Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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MYOSIN HEAVY CHAIN HOMOLOG
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of a myosin
heavy chain
homoiog and to the use of these sequences in the diagnosis, treatment, and
prevention of heart and
skeletal muscle disorders; developmental disorders; and cell proliferative
disorders including cancer.
BACKGROUND OF THE JINVENTION
Myosins are actin-activated ATPases, found in eukaryotic cells, that couple
hydrolysis of
ATP with motion. Myosin provides the motor function for muscle contraction and
intracellular
movements such as phagocytosis and rearrangement of cell contents during
mitotic cell division
(cytokinesis). Myosins are composed of one or two heavy chains and associated
light chains. Myosin
heavy chains contain an amino-terminal motor or head dornain, a neck that is
the site of light-chain
binding, and a carboxy-terminal tail domain. Conventional myosins, such as
those found in muscle
i5 tissue, are composed of two myosin heavy-chain subunits, each associated
with two light-chain
subunits that bind at the neck region and play a regulatory role.
Unconventional myosins, believed to
function in intracellular motion, may contain either one or two heavy chains
and associated light
chains. There is evidence for about 25 myosin heavy chain genes in
vertebrates; more than half of
them unconventional. Recently the myosins have been divided into 11 classes.
The heavy myosin chain head domain ends in an amino acid sequence which is
conserved in
most myosins. The neck domains of most myosin heavy Chains (MyHC) consist of a
variable number
of motifs with a conserved sequence believed to be the site for light-chain
binding. Calmoduiin or
calmoduiin-like proteins function as light chains. An unexpected degree of
variation has been
observed in the tail domains of different myosins. Severall unconventional
myosins contain domains
associated with signal transduction (Mooseker, M. et al. ( ll995) Annu. Rev.
Cell Dev. Biol. 11:633-
675).
Disorders of myosin function are involved in a variety of human diseases
including muscle
disorders, developmental disorders, and cancer. Two forms of myosin heavy
chain (alpha and beta)
have been observed in the mammalian ventricular myocardium. The speed with
which the heart
contracts is related to their relative expression, which suggests that
increased alpha MyHC expression
may be therapeutic in cardiovascular disease. Decline in atherosclerosis
resistance with age has been
related to downregulation of non-muscle MyHC (Amore, B. et al. (1996) J. Vasc.
Res. 33:442-453).
Aging has also been related to decreased class II MyHC expression and an
increase in Class l MyHC
expression {Larsson, L. et al. (1997) Acta Physiol. Scand. 159:81-89).
Mutations in genes coding for
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the beta-myosin heavy chain have been related to hypertrophic cardiomyopathy
(Marian, A.J. et al.
{I998) J. Cardiovasc. Electrophysiol. 9:88-99).
Growth and muscle defects are observed as a result of defects in MyHC
expression (Acakpo-
Satchivi, L.J. (1997) J. Cell Biol. 139:1219-1229). Abnormal MyHC expression
has been observed in
muscular dystrophy (Tidyman, W.E. et al. (1997) Dev. Dyn. 208:491-504).
Deafness associated with
disruption in organization of the hair cells of the inner ear results from a
mutant unconventional-
myosin gene (Probst, F.J. et al. (1998} Science 280:1444-1447).
Modulation of MyHC has been implicated in breast cancer (Ohyabu, I. et al.
(1998) Pathol.
Int. 48:433-439). The chromosomal aberration associated with acute myeloid
leukemia produces a
protein that contains the rod domain of the smooth muscle MyHC molecule
(Tanaka, Y. et al. (1998)
Oncogene 17:699-708). Myosin V is associated with transport of epidermal
melanocytes. The S91
mouse melanoma cell line shows a defect in transport of meianosomes {Brown,
D.A. et al. (1998) J.
Invest. Dermatol.l 10:428-437).
The discovery of a new myosin heavy chain homolag and the polynucleotides
encoding it
satisfies a need in the art by providing new compositions which are useful in
the diagnosis,
prevention, and treatment of heart and skeletal muscle disorders;
developmental disorders; and cell
proliferative disorders including cancer.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, human myosin heavy
chain
homolog, referred to as "MHCH:' In one aspect, the invention provides a
substantially purified
polypeptide comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: I
and fragments thereof. The invention also includes a polypeptide comprising an
amino acid sequence
that differs by one or more conservative amino acid substitutions from an
amino acid sequence
selected from the group consisting of SEQ ID NO:1 and fragments thereof.
The invention further provides a substantially purified variant having at
least 90% amino acid
identity to at least one of the amino acid sequences selected from the group
consisting of SEQ ID
NO:1 and fragments thereof. The invention also provides an isolated and
purified polynucleotide
encoding the polypeptide comprising an amino acid sequence selected from the
group consisting of
SEQ ID NO:I and fragments thereof. The invention also includes an isolated and
purified
poIynucleotide variant having at least 70% polynucleotide sequence identity to
the polynucleotide
encoding the polypeptide comprising an amino acid sequence ~setected from the
group consisting of
SEQ ID NO: I and fragments thereof.
Additionally, the invention provides an isolated arid purified polynucleotide
which hybridizes
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under stringent conditions to the polynucleotide encoding the polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1 and fragments
thereof. The invention
also provides an isolated and purified polynucleotide having a sequence which
is complementary to
the polynucleotide encoding the polypeptide comprising tl~~e amino acid
sequence selected from the
group consisting of SEQ ID NO:I and fragments thereof.
The invention also provides a method for detecting a polynucieotide in a
sample containing
nucleic acids, the method comprising the steps of (a) hybridizing the
complement of the
polynucleotide sequence to at least one of the poiynucleotides of the sample,
thereby forming a --
hybridization complex; and (b) detecting the hybridization complex, wherein
the presence of the
hybridization complex correlates with the presence of a polynucleotide in the
sample. In one aspect,
the method further comprises amplifying the polynucieotide prior to
hybridization.
The invention also provides an isolated and purified polynucieotide comprising
a
polynucleotide sequence selected from the group consisting of SEQ ID N0:2 and
fragments thereof.
The invention further provides an isolated and purified pol.ynucleotide
variant having at least 70%
poiynucleotide sequence identity to the polynucleotide sequence selected from
the group consisting of
SEQ ID N0:2 and fragments thereof. The invention also provides an isolated and
purified
polynucleotide having a sequence which is complementarfj to the polynucleotide
comprising a
polynucleotide sequence selected from the group consisting of SEQ ID N0:2 and
fragments thereof.
The invention further provides an expression vector containing at least a
fragment of the
polynucleotide encoding the polypeptide comprising an annino acid sequence
selected from the group
consisting of SEQ ID NO:1 and fragments thereof. In another aspect, the
expression vector is
contained within a host cell.
The invention also provides a method for producing a poiypeptide, the method
comprising the
steps of (a) culturing the host cell containing an expression vector
containing a polynucleotide of the
invention under conditions suitable for the expression of the polypeptide; and
(b) recovering the
polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a
substantially purified
polypeptide having the amino acid sequence selected from. the group consisting
of SEQ ID NO: i and
fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purifed antibody which binds to a polypeptide
selected from
the group consisting of SEQ ID NO:1 and fragments thereof. The invention also
provides a purified
agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder
associated with
decreased expression or activity of MHCH, the method comprising administering
to a subject in need
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WO 00J26372 PCTIUS99J26177
of such treatment an effective amount of a pharmaceutical composition
comprising a substantiaiIy
purified polypeptide having the amino acid sequence :elected from the group
consisting of SEQ iD
NO:1 and fragments thereof in conjunction with a suitable pharmaceutical
carrier,
The invention also provides a method for treating or preventing a disorder
associated with
increased expression or activity of MHCH, the method comprising administering
to a subject in need
of such treatment an effective amount of an antagonist of a polypeptide having
an amino acid
sequence selected from the group consisting of SEQ ID NO: I and fragments
thereof.
BRIEF DESCRIPTION OF THE FIGURES AND TABLE
Figures IA, 1B, 1C, ID, IE, and 1F show the amino acid sequence (SEQ ID NO:1)
and
nucleic acid sequence (SEQ ID N0:2) of MHCH. The alignment was produced using
MACDNASIS
PRO software (Hitachi Software Engineering, South San :Francisco CA).
Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I, 2J, and 2K show the amino acid
sequence
alignment among MHCH (Incyte Clone ID 1929760; SE(1 ID NO: l ), Caenorhabditis
elegans myosin
I heavy chain (GI 1279777; SEQ ID N0:3), and Helianthus annuus (sunflower)
unconventional
myosin I heavy chain (GI 2444174; SEQ ID N0:4), produced using the
multisequence alignment
program of LASERGENE software (DNASTAR, Madison WI).
Table 1 shows the tools, programs, and algorithms used to analyze MHCH, along
with
applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences;, and methods are described,
it is understood
that this invention is not limned to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, alt 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
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WO 00!26372 PCT/US99/26177
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein as:, 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 vi~.rtue of prior
invention.
DEFINITIONS
"MHCH" refers to the amino acid sequences of substantially purified MHCH
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, marine, equinE, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
MHCH. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of MHCH either by
directly interacting with
MHCH or by acting on components of the biological pathway in which MHCH
participates.
An "allelic variant" is an alternative form of the l;ene encoding MHCH.
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. Cornmon 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 MHCH include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as MHCH or a
poiypeptide with at least one functional characteristic of MHCH. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding MHCH, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
MHCH. 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 MHCH. Deliberate amino acid substitutions rr~ay 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 imrnunological activity of MHCH 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 hydrophiiicity values may include: asparagine and glutamine; and
serine and threonine.
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Amino acids with uncharged side chains having similar h;ydrophilicity values
may include: ieucine,
isoleucine, and valine; glycine and alanine; and phenylala.nine 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 MHCH. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of MHCH either by
IS directly interacting with MHCH or by acting on components of the biological
pathway in which
MHCH participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab'),, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind MHCH polypeptides can be prepared using intact
potypeptides or using
fragments containing small peptides of interest as the immunizing antigen. The
polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLH). T'he coupled peptide is
then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitape) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody
The term "antisense" refers to any composition containing a nucleic acid
sequence which is
complementary to the "sense" strand of a specific nucleic. acid sequence.
Antisense molecules may be
produced by any method including synthesis or transcription. Once introduced
into a cell, the
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complementary nucleotides combine with natural sequencca produced by the cell
to form duplexes
and to block either transcription or translation. The designation "negative"
or "minus" can refer to the
antisense strand, and the designation "positive" or "plus" c:an refer to the
sense strand.
The term ''biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise. ''immunologically
active" refers to the
capability of the natural, recombinant, or synthetic MHCH, 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 "complementarily" refer to the natural binding
of _.
poiynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the
t0 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 complementarily 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 MHCH or fragments of
MHCH may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid seqrsence which has been
resequenced to
resolve uncalled bases, extended using the XL-PCR kit (Pc~rkin-Elmer, Norwalk
CT) in the 5' andlor
the 3' direction, and resequenced, or which has been assembled from the
overlapping sequences of
one or more Incyte Clones and, in some cases, one or more public domain ESTs,
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI). Some sequences have been both extended and assembled to produce the
consensus sequence.
"Conservative amino acid substitutions" are those substitutions that, when
made, least
interfere with the properties of the original protein, i.e., the 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
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as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala GIy, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys AIa, Ser
Gin 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 F'he, 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, andlor (c} the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to the chemical modification of a polypeptide
sequence, or a
polynucieotide 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 MHCH or ths~ polynucleotide encoding MHCH
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment rnay comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from S 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 S, 10, 15,
20, 25, 30, 40, 50, 60, 75, 100, 350, 250 or at least 500 contiguous
nucleotides or amino acid residues
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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 liength that is
supported by the specification,
including the Sequence Listing, tables, and figures, may lbe encompassed by
the present embodiments.
A fragment of SEQ ID N0:2 comprises a region of unique polynucleotide sequence
that
specifically identifies. SEQ ID N0:2, for example, as distinct from any other
sequence in the same
genome. A fragment of SEQ ID N0:2 is useful, for example, in hybridization and
amplification
technologies and in analogous methods that distinguish SEQ ID N0:2 from
related polynucleotide
sequences. The precise length of a fragment of SEQ ID N0:2 and the region of
SEQ ID N0:2 to
which the fragment corresponds are routinely determinable by one of ordinary
skill in the art based an
the intended purpose for the fragment.
A fragment of SEQ ID NO:1 is encoded by a fral;ment of SEQ ID N0:2. A fragment
of SEQ
ID NO:1 comprises a region of unique amino acid sequence that specifically
identifies SEQ ID NO:1.
For example, a fragment of SEQ ID NO:1 is useful as an immunogenic peptide for
the development
of antibodies that specifically recognize SEQ ID NO: l . 'If he precise length
of a fragment of SEQ ID
NO:I and the region of SEQ ID NO:1 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
ar complete similarity. The word "identity" may substitute for the word
"similarity." A partially
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-specif c
binding is permitted, as reduced stringency conditions recjuire that the
binding of two sequences to
one another be a specific (i.e., a selective) interaction. The absence of non-
specifc 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 poiynucieotide
sequences, refer
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to the percentage of residue matches between at feast 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 ofthe CLUSTAL V algorithm as incorporated into the MEGALIGN version
3.12e
sequence alignment program. This program is part of the :LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR; Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
IO 8:189-191. For pairwise alignments of polynucieotide 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 (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Blot. 215:403-410),
which is available from
several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://wvv.ncbi.nlm.nih.~~ovfBLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. ''BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.govleorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and biastp (discussed
below). BLAST
programs are commonly used with gap and other parametc;rs set to default
settings. For example, to
compare two nucleotide sequences, one may use biastn with the "BLAST 2
Sequences" tool Version
2Ø9 (May-07-1999) set at default parameters. Such default parameters may be,
for example:
Matrix: BLOSUM62
Reyvard for match: 1
Penalty for mismatch: -2
Open Gap: .i and Extension Gap: 2 penalties
Gap x drop-off .50
Expect: 10
Word Size: I I
Filter: on
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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 ail 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
I5 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 miay 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=I, gap
penalty=3, window=5, and "diagonals saved"=5. The PA1vI250 matrix is selected
as the default
residue weight table. As with poiynucleotide 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: II acrd Extension Gap: I penalties
Gap x drop-off 50
Expect: l Q
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypepticle sequence,
CA 02349212 2001-05-03
WO 00!26372 PCT/US99/26177
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
(east 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 abort 6 kb to 10 Mb in size, and which contain all of the
elements required frnr
stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibodyv 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 determiinable by one of
ordinary skill in the art and
may be consistent among hybridization experiments, whereas wash conditions may
be varied among
experiments to achieve the desired stringency, and therefore hybridization
specificity. Permissive
annealing conditions occur, for example, at 68°C in the.presence of
about 6 x SSC, about 1% (w/v)
SDS, and about 100 ug/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 Cloning: A Laboratory Manual, 2°d ed., vol. 1-:3, Cold Spring
Harbor Press, Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between poiynucleotides of the
present invention
include wash conditions of 68°C in the presence of about ~0.2 x SSC and
about 0. I % SDS, for 1 hour.
12
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Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration
may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
denatured salmon sperm DNA at about 100-200 pg/ml. Organic solvent, such as
formamide at a
concentration of about 35-50% vlv, 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, particularliy 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.
i0 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., C°t: or
R°t analysis) or formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on
a substrate.
The terms "element" and "array element" in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
The term ''modulate" refers to a change in the activity of MHCH. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of MHCH.
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 GNA-tike 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
13
CA 02349212 2001-05-03
WO OOI26372 PCT/US99/26I77
sequence. Generally, operably linked DNA sequences ma.y 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 1DNA or RNA and stop
transcript elongation,
and may be pegylated to extend their lifespan in the cell.
"Probe" refers to nucleic acid sequences encoding MHCH, their complements, or
fragm_~nts
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 oligonuclc:otides, which may be
annealed to a target
poiynucleotide 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
specification, including the tables, figures, and Sequence I fisting, 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"d
ed., vol, t-3, Cold
Spring Harbor Press, Plainview NY; Ausubel et a1.,1987, current Protocols in
Molecular Biolo~v,
Greene Publ. Assoc. & Wiley-Intersciences, New York N'Y; 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).
Oligonucieotides 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
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CA 02349212 2001-05-03
WO 00/26372 PCT/US99/26177
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
specifc 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
I0 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 usefii) in hybridization
technologies, for
example, as PCR or sequencing primers, microarray elements, or specific probes
to identify fully or
f 5 partially complementary polynucleotides in a sample of nucleic acids.
Methods of oligonucleotide
selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
20 artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of" a portion of
the nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part o:f a vector that is
used, for example, to
25 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 immunologicai response in the mammal.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
30 acids encoding MHCH, or fragments thereof, or MHCH 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
CA 02349212 2001-05-03
WO 00126372 PCTIUS99/26177
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or ep.itope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least about 60% free,
preferably about 75% free, and most preferably about 90°ro 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, gets, tubing,
plates, polymers,
microparticles and capiitaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleoticles or polypeptides
are bound.
"Transformation" describes a process by which eacogenous DNA enters and
changes a
recipient cell. Transformation may occur under natural or' artiBciai
conditions according to various
methods well known in the art, and may rely on any known method for the
insertion of foreign nucleic
acid sequences~into a prokaryotic or eukaryotic host cell. The method for
transformation is selected
based on the type of host cell. being transformed and may include, but is not
limited to, viral infection,
electroporation, heat shock, lipofection, and particle bombardment. The term
"transformed" cells
includes stably transformed cells in which the inserted DNA is capable of
replication either as an
autonomously replicating plasmid or as part ofthe host chromosome, as well as
transiently
transformed cells which express the inserted DNA or RNA for limited periods of
time.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at Ieast 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 rnRNA processing.
The corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
16
CA 02349212 2001-05-03
WO 00/26372 PCTIUS99/26177
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. Poiymorphic variants also may
encompass "single nucleotide
S polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07
1999). set at default parameters. Such a pair of polypeptides may show, for
example, at least SO%, 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 polypeiptides.
THE INVENTION
1 S The invention is based an the discovery of a new ihuman myosin heavy chain
homolog
(MHCH), the pofynucleotides encoding MHCH, and the use of these compositions
for the diagnosis,
treatment, or prevention of heart and skeletal muscle disorders; developmental
disorders; and cell
proiiferative disorders including cancer.
Nucleic acids encoding the MHCH of the present invention were identified in
Incyte Clone
I929760H 1 from the colon tumor cDNA library (COLNTUT03) using a computer
search for
nucleotide and/or amino acid sequence alignments. A consensus sequence, SEQ ID
N0:2, was
derived from the following overlapping and/or extended nucleic acid sequences:
Incyte Clones
1929760H 1 and I 929760F6 {COLNTUT03}, 2418744F3 I;HNT3AZT01 }, 3229696X 11 F
1
(COTRNOT01), 3344480F6 (SPLNNOT09), 401389H1 ('TMLR3DT01), S111b81H1
(ENDITXTO1),
1451483H1 (PENITUT01), and shotgun sequence SBCA04642FI.
In one embodiment, the invention encompasses a poiypeptide comprising the
amino acid
sequence of SEQ ID NO:1, as shown in Figures lA, 1 B, 1 C, 1 D, 1 E, and 1 F.
MHCH is 6I2 amino
acids in length and has 7 potential casein kinase II phosphorylation sites at
residues S62, T146, T221,
S280, 5323, S390, and T546; and 6 potential protein kinase C phosphorylation
sites at residues S 19,
S140, S303, T441, 5555, and S563. The sequence from T'383 through M387 of MHCH
is 80%
identical to the conserved sequence found at the end of myosin head domains.
MHCH contains two
possible light-chain binding sites. The first, from 1410 through E421,
contains 4 out of 6 conserved
residues and the second, from I432 through K443, contains S out of 6 conserved
residues. PFAM
analysis shows that MHCH shares homology with a myosin head domain from
residue F51 to residue
17
CA 02349212 2001-05-03
WO 00/26372 PCT/US99/26177
L314. PRINTS analysis shows that MHCH shares homology with a myosin heavy
chain signature
motif from residue F51 to K79 and from residue F105 to C;133. MHCH has a
possible
transmembrane motif from residue W506 to P535. As shown in Figures 2A, 2B, 2C,
2D, 2E, 2F, 2G,
2H, 2I, 2J, and 2K, MHCH has chemical and structural similarity with
Caenorhabditis eleeans myosin
I heavy chain (GI 1279777; SEQ ID N0:3), and Helianthus annuus unconventional
myosin heavy
chain (GI 2444174; SEQ ID N0:4). MHCH and myosin I heavy chain share 23.2%
identity, and in
particular they share 39% identity from residue F51 to residue L314 of MHCH.
MHCH and
unconventional myosin I share 22.4% identity, and in partiicular they share
38% identity from residue
F51 to residue L314 of MHCH. A fragment of SEQ ID NO:2 from about nucleotide
122 to about
nucleotide 166 is useful in hybridization or amplification technologies to
identify SEQ ID N0:2 and
to distinguish between SEQ ID N0:2 and a related sequence. The encoded
potypeptide is useful, for
example, as an antigenic polypeptide. Northern analysis shows the expression
of this sequence in
various libraries, at least 65% of which are associated with cell
proliferation or cancer, at least 34% of
which are associated with the immune response, at least 2~l% of which are
associated with
gastrointestinal tissue, at least 24% of which are associated with
reproductive tissue, at least 13% of
which are associated with hematopoietic/immune tissue, at least 10% are
associated with
musculoskeletal tissue, and at least 10% are associated with nervous tissue.
The invention also encompasses MHCH variants. A preferred MHCH 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 MHCH amino acid sequence, and which contains at least
one functional or
structural characteristic of MHCH.
The invention also encompasses polynucleotides vvhich encode MHCH. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising the
sequence of SEQ
ID N0:2, which encodes MHCH.
The invention also encompasses a variant of a polynucleotide sequence encoding
MHCH. 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% polynucleotidc~ sequence identity
to the polynucleotide
sequence encoding MHCH. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising the sequence of SEQ ID N0:2 which has at
least about 70%, or
alternatively at least about 85%, or even at least about 95°io
polynucleotide sequence identity to a
nucleic acid sequence consisting of SEQ ID N0:2. Any one of the polynucleotide
variants described
above can encode an amino acid sequence which contains at~least one functional
or structural
characteristic of MHCH.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
18
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WO 00/26372 PCT/US99/26177
genetic code, a multitude of polynucleotide sequences encoding MHCH, 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 MHCH, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode MF-iCH and its variants are
generally capOble of
hybridizing to the nucleotide sequence of the naturally occurring MHCH under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding MHCH
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 MHCH and its derivatives without altf:ring 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 occrarring sequence.
The invention also encompasses production of Dr~IA sequences which encode MHCH
and
MHCH derivatives, or fragments thereof, entirety by syntlhetic 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 MHCH or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucieotide sequences, and, in particular, to
those shown in SEQ ID
N0:2 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-
S I 1.) Hybridization conditions, including annealing and wash conditions, are
described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase 1, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Perkin-
Elmer), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway
NJ); or
combinations of poiymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
l9
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WO 00/26372 PCTIUS99126177
automated with machines such as the M1CROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycier (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., Ausubei,
F.M. ( 1997) Short
Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, unit 7.7;
Meyers, R.A. ( 1995)
Molecular Biolosy and Biotechnology, Wiley VCH, New York NY, pp. 856-853.) _.
The nucleic acid sequences encoding MHCH may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
a1. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PC;R 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).
Additional3y, 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 maybe
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 b8°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 S' regions of genes, are preferablle 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.
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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 MHCH may be cloned in recombinant DNA molecules that direct
expression of
MHCH, or fragments or functional equivalents thereof, in appropriate host
cells. Due to the inherent
degeneracy of the genetic code, other DNA sequences which encode substantially
the same or a
functionally equivalent amino acid sequence may be produced and used to
express MHCH.
I S The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter MHCH-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, andlor expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
In another embodiment, sequences encoding MHC:H 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, MHCH 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. (i995) Science 269:202-204.) Automated synthesis may be
achieved using the
ABI 431 A peptide synthesizer (Perkin-Elmer). Additionally, the amino acid
sequence of MHCH, or
any part thereof, may be altered during direct synthesis and/or combined with
sequences from other
proteins, or any part thereof, to produce a variant polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, T. ( 19$4) Proteins, Structures and Molecular
Properties, WH Freeman, New
21
CA 02349212 2001-05-03
WO 00126372 PCT/US99J26I77
York NY.)
In order to express a biologically active MHCH, the nucleotide sequences
encoding MHCH
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 regul;~tory sequences,
such as enhancers,
constitutive and inducible promoters, and 5' and 3' untranslated regions in
the vector and in
polynucieotide sequences encoding MHCH. Such elements may vary in their
strength and specificity.
Specifc initiation signals may also be used to achieve more efficient
translation of sequences -.
encoding MHCH. Such signals include the ATG initiation codon and adjacent
sequences, e.g. the
Kozak sequence. In cases where sequences encoding MHCH 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
I S 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 MHCH and appropriate transcriptional and
transiational
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
LaboratorX Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-
17; Ausubel, F.M. et
al. (1995) Current Protocols in Molecular Biolor;y, John V~Jiley & 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 MHCH. 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 piasmids); or
animal cell systems. The invention is not limited by the host cell employed. .
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding MHCH. For example,
routine cloning;
subcloning, and propagation of polynucleotide sequences encoding MHCH can be
achieved using a
22
CA 02349212 2001-05-03
WO 00/26372 PCTIUS99/26I77
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding MHCH into the
vector's multiple
cloning site disrupts the IacZ gene, allowing a colorimetrnc 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 Eleeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of MHCHf are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of IvIHCH 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 producaion of MHCH. 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
nastoris. 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)
BioJTechnology 12:181-184.)
Plant systems may also be used for expression of MHCH. Transcription of
sequences
encoding MHCH may be driven viral promoters, e.g., the 35S and 19S promoters
of CaMV used
alone or in combination with the omega leader sequence i:rom TMV (Takamatsu,
N. (1987) EMBO d.
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 ai. (1984) EMBO J. 3:1671-
1680; Broglie, R. et aI.
(1984) Science 224:838-843; and Winter, J. et al. { 1991 } :EZesults 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 Technoioev
(1992) McGraw Hill, New York NY, pp. i91-196.)
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 MHCH
may be ligated into
an adenovirus transcription/transiation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential EI or E3 region of the viral genome may
be used to obtain
infective virus which expresses MHCH in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used. to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
23
CA 02349212 2001-05-03
WO 00/26372 PCTIUS99126177
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat. Genet.
I 5:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
MHCH in cell lines is preferred. For example, sequences encoding MHCH can be
transformed into
cell lines using expressian 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 stabty 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
IS include, but are not limited to, the herpes simplex virus thymidine kinase
and adenine
phosphoribosyltransferase genes, for use in th 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 aminoglycosidles 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., Har~tman, S.C. and
R.C. Mulligan ( 1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate 13-glucuronide, or
iuciferase 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 MHCH is inserted within a marker gene sequence, transformed
cells containing
sequences encoding MHCH can be identified by the absence of marker gene
function. Alternatively,
a marker gene can be placed in tandem with a sequence encoding MHCH under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
24
CA 02349212 2001-05-03
WO 00/26372 PCTIUS99/261??
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding MHCH
and that express
MHCH 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 techrriques 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 MHCH 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 MHCH 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) Seroloeical Methods. a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in lmmunoloey, Greene
Pub. Associates and
i5 Wiley-Interscience, New York NY; and Pound, J.D. (i99'r3) Immunochemica)
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 MHCH
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding MHCH, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding MHCH 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 polynucieotides which encode MHCH may be designed to contain signal
sequences which
direct secretion of MHCH through a prokaryotic or eukar/otic cell membrane.
CA 02349212 2001-05-03
WD Ot)126372 PCT/US99126177
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include. but are not limited to, acetylation, carboxylation,
glycosylation,
phosphoryiation, lipidation, and acylation. Post-translaticroal 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, HI:K293, 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 MHCH 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 MHCH protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of MHCH activity.
Heterologous protein and
1 S 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}, caimodulin 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,
calrnodulin, and metal-
chelate resins, respectively. FLAG, c-myc, and hemagglu~tinin (HA) enable
immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the MHCH encoding sequence and the
heterologous protein
sequence, so that MHCH 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 rnay also be used to facilitate
expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled MHCH may
he 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 MHCH may be produced not only b:y recombinant means, but also by
direct
peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra.
pp. 55-60.) Protein
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CA 02349212 2001-05-03
WO 00/26372 PCT/US99/26177
synthesis may be performed by manual techniques or by autamation. Automated
synthesis may be
achieved, for example, using the ABI 431 A peptide synthesizer (Perkin-Elmer).
Various fragments of
MHCH 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 MHCH and myosin heavy chain. In addition, the expression of MHCH is
closely
associated with cell proliferation or cancer, the immune response,
gastrointestinal tissue, reproductive
tissue, musculoskeletai tissue, and nervous tissue. Therefore, MHCH appears to
play a role in heart
and skeletal muscle disorders; developmental disorders; and cell proliferative
disorders including
cancer. In the treatment of disorders associated with increased MHCH
expression or activity, it is
desirable to decrease the expression or activity of MHCH. do the treatment of
disorders associated
with decreased MHCH expression or activity, it is desirable to increase the
expression or activity of
MHCH.
Therefore, in one embodiment, MHCH 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 MHCH. Examples of such disorders include, but are not limited to,
a heart or skeletal
muscle disorder such as angina, anaphylactic shock, arrhythmias, asthma,
Becker's muscular
dystrophy, cardiovascular shock, central core disease, Cushing's syndrome,
Duchenne's muscular
dystrophy, encephalopathy, epilepsy, hypertension, hypol;lycemia, Kearns-Sayre
syndrome, lactic
acidosis, migraine, infectious myositis, polymyositis, dennatomyositis,
inclusion body myositis,
myocardial infarction, myotonic dystrophy, myocarditis, myoclonic disorder,
ophthalmoplegia,
pheochromocytoma, and myopathies including cardiomyopathy, centronuclear
myopathy, ethanol
myopathy, lipid myopathy, mitochondrial myopathy nemaiine myopathy, and
thyrotaxic myopathy; a
developmental disorder such as renal tubular acidosis, anemia, Cushing's
syndrome, achondroplastic
dwa~sm, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis,
WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation), Smith-
Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia, hereditary
keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis,
hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea
and cerebral palsy,
spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract,
and sensorineural
hearing loss; and a cell proliferative disorder such as actinic keratosis,
arteriosclerosis,
atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue
disease (MCTD), myelofibrosis,
paroxysmal nocturnal hemoglobinuria, polycythemia vem, psoriasis, primary
thrombocythemia, and a
cancer including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma,
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CA 02349212 2001-05-03
WO OOI26372 PCTIUS99/26177
teratocarcinoma, and, in particular, a cancer of the adrenail 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, or uterus.
In another embodiment, a vector capable of expressing MHCH 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 MHCH including. but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
MHCH in conjunction with a suitable pharmaceutical carrier may be administered
to a subject too treat
or prevent a disorder associated with decreased expression or activity of MHCH
including, but not
limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of MHCH
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of MHCH including, but not limited to, those listed above.
In a further embodiment, an antagonist of MHCHf may be administered to a
subject to treat or
IS prevent a disorder associated with increased expression or activity of
MHCH. Examples of such
disorders include, hut are not limited to, those heart and skeletal muscle
disorders; developmental
disorders; and cell proliferative disorders including cancer described above.
In one aspect, an
antibody which specifically binds MHCH 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
MHCH.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding MHCH may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of MHCH 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 rnay be able to
achieve therapeutic
3U efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of MHCH may be produced using methods which are generally known
in the
art. In particular, purified MHCH may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind MHCH.
Antibodies to MHCH may
also be generated using methods that are well known in the art. Such
antibodies may include, but are
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CA 02349212 2001-05-03
WO 00/26372 PCT/US99/26I77
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 MHCH or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include;, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants
used in humans,
t0 BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
MHCH 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
viigopeptides, 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 MHCH amino
acids may be fused with those of another protein, such as 1KLH, and antibodies
to the chimeric
molecule may be produced.
Monoclonal antibodies to MHCH 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. ( I975) 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) Moi. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. ( 1984) Nature
312:604-608; and Takeda,
S. et al. ( 1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
MHCH-specific single
chain antibodies. Antibodies with related specificity; but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial irnmunoglobulin
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 pa.neis of highly
specific binding reagents as
29
CA 02349212 2001-05-03
WO 00126372 PCT/US99/2bI77
disclosed in the literature. (See, e.g., Orlandi; R. et al. ( t 989) Proc.
Natl. Acad. Sci. USA
86:3833-3837; Winter, G. et al. ( 1991 ) Nature 349:293-2f9.)
Antibody fragments which contain specific binding sites for MHCH may also be
generated.
For example, such fragments include, but are not limited to, F(ab'), fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 24b: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
polyclanal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
MHCH and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering MHCH epitopes is generallly used, but a
competitive binding assay
may also be employed (Pound, supra}.
Various methods such as Scatchard analysis in conjunction with
radioirnmunoassay
techniques may be used to assess the affinity of antibodies for MHCH. Affinity
is expressed as an
association constant, Ka, which is defned as the molar concentration ofMHCH-
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 MHCH epitopes, represents the average affnity, or
avidity, of the antibodies for
MHCH. The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular MHCH 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
MHCH-antibody complex must withstand rigorous manipulations. Low-affinity
antibody
preparations with Ka ranging from about 106 to 10' L/mole are preferred for
use in
immunopurification and similar procedures which ultimately require
dissociation of MHCH,
preferably in active form, from the antibody (Catty, D. (1988) Antibodies.
Volume I: A Practical
Approach, IRL Press, Washington, DC; Liddell, J.E. and Cryer, A. (1991) A
Practical Guide to
Monoclonal Antibodies, 3ohn 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 fdr certain
downstream applications. For
example, a polyclonai 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 MHCH-
CA 02349212 2001-05-03
WO 00/26372 PCT/US99/26177
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 ai. supra.)
In another embodiment of the invention, the polynucleotides encoding MHCH, ar
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, the
complement of the polynucleotide encoding MHCH 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 polynucIeotides encoding MHCH. Thus,
corttplementary molecules ar
fragments may be used to modulate MHCH 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
MHCH.
Expression vectors derived from retroviruses, adenoviruses, or herpes or
vaccinia viruses, or
from various bacterial plasmids, may be used for delivery c>f nucleotide
sequences to the targeted
organ, tissue, or cell population. Methods which are well I<;nown to those
skilled in the art can be used
to construct vectors to express nucleic acid sequences complementary to the
polynucleotides encoding
MHCH. (See, e.g., Sambrook, supra; Ausubel, 1995, suarai.)
Genes encoding MHCH can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding MHCH. 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. Transients 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 ar antisense molecules (DNA, RNA, or PNA) to the
control, 5', or
regulatory regions of the gene encoding MHCH. Oiigonucieotides 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 poiymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances
using triplex DNA have been described in the literature. {C~ee, e.g., Gee,
J.E. et ai. (1994) in Huber,
B.E. and B.I. Carr, Molecular and Immunolo i~c Approaches, Futura Publishing,
Mt. Kisco NY, pp.
163-177.) A complementary sequence or antisense molecule may also be designed
to block
3I
CA 02349212 2001-05-03
WO 00/2b372 PCTIUS99/2b177
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 endonucteolytic cleavage:
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding MHCH.
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 I S and 20
ribonucleotides,
corresponding to the region of the target gene containing t:he 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
IS by any method known in the art for the synthesis of nucleiic acid
molecules. These include techniques
for chemically synthesizing oligonucleotides such as solidl phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding MHCH. 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 inhexent 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.)
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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 relatea 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 MHCH,
antibodies to MHCH, and mimetics, agonists, antagonists, or inhibitors of
MHCH. The compositions
may be administered alone or in combination with at least one other agent,
such as a stabilizing..
compound, which may be administered in any sterile, biocompatible
pharmaceutical carrier including,
but not limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered
to a patient alone, or in combination with other agents, drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered
by any
number of routes including, but not limited to, oral, intravenous,
intramuscular, infra-arterial,
intrameduilary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal,
I S 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 Reminnton'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 far 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
33
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WO 00/26372 PCT/US99126177
solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone,
carbopol gel, polyethylene
glycol, andlor titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for
product identification or to
characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as well as soft, sealed capsules made of gelatin and a coating, such
as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or
binders; such as lactose or
starches, lubricants, such as talc or magnesium stearate, and, optionally,
stabilizers. In soft capsules,
the active compounds may be dissolved or suspended in suitable liquids, such
as fatty oils, liquid, or
liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hanks' solution, Ringer's
solution, or physiologically buffered saline. Aqueous injection suspensions
may contain substances
which increase the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or
dextran. Additionally, suspensions of the active compounds may be prepared as
appropriate oily
injection suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or
synthetic fatty acid esters, such as ethyl oleate, triglycerides, or
liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the suspension may
also contain suitable
stabilizers or agents to increase the solubility of the compounds and allow
for the preparation of
highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art.
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 ofthe following: 1 mM to 50 mM histidine, O.I% to 2% sucrose, and 2% to
7% mannitol, at a
pH range of 4.5 to 5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate
container and labeled for treatment of an indicated condition. For
administration of MHCH, such
labeling would include amount, frequency, and method of administration.
34
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WO 00/26372 PCT/US99/26177
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 close 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
MHCH or fragments thereof, antibodies of MHCH, and agonists, antagonists or
inhibitors of MHCH,
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be determined
by standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDS° (the dose therapeutically effective in 50% of the
population) or LDS° (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 LDS°JEDS°
ratio. Pharmaceutical compositions
which exhibit large therapeutic indices are preferred. The. data obtained from
cell culture assays and
animal studies are used to formulate a range of dosage for human use. The
dosage contained in such
compositions is preferably within a range of circulating concentrations that
includes the EDS° with
little or no toxicity. The dosage varies within this range diepending upon the
dosage form employed,
the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practiitioner, 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 ~cg, up to a total
dose of
about i 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.
CA 02349212 2001-05-03
WO 00/26372 PCT/US99/26177
DIAGNOSTICS
In another embodiment, antibodies which specifically bind MHCH may be used for
the
diagnosis of disorders characterized by expression of MHCH, or in assays to
monitor patients being
treated with MHCH or agonists, antagonists, or inhibitors of MHCH. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for MHCH include methods which utilize the antibody arnd a label to detect
MHCH in human body
fluids or in extracts of cells or tissues. The antibodies ma;y 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 MHCH, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
MHCH expression. Normal
or standard values for MHCH expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibody to MHCH under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of MHCH
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 MHCH may
be used
for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The poiynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of MHCH
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of MHCH, and to monitor regulation of MHCH levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding MHCI-I or closely related
molecules may be used
to identify nucleic acid sequences which encode MHCH. 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 MHCH, 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 MHCH encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:2 or from
genomic sequences including promoters, enhancers, and introns of the MHCH
gene.
36
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WO 00/26372 PCT/US99126177
Means for producing specific hybridization probe<_~ for DNAs encoding MHCH
include the
cloning of potynucleotide sequences encoding MHCH or MHCH 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
potymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 3=P or 355,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/6iotin coupling
systems, and the like.
Polynucleotide sequences encoding MHCH may be used far the diagnosis of
disorders -.
associated with expression of MHCH. Examples of such disorders include, but
are not limited to, a
heart or skeletal muscle disorder such as angina, anaphylactic shock,
arrhythmias, asthma, Becker's
muscular dystrophy, cardiovascular shock, central core disease, Cushing's
syndrome, Duchenne's
muscular dystrophy, encephalopathy, epilepsy, hypertension, hypoglycemia,
Kearns-Sayre syndrome,
lactic acidosis, migraine, infectious myositis, polymyositis,,
dermatomyositis, inclusion body myositis,
myocardial infarction, myotonic dystrophy, myocarditis, myoclonic disorder,
ophthalmoplegia,
pheochromocytoma, and myopathies including cardiomyo;pathy, centronuciear
myopathy, ethanol
myopathy, lipid myopathy, mitochondria) rnyopathy nemaline myopathy, and
thyrotoxic myopathy; a
deveiopmentai disorder such as renal tubular acidosis, anemia, Cushing's
syndrome, achondroplastic
dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal
dysgenesis, WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation), Smith-
Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia, hereditary
keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis,
hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea
and cerebral palsy,
spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract,
and sensorineural
hearing loss; and a cell proliferative disorder such as actinic keratosis,
arteriosclerosis,
atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue
disease (MCTD), myelofibrosis,
paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and a
cancer including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma,
teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder,
bone, bone marrow, brain,
breast, cervix; gall bladder, ganglia, gastrointestinal tract, (heart, kidney,
liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis,
thymus, thyroid, or uterus.
The polynucleotide sequences encoding MHCH 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 MHCH
expression. Such qualitative or quantitative methods are yell known in the
art.
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WO 00!26372 PCTlUS99/26177
In a particular aspect, the nucleotide sequences encoding MHCH may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding MHCH 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 sarniple is
significantly altered in comparison to a
control sample then the presence of altered levels of nucleotide sequences
encoding MHCH 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
MHCH, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding MHCH, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified lby comparing the
values obtained from
normal subjects with values from an experiment in which a known amount of a
substantially purified
polynucieotide is used. Standard values obtained in this nnanner 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 abnoraaal 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 dcaigned from the sequences
encoding
MHCH 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 MHCH, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
38
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WO flfl/26372 PCT/US99/26I77
MHCH, 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 rnay also be used to quantify the expression of MHCH include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer 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 infarmation may be used
to determine gene
i S 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; Baldeschweiier et al. ( 1995) PCT application W095/251 I
16; Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997} Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et ai. ( 1997) U.S. Patent No. 5,605,662.)
In another embodiment of the invention, nucleic acid sequences encoding MHCH
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 P 1
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 MHCH 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.
39
CA 02349212 2001-05-03
WO 00!26372 PCT/US99I26177
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 infarmation 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 1 iq22-
23, any sequences mapping
to that area may represent associated or regulatory genes far 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 locatian due to translocation,
inversion, etc., among
normal, carrier, or affected individuals.
In another embodiment of the invention, MHCH, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intraceilullarly. The
formation of binding complexes
between MHCH and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysers, 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 MHCH, or
fragments thereof,
and washed. Bound MHCH is then detected by methods well known in the art.
Purified MHCH 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 MHCH specifically compete with a test compound
for binding MHCH.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with MHCH.
In additional embodiments, the nucleotide sequences which encode MHCH 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
CA 02349212 2001-05-03
WO OOI26372 PCT/US99/26177
properties as the triplet genetic code and specific base pair interactions.
Without. further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U:S. Ser. No. [Attorney Docket No. PF-0621 P, fsled November 5,
1998], are hereby
expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
The COLNTUT03 library was constructed from tumorous sigmoidal colon tissue,
obtained
from a 62-year-old Caucasian male during a sigmoidectorny and permanent
colostomy. Pathology
indicated an invasive grade 2 (of 4) adenocarcinoma. Patiient history included
hyperlipidemia,
cataract disorder, and dermatitis. Previous surgeries included cholecystectomy
and repair of indirect
inguinal hernia. Family history included benign hypertension, atherosclerotic
coronary artery disease,
hyperlipidemia, breast cancer, and prostate cancer.
The frozen tissue was homogenized and lysed in guanidinium isathiocyanate
solution using a
Polytron-PT 3000 homogenizes (Brinkmann Instruments, Westbury NY). RNA was
isolated as per
Stratagene's RNA isolation protocol {Stratagene, La Jolla CA). RNA was
extracted twice with acid
phenol, precipitated with sodium acetate and ethanol, resuspended in RNase-
free water, and treated
with DNase. Poly (A+) RNA was isolated using the OL1GOTEX kit {QIAGEN Inc,
Valencia CA).
Poly (A+) RNA was used to construct the COLN'CUT03 cDNA library according to
the
recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies).
The cDNAs
were fractionated on a SEPHAROSE CL4B column (Ame;rsham Pharmacia Biotech),
and those
cDNAs exceeding 400 by were ligated into the plasmid pINCY (Incyte
Pharmaceuticals, Palo Alto
CA). Recombinant plasmids were transformed into DHScc competent cells (Life
Technologies).
II. Isolation of cDNA Clones
Plasmids were recovered from host cells by in vivo excision using the UNIZAP
vector system
(Stratagene) or by cell lysis. Plasmids were purified using at least one of
the following: a Magic or
WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep
purification kit (Edge
Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,
QIAWELL 8
Ultra Plasmid purifcation systems or the R.E.A.L. PREP 96 plasmid purification
kit from QIAGEN.
Following precipitation, plasmids were resuspended in 0.11 ml of distilled
water and stored, with or
41
CA 02349212 2001-05-03
WO 00/26372 PCT/US99/26177
without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell iysates using direct
link PCB 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
S 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 Analyses
cDNA sequencing reactions were processed using standard methods or high-
throughput
instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) thermal cycler or
the PTC-200
thermal cycler (MJ Research} in conjunction with the HYDRA microdispenser
(Bobbins 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).
I5 Electrophoretic separation of cDNA sequencing reactions and detection of
labeled poiynucleotides
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 poiynucleotide 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 1 summarizes the tools, programs, and algorithms used and provides
applicable
2S descriptions, references, and threshold parameters. The first column of
Table 1 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 clusta) algorithm as incorporated into the
MEGALIGN multisequence
alignment program {DNASTAR), which also calculates the percent identity
between aligned
42
CA 02349212 2001-05-03
WO 00/26372 PCT/US99126177
sequences.
The poiynucieotide sequences were validated by rc;moving 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 polynucieotide sequences using programs based on Phred,
Phrap, and Consed, and
were screened for open reading frames using programs bared on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences were translated to derive the
corresponding full length
amino acid sequences, and these full length sequences were subsequently
analyzed by querying
against databases such as the GenBank databases (describe;d above), SwissProt,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family
databases such
as PFAM. HMM is a probabilistic approach which analyzes consensus primary
structures of gene
families. (See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:36I-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
N0:2. 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, s.~ra,, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in nucleotide databases such as GenBank ar LIF'ESEQ (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 °/a maximum BLAST score
I00
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
43
CA 02349212 2001-05-03
WO 00I2b372 PCT/US99l26177
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 MNCH 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 the
description of the invention.
V. Extension of MHCH Encoding Pofynucleotides
The full length nucleic acid sequences of SEQ IDS N0:2 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
24 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 Mg'-~, (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 l: 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: fib°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 pl
P1COGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1 X TE
44
CA 02349212 2001-05-03
WO 00/26372 PCT/US99/26177
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 ~l to 10 ~I 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 Cvi3I cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech}. For
shotgun sequencing, the digested nucleotides were separated on tow
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC I 8
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Si:ratagene) 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 1: 94°C, 3 min; Step 2: 94°C, IS sec; Step 3:
50°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, '_. min;
Step 7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with
20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing
primers and the DYENAMIC DIRECT kit (Amersham Ph,armacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Eimer).
In like manner, the nucleotide sequences of SEQ ID N0:2 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 NO:~: are employed to screen cDNAs,
genomic
DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about
20 base pairs, is
specifically described, essentially the same procedure is used.with larger
nucleotide fragments.
Oligonucleotides are designed using state-of the-art software such as OLIGO
4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~cCi of [y-
'zP] adenosine
CA 02349212 2001-05-03
WO 00/26372 PCT/US99/26177
triphosphate (Amersham Pharmacia Biotech), and T4 poivnucleotide kinase
(DuPont NEN, Boston
MA). The labeled oiigonucleotides are substantially purified using a SEPHADEX
G-25 supe~ne
size exclusion dextran bead column (Amersham Pharmac~a 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 ofthe 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, Schieicher & Schuell, Durham I~IH). 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 sodiurn citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VII. Microarrays
A chemical coupling procedure and an ink jet device can be used to synthesize
array
elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.} An
array analogous to a dot
or slot blot may also be used to arrange and link elements to the surface of a
substrate using thermal,
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
rmages.
Full-length cDNAs, Expressed Sequence Tags (SSTs), 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 subsequ~,ent drying. (See,
e.g., Schena, M. et al.
(1995) Science 270:467-470; Shaion, 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 MHCH-encoding sequences, or any parts thereof,
are used
46
CA 02349212 2001-05-03
W0. 00126372 PCTIUS99I26177
to detect, decrease, or inhibit expression of naturally occurring MHCH.
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
oligonucieotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of MHCH. To
inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5' sequence
and used to prevent promoter binding to the coding sequence. To inhibit
translation, a complementary
oligonucleotide is designed to prevent ribosomal binding to the MHCH-encoding
transcript.
IX. Expression of MHCH
Expression and purification of MHCH is achieved using bacterial or virus-based
expression
l0 systems. For expression of MHCH 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 (tic) 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).
i5 Antibiotic resistant bacteria express MHCH upon induction with isopropyl
beta-D-
thiogalactopyranoside (IPTG). Expression of MHCH in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Auto ranhica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding MHCH by either homologous recombination or
bacterial-mediated
20 transposition involving transfer plasmid intermediates. Viral infectivity
is maintained and the strong
poiyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera fru iperda (Sf9) insect cells in most casea, 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. Acid. Sci. USA 91:3224-3227; Sandig, V. et ai. (
1996) Hum. Gene Ther.
25 7:1937-1945.)
In most expression systems, MHCH 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
Iysates. GST, a 26-
kilodalton enzyme from Schistosoma janonicum, enables the purification of
fusion proteins on
30 immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
MHCH at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity
purification using commerciaEly available monoclonal and polyclonal anti-FLAG
antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues, enables
purification on metal-chelate
47
CA 02349212 2001-05-03
WO 00/26372 PCT/U599126177
resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel ( 1995,
suara, ch. 10 and 16). Purified MHCH obtained by these methods can be used
directly in the
following activity assay.
X. Demonstration of MHCH Activity
The assay for MHCH activity is based upon the ability of MHCH to interact with
actinomyosin filaments in vitro (Ho, G. and R.L. Chisholna (1997) J. Biol.
Chem. 272:4522-4527).
Actin-activated ATPase is assayed in buffer A {10 mM Tris-HCI, pH 7.6, 25 mM
KC1, 5 mM MgCI,,
0.1 mM CaCI.,, i mM ATP), 0-10 pM MHCH, 0-10 p.M actin, and 50 ug/ml myosin.
Ca'-+-activated
ATPase is assayed in buffer B {20 mM Tris-HC1, pH 8.0, ;500 mM KCI, 10 mM
CaCh, I mM ATP),
0-10 pM MHCH, and SO pg/ml myosin. Reactions are incubated at room temperature
for 5 min and
then quenched with acid, and the liberated inorganic phosphate (P;) is
quantified following organic
extraction.
In vitro motility assays are performed as follows.. Myosin is diluted to 200
p.glml in buffer C
(25 mM imidazole, pH 7.4, 25 mM KCI, 4 mM MgCh, 1 mM EGTA, 10 mM
dithiothreitol), applied
to a flow cell coated with nitrocellulose, and blocked with buffer C
containing 0.5 mg/ml BSA
(C/BSA). A solution of phalloidin-labeled actin is perfused followed by 1 mM
ATP in C/BSA to
remove myosin heads that bind actin in a rigorous fashion. After washing with
C/BSA to remove the
excess nonfluorescent actin, a solution of rhodamine-phal:loidin-labeled actin
and MHCH in CJBSA is
introduced. Active movement is initiated at room temperature by introducing
C/BSA containing 1
mM ATP and oxygen scavenger enzymes. Microscopic images of moving myotubes are
tracked for
up to 30s, and translocation velocities calculated using the; myotube
centroids to establish initial and
final positions for 2s or 4s samples during the continuous movement.
XI. Functional Assays
MHCH function is assessed by expressing the sequences encoding MHCH at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen, Carlsbad CA),
both of which
contain the cytomegalovirus promoter. 5-10 acg of recombinant vector are
transiently transfected into
a human cell Eine, for example, an endothelial or hematop~oietic cell line,
using either liposome
formulations or electroporation. 1-2 ,ug 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-
48
CA 02349212 2001-05-03
WO 00/26372 PCT/US99/261 T1
based technique, is used to identify transfected cells expre ssing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured.by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of MHCH on gene expression can lbe assessed using highly
purified
populations of cells transfected with sequences encoding MHCH 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
IS cells using magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake
Success NY). mRNA can be purified from the cells using methods well known by
those of skill in the
art. Expression of mRNA encoding MHCH and other genes of interest can be
analyzed by northern
analysis or microarray techniques.
XII. Production of MHCH Specific Antibodies
MHCH substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.;
Harrington, M.G. {1990} Methods Enzymol. 182:488-495}, or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the MHCH 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 oo those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-.terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, s_unra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to ICLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbiits are immunized with
the oligopeptide-
ICLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-
MHCH activity by, for example, binding the peptide or MHCH to a substrate,
blocking with 1 % BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
a9
CA 02349212 2001-05-03
WO 00126372 PCT/US99/26177
XIII. Purification of Naturally Occurring MHCH Using Specific Antibodies
Naturally occurring or recombinant MHCH is substantially purified by
imrnunoaffinity
chromatography using antibodies specific for MHCH. An immunoafl inity column
is constructed by
covalently coupling anti-MHCH 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 MHCH are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorb;ance of MHCH (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/MHCH 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 MHCH is collected.
XIV. Identification of Molecules Which Interact with MHCH
MHCH, or biologically active fragments thereof, are labeled with 'ZSI Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
l5 previously arrayed in the wells of a multi-well plate are incubated with
the labeled MHCH, washed,
and any wells with labeled MHCH complex are assayed. Data obtained using
different concentrations
of MHCH are used to calculate values for the number, affinity, and association
of MHCH with the
candidate molecules.
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the invention.
Although the invention has been described in connection vrith 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.
CA 02349212 2001-05-03
WO 00/26372 PCTIUS99126177
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51
CA 02349212 2001-05-03
WO 00/26372 PCT/US99126177
c7
t0
N ,
'C U '~ :y N
,C ', ~ ~ ~-i ~ o C
x °' ~
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a~
52
CA 02349212 2001-05-03
WO 00/26372 PCTIUS99/26177
SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
TANG, Y. Tom
CORLEY, Neil C.
GORGONE, Gina A.
GUEGLER, . Karl J .
BAUGHN, Mariah R.
<120> MYOSIN HEAVY CHAIN HOMOLOG
<130> PF-0621 PCT --
<140> To Be Assigned
<141> Herewith
<150> 09/187,060; unassigned
<151> 1998-11-05; 1998-11-05
<160> 4
<170> PERL Program
<210> 1
<211> 612
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1929760CD1
<400> 1
Met Phe Cys Pro Pro Gln Val Ser Cys Ser Leu Ser Leu Met Pro
1 5 10 15
Arg Leu Pro Ser Ile Arg His Trp Gln Gly Pro Ser His Pro Gly
20 25 30
Phe Leu Gly Pro Leu Phe Pro Ile Cys Ser Leu Gln Trp Pro His
35 40 45
Gly Phe Ser Ala Ile Phe Pro Gly Leu Leu Asp Val Tyr Gly Phe
50 55 60
Glu Ser Phe Pro Asp Asn Ser Leu Glu Gln Leu C:ys Ile Asn Tyr
65 70 75
AIa Asn Glu Lys Leu Ghi Gln His Phe Val Ala His Tyr Leu Arg
80 85 90
Ala G1n Gln Glu Glu Tyr Ala Val Glu Gly Leu C~lu Trp Sex Phe
95 100 105
Ile Asn Tyr Gln Asp Asn Gln Pro Cys Leu Asp Leu Ile Glu Gly.
110 215 120
Ser Pro Ile Ser Ile Cys Ser Leu Ile Asn G1u Glu Cys Arg Leu
125 130 ~ 135
Asn Arg Pro Ser Ser Ala Arg Gln Leu Gln Thr Arg Ile Glu Thr
140 145 150
Ala Leu Ala Gly Ser Pro Cys Leu Gly His Asn Lys Leu Ser Arg
~/l l
CA 02349212 2001-05-03
WO 00/26372 PCT/US99/26177
Zs5 160 165
Glu Pro Ser Phe Ile Val Val His Tyr Ala Gly Pro Val Arg Tyr
170 175 180
His Thr Ala G1y Leu Val Glu Lys Asn Lys Asp Pro Ile Pro Pro
185 190 lg5
Glu Leu Thr Arg Leu Leu Gln Gln Ser Gln Asp Pro Leu Leu Met
200 205 210
Gly Leu Phe Pro Thr Asn Pro Lys Glu Lys Thr Gln Glu Glu Pro
215 220 225
Pro Gly Gln Ser Arg Ala Pro Val Leu Thr Val Val Ser Lys Phe
230 235 240
Lys Ala Ser Leu Glu Gln Leu Leu Gln Val Leu His Ser Thr Thr
245 250 255
Pro His Tyr Ile Arg Cys Ile Lys Pro Asn Ser Gln Gly Gln Ala
260 265 270
Gln Thr Phe Leu Gln Glu Glu Val Leu Ser Gln Leu Glu Ala Cys
275 280 285
Gly Leu Val Glu Thr Ile His IIe Ser Ala Ala Gly Phe Pro Ile
290 295 300
Arg Val Ser His Arg Asn Phe Val Glu Arg Tyr Lys Leu Leu Arg
305 310 315
Arg Leu His Pro Cys Thr Ser Ser Gly Pro Asp Ser Pro Tyr Pro
320 325 330
Ala Lys Gly Leu Pro Glu Trp Cys Pro His Sex Glu Glu Ala Thr
335 340 345
Leu Glu Pro Leu Ile Gln Asp Ile Leu His Thr Leu Pro Val Leu
350 355 360
Thr Gln Ala Ala Ala Ile Thr Gly Asp Ser Ala Glu Ala Met Pro
365 370 375
Ala Pro Met His Cys Gly Arg Thr Lys Val Phe Met Thr Asp Ser
380 385 390
Met Leu Glu Leu Leu Glu Cys Gly Arg Ala Arg Val Leu Glu Gln
395 400 405
Cys Ala Arg Cys Ile Gln Gly Gly Trp Arg Arg His Arg His Arg
410 415 420
Glu Gln GIu Arg Gln Trp Arg Ala Val Met Leu Ile Gln Ala Ala
425 430 435
Ile Arg Ser Trp Leu Thr Arg Lys His I1e Gln .Arg Leu His Ala
440 445 450
Ala Ala Thr Val Ile Lys Arg Ala Trp Gln Lys 'Trp Arg Ile Arg
455 460 465
Met Ala Cys Leu Ala Ala Lys Glu Leu Asp Gly 'Val Glu Glu Lys
470 475 480
His Phe Ser Gln Ala Pro Cys Ser Leu Ser Thr Ser Pro Leu Gln
485 490 495
Thr Arg Leu Leu Glu Ala Ile Ile Arg Leu Trp :Pro Leu Gly Leu
500 505 510
Val Leu Ala Asn Thr Ala Met Gly Val Gly Ser Phe Gln Arg Lys
515 520 525
Leu Val Val Trp Ala Cys Leu Gln Leu Pro Arg Gly Ser Pro Ser
530 535 540
Ser Tyr Thr Val Gln Thr Ala Gln Asp Gln Ala tily Val Thr Ser
545 550 555
Ile Arg Ala Leu Pro Gln Gly Ser Ile Lys Phe Isis Cys Arg Lys
560 565 570
2/11
CA 02349212 2001-05-03
WO OU/26372 PCT/US99/26177
Ser Pro Leu Arg Tyr Ala Asp Tle Cys Pro,Glu Pro Ser Pro Tyr
575 580 585
Ser Ile Thr Gly Phe Asn Gln Ile Leu Leu Glu Arg His Arg Leu
590 595 600
Ile His Val Thr Ser Ser A1a Phe Thr Gly Leu Gly
605 610
<210> 2
<211> 2109
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1929760CB1
<400> 2
tgatgctctg ggctgtcttc acacttcatt tgggtttcct gcttgctctg agctctacag 60
gggaatgggg tagagatggg agccaccttg ggtggagggt ggggaaggta tgttctgccc 120
accacaggtg tcatgctcac tcagcctgat gcccaggctg ccaagtataa ggcattggca 180
ggggcccagc caccctgggt tccttggtcc cctattcccc atctgctccc tgcagtggcc 240
ccatgggttc tctgccatct tcccaggcct gctggatgtg tatggatttg aatcatttcc 300
tgacaacagt ctggaacagt tgtgcatcaa ctacgccaat gagaagctgc agcagcattt 360
tgtggctcac tacctaaggg cccagcagga ggaatacgca gttgagggcc tggagtggtc 420
attcatcaac taccaggaca accagccctg tttggatctc attgagggaa gccccatcag 480
catctgctcc ctcataaatg aggaatgccg cctcaatcga cccagcagcg cacgccagct 540
ccagacacgc attgagactg ccctggcagg cagcccctgc ctgggccaca ataagctcag 600
ccgggagccc agcttcattg tggtgcatta tgcggggcct gtgcggtacc acacagcagg 660
cctggtggag aagaacaagg accctatccc acctgagctg accaggctcc tgcagcaatc 720
ccaggacccc ctgctcatgg ggctgtttcc tactaacccc aaagagaaga cccaggagga 780
accccctggc cagagcaggg cccctgtgtt gaccgtggtg tccaagttca aggcctcact 840
ggagcagctt ctgcaggtcc tacacagcac cacgccccac tacattcgct gcatcaagcc 900
caacagccag ggccaggcgc agacctttct ccaagaggag gtcctgagcc agctggaggc 960
ctgtggcctc gtggagacca tccatatcag tgctgctggc ttccccatcc gggtctctca 1020
ccgaaacttt gtagaacgat acaagttact aagaaggctt catccttgca catcctctgg 1080
ccccgacagc ccatatcctg ccaaagggct ccctgaatgg tgtccacaca gcgaggaagc 114 0
cacgcttgaa cctctcatcc aggacattct ccacactctg ccggtcctaa ctcaggcagc 1200
agccataact ggtgactcgg ctgaggccat gccagccccc atgcactgtg gcaggaccaa 1260
ggtgttcatg actgactcta tgctggagct tctggaatgt gggcgtgccc gggtgctgga 1320
gcagtgtgcc cgctgcatcc agggtggctg gaggcgacac cggcaccgag agcaggagcg 1380
gcagtggcgg gccgtcatgc tcatccaggc agccattcgt tcctggttaa ctcggaaaca 1440
catccagagg ctgcatgcag ctgccacagt catcaagcgt gcatggcaga agtggagaat 1500
cagaatggcc tgccttgctg ctaaagagct ggatggtgtg gaagaaaaac acttctctca 1560
agctccctgt tccctgagca ~cctcgccgct gcagaccagg ctcctggagg caataatccg 1620
cctctggccc ctgggactgg tcctggccaa tacggctatg ggtgtaggca gctttcagag 1680
gaaattagtg gtctgggctt gcctccagct ccccaggggc agccccagta gctacactgt 1740
ccagacagca caagaccagg ctggtgtcac gtccatccga gcgctgcctc agggatcgat 1800
aaagtttcac tgcagaaagt ctccactgcg gtatgctgac atctgccctg aaccttcacc 1860
ctacagcatt acaggcttta atcagattct gctggaaaga cacaggctga tccacgtgac 1920
ctcttctgcc ttcactgggc tggggtgatc cttggtgcct ttgtttccac aaggcctttt 1980
cctgccccct gccttgccaa agacatttaa tcagcacaca gctgccagac tattcccaca 2040
gtgctccaaa tgcacatgaa caacagtgac ggctccagcc ttcgacccag agccccgtgc 2100
ccagtgcgt 2109
3/11
CA 02349212 2001-05-03
WO 00/26372 PCT/US99/26177
<210> 3
<211> 1839
<212> PRT
<213> Caenorhabditis elegans
<300>
<308> GenBank TD No: 81279777
<400> 3
Met Phe Asn Tyr Ser Lys Ile Phe Gln Ile His Arg Ala Cys Ser
1 5. 10 15
Pro Asn Arg Lys Lys Ile Gly Ser Ile Gln Tyr Gly Arg Arg Arg
20 25 30
His Ser Trp Gln Gly Pro Val Val Pro Ala Ala Lys Leu Gln Val
35 40 4S
Leu Ile Lys Gly Val Arg Ile Trp His Arg His Pro Thr Leu Val
50 55 60
Trp Ile Gly Ala Thr Leu Glu Glu Asp Ile Thr Phe Gln Thr Arg
6S 70 75
Asn Val Arg Ile Arg Leu Glu Asp Asp Thr Glu Val Glu Tyr Ala
80 8S 90
Ile Lys Ser Leu Asp Gln Leu Pro Phe Leu Arg Asn Pro Ala Phe
95 100 105
Leu Val Gly Lys Asp Asp Leu Thr Leu Leu Ser Tyr Leu His Glu
110 115 120
Pro Ala Val Leu His Asn Leu Gln Val Arg Phe Val Lys Gly Ser
125 130 13S
Ser Ile Tyr Thr Tyr Cys Gly Ile Val Leu Val Ala Ile Asn Pro
140 145 150
Tyr Ala Asp Cys Ser His Ile Tyr Gly Glu Glu I:le Ile Gln Val
155 160 165
Tyr Arg Gly Ala Gly Lys Ser Ala Arg Glu Met Asp Pro His Ile
170 175 180
Phe Ala Val Ala Glu Glu Ala His Phe Asp Met C~ly Ala Phe Gly
185 190 195
Lys Ser Gln Ser Ile Ile Val Ser Gly Glu Ser C;ly Ala Gly Lys
200 205 210
Thr Val Ser Ala Lys Phe Val Met Arg Tyr Leu Ala Ser Val Ala
215 220 225
Ala Ser Lys Thr Arg Asn Gly Gly Thr Thr Ser I:le Glu Ala Arg
230 235 240
Val Leu Ala Ser Asn Pro Ile Met Glu Ser Ile Gly Asn Ala Lys
245 250 255
Thr Ile Arg Asn Asp Asri Ser Ser Arg Phe Gly Lys Phe Ile Gln
260 265 270
Ile Asn Phe Cys Glu Arg Gly Arg Arg Ile Val Gly Ala Glu Met
27S 280 285
Lys Thr Tyr Leu Leu Glu Lys Ser Arg l.eu Val Phe Gln Ala Pro
290 295 300
Gly Glu Arg Asn Tyr His Ile Phe Tyr Gln Leu C'.ys Ala Ala Arg
305 310 315
Asn His Gln Val Leu Lys Asp Leu His Leu Gly Pro Cys Glu Ser
320 325 330
Tyr Ser Tyr Leu Thr Gln Gly Gly Asp Ser Arg Ile Pro Gly Val
4/1 l
CA 02349212 2001-05-03
WO 00/26372 PCT/US99l26177
335 340 345
Asp Asp Lys Ala Asp Phe Glu Ala Leu Leu Lys Ala Leu Gln Leu
350 35S 360
Leu Gly Phe Asp Glu Lys Gln Met Ser Asp Val :Phe Arg Leu Leu
365 370 375
Ala Gly Leu Leu Leu Leu Gly Asn Val His Phe Glu Asn Gly Glu
380 385 390
Gly Ser Ser Ala Val Ser Ala Ser Ser Cys Gln Glu Ile Ser Arg
395 400 405
Leu Cys Arg Glu Phe Trp Lys Ile Ser Glu Ser i~sp Leu Arg Ile
410 415 420
Trp Leu Thr Arg Arg Glu Ile Arg Ala Val Asn Glu ile Val Thr
425 430 435
Lys Pro Leu Thr Lys Asn Glu Ala Val Arg Ser i~rg Asp Ala Leu
440 445 450
Thr Lys Met Leu Tyr Ser His Leu Phe Gly Trp 7~eu Val Asp Lys
455 460 465
Ile Asn Glu Ala Leu Asn Glu Lys Asp Lys Leu i~sp Gly Thr Asn
470 475 480
G1n Lys Lys Arg Pro Asp Arg Phe Ile Gly Val heu Asp Ile Tyr
485 490 495
Gly Phe Glu Thr Phe Asp Val Asn Ser Phe Glu Gln Phe Ser Ile
500 505 510
Asn Tyr Ala Asn Glu Lys Leu Gln Gln Gln Phe Asn Gln His Val
515 520 525
Phe Lys Leu Glu Gln Glu Glu Tyr Ile Arg Glu Glu Ile Glu Trp
530 535 540
Val Arg Val Asp Phe His Asp Asn Gln Pro Ala :Cle Asp Leu Ile
545 550 555
Glu Gly Pro Val Gly Met Ile Asn Leu Leu Asp t~lu Gln Cys Lys
560 565 570
Arg Leu Asn Gly Sex Asp Ala Asp Trp Leu Ser Gln Leu Gln Asn
575 S80 585
Ser Thr Glu Leu Lys Arg Asn Pro Gln Leu Ala J?he Pro Lys Val
590 595 600
Arg Ser Asn Asp Phe Ile Val Arg His Phe Ala l~l.a Asp Val Thr
605 610 615
Tyr Sex Thr Asp Gly Phe Val Glu Lys Asn Arg Asp Ala Ile Gly
620 625 630
Glu Gln Leu Leu Asp Val Val Val Ala Ser Lys Phe Pro Phe Ile
635 640 645
Arg Thr Val Ile Gly Ser Thr Ala Pro Thr Ser Val Ser Ser Ser
650 655 660
Ser Ser Ser Ser Thr Pro Gly Lys Arg Thr Ile hys Lys Thr Val
665 ' 670 675
AIa Ser Gln Phe Arg Asp Ser Leu Lys Glu Leu Met Ser Va1 Leu
680 685 690
Cys Ser Thr Arg Pro His Tyr Val Arg Cys Ile hys Pro Asn Asp
695 700 705
Ser Lys Ile Sex Phe Asp Phe Glu Pro Lys Arg Ala Ile Gln Gln
710 715 720'
Leu Arg Ala Cys Gly Val Leu Glu Thr Va1 Arg 3:1e Ser Ala Ala
725 730 735
Gly Phe Pro Ser Arg Tyr Pro fiyr Glu Glu Phe Ala Arg Arg Tyr
740 745 750
S/11
CA 02349212 2001-05-03
WO 00/26372 PCT/US99126I77
Arg Val Ile Tyr Thr Lys Glu Ala Ala Leu Trp Arg Asp Lys Pro
755 760 765
Lys Gln Phe Ala Glu Leu Ala Cys Gln Gln Cys Leu Glu Glu Gly
770 775 780
Lys Tyr Ala Val Gly Lys Thr Lys Ile Phe Leu Arg Thr G1y Gln
785 790 795
Val Ala Val Leu Glu Arg Val Arg Leu Asp Thr Leu Ala Ala Ala
800 805 810
Ala Thr Val Ile Gln Lys Met Trp Lys Gly Phe Leu Ala Arg Arg
815 820 825
Lys Tyr Glu Thr Met Arg Arg Ser Leu Leu Ile Val Gln A1a Ser
830 835 840
Leu Lys Ala Phe Leu Ala Phe Arg Arg Ile Lys Tyr Leu Gln Met
845 850 855
His Arg Ala Val Ile Val Met Gln Ser Ala Val Arg Gly Tyr Leu
860 865 870
Glu Arg Arg Lys Tyr Glu Gln Ile Arg Asp Ser Ile Ile Gly Ile
8?5 880 885
Gln Ala Met Phe Lys Ala Asn Arg Val Arg Arg Tyr Val Glu Lys
890 895 900
Leu Arg Tyr Glu Lys Ser Ala Tle Thr Ile Gln Ala Ala Trp Arg
905 910 915
Gly Tyr Leu Ala Arg Arg Glu Gln Ile Ala Asn Arg Lys Lys Val
920 925 930
Val Met Val Gln Cys Ala Val Arg Lys Trp Leu Ala Lys Arg Arg
935 940 945
Leu Arg Glu Leu Lys Ile Glu Ala Arg Ser Val Gly His Leu Gln
950 955 960
Lys Leu Asn Thr G1y Leu Glu Asn Lys Ile Ile Glu Leu Gln Met
965 970 975
Arg Leu Asp Ile Ala Asn Ala Arg Thr Lys Glu Glu Ala Glu Lys
980 985 990
Phe Ala Thr Ala Ser Lys Asn Leu Gln Lys Thr Lys Ala Asp Leu
995 1000 1005
A1a Met Met Glu Ala Glu Arg Leu Thr Leu Leu Glu Ala Arg Asn
1010 1015 1020
Arg Val Glu Val Leu Gln Glu Glu Val Glu Arg Leu Glu Thr Glu
1025 1030 1035
Cys Asp Leu Lys Glu Ala Gln Arg Gly Gly Met Glu Thr Lys Met
1040 1045 1050
Val Glu Leu Gln Ser Arg Leu Asp Gln Phe Gln Met Gln Ser Glu
1055 1060 1065
Ser Gly Gln Thr Ile Val Glu Leu Thr Glu Gln Leu Glu Lys Ala
1070 1075 1080
Lys Ala Asp Arg Val Leu Trp Asp Glu Glu Arg Gln Arg Met Glu
1085 1090 1095
Ala Ala Leu Asn Thr Glu Arg Ser Ala Arg Asn Ala Leu Asp Ala
1100 1105 1110
Glu Met Ala Ala Met Arg Glu Gln Leu Met Lys Asn Val Asp Leu
11IS 1120 1125
Phe Glu Ser Ser Thr Phe Gln Lys Arg Pro Ser Gln Lys Lys Asn
1130 1135 1140
Arg Asp Asp Asp Ser Cys Ser Arg Thr Thr Ser ASn Leu Ser Gln
1145 1150 1155
Leu Thr Gly Ser Phe Thr Ala Glu Thr Ile Asn Gly Val His Ser
6/11
CA 02349212 2001-05-03
WO 00/26372 PCTIUS99126177
II60 1165 1170
Thr Ser Arg Gly Ser Pro Glu Val Leu Leu Asp Asn Met Ala Ser
1175 1180 1185
Thr Phe Glu Gln Leu Arg Met Ile Asn Asp Leu Arg Gln Arg Asn
1190 1195 1200
Glu His Cys Gln Arg Glu Thr Giu Arg Met Lys Ala Ile Ile Glu
1205 1210 1215
Ala Ser Thr Leu Ile Glu Thr Leu Asp Lys Lys Thr Ser Leu Lys
1220 1225 1230
Ala Phe Glu Ser Ile Arg Val Gly Glu Leu Glu Gly Ala Tyr Asn
1235 1240 1245
Arg Leu Lys Asn Asp Met Glu Arg Leu Val Ser Gly Glu Asn Gly
1250 1255 1260 ..
AIa Thr His Ser Val Phe Glu Arg Ile Met Glu Glu Asn Glu Arg
1265 1270 1275
Leu Arg Glu Glu Ala Val Glu Leu Arg Sex Met Leu Ser Ser His
1280 1285 1290
Phe Glu Lys Gln Ser Val Ala Gly Ser Ser Gly Tyr Arg Arg Ser
1295 1300 1305
Pro Arg Pro Asp Ser Gly His Cys Ser Gly Ala Asp Ser Glu Asp
1310 1315 1320
Gly Ser Ser Gly Ala Asp Leu Glu Glu Asp Leu Cys Ile Glu Arg
1325 133 0 1335
Gln Cys Arg His Leu Lys Asn Leu Ala Glu Asn Leu Thr Lys Met
1340 1345 1350
Leu Thr Asn Gln Asn Leu Glu IIe Glu Arg Leu Gln Gln Gln Leu
1355 1360 1365
Arg Phe Ser Glu Ser Gln Thr Val Phe Arg Pro Ser Asp Cys Ser
1370 1375 1380
Leu Asp Glu Ala Val Arg Gly Ala His Lys Gln Thr Gln Leu Leu
1385 1390 1395
Ala Gln Gln Asn Met Asp Leu Asn Asp Lys Leu Thr Arg Gln Ser
1400 1405 1410
Glu Glu Leu Ala Glu Ala Arg Ala Gln Leu Arg Gly Tyr Ser Gly
1415 1420 1425
Pro Leu Gly Leu Glu Asn Ala Ser Asp Glu Glu Ile Ile Arg Leu
1430 1435 1440
Glu Ala Phe Glu Lys Gly Sex Ile Lys His Ser Gly Phe Leu Glu
1445 1450 1455
Val Tyr Asn val Pro Glu Phe Ala Arg Ile Ile Val Cys Glu Leu
1460 1465 1470
Lys Pro Thr Leu Ala Arg Leu Leu Thr Lys Asn Leu Pro Ala Tyr
1475 1480 1485
Leu Leu Val Ala Ala Phe Arg Asn His Asp Glu Lys Arg Asp Glu
1490 1495 1500
Thr Ala Leu Thr Gly Leu Phe Ser Ser Val His Leu Val Leu Lys
1505 1510 1515
Asp Thr Ile Ser Arg Sex His Asp Leu Asp Leu Leu Ser Leu Trp
1520 1525 1530
Leu Val Asn Leu Trp Arg Leu Phe Asn Leu Leu_Arg Gln Tyr Ser
1535 1540 1545
Gly Glu Asp Ser Gln Pro Glu Trp His Val Ala Asn Thr GIu Thr
1550 1555 1560
Gln Asn Ser Tyr Arg Phe Lys Ala Tyr Asp Val Ala Pro Ile Arg
1565 1570 1575
7/1 ~
CA 02349212 2001-05-03
WO 00/2b372 PCTIUS99l2b177
Asp Gln Leu Lys Leu Arg Ile Glu Glu Cys Tyr Thr Ser Leu Met
1580 1585 1590
Lys Lys Ala Ile Glu His Val Leu Sex Pro Lys Ile Val Pro Gly
1595 1600 1605
Ile Leu Gln His Glu Ser Ser Ser Asp Leu Met Thr Ala Gly Gln
1610 1615 1620
Glu Arg Arg Asp Arg Asn Ser Gly Ser Val Glu Ser Gln Arg Lys
1625 1630 1635
Ser Leu Asp Asp Leu Leu Gln Phe Met Glu Ile Val His Thr Lys
1640 1645 1650
Leu Thr Thr Tyr Gly Gly Asp Asp Ile Val Val hys Gln Val Ile
1655 1660 1665
Gly Gln Met Ala Arg Trp Met Cys Ala Leu Ala heu Asn Tyr Met -.
1670 1675 1680
Met Phe Arg Arg Glu Leu Cys Asn Phe Glu Lys Ala Ile Gln Ile
1685 1690 1695
Lys His Asn Val Thr Gln Ile Gln Asn Trp Leu Asn Ala Lys Gly
1700 1705 1710
Leu Ser Asp Cys Arg Asp His Phe Glu Pro Leu Val Gln Ala Cys
1715 1720 1725
His Leu Leu Gln Ser Arg Lys Asp Pro Ser Asn Leu Asp Thr Leu
1730 1735 1740
Cys Gly Glu Met Thr Ser Arg Leu Lys Pro Arg Gln Val Val Ala
1745 1750 1755
Ile Leu Gln His Tyr Asp Pro Ser Asp Glu Met Glu Asp Gly Leu
1760 1765 1770
Ser Pro Glu Phe Leu Val Gln Ile Gln Lys Lys Leu Asn Glu Arg
1775 1780 1785
Ala Ile Ala Asn Asn Asp Pro Ile Glu Asp Lys Asp Lys Leu Ile
1790 1795 1800
Met Leu Gly Thr Tyr Leu Pro Pro Phe Asp Thr Gln Pro Phe Ser
1805 1810 181-5
Tyr Ser Asp Phe Pro Leu Glu Thr Leu Ser Leu IPro Ser Cys Leu
1820 1825 1830
His Met Gln Ser Val Cys Arg Leu Val
1835
<210> 4
<211> 1120
<212> PRT
<213> Helianthus annuus
<300>
<308> GenBank ID No: g2444174
<400> 4
Met Asp Arg Val Val Asp Asp Asp Ser Pro Tyr Gly Gln Gly Ser
1 5 10 15
Ser Phe Leu Leu Asn Asp Arg Pro Ser Val Asp Asp Val Asn Asp
20 25 30
Asp Asp Asp Ala Asp Val Asn Pro Ser Val Ser :~la Gln Gly Ser
35 40 45
Val Leu Gly Ser Trp Gly Asn Lys Lys Trp Gly ,Asp Thr Ala Ser
8/11
CA 02349212 2001-05-03
WO 00!26372 PCT/US99/26177
50 55 60
Tyr Ile Ala Lys Lys Lys Leu Gln Ser Trp Phe Gln Thr Ser Asp
65 70 75
Gly Asn Trp Glu Leu Ala Lys Ile Leu Ser Ile Thr Gly Ser Glu
80 85 90
Ser Leu Met Ser Leu Ser Glu Glu Lys Val Leu :Lys Val Ser Ser
95 . 100 105
Asp Ser Leu Leu Pro Ala Asn Pro Glu Ile Leu Asp Gly Val Asp
110 115 120
Asp Leu Met Gln Leu Ser Tyr Leu Asn Glu Pro ;5er Val Leu Tyr
125 130 135
Asn Leu Gln Tyr Arg Tyr Asp Arg Asp Met Ile 'Pyr Ser Lys Ala
140 145 150 -.
Gly Pro Val Leu Val Ala Ile Asn Pro Phe Lys :Lys Ile Pro Leu
155 160 165
Tyr Gly Sex Asp Tyr Ile Glu Ala Tyr Lys Arg :Lys Ser Ile Asp
170 175 180
Asn Pro His Val Tyr Ala Ile Ala Asp Thr Ala Ile Arg Glu Met
185 190 195
Ile Arg Asp Glu Val Asn Gln Ser Ile Vai Ile Ser Gly Glu Ser
200 205 210
Gly Ala Gly Lys Thr Glu Thr Pro Lys I1e Ala IHet Gln Tyr Leu
215 220 225
Ala Ala Leu Gly Gly Gly Asp Ala Arg G1u Ser Gly Ile Leu Ser
230 235 240
His Asn Gly Cys Arg Thr Pro Arg Arg Ala Glu Ala Phe Gly Asn
245 250 255
Ala Lys Thr Ser Arg Asp Asn Asn Ser Ser Arg Ile Gly Lys Leu
260 265 270
Ile Glu Ile His Phe Ser Glu Thr Gly Lys Ile Ser Gly Ala Lys
275 280 285
Ile Gln Thr Phe Leu Leu Glu Lys Ser Arg Val 'Val Gln Cys Thr
290 295 300
Asp Gly Glu Arg Ser Tyr His Ser Phe Tyr Gln :Geu Cys Ala Gly
305 310 315
Ala Pro Pro Ser Leu Arg Glu Lys Leu Asn Leu Lys Ser Ala Arg
320 325 330
Glu Tyr Lys Tyr Phe Gln Gln Ser Thr Cys Tyr Ser Ile Asn Gly
335 340 345
Val Asp Asp Ala Glu Glu Phe Arg Val Val Val Glu Ala Leu Asp
350 355 360
Ala Val His Val Ser Lys Glu Asn Gln Glu Asn .Ala Phe Ala Met
365 370 375
Leu Ala Aia Val Leu Trp Leu Gly Asn Val Thr Phe Ser Ile Val
380 385 390
Asp Asn Glu Asn His Val Glu Pro Ile Ile Asp .Asp Ala Leu Leu
395 400 405
Asn Val Ala Lys Leu Ile Gly Cys Glu Ala Asp .Asp Leu Lys Leu
410 415 420
Ala Leu Ser Thr Arg Asn Met Lys Val Gly Asn .Asp Ile Ile Val
425 430 435
Gln Lys Leu Thr Leu Ala Gln Ala Ile Asp Thr .Arg. Asp Ala Leu
440 445 450
Ala Lys Ser Ile Tyr Ser Cys Leu Phe Asp Trp :Leu Val Glu G1n
455 460 465
9/11
CA 02349212 2001-05-03
WO 00/26372 PCT/US99/26I77
Ile Asn Lys Ser Leu Ala Val Gly Lys Arg Arg 'Phr Gly Arg Ser
470 475 480
Ile Ser Ile Leu Asp Ile Tyr Gly Phe Glu Ser :Phe Asp Val Asn
485 490 495
Ser Phe Glu Gln Phe Cys Ile Asn Tyr Ala Asn Glu Arg Leu Gln
500 505 510
Gln His Phe Asn Arg His Leu Phe Lys Leu Glu Gln Glu Glu Tyr
515 520 525
Ile Gln Asp Gly Ile Asp Trp Ala Lys Val Asp :Phe Glu Asp Asn
530 535 540
Gln Asp Cys Leu Asn Leu Phe Glu Lys Lys Pro :Leu Gly Leu Met
545 550 555
Thr Leu Leu Asp Glu Glu Ser Thr Phe Pro Asn (.;ly Thr Asp Met -.
560 565 5T0
Thr Phe Ala Thr Lys Leu Lys Gln His Leu Lys Thr Asn Ser Cys
575 580 585
Phe Arg Gly Glu Arg Gly Lys Ala Phe Thr Val l3is His Tyr Ser
590 595 600
Gly Glu VaI Thr Tyr Asp Thr Ser Gly Phe Leu Glu Lys Asn Arg
605 610 615
Asp Leu Leu His Leu Asp Ser Ile Gln Leu Leu Ser Ser Cys Thr
620 625 630
Cys Glu Leu Pro Gln Ala Phe Ala Ser Asn Met 7Leu Ser Leu Ser
635 640 ~ 645
Glu Lys Pro Val Pro Gly Pro Leu His Lys Ser Gly Gly Ala Asp
650 65S 660
Ser Gln Lys Leu Ser Val Val Thr Lys Phe Lys t=ly Gln Leu Phe
665 670 675
Gln Leu Met Gln Arg Leu Glu Ser Thr Thr Pro Iiis Phe Ile Arg
680 685 690
Cys Ile Lys Pro Asn Asn Ser Gln Ser Pro Gly :Cle Tyr His Gln
695' 700 705
Gly Leu Val Leu Gln Gln Leu Arg Cys Cys Gly Val Leu Glu Val
710 715 720
Val Arg Ile Ser Arg Ser Gly Phe Pro Thr Arg Pdet Ser His Gln
725 730 73S
Lys Phe Ala Arg Arg Tyr Gly Phe Leu Leu Leu <slu His Val Ala
740 745 750
Ser Gln Asp Pro Leu Ser Val Ser Val Ala Ile Leu His Gln Phe
755 760 765
Asp Ile Leu Pro Glu Met Tyr Gln Ile GIy Tyr '.Chr Lys Leu Phe
770 775 780
Phe Arg Thr Gly Gln Ile Gly Lys Leu Glu Asp '.Phr Arg Asn Arg
785 790 795
Thr Leu Asn Gly Ile Leu Arg Val Gln Ser Cys Phe Arg Gly His
800 805 810
Lys Ala Arg Gln Tyr Met Lys Glu Leu Lys Arg Gly Ile Phe Asn
815 820 825
Leu Gln Ala Phe Ala Arg Gly Glu Lys Thr Arg Lys Glu Phe Ala
830 835 840
Ile Leu Val His Arg His Arg Ala Ala Val His :fle Gln Lys His
845 850 855
Ile Lys AIa Lys Ile Sex Lys Lys Arg Phe Glu Asp Val His Gly
860 865 870
Ala Thr Ile Thr Leu Gln Aia Val Ile Arg Gly 'Prp Leu Val Arg
10/11
CA 02349212 2001-05-03
WO 00126372 PCTIUS99i26177
875 880 885
Arg Cys Ser Gly Asp Ile Ala Leu Leu Gln Phe Gly Ser Gly Lys
890 895 900
Gly Asn Gly Ser Asp GIu Val Leu Val Lys Ser Ser Tyr Leu Ala
905 910 915
Glu Leu Gln Arg Arg Ile Leu Lys Ala Glu Ala Gly Leu Arg G1u
920 925 930
Lys Glu Glu Glu Asn Asp Ile Leu His Gln Arg Leu Gln Gln Tyr
935 940 945
Glu Asn Arg Trp Ser GIu Tyr G1u Leu Lys Met Lys Ser Met Glu
950 955 960
Glu Val Trp Gln Lys Gln Met Arg Ser Leu Gln Ser Ser Leu Ser
965 970 975
Ile Ala Lys Lys Ser Leu Ser Tyr Asp Asp Ser Glu Arg Asn Ser
980 985 990
Asp Ala Ser Ile Asn Thr Ala Asn Asp Glu Thr Asn Pro Pro Trp
995 1000 1005
Asp Ala Ala Thr Asn Gly Arg Arg Asn Gly Val Glu Asn Val Arg
1010 1015 1020
Pro Met Ser Ala Gly Leu Ser Val Ile Sex Arg Leu Ala Glu Glu
1025 1030 1035
Phe Glu Gln Arg Ser Gln Val Phe Gly Asp Asp Ala Lys Phe Leu
1040 1045 1050
Val Glu Val Lys Ser Gly Gln Val Glu Ala Asn Leu Asn Pro Asp
1055 1060 1065
His Glu Leu Arg Arg Leu Lys Gln Met Phe Glu G1y Trp Lys Lys
1070 1075 1080
Asp Tyr Thr. Ala Arg Leu Arg Glu Thr Lys Val Ile Leu Asn Lys
1085 1090 1095
Leu Gly His Glu Asp Gly Asp Gly Glu Lys Gly Lys Lys Lys Trp
1100 1105 1110
Trp Gly Arg Leu Asn Ser Ser Arg Val Asn
1115 1120
1 ill 1
