of
the structural support
of DNA and RNA, respectively. Third, carbohydrate modifications are added to
secreted and
membrane proteins and lipids as they traverse the secretory pathway. Indeed, 2-
10% of the content of
eukaryotic cell membranes are contributed by oligosaccharides on membrane
glycoproteins and
glycolipids. Oligosaccharide modifications of carbohydrates create great
structural diversity.
Modifications on glycoproteins and glycolipids are mostly located on the
extracellular side of the
plasma membrane and are important for intercellular recognition (Stryer, L. (
1988) Biochemistry,
W.H. Freeman and Company, New York NY, pp. 298-299, 331-347).
N- and O-linked oligosaccharides are transferred to proteins and modified in a
series of
enzymatic reactions that occurs in the endoplasmic reticulum (ER) and Golgi.
Oligosaccharides
stabilize a protein during and after folding, orient the protein in the
membrane, improve the protein's
solubility, and act as a signal for lysosome targeting. Glycolipids, along
with phospholipids and
cholesterol, form the membrane of cells. Examples of glycolipids include blood
group antigens on
erythrocytes and gangliosides in the myelin sheath of neurons (Lodish, H. et
al. ( 1995) Molecular
Cell Bioloey, Scientific American Books, New York NY, pp. 612-615).
Carbohydrates also form glycosaminoglycans (GAGS), which are linear unbranched
polysaccharides composed of repetitive disaccharide units. GAGS exist free or
as part of
proteoglycans, large molecules composed of a core protein attached to one or
more GAGs. GAGS are
found on the cell surface, inside cells, and in the extracellular matrix. The
GAG hyaluronan is
abundant in synovial fluid (Pitsillides, A.A. et al. ( 1993) Int. J. Exp.
Pathol. 74:27-34).
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Proteoglycans in the extracellular matrix of connective tissues such as
cartilage are essential for
distributing the load in weight-bearing joints. Cell-surface-attached
proteoglycans anchor cells to the
extracellular matrix. Both extracellular and cell-surface proteoglycans bind
growth factors,
facilitating their binding to cell-surface receptors and subsequent triggering
of signal transduction
pathways (Lodish, supra, pp. 1139-1142).
Man9 mannosidase is an a 1,2-mannosidase (glycosyl hydrolase) involved in the
early
processing of N-linked oligosaccharides. This enzyme catalyzes the specific
cleavage of a 1,2-
mannosidic linkages in Man9-(GIcNAc)2 and Mans-(GIcNAc)2. Multiple a 1,2-
mannosidases have
been identified in mammalian cells and may be needed for the processing of
distinct classes of N-
glycoproteins. Man9 mannosidase is a Type II membrane protein with a short
cytoplasmic tail, a
single transmembrane domain, and a large luminal catalytic domain. The pig
liver Man9 mannosidase
is localized to the ER and transient vesicles while the human kidney Man9
mannosidase is localized to
the Golgi (Bause, E. et al. (1993) Eur. J. Biochem. 217:535-540; Bieberich, E.
and E. Bause (1995)
Eur. J. Biochem. 233:644-649).
Transferases participate in reactions essential to the synthesis and
degradation of cellular
components such as carbohydrates. For example, galactosyltransferase catalyzes
the reaction
producing galactose beta-1,4-N-acetylglucosamine, has a role in lactose
synthesis, and, as a
component of the plasma membrane, may function in intracellular recognition
and/or adhesion
(Masri, K.A. et al. (1988) Biochem. Biophys. Res. Commun. 157:657-663).
Synthetases are another class of carbohydrate-modifying enzymes that have
critical roles in
proper cell functioning. For example, synthesis of sialylated glycoconjugates
requires the synthesis
of cytidine 5 =monophosphate N-acetylneuraminic acid (CMP-NeuSAc), a reaction
catalyzed by
CMP-NeuSAc synthetase (Munster, A.K. et al. (1998) Proc. Natl. Acad. Sci. USA
95:9140-9145).
Sialic acids of cell surface glycoproteins and glycolipids contribute to
proper structure and function in
a variety of tissues.
Another class of carbohydrate-modifying enzymes is the glucosidases that
catalyze the
release of glucose from carbohydrates through hydrolysis of the glycosidic
link in various glucosides.
The inherited disorder type I Gaucher disease, characterized by hematologic
abnormalities, can be
detected in a heterozygous or homozygous individual through an assay of
leukocyte beta-glucosidase
levels (Raghavan, S.S. et al. ( 1980) Am. J. Hum. Genet. 32:158-173).
Carbohydrate metabolism is altered in several other disorders. Diabetes
mellitus is
characterized by abnormally high blood glucose (hyperglycemia). Type I
diabetes results from an
autoimmune-related loss of pancreatic insulin-secreting cells. Type II
diabetes results from insulin
resistance and impaired insulin secretory response to glucose, and is
associated with obesity.
Hypoglycemia, or abnormally low blood glucose levels, has several causes
including drug use,
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genetic deficiencies in carbohydrate metabolism enzymes, cancer, liver
disease, and renal disease
(Berkow, R. et al. (1992) The Merck Manual of Diagnosis and Therapy, Internet
Edition, Section 8,
Chapter 91, Diabetes Mellitus, Hypoglycemia).
Changes in glycosaminoglycan (GAG) levels are associated with several
autoimmune
diseases. Both increases and decreases in various GAGS occur in patients with
autoimmune thyroid
disease and autoimmune diabetes mellitus. Antibodies to GAGS were found in
patients with systemic
lupus erythematosus and autoimmune thyroid disease (Hansen, C. et al. ( 1996)
Clin. Exp. Rheum. 14
(Suppl. 15):S59-S67).
Carbohydrate metabolism is associated with cancer. Reduced GAG and
proteoglycan
expression is associated with human lung carcinomas (Nackaerts, K. et al. (
1997) Int. J. Cancer
74:335-345). The carbohydrate determinants, sialyl Lewis A and sialyl Lewis X,
are frequently
expressed on human cancer cells. These determinants, ligands for the cell
adhesion molecule E-
selectin, are involved in the adhesion of cancer cells to vascular endothelium
and contribute to
hematogenous metastasis of cancer (Kannagi, R. ( 1997) Glycoconj. J. 14:577-
584). Alterations of the
N-linked carbohydrate core structure of cell surface glycoproteins are linked
to colon and pancreatic
cancers (Schwarz, R.E. et al. (1996) Cancer Lett. 107:285-291). Reduced
expression of the Sda blood
group carbohydrate structure in cell surface glycolipids and glycoproteins is
observed in
gastrointestinal cancer (Dohi, T. et al. (1996) Int. J. Cancer 67:626-631).
The discovery of new carbohydrate-modifying 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 carbohydrate metabolism disorders,
autoimmune/inflammatory
disorders, and cancer.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, carbohydrate-modifying enzymes,
referred to
collectively as "CME" and individually as "CME-1," "CME-2," "CME-3," "CME-4,"
and "CME-5."
In one aspect, the invention provides an isolated polypeptide comprising a) an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-5, b) a naturally occurring
amino acid sequence
' having at least 90% sequence identity to an amino acid sequence selected
from the group consisting
of SEQ ID NO:1-5, c) a biologically active fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-5, or d) an immunogenic fragment of an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-5. In one alternative, the
invention provides an
isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-5.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising
a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-5,
b) a naturally
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occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-5, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-5, or d) an
immunogenic fragment
of an amino acid sequence selected from the group consisting of SEQ 1D NO:1-5.
In one alternative,
the polynucleotide is selected from the group consisting of SEQ 1D N0:6-10.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
a) an amino acid
sequence selected from the group consisting of SEQ )D NO:1-5, b) a naturally
occurring amino acid
sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-5, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-5, or d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-5. In one
alternative, the invention
provides a cell transformed with the recombinant polynucleotide. In another
alternative, the invention
provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising a)
an amino
acid sequence selected from the group consisting of SEQ ID NO:1-5, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the
group consisting of SEQ ID NO: l-5, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-5, or d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-5. The method
comprises a)
culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide comprising a) an amino acid sequence selected from the group
consisting of SEQ 1D
NO:1-5, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID NO:1-5, c) a
biologically active
fragment of an amino acid sequence selected from the group consisting of SEQ
)D NO:1-5, or d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-5.
The invention further provides an isolated polynucleotide comprising a) a
polynucleotide
sequence selected from the group consisting of SEQ ID N0:6-10, b) a naturally
occurring
polynucleotide sequence having at least 70% sequence identity to a
polynucleotide sequence selected
from the group consisting of SEQ ID N0:6-10, c) a polynucleotide sequence
complementary to a), or
d) a polynucleotide sequence complementary to b). In one alternative, the
polynucleotide comprises
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at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
comprising a) a
polynucleotide sequence selected from the group consisting of SEQ ID N0:6-10,
b) a naturally
occurring polynucleotide sequence having at least 70% sequence identity to a
polynucleotide
sequence selected from the group consisting of SEQ ID N0:6-10, c) a
polynucleotide sequence
complementary to a), or d) a polynucleotide sequence complementary to b). The
method comprises a)
hybridizing the sample with a probe comprising at least 16 contiguous
nucleotides comprising a
sequence complementary to said target polynucleotide in the sample, and which
probe specifically
hybridizes to said target polynucleotide, under conditions whereby a
hybridization complex is formed
between said probe and said target polynucleotide, and b) detecting the
presence or absence of said
hybridization complex, and optionally, if present, the amount thereof. In one
alternative, the probe
comprises at least 30 contiguous nucleotides. In another alternative, the
probe comprises at least 60
contiguous nucleotides.
The invention further provides a pharmaceutical composition comprising an
effective amount
of a polypeptide comprising a) an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-5, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an
amino acid sequence selected from the group consisting of SEQ m NO:1-5, c) a
biologically active
fragment of an amino acid sequence selected from the group consisting of SEQ
D7 NO:1-5, or d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-5, and a pharmaceutically acceptable excipient. The invention
additionally provides a method
of treating a disease or condition associated with decreased expression of
functional CME,
comprising administering to a patient in need of such treatment~the
pharmaceutical composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising a) an amino acid sequence selected from
the group consisting of
SEQ m NO:1-5, b) a naturally occurring amino acid sequence having at least 90%
sequence identity
to an amino acid sequence selected from the group consisting of SEQ m NO:1-5,
c) a biologically
active fragment of an amino acid sequence selected from the group consisting
of SEQ ID NO:1-5, or
d) an immunogenic.fragment of an amino acid sequence selected from the group
consisting of SEQ
ID NO:1-5. The method comprises a) exposing a sample comprising the
polypeptide to a compound,
and b) detecting agonist activity in the sample. In one alternative, the
invention provides a
pharmaceutical composition comprising an agonist compound identified by the
method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional CME, comprising
administering to a patient in need of such treatment the pharmaceutical
composition.
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Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising a) an amino acid sequence selected
from the group
consisting of SEQ ID NO: l-5, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ 117 NO:1-5, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-5, or d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-5. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a pharmaceutical composition comprising an antagonist
compound identified by
the method and a pharmaceutically acceptable excipient. In another
alternative, the invention
provides a method of treating a disease or condition associated with
overexpression of functional
CME, comprising administering to a patient in need of such treatment the
pharmaceutical
composition.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID N0:6-10, the method
comprising a) exposing
a sample comprising the target polynucleotide to a compound, and b) detecting
altered expression of
the target polynucleotide.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs),
clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments
used to assemble full-
length sequences encoding CME.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of CME.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-
specific
expression patterns of each nucleic acid sequence as determined by northern
analysis; diseases,
disorders, or conditions associated with these tissues; and the vector into
which each cDNA was
cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA clones
encoding CME were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze CME, along
with
applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
6
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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
1o 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
"CME" refers to the amino acid sequences of substantially purified CME
obtained from any
species, particularly a mammalian species, including bovine, ovine, porcine,
murine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
CME. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of CME either by directly
interacting with
CME or by acting on components of the biological pathway in which CME
participates.
An "allelic variant" is an alternative form of the gene encoding CME. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding CME include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as CME or a
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polypeptide with at least one functional characteristic of CME. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding CME, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
CME. 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 CME. 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 CME 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 polymerase chain reaction (PCR)
technologies well
known in the art. '
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of CME. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of CME either by
directly interacting with CME or by acting on components of the biological
pathway in which CME
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind CME polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide
used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived
from the translation of
RNA, or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly
used Garners that are chemically coupled to peptides include bovine serum
albumin, thyroglobulin,
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and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to
immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
strand of a specific nucleic acid sequence. Antisense compositions may include
DNA; RNA; peptide
nucleic acid (PNA); oligonucleotides having modified backbone linkages such as
phosphorothioates,
methylphosphonates, or benzylphosphonates; oligonucleotides having modified
sugar groups such as
2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having
modified bases such
as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense
molecules may be
produced by any method including chemical synthesis or transcription. Once
introduced into a cell,
the complementary antisense molecule base-pairs with a naturally occurring
nucleic acid sequence
produced by the cell to form duplexes which block either transcription or
translation. The
designation "negative" or "minus" can refer to the antisense strand, and the
designation "positive" or
"plus" can refer to the sense strand of a reference DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" refers to the
capability of the natural, recombinant, or synthetic CME, or of any
oligopeptide thereof, to induce a
specific immune response in appropriate animals or cells and to bind with
specific antibodies.
The terms "complementary" and "complementarity" 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'." Complementarity 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 complementarity exists between the single stranded molecules. The degree
of complementarity
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 CME or fragments of
CME may be
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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
Wn. 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 structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile> Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
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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, acyl, hydroxyl, or amino group.
A derivative
polynucleotide encodes a polypeptide which retains at least one biological or
immunological function
of the natural molecule. A derivative polypeptide is one modified by
glycosylation, pegylation, or
any similar process that retains at least one biological or immunological
function of the polypeptide
from which it was derived.
A "fragment" is a unique portion of CME or the polynucleotide encoding CME
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example,
a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide)
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID 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 fragment.
A fragment of SEQ ID NO:1-5 is encoded by a fragment of SEQ ID N0:6-10. A
fragment 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 antibodies 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
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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 may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison Wn. CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp ( 1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. ( 1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequence pairs.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBn Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
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Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
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: 5 and Extension Gap: 2 penalties
Gap x drop-off:' S0
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 and 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
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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: I penalties
Gap x drop-off. 50
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
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1 % (w/v) SDS, and about 100 pg/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 (Tm) 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 Clonins: 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-SO% v/v, may also be used under
particular
circumstances, such as for RNA:DNA hybridizations. Useful variations on these
wash conditions
will be readily apparent to those of ordinary skill in the art. Hybridization,
particularly under high
stringency conditions, may be suggestive of evolutionary similarity between
the nucleotides. Such
similarity is strongly indicative of a similar role for the nucleotides and
their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of CME
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment
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of CME which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of 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.
The term "modulate" refers to a change in the activity of CME. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of CME.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
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 CME, 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 polymerise enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerise chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
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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 a1.,1987, Current Protocols in
Molecular Biolo~y,
Greene Publ. Assoc. & Wiley-hitersciences, New York NY; Innis et al., 1990,
PCR Protocols. A
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 TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MIT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or patrtially 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
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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
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.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding CME, or fragments thereof, or CME 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 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino 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 occur under natural or artificial
conditions according to various
methods well known in the art, and may rely on any known method for the
insertion of foreign
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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 "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
IS cyanobacteria, fungi, and plants and animals. The isolated DNA of the
present invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook et al. (1989),
sera.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
95% or at least 98% or
greater sequence identity over a certain defined length. A variant may be
described as, for example,
an "allelic" (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may
have significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to 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 polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
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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 carbohydrate-modifying
enzymes
(CME), the polynucleotides encoding CME, and the use of these compositions for
the diagnosis,
treatment, or prevention of carbohydrate metabolism disorders,
autoimmune/inflammatory disorders,
and cancer.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
CME. 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 CME 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 CME and are useful as fragments in hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
shows homologous sequences as identified by BLAST analysis; and column 7 shows
analytical
methods and in some cases, searchable databases to which the analytical
methods were applied. The
methods of column 7 were used to characterize each polypeptide through
sequence homology and
protein motifs.
The columns of Tabte 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding CME. The first column of Table 3
lists the nucleotide
SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1.
These fragments
are useful, for example, in hybridization or amplification technologies to
identify SEQ B7 N0:6-10
and to distinguish between SEQ ID N0:6-10 and related polynucleotide
sequences. The polypeptides
encoded by these fragments are useful, for example, as immunogenic peptides.
Column 3 lists tissue
categories which express CME as a fraction of total tissues expressing CME.
Column 4 lists
CA 02369342 2001-10-09
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diseases, disorders, or conditions associated with those tissues expressing
CME as a fraction of total
tissues expressing CME. Column 5 lists the vectors used to subclone each cDNA
library.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding CME 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 CME variants. A preferred CME 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 CME amino acid sequence, and which contains at least one
functional or structural
characteristic of CME.
The invention also encompasses polynucleotides which encode CME. 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 CME. The
polynucleotide sequences
of SEQ ID N0:6-10, as presented in the Sequence Listing, embrace the
equivalent RNA sequences,
wherein occurrences of the nitrogenous base thymine are replaced with uracil,
and the sugar backbone
is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
CME. 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 CME. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:6-
10 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 of the polynucleotide variants described above can
encode an amino acid
sequence which contains at least one functional or structural characteristic
of CME.
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 CME, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring CME, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode CME and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring CME under
appropriately selected
21
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding CME 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 CME and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater
half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode CME
and CME
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 CME or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:6-10 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
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Perkin-
Elmer), thermostable T7 polymerise (Amersham Pharmacia Biotech, Piscataway
NJ), or
combinations of polymerises 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) Short
Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, unit 7.7;
Meyers, R.A. ( 1995)
Molecular Biology and Biotechnolo~y, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding CME may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
22
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. ( 1993) PCR Methods
Applic. 2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. ( 1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments
adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme
digestions and ligations may be used to insert an engineered double-stranded
sequence into a region
of unknown sequence before performing PCR. Other methods which may be used to
retrieve
unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991)
Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries
(Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need
to screen libraries
and is useful in finding intron/exon junctions. For all PCR-based methods,
primers may be designed
using commercially available software, such as OLIGO 4.06 Primer Analysis
software (National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, 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 CME may be cloned in recombinant DNA molecules that direct
expression of CME, or
23
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
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 CME.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter CME-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of CME, such as its biological or enzymatic
activity or its ability to
bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding CME 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, CME 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 CME, 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.
24
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier ( 1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, T. ( 1984) Proteins, Structures and
Molecular Properties, WH
Freeman, New York NY.)
In order to express a biologically active CME, the nucleotide sequences
encoding CME 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 CME. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
CME. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding CME 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 CME and appropriate transcriptional and
translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. ( 1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. ( 1995)
Current Protocols in Molecular Bioloav, 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 CME. 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
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
upon the use intended for polynucleotide sequences encoding CME. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding CME can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding CME into the
vector's multiple cloning
site disrupts the lacZ gene, alloying 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 CME are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of CME may be used. For
example, vectors
containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of CME. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomvces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, G.A. et al. ( 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 CME. Transcription of
sequences encoding
CME may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N. ( 1987)
EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Broglie, R. et al.
( 1984) Science 224:838-843; and Winter, J. et al. ( 1991 ) Results Probl.
Cell Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of
Science and Technoloey
(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 CME
may be ligated into an
adenovirus transcription/translation complex consisting of the late promoter
and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses CME 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.
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CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
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., Harnngton, J.J.
et al. ( 1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of CME in cell lines is preferred. For example, sequences encoding CME 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), B glucuronidase and its substrate B-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding CME is inserted within a marker gene sequence, transformed
cells containing
sequences encoding CME can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding CME under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
27
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding CME and
that express
CME 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 CME 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 CME 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 Immunoloey, 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 CME
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding CME, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison Wn, 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 CME 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 CME may be designed to contain signal
sequences which
direct secretion of CME through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
28
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding CME may be ligated to a heterologous sequence resulting in
translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric CME protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of CME activity.
Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase (GST),
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their
cognate fusion proteins on immobilized glutathione, maltose, phenylarsine
oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffmity
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 CME encoding sequence and the
heterologous protein
sequence, so that CME may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel ( 1995, supra, ch. 10).
A variety of commercially available kits may also be used to facilitate
expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled CME may be
achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
Fragments of CME 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
29
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
CME 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 CME and carbohydrate-modifying enzymes. In addition, the
expression of CME
is closely associated with cardiovascular, developmental, gastrointestinal,
hematopoietic/immune,
nervous, reproductive, and urologic tissues. Therefore, CME appears to play a
role in carbohydrate
metabolism disorders, autoimmune/inflammatory disorders, and cancers. In the
treatment of
disorders associated with increased CME expression or activity, it is
desirable to decrease the
expression or activity of CME. In the treatment of disorders associated with
decreased CME
expression or activity, it is desirable to increase the expression or activity
of CME.
Therefore, in one embodiment, CME 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 CME.
Examples of such disorders include, but are not limited to, a carbohydrate
metabolism disorder such
as diabetes, insulin-dependent diabetes mellitus, non-insulin-dependent
diabetes mellitus,
, hypoglycemia, glucagonoma, galactosemia, hereditary fructose intolerance,
fructose-1,6-diphosphatase deficiency, obesity, congenital type II
dyserythropoietic anemia,
mannosidosis, neuraminidase deficiency, galactose epimerase deficiency,
glycogen storage diseases,
lysosomal storage diseases, fructosuria, pentosuria, and inherited
abnormalities of pyruvate
metabolism; an autoimmune/inflammatory disorder such as acquired
immunodeficiency syndrome
(AIDS), Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune
thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease,
atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel
syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial
inflammation,
osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's
syndrome, rheumatoid
arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic
lupus erythematosus,
systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis,
Werner syndrome,
complications of cancer, hemodialysis, and extracorporeal circulation, viral,
bacterial, fungal,
parasitic, protozoal, and helminthic infections, and trauma; and a cancer 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.
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
In another embodiment, a vector capable of expressing CME 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 CME including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
CME 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 CME
including, but not
limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of CME
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of CME including, but not limited to, those listed above.
In a further embodiment, an antagonist of CME may be administered to a subject
to treat or
prevent a disorder associated with increased expression or activity of CME.
Examples of such
disorders include, but are not limited to, those carbohydrate metabolism
disorders,
autoimmune/inflammatory disorders, and cancers described above. In one aspect,
an antibody which
specifically binds CME 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 CME.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding CME may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of CME 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 CME may be produced using methods which are generally known
in the art.
In particular, purified CME may be used to produce antibodies or to screen
libraries of
pharmaceutical agents to identify those which specifically bind CME.
Antibodies to CME may also
be generated using methods that are well known in the art. Such antibodies may
include, but are not
limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab
fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are 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 CME or with any fragment or
oligopeptide thereof
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WO 00/63351 PCT/US00/10882
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among
adjuvants used in
humans, BCG (bacilli Calmette-Guerin) and Corynebacterium ~arvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to CME
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 CME
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 CME 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 314:452-4.54.) Alternatively, techniques described
for the production of single
chain antibodies may be adapted, using methods known in the art, to produce
CME-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 CME may also be
generated.
For example, such fragments include, but are not limited to, F(ab')Z 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
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WO 00/63351 PCT/US00/10882
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
CME and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering CME epitopes is generally used, but a
competitive binding assay may
also be employed (Pound, su ra .
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for CME. Affinity
is expressed as an
association constant, Ka, which is defined as the molar concentration of CME-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium conditions.
The Ka determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple CME epitopes, represents the average affinity, or
avidity, of the antibodies for
CME. The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular CME epitope, represents a true measure of affinity. High-affinity
antibody preparations
with Ka ranging from about 109 to 10'Z L/mole are preferred for use in
immunoassays in which the
CME-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 CME, preferably in active
form, from the
antibody (Catty, D. (1988) Antibodies. Volume I: A Practical Approach, IRI.
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/ml, is generally employed in procedures
requiring precipitation
of CME-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 CME, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
the complement of the
polynucleotide encoding CME 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
33
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
polynucleotides encoding CME. Thus, complementary molecules or fragments may
be used to
modulate CME 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
CME.
Expression vectors derived from retroviruses, adenoviruses, or herpes or
vaccinia viruses, or
from various bacterial plasmids, may be used for delivery of nucleotide
sequences to the targeted
organ, tissue, or cell population. Methods which are well known to those
skilled in the art can be
used to construct vectors to express nucleic acid sequences complementary to
the polynucleotides
encoding CME. (See, e.g., Sambrook, supra; Ausubel, 1995, supra.)
Genes encoding CME can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding CME. 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 CME. 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 Immunolo ig c 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 CME.
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,
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CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding CME. Such DNA sequences may be incorporated into a wide
variety of vectors
with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these
cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines,
cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine,
queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine,
cytidine, guanine, thymine, and uridine which are not as easily recognized by
endogenous
endonucleases.
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
pharmaceutical
or sterile composition, in conjunction with a pharmaceutically acceptable
carrier, for any of the
therapeutic effects discussed above. Such pharmaceutical compositions may
consist of CME,
antibodies to CME, and mimetics, agonists, antagonists, or inhibitors of CME.
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 Garner including,
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
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, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain suitable
pharmaceutically-acceptable carriers comprising excipients and auxiliaries
which facilitate processing
of the active compounds into preparations which can be used pharmaceutically.
Further details on
techniques for formulation and administration may be found in the latest
edition of Remin on'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.
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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
S dextran. Additionally, suspensions of the active compounds may be prepared
as appropriate oily
injection suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil,
or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or
liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the suspension may
also contain suitable
stabilizers or agents to increase the solubility of the compounds and allow
for the preparation of
highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
barner 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, 0.1% 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 CME, 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 CME
or fragments thereof, antibodies of CME, and agonists, antagonists or
inhibitors of CME, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
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WO 00/63351 PCT/US00/10882
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio.
Pharmaceutical compositions
which exhibit large therapeutic 'indices are preferred. The data obtained from
cell culture assays and
animal studies are used to formulate a range of dosage for human use. The
dosage contained in such
compositions is preferably within a range of circulating concentrations that
includes the EDSO with
little or no toxicity. The dosage varies within this range depending upon the
dosage form employed,
the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4
days, every week, or biweekly depending on the half life and clearance rate of
the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 fig, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind CME may be used for
the
diagnosis of disorders characterized by expression of CME, or in assays to
monitor patients being
treated with CME or agonists, antagonists, or inhibitors of CME. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for CME include methods which utilize the antibody and a label to detect CME
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 CME, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
CME expression. Normal
or standard values for CME expression are established by combining body fluids
or cell extracts
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WO 00/63351 PCT/US00/10882
taken from normal mammalian subjects, for example, human subjects, with
antibody to CME under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of CME
expressed in subject,
control, and disease samples from biopsied tissues are compared with the
standard values. Deviation
between standard and subject values establishes the parameters for diagnosing
disease.
In another. embodiment of the invention, the polynucleotides encoding CME 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 CME
may be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
CME, and to monitor regulation of CME levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding CME or closely related
molecules may be used to
identify nucleic acid sequences which encode CME. 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 CME, 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 CME encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ m N0:6-
10 or from
genomic sequences including promoters, enhancers, and introns of the CME gene.
Means for producing specific hybridization probes for DNAs encoding CME
include the
cloning of polynucleotide sequences encoding CME or CME 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
polymerises and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as'ZP or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding CME may be used for the diagnosis of
disorders
associated with expression of CME. Examples of such disorders include, but are
not limited to, a
carbohydrate metabolism disorder such as diabetes, insulin-dependent diabetes
mellitus,
non-insulin-dependent diabetes mellitus, hypoglycemia, glucagonoma,
galactosemia, hereditary
fructose intolerance, fructose-1,6-diphosphatase deficiency, obesity,
congenital type II
dyserythropoietic anemia, mannosidosis, neuraminidase deficiency, galactose
epimerase deficiency,
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glycogen storage diseases, lysosomal storage diseases, fructosuria,
pentosuria, and inherited
abnormalities of pyruvate metabolism; an autoimmune/inflammatory disorder such
as acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory
distress syndrome,
allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis, autoimmune
hemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact
dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia
with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic
gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis,
Werner syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral,
bacterial, fungal, parasitic, protozoal, and helminthic infections, and
trauma; and a cancer 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 . The
polynucleotide sequences encoding CME 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 CME
expression. Such qualitative or quantitative methods are well known in the
art.
In a particular aspect, the nucleotide sequences encoding CME may be useful in
assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding CME 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 CME 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 CME,
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
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
fragment thereof, encoding CME, under conditions suitable for hybridization or
amplification.
Standard hybridization may be quantified by comparing the values obtained from
normal subjects
with values from an experiment in which a known amount of a substantially
purified polynucleotide
is used. Standard values obtained in this manner may be compared with values
obtained from
samples from patients who are symptomatic for a disorder. Deviation from
standard values is used to
establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer. '
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding CME
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 CME, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
CME, 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 CME include
radiolabeling or
biotinylating nucleotides, coampli~cation of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. ( 1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. ( 1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer of interest is
presented in various dilutions and a spectrophotometric or colorimetric
response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as targets in a
microarray. The microarray
can be used to monitor the expression level of large numbers of genes
simultaneously and to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
41
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
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; Baldeschweilei et al. (1995) PCT application W095/251116;
Shalon, D. et al.
( 1995) PCT application W095/35505; Heller, R.A. et al. ( 1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. ( 1997) U.S. Patent No. 5,605,662.)
In another embodiment of the invention, nucleic acid sequences encoding CME
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. The
sequences may be mapped to a particular chromosome, to a specific region of a
chromosome, or to
artificial chromosome constructions, e.g., human artificial chromosomes
(HACs), yeast artificial
chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1
constructions, or single
chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price,
C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7:149-
154.)
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, supra,
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 CME on a physical chromosomal map and a specific
disorder, or a
predisposition to a specific disorder, may help define the region of DNA
associated with that
disorder. The nucleotide sequences of the invention may be used to detect
differences in gene
sequences among normal, carrier, and affected individuals.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the 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 crudely
localized by genetic
linkage to a particular genomic region, e.g., ataxia-telangiectasia to l 1q22-
23, any sequences mapping
to that area may represent associated or regulatory genes for further
investigation. (See, e.g., Gatti,
R.A. et al. ( 1988) Nature 336:577-580.) The nucleotide sequence of the
subject invention may also
be used to detect differences in the chromosomal location due to
translocation, inversion, etc., among
normal, Garner, or affected individuals.
In another embodiment of the invention, CME, its catalytic or immunogenic
fragments, or
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CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
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 CME and the agent being tested may be measured.
Another technique foi drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, 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 CME, or
fragments thereof,
and washed. Bound CME is then detected by methods well known in the art.
Purified CME 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
antibodies capable of binding CME specifically compete with a test compound
for binding CME. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with CME.
In additional embodiments, the nucleotide sequences which encode CME may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. No. 60/130,383, are hereby expressly incorporated by
reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was Rurchased from Clontech or isolated from tissues described in Table 4.
Some
tissues were homogenized and lysed in guanidinium isothiocyanate, while others
were homogenized
and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL
(Life Technologies), a
monophasic solution of phenol and guanidine isothiocyanate. The resulting
lysates were centrifuged
over CsCI cushions or extracted with chloroform. RNA was precipitated from the
lysates with either
isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
43
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purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCR1PT plasmid (Stratagene), PSPORTI plasmid (Life Technologies),
pcDNA2.1 plasmid
(Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Pharmaceuticals, Palo Alto
CA). Recombinant
plasmids were transformed into competent E. coli cells including XLI-Blue, XL1-
BIueMRF, or
SOLR from Stratagene or DHSa, DHIOB, or ElectroMAX DHIOB 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,
Q1AWELL 8
Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid
purification kit from QIAGEN.
Following precipitation, plasmids were resuspended in 0.1 ml of distilled
water and stored, with or
without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
cDNA sequencing reactions were processed using standard methods or high-
throughput
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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,
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, and algorithms used and provides
applicable
descriptions, references, and threshold parameters. The first column of Table
5 shows the tools,
programs, and algorithms used, the second column provides brief descriptions
thereof, the third
column presents appropriate references, all of which are incorporated by
reference herein in their
entirety, and the fourth column presents, where applicable, the scores,
probability values, and other
parameters used to evaluate the strength of a match between two sequences (the
higher the score, the
greater the homology between two sequences). Sequences were analyzed using
MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software
(DNASTAR). Polynucleotide and polypeptide sequence alignments were generated
using the default
parameters specified by the clustal algorithm as incorporated into the
MEGALIGN multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned
sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then queried
against a selection of public databases such as the GenBank primate, rodent,
mammalian, vertebrate,
and eukaryote databases, and BLOCKS, PRINTS, DOMO> PRODOM, and PFAM to acquire
annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences
were assembled
into full length polynucleotide sequences using programs based on Phred,
Phrap, and Consed, 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
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
amino acid sequences, and these full length sequences were subsequently
analyzed by querying
against databases such as the GenBank databases (described above), Swissl'rot,
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
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:
% sequence identity x % maximum BLAST score
100
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. For example, with a product score of 40, the
match will be exact
within a 1 % to 2% error, and, with a product score of 70, the match will be
exact. Similar molecules
are usually identified by selecting those which show product scores between 15
and 40, although
lower scores may identify related molecules.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding CME occurred. Analysis involved the categorization of
cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease/condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
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.
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V. Extension of CME 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
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,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)ZS04,
and ~i-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (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 pl
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,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 /.cl to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose mini-gel to determine which reactions were
successful in extending
the sequence. ,
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation 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 relegated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
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WO 00/63351 PCT/US00/10882
Pharmacia Biotech), treated with Pfu DNA polymerase (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 Garb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step I: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide ( I :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 ~Ci of
[y-32P~ adenosine triphosphate (Amersham Pharmacia Biotech); and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 supe~ne 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 room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VII. Microarrays
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A chemical coupling procedure and an ink jet device can be used to synthesize
array
elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An
array analogous to a
dot or slot blot may also be used to arrange and link elements to the surface
of a substrate using
thermal, UV, chemical, or mechanical bonding procedures. A typical array may
be produced by hand
or using available methods and machines and contain any appropriate number of
elements. After
hybridization, nonhybridized probes are removed and a scanner used to
determine the levels and
patterns of fluorescence. The degree of complementarity and the relative
abundance of each probe
which hybridizes to an element on the microarray may be assessed through
analysis of the scanned
images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may
comprise
the elements of the microarray. Fragments suitable for hybridization can be
selected using software
well known in the art such as 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 CME-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring CME. Although
use of oligonucleotides
comprising from about 15 to 30 base pairs is described, essentially the same
procedure is used with
smaller or with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of CME. To
inhibit transcription, a
complementary oligonucleotide is designed from the most unique S' 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 CME-encoding transcript.
IX. Expression of CME
Expression and purification of CME is achieved using bacterial or virus-based
expression
systems. For expression of CME 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).
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Antibiotic resistant bacteria express CME upon induction with isopropyl beta-D-
thiogalactopyranoside (IPTG). Expression of CME in eukaryotic cells is
achieved by infecting insect
or mammalian cell lines with recombinant AutoQraphica californica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovinas. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding CME by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, CME is synthesized as a fusion protein with, e.g.,
glutathione S-
transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-
kilodalton enzyme from Schistosoma iaponicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
CME 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 CME obtained by these methods can be used
directly in the following
activity assay. -
X. Demonstration of CME Activity
Galactosyltransferase activity in CME is determined by measuring the transfer
of galactose
from UDP-galactose to a GIcNAc-terminated oligosaccharide chain in a
radioactive assay (Hennet, T.
et al. (1998) J. Biol. Chem. 273:58-65). An aliquot of CME is incubated with
14 pl of assay stock
solution ( 180 mM sodium cacodylate, pH 6.5, 1 mg/ml bovine serum albumin,
0.26 mM UDP-
galactose, 2 pl of UDP-['H]galactose), I pl of MnClz (500 mM), and 2.5 pl of
GIcNAc[30-(CH=)8
COZMe (37 mg/ml in dimethyl sulfoxide) for 60 minutes at 37°C. The
reaction is quenched by the
addition of 1 ml of water and loaded on a C 18 Sep-Pak cartridge (Waters), and
the column is washed
twice with 5 ml of water to remove unreacted UDP-['H]galactose. The
['H]galactosylated
GIcNAc(i0-(CHZ)a COzMe remains bound to the column during the water washes and
is eluted with 5
ml of methanol. Radioactivity in the eluted material is measured by liquid
scintillation counting and
is proportional to CME galactosyltransferase activity.
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
Mannosidase activity in CME is demonstrated by the ability to release mannose
from Man9
(GIcNAc)Z oligosaccharide (Schweden, J. et al. ( 1986) Eur. J. Biochem.
157:563-570). CME, in 200
mM phosphate buffer, pH 6.5 and 1% Triton X-100, is mixed with
["C](Man9)(GIcNAc)2 (2-3 x 10'
cpm) in a final volume of 30 pl at 37°C for 60 minutes. The reaction is
terminated by the addition of
30 pl glacial acetic acid. The amount of liberated ['4C]mannose, analyzed by
paper chromatography
in 2-propanol/acetic acid/water (29/4/9, by volume), is proportional to the
activity of CME in the
starting sample.
XI. Functional Assays
CME function is assessed by expressing the sequences encoding CME at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid
(Invitrogen), both of which
contain the cytomegalovirus promoter. 5-10 ~cg 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 Onmerod, M.G. ( 1994) Flow Cytometry, Oxford, New York NY.
The influence of CME on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding CME 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.
51
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
Expression of mRNA encoding CME and other genes of interest can be analyzed by
northern analysis
or microarray techniques.
XII. Production of CME Specific Antibodies
CME substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. ( 1990) Methods Enzymol. I 82:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the CME 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, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
IS immunogenicity. (See, e.g., Ausubel, 1995, su ra.) Rabbits are immunized
with the oligopeptide-
KLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-
CME activity by, for example, binding the peptide or CME 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 CME Using Specific Antibodies
Naturally occurring or recombinant CME is substantially purified by
immunoaffinity
chromatography using antibodies specific for CME. An immunoaffinity column is
constructed by
covalently coupling anti-CME 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 CME are passed over the immunoaffinity column, and the column
is
washed under conditions that allow the preferential absorbance of CME (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/CME 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 CME is collected.
XIV. Identification of Molecules Which Interact with CME
CME, or biologically active fragments thereof, are labeled with'z5I 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 CME, washed, and
any wells with labeled CME complex are assayed. Data obtained using different
concentrations of
CME are used to calculate values for the number, affinity, and association of
CME with the candidate
52
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
molecules.
Alternatively, molecules interacting with CME are analyzed using the yeast two-
hybrid
system as described in Fields, S. and O. Song ( 1989, Nature 340:245-246), or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
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.
53
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
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CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
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CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
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CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
LAL, Preeti
YUE, Henry
TANG, Y. Tom
HILLMAN, Jennifer L.
BAUGHN, Mariah R.
YANG, Junming
<120> CARBOHYDRATE-MODIFYING ENZYMES
<130> PF-0687 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/130,383
<151> 1999-04-21
<160> 10
<170> PERL Program
<210> 1
<211> 434
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 000422CD1
<400> 1
Met Asp Ser Val Glu Lys Gly Ala Ala Thr Ser Val Ser Asn Pro
1 5 10 15
Arg Gly Arg Pro Ser Arg Gly Arg Pro Pro Lys Leu Gln Arg Asn
20 25 30
Ser Arg Gly Gly Gln Gly Arg Gly Val Glu Lys Pro Pro His Leu
35 40 45
Ala Ala Leu Ile Leu Ala Arg Gly Gly Ser Lys Gly Ile Pro Leu
50 55 60
Lys Asn Ile Lys His Leu Ala Gly Val Pro Leu Ile Gly Trp Val
65 70 75
Leu Arg Ala Ala Leu Asp Ser Gly Ala Phe Gln Ser Val Trp Val
80 85 90
Ser Thr Asp His Asp Glu Ile Glu Asn Val Ala Lys Gln Phe Gly
95 100 105
Ala Gln Val His Arg Arg Ser Ser Glu Val Ser Lys Asp Ser Ser
110 115 120
Thr Ser Leu Asp Ala Ile Ile Glu Phe Leu Asn Tyr His Asn Glu
125 130 135
Val Asp Ile Val Gly Asn Ile Gln Ala Thr Ser Pro Cys Leu His
140 145 150
Pro Thr Asp Leu Gln Lys Val Ala Glu Met Ile Arg Glu Glu Gly
155 160 165
Tyr Asp Ser Val Phe Ser Val Val Arg Arg His Gln Phe Arg Trp
170 175 180
Ser Glu Ile Gln Lys Gly Val Arg Glu Val Thr Glu Pro Leu Asn
185 190 195
Leu Asn Pro Ala Lys Arg Pro Arg Arg Gln Asp Trp Asp Gly Glu
1/10
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
200 205 210
Leu Tyr Glu Asn Gly Ser Phe Tyr Phe Ala Lys Arg His Leu Ile
215 220 225
Glu Met Gly Tyr Leu Gln Gly Gly_Lys Met Ala Tyr Tyr Glu Met
230 235 240
Arg Ala Glu His Ser Val Asp Ile Asp Val Asp Ile Asp Trp Pro
245 250 255
Ile Ala Glu Gln Arg Val Leu Arg Tyr Gly Tyr Phe Gly Lys Glu
260 265 270
Lys Leu Lys Glu Ile Lys Leu Leu Val Cys Asn Ile Asp Gly Cys
275 280 285
Leu Thr Asn Gly His Ile Tyr Val Ser Gly Asp Gln Lys Glu Ile
290 295 300
Ile Ser Tyr Asp Val Lys Asp Ala Ile Gly Ile Ser Leu Leu Lys
305 310 315
Lys Ser Gly Ile Glu Val Arg Leu Ile Ser Glu Arg Ala Cys Ser
320 325 330
Lys Gln Thr Leu Ser Ser Leu Lys Leu Asp Cys Lys Met Glu Val
335 340 345
Ser Val Ser Asp Lys Leu Ala Val Val Asp Glu Trp Arg Lys Glu
350 355 360
Met Gly Leu Cys Trp Lys Glu Val Ala Tyr Leu Gly Asn Glu Val
365 370 375
Ser Asp Glu Glu Cys Leu Lys Arg Val Gly Leu Ser Gly Ala Pro
380 385 390
Ala Asp Ala Cys Ser Thr Ala Gln Lys Ala Val Gly Tyr Ile Cys
395 400 405
Lys Cys Asn Gly Gly Arg Gly Ala Ile Arg Glu Phe Ala Glu His
410 415 420
Ile Cys Leu Leu Met Glu Lys Val Asn Asn Ser Cys Gln Lys
425 430
<210> 2
<211> 302
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 983984CD1
<400> 2
Met Lys Ala Pro Gly Arg Leu Val Leu Ile Ile Leu Cys Ser Val
1 5 10 15
Val Phe Ser Ala Val Tyr Ile Leu Leu Cys Cys Trp Ala Gly Leu
20 25 30
Pro Leu Cys Leu Ala Thr Cys Leu Asp His His Phe Pro Thr Gly
35 40 45
Ser Arg Pro Thr Val Pro Gly Pro Leu His Phe Ser Gly Tyr Ser
50 55 60
Ser Val Pro Asp Gly Lys Pro Leu Val Arg Glu Pro Cys Arg Ser
65 70 75
Cys Ala Val Val Ser Ser Ser Gly Gln Met Leu Gly Ser Gly Leu
80 85 90
Gly Ala Glu Ile Asp Ser Ala Glu Cys Val Phe Arg Met Asn Gln
95 100 105
Ala Pro Thr Val Gly Phe Glu Ala Asp Val Gly Gln Arg Ser Thr
110 115 120
Leu Arg Val Val Ser His Thr Ser Val Pro Leu Leu Leu Arg Asn
125 130 135
Tyr Ser His Tyr Phe Gln Lys Ala Arg Asp Thr Leu Tyr Met Val
2/10
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
140 145 150
Trp Gly Gln Gly Arg His Met Asp Arg Val Leu Gly Gly Arg Thr
155 160 165
Tyr Arg Thr Leu Leu Gln Leu Thr Arg Met Tyr Pro Gly Leu Gln
170 175 180'
Val Tyr Thr Phe Thr Glu Arg Met Met Ala Tyr Cys Asp Gln Ile
185 190 195
Phe Gln Asp Glu Thr Gly Lys Asn Arg Arg Gln Ser Gly Ser Phe
200 205 210
Leu Ser Thr Gly Trp Phe Thr Met Ile Leu Ala Leu Glu Leu Cys
215 220 225
Glu Glu Ile Val Val Tyr Gly Met Val Ser Asp Ser Tyr Cys Arg
230 235 240
Glu Lys Ser His Pro Ser Val Pro Tyr His Tyr Phe Glu Lys Gly
245 250 255
Arg Leu Asp Glu Cys Gln Met Tyr Leu Ala His Glu Gln Ala Pro
260 265 270
Arg Ser Ala His Arg Phe Ile Thr Glu Lys Ala Val Phe Ser Arg
275 280 285
Trp Ala Lys Lys Arg Pro Ile Val Phe Ala His Pro Ser Trp Arg
290 295 300
Thr Glu
<210> 3
<211> 578
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2210054CD1
<400> 3
Met Pro Phe Arg Leu Leu Ile Pro Leu Gly Leu Leu Cys Ala Leu
1 5 10 15
Leu Pro Gln His His Gly Ala Pro Gly Pro Asp Gly Ser Ala Pro
20 25 30
Asp Pro Ala His Tyr Arg Glu Arg Val Lys Ala Met Phe Tyr His
35 40 45
Ala Tyr Asp Ser Tyr Leu Glu Asn Ala Phe Pro Phe Asp Glu Leu
50 55 60
Arg Pro Leu Thr Cys Asp Gly His Asp Thr Trp Gly Ser Phe Ser
65 70 75
Leu Thr Leu Ile Asp Ala Leu Asp Thr Leu Leu Ile Leu Gly Asn
80 85 90
Val Ser Glu Phe Gln Arg Val Val Glu Val Leu Gln Asp Ser Val
95 100 105
Asp Phe Asp Ile Asp Val Asn Ala Ser Val Phe Glu Thr Asn Ile
110 115 120
Arg Val Val Gly Gly Leu Leu Ser Ala His Leu Leu Ser Lys Lys
125 130 135
Ala Gly Val Glu Val Glu Ala Gly Trp Pro Cys Ser Gly Pro Leu
140 145 150
Leu Arg Met Ala Glu Glu Ala Ala Arg Lys Leu Leu Pro Ala Phe
155 160 165
Gln Thr Pro Thr Gly Met Pro Tyr Gly Thr Val Asn Leu Leu His
170 175 180
Gly Val Asn Pro Gly Glu Thr Pro Val Thr Cys Thr Ala Gly Ile
185 190 195
Gly Thr Phe Ile Val Glu Phe Ala Thr Leu Ser Ser Leu Thr Gly
200 205 210
3/10
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
Asp Pro Val Phe Glu Asp Val Ala Arg Val Ala Leu Met Arg Leu
215 220 225
Trp Glu Ser Arg Ser Asp Ile Gly Leu Val Gly Asn His Ile Asp
230 235 240
Val Leu Thr Gly Lys Trp Val Ala Gln Asp Ala Gly Ile Gly Ala
245 250 255
Gly Val Asp Ser Tyr Phe Glu Tyr Leu Val Lys Gly Ala Ile Leu
260 265 270
Leu Gln Asp Lys Lys Leu Met Ala Met Phe Leu Glu Tyr Asn Lys
275 280 285
Ala Ile Arg Asn Tyr Thr Arg Phe Asp Asp Trp Tyr Leu Trp Val
290 295 300
Gln Met Tyr Lys Gly Thr Val Ser Met Pro Val Phe Gln Ser Leu
305 310 315
Glu Ala Tyr Trp Pro Gly Leu Gln Ser Leu Ile Gly Asp Ile Asp
320 325 330
Asn Ala Met Arg Thr Phe Leu Asn Tyr Tyr Thr Val Trp Lys Gln
335 340 345
Phe Gly Gly Leu Pro Glu Phe Tyr Asn Ile Pro Gln Gly Tyr Thr
350 355 360
Val Glu Lys Arg Glu Gly Tyr Pro Leu Arg Pro Glu Leu Ile Glu
365 370 375
Ser Ala Met Tyr Leu Tyr Arg Ala Thr Gly Asp Pro Thr Leu Leu
380 385 390
Glu Leu Gly Arg Asp Ala Val Glu Ser Ile Glu Lys Ile Ser Lys
395 400 405
Val Glu Cys Gly Phe Ala Thr Ile Lys Asp Leu Arg Asp His Lys
410 415 420
Leu Asp Asn Arg Met Glu Ser Phe Phe Leu Ala Glu Thr Val Lys
425 430 435
Tyr Leu Tyr Leu Leu Phe Asp Pro Thr Asn Phe Ile His Asn Asn
440 445 450
Gly Ser Thr Phe Asp Thr Val Ile Thr Pro Tyr Gly Glu Cys Ile
455 460 465
Leu Gly Ala Gly Gly Tyr Ile Phe Asn Thr Glu Ala His Pro Ile
470 475 480
Asp Pro Ala Ala Leu His Cys Cys Gln Arg Leu Lys Glu Glu Gln
485 490 495
Trp Glu Val Glu Asp Leu Met Arg Glu Phe Tyr Ser Leu Lys Arg
500 505 510
Ser Arg Ser Lys Phe Gln Lys Asn Thr Val Ser Ser Gly Pro Trp
515 520 525
Glu Pro Pro Ala Arg Pro Gly Thr Leu Phe Ser Pro Glu Asn His
530 535 540
Asp Gln Ala Arg Glu Arg Lys Pro Ala Lys Gln Lys Val Pro Leu
545 550 555
Leu Ser Cys Pro Ser Gln Pro Phe Thr Ser Lys Leu Ala Leu Leu
560 565 570
Gly Gln Val Phe Leu Asp Ser Ser
575
<210> 4
<211> 461
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2618358CD1
<400> 4
4/10
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
Met Gly Gly Ser Thr Ala Ala Tyr Gln Val Glu Gly Gly Trp Asp
1 5 10 15
Ala Asp Gly Lys Gly Pro Cys Val Trp Asp Thr Phe Thr His Gln
20 25 30
Gly Gly Glu Arg Val Phe Lys Asn Gln Thr Gly Asp Val Ala Cys
35 40 45
Gly Ser Tyr Thr Leu Trp Glu Glu Asp Leu Lys Cys Ile Lys Gln
50 55 60
Leu Gly Leu Thr His Tyr Arg Phe Ser Leu Ser Trp Ser Arg Leu
65 70 75
Leu Pro Asp Gly Thr Thr Gly Phe Ile Asn Gln Lys Gly Ile Asp
80 85 90
Tyr Tyr Asn Lys Ile Ile Asp Asp Leu Leu Lys Asn Gly Val Thr
95 100 105
Pro Ile Val Thr Leu Tyr His Phe Asp Leu Pro Gln Thr Leu Glu
110 115 120
Asp Gln Gly Gly Trp Leu Ser Glu Ala Ile Ile Glu Ser Phe Asp
125 130 135
Lys Tyr Ala Gln Phe Cys Phe Ser Thr Phe Gly Asp Arg Val Lys
140 145 150
Gln Trp Ile Thr Ile Asn Glu Ala Asn Val Leu Ser Val Met Ser
155 160 165
Tyr Asp Leu Gly Met Phe Pro Pro Gly Ile Pro His Phe Gly Thr
170 175 180
Gly Gly Tyr Gln Ala Ala His Asn Leu Ile Lys Ala His Ala Arg
185 190 195
Ser Trp His Ser Tyr Asp Ser Leu Phe Arg Lys Lys Gln Lys Gly
200 205 210
Met Val Ser Leu Ser Leu Phe Ala Val Trp Leu Glu Pro Ala Asp
215 220 225
Pro Asn Ser Val Ser Asp Gln Glu Ala Ala Lys Arg Ala Ile Thr
230 235 240
Phe His Leu Asp Leu Phe Ala Lys Pro Ile Phe Ile Asp Gly Asp
245 250 255
Tyr Pro Glu Val Val Lys Ser Gln Ile Ala Ser Met Ser Gln Lys
260 265 270
Gln Gly Tyr Pro Ser Ser Arg Leu Pro Glu Phe Thr Glu Glu Glu
275 280 285
Lys Lys Met Ile Lys Gly Thr Ala Asp Phe Phe Ala Val Gln Tyr
290 295 300
Tyr Thr Thr Arg Leu Ile Lys Tyr Gln Glu Asn Lys Lys Gly Glu
305 310 315
Leu Gly Ile Leu Gln Asp Ala Glu Ile Glu Phe Phe Pro Asp Pro
320 325 330
Ser Trp Lys Asn Val Asp Trp Ile Tyr Val Val Pro Trp Gly Val
335 340 345
Cys Lys Leu Leu Lys Tyr Ile Lys Asp Thr Tyr Asn Asn Pro Val
350 355 360
Ile Tyr Ile Thr Glu Asn Gly Phe Pro Gln Ser Asp Pro Ala Pro
365 370 375
Leu Asp Asp Thr Gln Arg Trp Glu Tyr Phe Arg Gln Thr Phe Gln
380 385 390
Glu Leu Phe Lys Ala Ile Gln Leu Asp Lys Val Asn Leu Gln Val
395 400 405
Tyr Cys Ala Trp Ser Leu Leu Asp Asn Phe Glu Trp Asn Gln Gly
410 415 420
Tyr Ser Ser Arg Phe Gly Leu Phe His Val Asp Phe Glu Asp Pro
425 430 435
Ala Arg Pro Arg Val Pro Tyr Thr Ser Ala Lys Glu Tyr Ala Lys
440 445 450
Ile Ile Arg Asn Asn Gly Leu Glu Ala His Leu
455 460
5/10
CA 02369342 2001-10-09
WO 00/63351 PCT/ITS00/10882
<210> 5
<211> 529
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2912330CD1
<400> 5
Met Ser Met Lys Trp Thr Ser Ala Leu Leu Leu Ile Gln Leu Ser
1 5 10 15
Cys Tyr Phe Ser Ser Gly Ser Cys Gly Lys Val Leu Val Trp Pro
20 25 30
Thr Glu Phe Ser His Trp Met Asn Ile Lys Thr Ile Leu Asp Glu
35 40 45
Leu Val Gln Arg Gly His Glu Val Thr Val Leu Ala Ser Ser Ala
50 55 60
Ser Ile Ser Phe Asp Pro Asn Ser Pro Ser Thr Leu Lys Phe Glu
65 70 75
Val Tyr Pro Val Ser Leu Thr Lys Thr Glu Phe Glu Asp Ile Ile
80 85 90
Lys Gln Leu Val Lys Arg Trp Ala Glu Leu Pro Lys Asp Thr Phe
95 100 105
Trp Ser Tyr Phe Ser Gln Val Gln Glu Ile Met Trp Thr Phe Asn
110 115 120
Asp Ile Leu Arg Lys Phe Cys Lys Asp Ile Val Ser Asn Lys Lys
125 13 0 13 5
Leu Met Lys Lys Leu Gln Glu Ser Arg Phe Asp Val Val Leu Ala
140 145 150
Asp Ala Val Phe Pro Phe Gly Glu Leu Leu Ala Glu Leu Leu Lys
155 160 165
Ile Pro Phe Val Tyr Ser Leu Arg Phe Ser Pro Gly Tyr Ala Ile
170 175 180
Glu Lys His Ser Gly Gly Leu Leu Phe Pro Pro Ser Tyr Val Pro
185 190 195
Val Val Met Ser Glu Leu Ser Asp Gln Met Thr Phe Ile Glu Arg
200 205 210
Val Lys Asn Met Ile Tyr Val Leu Tyr Phe Glu Phe Trp Phe Gln
215 220 225
Ile Phe Asp Met Lys Lys Trp Asp Gln Phe Tyr Ser Glu Val Leu
230 235 240
Gly Arg Pro Thr Thr Leu Ser Glu Thr Met Ala Lys Ala Asp Ile
245 250 255
Trp Leu Ile Arg Asn Tyr Trp Asp Phe Gln Phe Pro His Pro Leu
260 265 270
Leu Pro Asn Val Glu Phe Val Gly Gly Leu His Cys Lys Pro Ala
275 280 285
Lys Pro Leu Pro Lys Glu Met Glu Glu Phe Val Gln Ser Ser Gly
290 295 300
Glu Asn Gly Val Val Val Phe Ser Leu Gly Ser Met Val Ser Asn
305 310 315
Thr Ser Glu Glu Arg Ala Asn Val Ile Ala Ser Ala Leu Ala Lys
320 325 330
Ile Pro Gln Lys Val Leu Trp Arg Phe Asp Gly Asn Lys Pro Asp
335 340 345
Thr Leu Gly Leu Asn Thr Arg Leu Tyr Lys Trp Ile Pro Gln Asn
350 355 360
Asp Leu Leu Gly His Pro Lys Thr Lys Ala Phe Ile Thr His Gly
365 370 375
Gly Met Asn Gly Ile Tyr Glu Ala Ile Tyr His Gly Val Pro Met
6/10
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
380 385 390
Val Gly Val Pro Ile Phe Gly Asp Gln Leu Asp Asn Ile Ala His
395 400 405
Met Lys Ala Lys Gly Ala Ala Val Glu Ile Asn Phe Lys Thr Met
410 415 420
Thr Ser Glu Asp Leu Leu Arg Ala Leu Arg Thr Val Ile Thr Asp
425 430 435
Ser Ser Tyr Lys Glu Asn Ala Met Arg Leu Ser Arg Ile His His
440 445 450
Asp Gln Pro Val Lys Pro Leu Asp Arg Ala Val Phe Trp Ile Glu
455 460 465
Phe Val Met Arg His Lys Gly Ala Lys His Leu Arg Ser Ala Ala
470 475 480
His Asp Leu Thr Trp Phe Gln His Tyr Ser Ile Asp Val Ile Gly
485 490 495
Phe Leu Leu Thr Cys Val Ala Thr Ala Ile Phe Leu Phe Thr Lys
500 505 510
Cys Phe Leu Phe Ser Cys Gln Lys Phe Asn Lys Thr Arg Lys Ile
515 520 525
Glu Lys Arg Glu
<210> 6
<211> 1772
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 000422CB1
<400> 6
cggcgggcac tgccaggcgg ggatcgggcg gcgccgagct gaggtggtga gggactagct 60
cccggatgtg gagaagctgg ggagaaggcg tgggaggaag atggactcgg tggagaaggg 120
ggccgccacc tccgtctcca acccgcgggg gcgaccgtcc cggggccggc cgccgaagct 180
gcagcgcaac tctcgcggcg gccagggccg aggtgtggag aagcccccgc acctggcagc 240
cctaattctg gcccggggag gcagcaaagg catccccctg aagaacatta agcacctggc 300
gggggtcccg ctcattggct gggtcctgcg tgcggccctg gattcagggg ccttccagag 360
tgtatgggtt tcgacagacc atgatgaaat tgagaatgtg gccaaacaat ttggtgcaca 420
agttcatcga agaagttctg aagtttcaaa agacagctct acctcactag atgccatcat 480
agaatttctt aattatcata atgaggttga cattgtagga aatattcaag ctacttctcc 540
atgtttacat cctactgatc ttcaaaaagt tgcagaaatg attcgagaag aaggatatga 600
ttctgttttc tctgttgtga gacgccatca gtttcgatgg agtgaaattc agaaaggagt 660
tcgtgaagtg accgaacctc tgaatttaaa tccagctaaa cggcctcgtc gacaagactg 720
ggatggagaa ttatatgaaa atggctcatt ttattttgct aaaagacatt tgatagagat 780
gggttacttg cagggtggaa aaatggcata ctatgaaatg cgagctgaac atagtgtgga 840
tatagatgtg gatattgatt ggcctattgc agagcaaaga gtattaagat atggctattt 900
tggcaaagag aagcttaagg aaataaaact tttggtttgc aatattgatg gatgtctcac 960
caatggccac atttatgtat caggagacca aaaagaaata atatcttatg atgtaaaaga 1020
tgctattggg ataagtttat taaagaaaag tggtattgag gtgaggctaa tctcagaaag 1080
ggcctgttca aagcagacgc tgtcttcttt aaaactggat tgcaaaatgg aagtcagtgt 1140
atcagacaag ctagcagttg tagatgaatg gagaaaagaa atgggcctgt gctggaaaga 1200
agtggcatat cttggaaatg aagtgtctga tgaagagtgc ttgaagagag tgggcctaag 1260
tggcgctcct gctgatgcct gttctactgc ccagaaggct gttggataca tttgcaaatg 1320
taatggtggc cgtggtgcca tccgagaatt tgcagagcac atttgcctac taatggaaaa 1380
ggttaataat tcatgccaaa aatagaaatt agcgtaatat tgagaaaaaa atgatacagc 1440
cttcttcagc cagtttgctt ttatttttga ttaagtaaat tccatgttgt aatgttacag 1500
agagtgtgat ttggtttgtg atatatatat attgtgctct acttttctct ttacgcaaga 1560
taattattta gagactgatt acagtctttc tcagattttt agtaaatgca agtaagaaca 1620
tcatcaaagt tcactttgta ttgtaccctg taaaactgtg tgtttgtgtg ctttcaaaga 1680
tgttgggatt ttatttatct ggggacagtg tgtatggtaa gacatgccct tctattaata 1740
aaactacatt tctcaaactt gaaaaaaaaa as 1772
7/10
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
<210> 7
<211> 1416
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 1398
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 983984CB1
<400> 7
tctggccgcg cggatcagct tccagcccag tcggcccggc ccgggggcca tggagctccg 60
agcggcggat cgcgagcctc ctgcgaaccc cagcctgcac gcccggttag cattcggccg 120
ggagatgcgg cagtggaatc tggaagggcg gtgaaaaacc tacgtcctgc cctcgcccgg 180
cctctccatt cgtcccccgg gtagagaggt gcccggctcc caccccttcc cagccccagc 240
cctggagaca gcagccccta gactactgag ggacagcgac agcatgaagg ctccgggtcg 300
gctcgtgctc atcatcctgt gctccgtggt cttctctgcc gtctacatcc tcctgtgctg 360
ctgggccggc ctgcccctct gcctggccac ctgcctggac caccacttcc ccacaggctc 420
caggcccact gtgccgggac ccctgcactt cagtggatat agcagtgtgc cagatgggaa 480
gccgctggtc cgcgagccct gccgcagctg tgccgtggtg tccagctccg gccaaatgct 540
gggctcaggc ctgggtgctg agatcgacag tgccgagtgc gtgttccgca tgaaccaggc 600
gcccaccgtg ggctttgagg cggatgtggg ccagcgcagc accctgcgtg tcgtctcaca 660
cacaagcgtg ccgctgctgc tgcgcaacta ttcacactac ttccagaagg cccgagacac 720
gctctacatg gtgtggggcc agggcaggca catggaccgg gtgctcggcg gccgcaccta 780
ccgcacgctg ctgcagctca ccaggatgta ccccggcctg caggtgtaca ccttcacgga 840
gcgcatgatg gcctactgcg accagatctt ccaggacgag acgggcaaga accggaggca 900
gtcgggctcc ttcctcagca ccggctggtt caccatgatc ctcgcgctgg agctgtgtga 960
ggagatcgtg gtctatggga tggtcagcga cagctactgc agggagaaga gccacccctc 1020
agtgccttac cactactttg agaagggccg gctagatgag tgtcagatgt acctggcaca 1080
cgagcaggcg ccccgaagcg cccaccgctt catcactgag aaggcggtct tctcccgctg 1140
ggccaagaag aggcccatcg tgttcgccca tccgtcctgg aggactgagt agcttccgtc 1200
gtcctgccag ccgccatgcc gttgcggagg cctccgggat gtcccatccc aagccatcac 1260
actccacaaa aacatttaat ttatggttcc tgcctcctgc cacgtgctgg gtggacctaa 1320
aggttccttc ccaccccatt ctggccgaca tttggagcca tctcaggcct ccactccctg 1380
agtaattcat ggcatttngg gggctcaccc acctac 1416
<210> 8
<211> 1889
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2210054CB1
<400> 8
gacgtagcgg aagaaaccgc agcagctccc aggatgaact ggttgcagtg gctgctgctg 60
ctgcgggggc gctgagagga cacgagctct atgcctttcc ggctgctcat cccgctcggc 120
ctcctgtgcg cgctgctgcc tcagcaccat ggtgcgccag gtcccgacgg ctccgcgcca 180
gatcccgccc actacaggga gcgagtcaag gccatgttct accacgccta cgacagctac 240
ctggagaatg cctttccctt cgatgagctg cgacctctca cctgtgacgg gcacgacacc 300
tggggcagtt tttctctgac tctaattgat gcactggaca ccttgctgat tttggggaat 360
gtctcagaat tccaaagagt ggttgaagtg ctccaggaca gcgtggactt tgatattgat 420
gtgaacgcct ctgtgtttga aacaaacatt cgagtggtag gaggactcct gtctgctcat 480
ctgctctcca agaaggctgg ggtggaagta gaggctggat ggccctgttc cgggcctctc 540
ctgagaatgg ctgaggaggc ggcccgaaaa ctcctcccag cctttcagac ccccactggc 600
8/10
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
atgccatatg gaacagtgaa cttacttcat ggcgtgaacc caggagagac ccctgtcacc 660
tgtacggcag ggattgggac cttcattgtt gaatttgcca ccctgagcag cctcactggt 720
gacccggtgt tcgaagatgt ggccagagtg gctttgatgc gcctctggga gagccggtca 780
gatatcgggc tggtcggcaa ccacattgat gtgctcactg gcaagtgggt ggcccaggac 840
gcaggcatcg gggctggcgt ggactcctac tttgagtact tggtgaaagg agccatcctg 900
cttcaggata agaagctcat ggccatgttc ctagagtata acaaagccat ccggaactac 960
acccgcttcg atgactggta cctgtgggtt cagatgtaca aggggactgt gtccatgcca 1020
gtcttccagt ccttggaggc ctactggcct ggtcttcaga gcctcattgg agacattgac 1080
aatgccatga ggaccttcct caactactac actgtatgga agcagtttgg ggggctcccg 1140
gaattctaca acattcctca gggatacaca gtggagaagc gagagggcta cccacttcgg 1200
ccagaactta ttgaaagcgc aatgtacctc taccgtgcca cgggggatcc caccctccta 1260
gaactcggaa gagatgctgt ggaatccatt gaaaaaatca gcaaggtgga gtgcggattt 1320
gcaacaatca aagatctgcg agaccacaag ctggacaacc gcatggagtc gttcttcctg 1380
gccgagactg tgaaatacct ctacctcctg tttgacccaa ccaacttcat ccacaacaat 1440
gggtccacct tcgacacggt gatcaccccc tatggggagt gcatcctggg ggctgggggg 1500
tacatcttca acacagaagc tcaccccatc gaccctgccg ccctgcactg ctgccagagg 1560
ctgaaggaag agcagtggga ggtggaggac ttgatgaggg aattctactc tctcaaacgg 1620
agcaggtcga aatttcagaa aaacactgtt agttcggggc catgggaacc tccagcaagg 1680
ccaggaacac tcttctcacc agaaaaccat gaccaggcaa gggagaggaa gcctgccaaa 1740
cagaaggtcc cacttctcag ctgccccagt cagcccttca cctccaagtt ggcattactg 1800
ggacaggttt tcctagactc ctcataacca ctggataatt tttttatttt tatttttttg 1860
aggctaaact ataataaatt gcttttggt 1889
<210> 9
<211> 2135
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2618358CB1
<400> 9
ctcctctggc caaagggtgc tctgcttctg gcagctgaag atcccagtag acagcttctt 60
aaaccatggc tttccctgca ggatttggat gggcggcagc actgcagctt atcaagtaga 120
aggaggctgg gatgcagatg gaaaaggccc ttgtgtctgg gacacattta ctcatcaggg 180
aggagagaga gttttcaaga accagactgg cgatgtagct tgtggcagct acactctgtg 240
ggaggaagat ttgaaatgta tcaaacagct tggattgact cattaccgct tctctctttc 300
ctggtcacgt ctgttacctg atgggacgac aggtttcatc aaccagaaag gaattgatta 360
ttacaacaag atcatcgatg atttgttaaa aaatggggtt actcccattg tgaccctcta 420
ccactttgat ttgcctcaga ctttagaaga ccaaggaggt tggttgtcag aggcaatcat 480
tgaatccttt gacaaatatg ctcagttttg cttcagtacc tttggggatc gtgtcaagca 540
gtggatcacc ataaatgaag ctaatgttct ttctgtgatg tcatatgact taggtatgtt 600
tcctccgggt atccctcact ttgggactgg aggttatcag gcagctcata atttgattaa 660
ggctcatgcc agatcctggc acagctatga ttccttattt cgaaaaaagc agaaaggtat 720
ggtgtctcta tcactttttg cggtctggtt ggaaccagca gatcccaact cagtgtctga 780
ccaggaagct gctaaaagag ccatcacttt ccatctggat ttatttgcta aacccatatt 840
catcgatggt gattatcctg aagttgtcaa gtctcagatt gcctccatga gtcaaaagca 900
aggctatcca tcatcgaggc ttccagaatt cactgaagaa gagaagaaaa tgatcaaagg 960
cactgctgat ttttttgctg tgcaatatta tacaactcgc ttaatcaagt accaggagaa 1020
caagaaagga gaactaggta ttctccagga tgcggaaatt gaattttttc cagatccatc 1080
ttggaaaaat gtggattgga tctacgtggt accatgggga gtatgtaaac tactgaaata 1140
tattaaggat a~atataata accctgtaat ttacatcact gagaatgggt ttccccagag 1200
tgacccagcg cctcttgatg acactcaacg ctgggagtat ttcagacaaa catttcagga 1260
actgttcaaa gctatccaac ttgataaagt caatcttcaa gtatattgtg catggtctct 1320
tctggataac tttgagtgga accagggata cagcagccgg tttggtctct tccacgttga 1380
ttttgaagac ccagctagac cccgagtccc ttacacatcg gccaaggaat atgccaagat 1440
catccgaaac aatggccttg aagcacatct gtaggcaaga tggctgagaa atacaggaga 1500
ggcgtctgct tttggaaagg aaatctgctt tggtgatgat ctttcaggca atctcaactt 1560
acttctttaa tcaacattta atatcaatgg atctgtgatt aaaaggtctg aatatgtaat 1620
gcctcgtgaa gtatttaata atggccttta tttgtatttg gatcaatgag gtttttaaaa 1680
9/10
CA 02369342 2001-10-09
WO 00/63351 PCT/US00/10882
aaaatggaag agaaaaccac taaccttgat ttttgtattg caaaatcaga tagacctgga 1740
aacataaatt taaatcctta gacatttttc tagaaaaaaa tgcaaagttt ataaagatga 1800
tacaaccatg atttgcaact gtaacaggag accatttatt ataagcgtac ctgtttgtga 1860
acttaattat tctgattcca taagctgttt ttgcttaggt gatccactgc catgtgatcc 1920
ataatttttc tacataaaaa atcaaagtta aaagtcacat tatacagtta tgcattcatt 1980
tcaacaaaat agtgaattga taatctactt gttaatatat tcggcccata ttttgtgtgt 2040
ttggacaagt acatctccct tttgcctaat gaacttttga aaaataataa aataatagaa 2100
taaattagac tttgaatggc aaaaaaaaaa aaaaa 2135
<210> 10
<211> 1650
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2912330CB1
<400> 10
agcaactgga aaacaagcat tgcattgcat caggatgtct atgaaatgga cttcagctct 60
tctcctgata cagctgagct gttactttag ctctgggagt tgtggaaagg tgctggtgtg 120
gcccacagaa ttcagccact ggatgaatat aaagacaatc ctggatgaac ttgtccagag 180
aggtcatgag gtgactgtat tggcatcttc agcttccatt tctttcgatc ccaacagccc 240
atctactctt aaatttgaag tttatcctgt atctttaact aaaactgagt ttgaggatat 300
tatcaagcag ctggttaaga gatgggcaga acttccaaaa gacacatttt ggtcatattt 360
ttcacaagta caagaaatca tgtggacatt taatgacata cttagaaagt tctgtaagga 420
tatagtttca aataagaaac ttatgaagaa actacaggag tcaagatttg atgttgttct 480
tgcagatgct gttttcccct ttggtgagct gctggccgag ttacttaaaa taccctttgt 540
ctacagcctc cgcttctctc ctggctacgc aattgaaaag catagtggag gacttctgtt 600
ccctccttcc tatgtgcctg ttgttatgtc agaactaagt gaccaaatga ctttcataga 660
gagggtaaaa aatatgatct atgtgcttta ttttgaattt tggttccaaa tatttgacat 720
gaagaagtgg gatcagttct acagtgaagt tctaggaaga cccactacgt tatctgagac 780
aatggcaaaa gctgacatat ggcttattcg aaactactgg gattttcaat ttcctcaccc 840
actcttacca aatgttgagt tcgttggagg actccactgc aaacctgcca aacccctacc 900
gaaggaaatg gaagagtttg tccagagctc tggagaaaat ggtgttgtgg tgttttctct 960
ggggtcgatg gtcagtaaca cgtcagaaga aagggccaat gtaattgcat cagcccttgc 1020
caagatccca caaaaggttc tgtggagatt tgatgggaat aaaccagata ctttaggact 1080
caatactcgg ctgtacaagt ggatacccca gaatgatctt cttggtcatc ccaaaaccaa 1140
agcttttatc actcatggtg gaatgaatgg gatctatgaa gctatttacc atggggtccc 1200
tatggtggga gttcccatat ttggtgatca gcttgataac atagctcaca tgaaggccaa 1260
aggagcagct gtagaaataa acttcaaaac tatgacaagc gaagatttac tgagggcttt 1320
gagaacagtc attaccgatt cctcttataa agagaatgct atgagattat caagaattca 1380
ccatgatcaa cctgtaaagc ccctagatcg agcagtcttc tggatcgagt ttgtcatgcg 1440
ccacaaagga gccaagcacc tgcgatcagc tgcccatgac ctcacctggt tccagcacta 1500
ctctatagat gtgattgggt tcctgctgac ctgtgtggca actgctatat tcttgttcac 1560
aaaatgtttt ttattttcct gtcaaaaatt taataaaact agaaagatag aaaagaggga 1620
atagatcttt ccaaattcaa gaaagacctg 1650
10/10
