Note: Descriptions are shown in the official language in which they were submitted.
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LYSINE-RICH STATHERIN PROTEIN
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of a lysine-
rich statherin
protein and to the use of these sequences in the diagnosis, treatment, and
prevention of neoplastic and
autoimmune/inflammatory disorders, and infectious diseases.
BACKGROUND OF THE INVENTION
Low molecular weight proteins, polypeptide chains that are less than about one
hundred
amino acid residues (aa), have become the focus of investigation as components
of important
molecular and biochemical pathways during cell growth, tissue differentiation,
inflammation
disorders, and the immune response. Examples of such proteins are histatins
and statherins.
Human histatins are a family of low molecular weight (51 to 57 aa), neutral to
very basic,
histidine-rich salivary proteins. Histatins are believed to function as part
of the nonimmune host
defense system in the oral cavity. Salivary histatins (HSTs) are potent in
vitro antifungal and
antibacterial agents and have great promise as therapeutic agents in humans
with oral candidiasis.
Candida albicans is the predominant species of yeast isolated from patients
with oral candidiasis,
which is frequently a symptom of human immunodeficiency virus (HIV) infection
and is a criterion
for staging and progression of AIDS. 'z5I-HST binding studies indicated that
C. albicans expressed a
class of saturable binding sites, numbering 8.6 x 105 sites/cell. HSTs are
also effective in killing
another medically important opportunistic fungal pathogen, Cryptococcus
neoformans, which has
become a new threat among immunocompromised patients, including those with
AIDS (Tsai, H. and
L.A. Bobek (1997) Biochim. Biophys. Acta 1336:367-369).
Human statherin (STATH) is a low-molecular weight (43 aa) acidic
phosphoprotein that acts
as an inhibitor of precipitation of calcium phosphate salts in the oral
cavity. STATH also has affinity
to hydroxyapatite and interacts with oral bacteria on absorption to
hydroxyapatite (Lamkin, M.S. and
F.G. Oppenheim (1993) Crit. Rev. Oral. Biol. Med. 4:251-259). T'he HST and
STATH genes
show nearly identical overall gene structures. The HST and STATH loci exhibit
77%-81 % sequence
identity in intron DNA and 80%-88% sequence identity in noncoding exons but
only 38%-43%
sequence identity in the protein-coding regions of exons 4 and 5. It has been
suggested that HST and
STATH belong to a single gene family exhibiting accelerated evolution between
the HST and STATH
coding sequences (Sabatini, L.M. et al. (1993) Mol. Biol. Evol. 10:497-511).
The discovery of a new lysine-rich statherin protein and the polynucleotides
encoding it
satisfies a need in the art by providing new compositions which are useful in
the diagnosis,
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prevention, and treatment of neoplastic and autoimmune/inflammatory disorders,
and infectious
diseases.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, lysine-rich
statherin protein,
referred to as "LRSP." In one aspect, the invention provides a substantially
purified polypeptide
comprising an amino acid sequence selected from the group consisting of SEQ ID
NO:1 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.
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:I 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:1 and fragments thereof. The invention also includes an isolated and
purified
polynucleotide variant having at least 70% polynucleotide sequence identity to
the polynucleotide
encoding the polypeptide comprising an amino acid sequence selected from the
group consisting of
SEQ ID NO:1 and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide
which hybridizes
under stringent conditions to the polynucleotide encoding the polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1 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 the amino acid sequence
selected from the
group consisting of SEQ ID NO:1 and fragments thereof.
The invention also provides a method for detecting a polynucleotide in a
sample containing
nucleic acids, the method comprising the steps of: (a) hybridizing the
complement of the
polynucleotide sequence to at least one of the polynucleotides of the sample,
thereby forming a
hybridization complex; and (b) detecting the hybridization complex, wherein
the presence of the
hybridization complex correlates with the presence of a polynucleotide in the
sample. In one aspect,
the method further comprises amplifying the polynucleotide prior to
hybridization.
The invention also provides an isolated and purified polynucleotide comprising
a
polynucleotide sequence selected from the group consisting of SEQ ID N0:2 and
fragments thereof.
The invention further provides an isolated and purified polynucleotide variant
having at least 70%
polynucleotide sequence identity to the polynucleotide sequence selected from
the group consisting of
2
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SEQ ID N0:2 and fragments thereof. The invention also provides an isolated and
purified
polynucleotide having a sequence which is complementary to the polynucleotide
comprising a
polynucleotide sequence selected from the group consisting of SEQ ID N0:2 and
fragments thereof.
The invention further provides an expression vector containing at least a
fragment of the
polynucleotide encoding the polypeptide comprising the amino acid sequence of
SEQ ID NO:1. In
another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method
comprising the
steps of: (a) culturing the host cell containing an expression vector
containing a polynucleotide of the
invention under conditions suitable for the expression of the polypeptide; and
(b) recovering the
polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a
substantially purified
polypeptide having the amino acid sequence selected from the group consisting
of SEQ ID NO:1 and
fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a
polypeptide selected from
the group consisting of SEQ ID NO:1 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 LRSP, the method comprising administering
to a subject in need
of such treatment an effective amount of a pharmaceutical composition
comprising a substantially
purified polypeptide having the amino acid sequence selected from the group
consisting of SEQ ID
NO: l 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 LRSP, the method comprising administering
to a subject in need of
such treatment an effective amount of an antagonist of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ ID NO:1 and fragments thereof.
BRIEF DESCRIPTION OF THE FIGURES AND TABLE
Figures lA, 1B, and 1C show the amino acid sequence (SEQ ID NO:1) and nucleic
acid
sequence (SEQ ID N0:2) of LRSP. The alignment was produced using MACDNASIS PRO
software
(Hitachi Software Engineering, South San Francisco CA).
Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H show the nucleotide sequence
alignment
between LRSP (Incyte Clone ID 2820214; SEQ ID N0:2), human statherin (GI
338507; SEQ ID
NO:S), and human basic histidine-rich protein (GI 179465; SEQ ID N0:6),
produced using the
multisequence alignment program of LASERGENE software (DNASTAR, Madison WI).
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Figure 3 shows the amino acid sequence alignment between LRSP (Incyte Clone ID
2820214;
SEQ ID NO:1 ), human statherin (GI 338508; SEQ ID N0:3), and human basic
histidine-rich protein
(GI 179466; 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 LRSP, along
with
applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"LRSP" refers to the amino acid sequences of substantially purified LRSP
obtained from any
species, particularly a mammalian species, including bovine, ovine, porcine,
murine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
LRSP. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of LRSP either by
directly interacting with
LRSP or by acting on components of the biological pathway in which LRSP
participates.
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An "allelic variant" is an alternative form of the gene encoding LRSP. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding LRSP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as LRSP or a
polypeptide with at least one functional characteristic of LRSP. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding LRSP, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
LRSP. The encoded protein may also be "altered," and may contain deletions,
insertions, or.
IS substitutions of amino acid residues which produce a silent change and
result in a functionally
equivalent LRSP. Deliberate amino acid substitutions may be made on the basis
of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the
residues, as long as the biological or immunological activity of LRSP is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and ''amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to an amino acid
sequence of a naturally
occurring protein molecule, "amino acid sequence" and like terms are not meant
to limit the amino
acid sequence to the complete native amino acid sequence associated with the
recited protein
molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of LRSP. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
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molecules, or any other compound or composition which modulates the activity
of LRSP either by
directly interacting with LRSP or by acting on components of the biological
pathway in which LRSP
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F{ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind LRSP polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used
to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from
the translation of RNA,
or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly used
carriers that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and
keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid
sequence which is
complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules may be
produced by any method including synthesis or transcription. Once introduced
into a cell, the
complementary nucleotides combine with natural sequences produced by the cell
to form duplexes
and to block either transcription or translation. The designation "negative"
or "minus" can refer to the
antisense strand, and the designation "positive" or "plus" can refer to the
sense strand.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" refers to the
capability of the natural, recombinant, or synthetic LRSP, or of any
oligopeptide thereof, to induce a
specific immune response in appropriate animals or cells and to bind with
specific antibodies.
The terms "complementary" and "complementarity" refer to the natural binding
of
polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the
complementary sequence "3' T-C-A 5'." Complementarity between two single-
stranded molecules
may be "partial," such that only some of the nucleic acids bind, or it may be
"complete," such that
total complementarity exists between the single stranded molecules. The degree
of complementarity
between nucleic acid strands has significant effects on the efficiency and
strength of the hybridization
between the nucleic acid strands. This is of particular importance in
amplification reactions, which
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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 LRSP or fragments of
LRSP may be '
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
resequenced to
resolve uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk
CT) in the 5' and/or
the 3' direction, and resequenced, or which has been assembled from the
overlapping sequences of
one or more Incyte Clones and, in some cases, one or more public domain ESTs,
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI). Some sequences have been both extended and assembled to produce the
consensus sequence.
"Conservative amino acid substitutions" are those substitutions that, when
made, least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Z-t.p Phe, Tyr
a0 Tyr His, Phe, Trp
7
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Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to the chemical modification of a polypeptide
sequence, or a
polynucleotide sequence. Chemical modifications of a polynucleotide sequence
can include, for
example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
A derivative
polynucleotide encodes a polypeptide which retains at least one biological or
immunological function
of the natural molecule. A derivative polypeptide is one modified by
glycosylation, pegylation, or any
similar process that retains at least one biological or immunological function
of the polypeptide from
which it was derived.
A "fragment" is a unique portion of LRSP or the polynucleotide encoding LRSP
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10, 15,
20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid residues
in length. Fragments may be preferentially selected from certain regions of a
molecule. For example,
a polypeptide fragment may comprise a certain length of contiguous amino acids
selected from the
first 250 or 500 amino acids (or first 25% or 50% of a polypeptide) as shown
in a certain defined
sequence. Clearly these lengths are exemplary, and any length that is
supported by the specification,
including the Sequence Listing, tables, and figures, may be encampassed 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 on
the intended purpose for the fragment.
A fragment of SEQ ID NO:1 is encoded by a.fragment 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.
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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 . The precise length of
a fragment of SEQ ID
NO:1 and the region of SEQ ID NO: I to which the fragment corresponds are
routinely determinable
by one of ordinary skill in the art based on the intended purpose for the
fragment.
The term "similarity" refers to a degree of complementarity. There may be
partial similarity
or complete similarity. The word "identity" may substitute for the word
"similarity." A partially
complementary sequence that at least partially inhibits an identical sequence
from hybridizing to a
target nucleic acid is referred to as "substantially similar." The inhibition
of hybridization of the
completely complementary sequence to the target sequence may be examined using
a hybridization
assay (Southern or northern blot, solution hybridization, and the like) under
conditions of reduced
stringency. A substantially similar sequence or hybridization probe will
compete for and inhibit the
binding of a completely similar (identical) sequence to the target sequence
under conditions of
reduced stringency. This is not to say that conditions of reduced stringency
are such that non-specific
binding is permitted, as reduced stringency conditions require that the
binding of two sequences to
one another be a specific (i.e., a selective) interaction. The absence of non-
specific binding may be
tested by the use of a second target sequence which lacks even a partial
degree of complementarity
(e.g., less than about 30% similarity or identity). In the absence of non-
specific binding, the
substantially similar sequence or probe will not hybridize to the second non-
complementary target
sequence.
The phrases "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
ai. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequence pairs.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
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is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. ( 1990) J. Mol. Biol. 215:403-410),
which is available from
several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www ncbi nlm.nih.~ovBLASTI. 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:/hvww ncbi
nlm.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø9 (May-07-1999) set at default parameters. Such default parameters may be,
for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off: 50
Expect: l0
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least S0, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
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alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the
hydrophobicity and acidity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=s, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø9
(May-07-1999) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
I S Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off. 50
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for
stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino
acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
11
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hybridization is an indication that two nucleic acid sequences share a high
degree of identity. Specific
hybridization complexes form under permissive annealing conditions and remain
hybridized after the
"washing" step(s). The washing steps) is particularly important in determining
the stringency of the
hybridization process, with more stringent conditions allowing less non-
specific binding, i.e., binding
between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for
annealing of nucleic acid sequences are routinely determinable by one of
ordinary skill in the art and
may be consistent among hybridization experiments, whereas wash conditions may
be varied among
experiments to achieve the desired stringency, and therefore hybridization
specificity. Permissive
annealing conditions occur, for example, at 68°C in the presence of
about 6 x SSC, about 1% (w/v)
SDS, and about 100 pg/ml denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Generally, such wash temperatures
are selected to be about
5°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook et al., 1989,
Molecular 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 polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1% SDS, for 1 hour.
Alternatively, temperatures of about 65°C, b0°C, 55°C, or
42°C may be used. SSC concentration
may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1
%. Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
denatured salmon sperm DNA at about 100-200 pg/ml. Organic solvent, such as
formamide at a
concentration of about 35-50% v/v, may also be used under particular
circumstances, such as for
RNA:DNA hybridizations. Useful variations on these wash conditions will be
readily apparent to
those of ordinary skill in the art. Hybridization, particularly under high
stringency conditions, may be
suggestive of evolutionary similarity between the nucleotides. Such similarity
is strongly indicative
of a similar rote for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., 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
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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 LRSP. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of LRSP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with the second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Generally, operably linked DNA sequences may be in close proximity
or contiguous and,
where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation,
and may be pegylated to extend their lifespan in the cell.
"Probe" refers to nucleic acid sequences encoding LRSP, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
13
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identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook et al., 1989, Molecular Cloning.: A Laboratory_Manual,
2"° ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel et al.,1987, Current Protocols in
Molecular Biolo~y,
Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis et al., 1990,
PCR Protocols. A
Guide to Methods and Applications, Academic Press, San Diego CA. PCR primer
pairs can be
derived from a known sequence, for example, by using computer programs
intended for that purpose
such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead Institute/MIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library,'' in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for
example, as PCR or sequencing primers, microarray elements, or specific probes
to identify fully or
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partially complementary polynucleotides in a sample of nucleic acids. Methods
of oligonucleotide
selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding LRSP, or fragments thereof, or LRSP itself, may comprise a
bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least about 60% free,
preferably about 75% free, and most preferably about 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or
nucleotides by
different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
CA 02348046 2001-04-20
WO 00/24??9 PCT/US99/24046
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
"Transformation" describes a process by which exogenous DNA enters and changes
a
recipient cell. Transformation may occur under natural or artificial
conditions according to various
methods well known in the art, and may rely on any known method for the
insertion of foreign nucleic
acid sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected
based on the type of host cell being transformed and may include, but is not
limited to, viral infection,
electroporation, heat shock, lipofection, and particle bombardment. The term
"transformed" cells
includes stably transformed cells in which the inserted DNA is capable of
replication either as an
autonomously replicating plasmid or as part of the host chromosome, as well as
transiently
transformed cells which express the inserted DNA or RNA for limited periods of
time.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
95% or at least 98% or
greater sequence identity over a certain defined length. A variant may be
described as, for example,
an "allelic" (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may
have significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides generally will have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleatide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least
98% or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
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The invention is based on the discovery of a new human lysine-rich statherin
protein (LRSP),
the polynucleotides encoding LRSP, and the use of these compositions for the
diagnosis, treatment, or
prevention of neoplastic and autoimmune/inflammatory disorders, and infectious
diseases.
Nucleic acids encoding the LRSP of the present invention were identified in
Incyte Clone
2820214 from the breast cDNA library (BRSTNOT14) 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
2820214H1 and
282021476 (BRSTNOT14), 3600320F6 (DRGTNOTO1), 4254615H1 (BSCNNOT03), 2656117F6
(THYMNOT04), and 1325717F6 (LPARNOT02).
In one embodiment, the invention encompasses a polypeptide comprising the
amino acid
sequence of SEQ ID NO:1, as shown in Figures lA, 1B, and 1C. LRSP is 95 amino
acids in length;
has two potential N-glycosylation sites at residues N24 and N76, one potential
casein kinase II
phosphorylation site at residue 715, and one potential protein kinase C
phosphorylation site at residue
529; and is rich in basic (lysine, arginine, and histidine) and aliphatic
amino acid residues, containing
21% and 26% ofthese, respectively. As shown in Figures 2A, 2B, 2C, 2D, 2E, 2F,
2G, and 2H, the
nucleotide sequence encoding LRSP (Incyte Clone ID 2820214; SEQ ID N0:2) has
chemical
similarity with the nucleotide sequence encoding human statherin (GI 338507;
SEQ ID NO:S) and
with the nucleotide sequence encoding human basic histidine-rich protein (GI
179465: SEQ ID
N0:6). In particular, the region of the nucleotide sequence encoding LRSP from
about nucleotide
1059 to about nucleotide 1331 is similar to a region of the nucleotide
sequence of human statherin
(94% identity) and LRSP is therefore considered a splice variant. In addition
the region of the
nucleotide sequence encoding LRSP from about nucleotide 1122 to about
nucleotide 1331 is similar
to a region of the nucleotide sequence of human basic histidine-rich protein
(68% identity). As shown
in Figure 3, LRSP (Incyte Clone ID 2820214; SEQ ID NO:1 ) has chemical and
structural similarity
with human statherin (GI 338508; SEQ ID N0:3) and human basic histidine-rich
protein (GI 179466:
SEQ ID N0:4). In particular, LRSP and human statherin share 14% identity, and
conserved residues
at F3, D20, P45, P48, and P61 in LRSP. In addition, LRSP and human basic
histidine-rich protein
have similar isoelectric points, 10.5 and 10.4, respectively. A fragment of
SEQ ID N0:2 from about
nucleotide 865 to about nucleotide 918 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. Northern
analysis shows the expression of this sequence in various libraries, at least
45% of which are
associated with cancer, at least 20% of which are associated with developing
and proliferating tissues
or cells, and at least 25% of which are associated with the
immune/inflammatory response. Of
particular note is the expression of LRSP in brain and pituitary (30%), gut
(25%), and reproductive
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WO 00/24779 PCT/US99/24046
(15%) tissues. In addition, these three tissue types are associated with
disorders and conditions
involving physiological stress.
The invention also encompasses LRSP variants. A preferred LRSP 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 LRSP amino acid sequence, and which contains at least
one functional or
structural characteristic of LRSP.
The invention also encompasses polynucleotides which encode LRSP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising the
sequence of SEQ
ID N0:2, which encodes LRSP.
The invention also encompasses a variant of a polynucleotide sequence encoding
LRSP. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at
least about 85%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding LRSP. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:2
which has at least about 70%, or alternatively at least about 85%, or even at
least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the
group consisting of
SEQ ID N0:2. Any one of the polynucleotide variants described above can encode
an amino acid
sequence which contains at least one functional or structural characteristic
of LRSP.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding LRSP, 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 LRSP, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode LRSP and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring LRSP under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding LRSP 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 LRSP and its derivatives without altering the encoded amino
acid sequences
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WO 00/24779 PCT/US99/24046
include the production of RNA transcripts having more desirable properties,
such as a greater
half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode LRSP
and
LRSP derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell systems
using reagents well known in the art. Moreover, synthetic chemistry may be
used to introduce
mutations into a sequence encoding LRSP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:2 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-
511.) Hybridization conditions, including annealing and wash conditions, are
described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
I S the embodiments of the invention. The methods may employ such enzymes as
the Klenow fragment
of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Perkin-
Elmer), thermostable T7 polymerise (Amersham Pharmacia Biotech, Piscataway
NJ), or
combinations of polymerises and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Perkin-Elmer). Sequencing is then carried out using either the ABI 373 or 377
DNA sequencing
system (Perkin-Elmer), the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics,
Sunnyvale CA), or other systems known in the art. The resulting sequences are
analyzed using a
variety of algorithms which are well known in the art. (See, e.g., Ausubel,
F.M. ( 1997) Short
Protocols in Molecular BioloQV, John Wiley & Sons, New York NY, unit 7.7;
Meyers, R.A. (1995)
Molecular Biolo~r and Biotechnolosw, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding LRSP 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
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CA 02348046 2001-04-20
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a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. ( 1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al.
( 1991 ) PCR Methods Applic. 1:111-119.) In this method, multiple restriction
enzyme digestions and
ligations may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991 ) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 Primer Analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOT'YPER 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 LRSP may be cloned in recombinant DNA molecules that direct
expression of LRSP,
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 LRSP.
The nucleotide sequences of the present invention can be engineered using
methods generally
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
known in the art in order to alter LRSP-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
In another embodiment, sequences encoding LRSP may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
aI. ( 1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.)
Alternatively, LRSP itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solid-phase
techniques. (See, e.g.,
Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be
achieved using the
ABI 431A peptide synthesizer (Perkin-Elmer). Additionally, the amino acid
sequence of LRSP, or
any part thereof, may be altered during direct synthesis and/or combined with
sequences from other
proteins, or any part thereof, to produce a variant polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier ( 1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular
Properties, WH Freeman, New
York NY.)
In order to express a biologically active LRSP, the nucleotide sequences
encoding LRSP or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in
a suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
encoding LRSP. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
LRSP. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding LRSP and its initiation codon and upstream regulatory
sequences are inserted
into the appropriate expression vector, no additional transcriptional or
translational control signals
may be needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted,
exogenous translational control signals including an in-frame ATG initiation
codon should be
provided by the vector. Exogenous translational elements and initiation codons
may be of various
origins, both natural and synthetic. The efficiency of expression may be
enhanced by the inclusion of
21
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
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 LRSP and appropriate transcriptional and
translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Clonin~~, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding LRSP. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding LRSP. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding LRSP can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORTI
plasmid (Life Technologies). Ligation of sequences encoding LRSP into the
vector's multiple cloning
site disrupts the IacZ gene, allowing a colorimetric screening procedure for
identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster ( 1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of LRSP are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of LRSP may be used.
For example, vectors
containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of LRSP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharom~ces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and
Scorer, C.A. et al. (1994)
Bio/Technology 12:181-184.)
2z
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
Plant systems may also be used for expression of LRSP. 'Transcription of
sequences encoding
LRSP may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J.
6:307-311 ). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Brogue, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of
Science and Technoloev
(1992) McGraw Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding LRSP
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E 1 or E3 region of the viral genome
may be used to obtain
infective virus which expresses LRSP in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to I 0 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
LRSP in cell lines is preferred. For example, sequences encoding LRSP can be
transformed into cell
lines using expression vectors which may contain viral origins of replication
and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer resistance
to a selective agent, and its presence allows growth and recovery of cells
which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and apr cells, respectively.
(See, e.g., Wigler, M. et
23
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
al. ( 1977) Cell 11:223-232; Lowy, I. et al. ( 1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (I980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan ( 1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding LRSP is inserted within a marker gene sequence, transformed
cells containing
sequences encoding LRSP can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding LRSP under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding LRSP
and that express
LRSP may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
Immunological methods for detecting and measuring the expression of LRSP 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 (FRCS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on LRSP 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) Serolo;~ical Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
24
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
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 LRSP
include oligolabeiing, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding LRSP, 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 LRSP may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
containing polynucleotides which encode LRSP may be designed to contain signal
sequences which
direct secretion of LRSP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression ofthe
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding LRSP 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 LRSP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of LRSP activity.
Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase (GST),
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their cognate
fusion proteins on immobilized glutathione, maltose, phenylarsine oxide,
calmodulin, and metal-
chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the LRSP encoding sequence and the
heterologous protein
sequence, so that LRSP may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel (1995, supra, ch. 10).
A variety of commercially available kits may also be used to facilitate
expression and purification of
fusion proteins.
In a further embodiment of the. invention, synthesis of radiolabeled LRSP may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 3sS-methionine.
Fragments of LRSP may be produced not only by recombinant means, but also by
direct
peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra,
pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation. Automated
synthesis may be
achieved, for example, using the ABI 431A peptide synthesizer (Perkin-Elmer).
Various fragments of
LRSP 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 LRSP, and lysine-rich statherin protein and basic histidine-rich
protein. In addition, the
expression of LRSP is closely associated with cancer and the
immune/inflammatory response and
with disorders and conditions involving physiological stress. Therefore, LRSP
appears to play a role
in neoplastic and autoimmune/inflammatory disorders, and infectious diseases.
In the treatment of
disorders associated with increased LRSP expression or activity, it is
desirable to decrease the
expression or activity of LRSP. In the treatment of disorders associated with
decreased LRSP
expression or activity, it is desirable to increase the expression or activity
of LRSP.
Therefore, in one embodiment, LRSP 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 LRSP.
Examples of such disorders include, but are not limited to, an
autoimmune/inflammatory disorder
such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult
respiratory distress
26
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis,
cholecystitis, contact dermatitis,
Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetaiis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections, and trauma; and an
infectious disease, such as
infections by bacterial agents classified as pneumococcus, staphylococcus,
streptococcus, bacillus,
corynebacterium, clostridium, meningococcus, gonococcus, listeria, moraxella,
kingella, haemophilus,
legionella, bordetella, gram-negative enterobacterium including shigella,
salmonella, and
campylobacter, pseudomonas, vibrio, brucella, francisella, yersinia,
bartoneila, norcardium,
actinomyces, mycobacterium, spirochaetale, rickettsia, chlamydia, and
mycoplasma; and infections by
fungal agents classified as aspergillus, blastomyces, dermatophytes,
cryptococcus, coccidioides,
malasezzia, histoplasma, and other fungal agents causing various mycoses.
In another embodiment, a vector capable of expressing LRSP 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 LRSP including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
LRSP in conjunction with a suitable pharmaceutical carrier may be administered
to a subject to treat
or prevent a disorder associated with decreased expression or activity of LRSP
including, but not
limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of LRSP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of LRSP including, but not limited to, those listed above.
In a further embodiment, an antagonist of LRSP may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of LRSP.
Examples of such
disorders include, but are not limited to, a neoplastic disorder, such as,
adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular,
cancers of the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
prostate, salivary glands, skin,
27
CA 02348046 2001-04-20
WO 00/24779 PCT/ETS99/2404b
spleen, testis, thymus, thyroid, and uterus; an autoimmune/inflammatory
disorder; and an infectious
disease. In one aspect, an antibody which specifically binds LRSP 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 LRSP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding LRSP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of LRSP including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of LRSP may be produced using methods which are generally known
in the art.
In particular, purified LRSP may be used to produce antibodies or to screen
libraries of
pharmaceutical agents to identify those which specifically bind LRSP.
Antibodies to LRSP may also
be generated using methods that are well known in the art. Such antibodies may
include, but are not
limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab
fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with LRSP or with any fragment or
oligopeptide thereof .
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunologieal response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants
used in humans,
BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
LRSP have an amino acid sequence consisting of at least about S amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein and contain the
entire amino acid sequence of a small, naturally occurring molecule. Short
stretches of LRSP amino
acids may be fused with those of another protein, such as KLH, and antibodies
to the chimeric
28
CA 02348046 2001-04-20
WO 00/24779 PCT/ITS99/24046
molecule may be produced.
Monoclonal antibodies to LRSP may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. ( 1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
LRSP-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g.,
Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for LRSP may also be
generated.
For example, such fragments include, but are not limited to, Flab")z fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
LRSP and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering LRSP epitopes is generally used, but a
competitive binding assay may
also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
29
CA 02348046 2001-04-20
WO 00/24779 PCT/US99124046
techniques may be used to assess the affnity of antibodies for LRSP. Affinity
is expressed as an
association constant, K" which is defined as the molar concentration of LRSP-
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 LRSP epitopes, represents the average affinity, or
avidity, of the antibodies for
LRSP. The Ke determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular LRSP 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
LRSP-antibody complex must withstand rigorous manipulations. Low-affinity
antibody preparations
with Ke ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of LRSP, preferably in active
form, from the
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington, DC;
Liddell, J.E. and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably
5-10 mg specific antibody/ml, is generally employed in procedures requiring
precipitation of LRSP-
antibody complexes. Procedures for evaluating antibody specificity, titer, and
avidity, and guidelines
for antibody quality and usage in various applications, are generally
available. (See, e.g., Catty,
supra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding LRSP, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
the complement of the
polynucleotide encoding LRSP may be used in situations in which it would be
desirable to block the
transcription of the mRNA. In particular, cells may be transformed with
sequences complementary to
polynucleotides encoding LRSP. Thus, complementary molecules or fragments may
be used to
modulate LRSP 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
LRSP.
Expression vectors derived from retroviruses, adenoviruses, or herpes or
vaccinia viruses, or
from various bacterial plasmids, may be used for delivery of nucleotide
sequences to the targeted
organ, tissue, or cell population. Methods which are well known to those
skilled in the art can be used
to construct vectors to express nucleic acid sequences complementary to the
polynucleotides encoding
LRSP. (See, e.g., Sambrook, supra; Ausubel, 1995, s_u~ra.)
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
Genes encoding LRSP can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding LRSP. Such
constructs may be used to introduce untranslatable sense or antisense
sequences into a cell. Even in
the absence of integration into the DNA, such vectors may continue to
transcribe RNA molecules
until they are disabled by endogenous nucleases. Transient expression may last
for a month or more
with a non-replicating vector, and may last even longer if appropriate
replication elements are part of
the vector system.
As mentioned above, modifications of gene expression can be obtained by
designing
complementary sequences or antisense molecules (DNA, RNA, or PNA) to the
control, S', or
regulatory regions of the gene encoding LRSP. Oligonucleotides derived from
the transcription
initiation site, e.g., between about positions -10 and +10 from the start
site, may be employed.
Similarly, inhibition can be achieved using triple helix base-pairing
methodology. Triple helix pairing
is useful because it causes inhibition of the ability of the double helix to
open sufficiently for the
binding of polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances
using triplex DNA have been described in the literature. (See, e.g., Gee, J.E.
et al. (1994) in Huber,
B.E. and B.I. Carr, Molecular and Lmmunolo i~ c Approaches, Futura Publishing,
Mt. Kisco NY, pp.
163-177.) A complementary sequence or antisense molecule may also be designed
to block
translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding LRSP.
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,
GIJLJ, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
31
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WO 00/24779 PCT/US99/24046
sequences encoding LRSP. Such DNA sequences may be incorporated into a wide
variety of vectors
with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these
cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines,
cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such' as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. ( 1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
pharmaceutical
or sterile composition, in conjunction with a pharmaceutically acceptable
carrier, for any of the
therapeutic effects discussed above. Such pharmaceutical compositions may
consist of LRSP,
antibodies to LRSP, and mimetics, agonists, antagonists, or inhibitors of
LRSP. 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, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain suitable
pharmaceutically-acceptable carriers comprising excipients and auxiliaries
which facilitate processing
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WO 00/24779 PCT/US99/24046
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 Remineton'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, gets, syrups, slurries, suspensions, and the like, for
ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combining
active
compounds with solid excipient and processing the resultant mixture of
granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be
added, if desired. Suitable
excipients include carbohydrate or protein fillers, such as sugars, including
lactose, sucrose, mannitol,
and sorbitol; starch from corn, wheat, rice, potato, or other plants;
cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums,
including arabic and
tragacanth; and proteins, such as gelatin and collagen. If desired,
disintegrating or solubilizing agents
may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and
alginic acid or a salt thereof,
such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as
concentrated sugar
solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone,
carbopol gel, polyethylene
glycol, and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for
product identification or to
characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as well as soft, sealed capsules made of gelatin and a coating, such
as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or
binders, such as lactose or
starches, lubricants, such as talc or magnesium stearate, and, optionally,
stabilizers. In soft capsules,
the active compounds may be dissolved or suspended in suitable liquids, such
as fatty oils, liquid, or
liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hanks' solution, Ringer's
solution, or physiologically buffered saline. Aqueous injection suspensions
may contain substances
which increase the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or
dextran. Additionally, suspensions of the active compounds may be prepared as
appropriate oily
injection suspensions. Suitable 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
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WO 00/24779 PCTNS99/24046
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 of the following: 1 mM to 50 mM histidine, 0.1 % to 2% sucrose, and 2%
to 7% mannitol, at a
pH range of 4.5 to 5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate
container and labeled for treatment of an indicated condition. For
administration of LRSP, such
labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein
the active ingredients are contained in an effective amount to achieve the
intended purpose. The
determination of an effective dose is well within the capability of those
skilled in the art.
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, or pigs.
An animal model may also be used to determine the appropriate concentration
range and route of
administration. Such information can then be used to determine useful doses
and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example LRSP
or fragments thereof, antibodies of LRSP, and agonists, antagonists or
inhibitors of LRSP, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or 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 LDso/ED3o 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
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WO 00/24779 PCT/US99/24046
compositions is preferably within a range of circulating concentrations that
includes the EDSO with
little or no toxicity. The dosage varies within this range depending upon the
dosage form employed,
the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4
days, every week, or biweekly depending on the half life and clearance rate of
the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 ~sg, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind LRSP may be used for
the
diagnosis of disorders characterized by expression of LRSP, or in assays to
monitor patients being
treated with LRSP or agonists, antagonists, or inhibitors of LRSP. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for LRSP include methods which utilize the antibody and a label to detect LRSP
in human body fluids
or in extracts of cells or tissues. The antibodies may be used with or without
modification, and may
be labeled by covalent or non-covalent attachment of a reporter molecule. A
wide variety of reporter
molecules, several of which are described above, are known in the art and may
be used.
A variety of protocols for measuring LRSP, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
LRSP expression. Normal
or standard values for LRSP expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibody to LRSP under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of LRSP
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.
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In another embodiment of the invention, the polynucleotides encoding LRSP may
be used for
diagnostic purposes. The polynucieotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of LRSP
may be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
LRSP, and to monitor regulation of LRSP levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding LRSP or closely related
molecules may be used to
identify nucleic acid sequences which encode LRSP. 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 LRSP, 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 LRSP 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 LRSP
gene.
Means for producing specific hybridization probes for DNAs encoding LRSP
include the
cloning of polynucleotide sequences encoding LRSP or LRSP derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 355,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding LRSP may be used for the diagnosis of
disorders
associated with expression of LRSP. Examples of such disorders include, but
are not limited to, a
neoplastic disorder, such as, adenocarcinoma, leukemia, lymphoma, melanoma,
myeloma, sarcoma,
teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder,
bone, bone marrow, brain,
breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis,
thymus, thyroid, and uterus;
an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis,
anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis, bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis,
dermatomyositis, diabetes
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WO 00/24779 PCT/US99/24046
mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis, erythema
nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout,
Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple
sclerosis, myasthenia
gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis,
psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner syndrome, complications of cancer, hemodiaiysis, and
extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic
infections, and trauma; and an
infectious disease, such as infections by bacterial agents classified as
pneumococcus, staphylococcus,
streptococcus, bacillus, corynebacterium, clostridium, meningococcus,
gonococcus, listeria,
rnoraxella, kingella, haemophilus, legionella, bordetella, gram-negative
enterobacterium including
shigella, salmonella, and campylobacter, pseudomonas, vibrio, brucella,
francisella, yersinia,
bartonella, norcardium, actinomyces, mycobacterium, spirochaetale, rickettsia,
chlamydia, and
mycoplasma; and infections by fungal agents classified as aspergillus,
blastomyces, dermatophytes,
cryptococcus, coccidioides, malasezzia, histoplasma, and other fungal agents
causing various
mycoses. The polynucleotide sequences encoding LRSP 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 LRSP expression. Such qualitative or quantitative methods are well
known in the art.
In a particular aspect, the nucleotide sequences encoding L RSP may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding LRSP may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a
suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to a
control sample then the presence of altered levels of nucleotide sequences
encoding LRSP 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 LRSP,
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 LRSP, under conditions suitable for hybridization
or amplification.
Standard hybridization may be quantified by comparing the values obtained from
normal subjects
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WO 00/24779 PCT/US99/24046
with values from an experiment in which a known amount of a substantially
purified polynucleotide is
used. Standard values obtained in this manner may be compared with values
obtained from samples
from patients who are symptomatic for a disorder. Deviation from standard
values is used to establish
the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding LRSP
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 LRSP, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
LRSP, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
Methods which may also be used to quantify the expression of LRSP 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 coiorimetric
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
simuitaneously and to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, and to develop and
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WO 00/24779 PCT/US99/24046
monitor the activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. ( 1995) U.S. Patent No. 5,474,796; Schena, M. et al. (
1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalon, D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997). Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
In another embodiment of the invention, nucleic acid sequences encoding LRSP
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. The
sequences may be mapped to a particular chromosome, to a specific region of a
chromosome, or to
artificial chromosome constructions, e.g., human artificial chromosomes
(HACs), yeast artificial
chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1
constructions, or single
chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price,
C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7:149-
154.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome
mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al. (
1995) in Meyers, supra,
pp. 965-968.) Examples of genetic rnap 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 LRSP on a physical chromosomal map and a
specific disorder, or a
predisposition to a specific disorder, rnay help define the region of DNA
associated with that disorder.
The nucleotide sequences of the invention may be used to detect differences in
gene sequences among
normal, carrier, and affected individuals.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the number or arm of a particular human
chromosome is not
known. New sequences can be assigned to chromosomal arms by physical mapping.
This provides
valuable information to investigators searching for disease genes using
positional cloning or other
gene discovery techniques. Once the disease or syndrome has been crudely
localized by genetic
linkage to a particular genomic region, e.g., ataxia-telangiectasia to l 1q22-
23, any sequences mapping
to that area may represent associated or regulatory genes for further
investigation. (See, e.g., Gatti,
R.A. et al. ( 1988) Nature 336:577-580.) The nucleotide sequence of the
subject invention may also be
used to detect differences in the chromosomal location due to translocation,
inversion, etc., among
normal, carrier, or affected individuals.
In another embodiment of the invention, LRSP, its catalytic or immunogenic
fragments, or
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WO 00/24779 PCT/US99/24046
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between LRSP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. ( 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 LRSP, or
fragments thereof,
and washed. Bound LRSP is then detected by methods well known in the art.
Purified LRSP 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 LRSP specifically compete with a test compound
for binding LRSP. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with LRSP.
In additional embodiments, the nucleotide sequences which encode LRSP may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are,
therefore, to be construed as merely illustrative, and not limitative of the
remainder of the disclosure
in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. No. 60/155,209, are hereby expressly incorporated by
reference.
EXAMPLES
I. Construction of cDNA Libraries
The BRSTNOT14 library was constructed using RNA isolated from breast tissue
removed
from a 62-year-old Caucasian female during a unilateral extended simple
mastectomy. Pathology for
the associated tumor tissue indicated an invasive grade 3 (of 4), nuclear
grade 3 (of 3)
adenocarcinoma. Patient history included a benign colon neoplasm,
hyperlipidemia, cardiac
dysrhythmia, and obesity. Family history included cardiovascular and
cerebrovascular disease and
colon, ovary and lung cancer.
CA 02348046 2001-04-20
WO 00124779 PCT/US99/24046
The frozen tissue was homogenized and lysed in TR1ZOL reagent (1 g tissue/10
ml TRIZOL;
Life Technologies), a monophasic solution of phenol and guanidine
isothiocyanate, using a
Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury NY).
After a brief
incubation on ice, chloroform was added ( 1:5 v/v) and the lysate was
centrifuged. The upper
chloroform layer was removed to a tiesh tube and the RNA extracted with
isopropanol, resuspended
in DEPC-treated water, and treated with DNase for 25 min at 37°C.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, su ra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were Iigated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), or
pINCY (Incyte
Pharmaceuticals, Palo Alto CA). Recombinant plasmids were transformed into
competent E. coli
cells including XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH10B,
or
ElectroMAX DH 1 OB from Life Technologies.
II. Isolation of cDNA Clones
Plasmids were recovered from host cells by in vivo excision using the UNIZAP
vector system
(Stratagene) or by cell lysis. Plasmids were purified using at least one of
the following: a Magic or
WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep
purification kit (Edge
Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,
QIAWELL 8
Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid
purification kit from QIAGEN.
Following precipitation, plasmids were resuspended in 0.1 ml of distilled
water and stored, with or
without lyophilization, at 4°C.
41
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
cDNA sequencing reactions were processed using standard methods or high-
throughput
instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) thermal cycler or
the PTC-200
thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser
(Robbins Scientific) or
the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-
Elmer).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Perkin-Elmer) in conjunction with
standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the techniques
disclosed in Example V.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 1 summarizes the tools, programs, and algorithms used and provides
applicable
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 clustal algorithm as incorporated into the
MEGALIGN multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned
sequences.
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CA 02348046 2001-04-20
WO 00/Z4779 PCT/US99/24046
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then queried
against a selection of public databases such as the GenBank primate, rodent,
mammalian, vertebrate,
and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences
were assembled
into full length polynucleotide sequences using programs based on Phred,
Phrap, and Consed, and
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences were translated to derive the
corresponding full length
amino acid sequences, and these full length sequences were subsequently
analyzed by querying
against databases such as the GenBank databases (described above), SwissProt,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family
databases such
as PFAM. HMM is a probabilistic approach which analyzes consensus primary
structures of gene
families. (See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide and
amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:2. 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, supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in nucleotide databases such as GenBank or LIFESEQ (Incyte
Pharmaceuticals). This
analysis is much faster than multiple membrane-based hybridizations. In
addition, the sensitivity of
the computer search can be modified to determine whether any particular match
is categorized as
exact or similar. The basis of the search is the product score, which is
defined as:
sequence identity x % maximum BLAST score
100
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. For example, with a product score of 40, the
match will be exact within
a I% to 2% error, and, with a product score of 70, the match will be exact.
Similar molecules are
usually identified by selecting those which show product scores between 15 and
40, although lower
43
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
scores may identify related molecules.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding LRSP 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 LRSP Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID 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
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)~SO,,
and (i-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerise (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, I min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94 °C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 pl
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 pl of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
44
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 to 10 ~cl aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose mini-gel to determine which reactions were
successful in extending
the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, individual colonies were picked and cultured
overnight at 37°C in 384-
well plates in LB/2x carb 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, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with
20% dimethysulfoxide ( 1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID N0:2 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 1D N0:2 are employed to screen cDNAs,
genomic
DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about
20 base pairs, is
specifically described, essentially the same procedure is used with larger
nucleotide fragments.
Oligonucleotides are designed using state-of the-art software such as OLIGO
4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~Ci of [y-
3zP] adenosine
triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase
(DuPont NEN, Boston
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superfine
size exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot
containing 10'
counts per minute of the labeled probe is used in a typical membrane-based
hybridization analysis of
human genomic DNA digested with one 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, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VII. Microarrays
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, su ra.) An
array analogous to a dot
or slot blot may also be used to arrange and link elements to the surface of a
substrate using thermal,
UV, chemical, or mechanical bonding procedures. A typical array may be
produced by hand or using
available methods and machines and contain any appropriate number of elements.
After
hybridization, nonhybridized probes are removed and a scanner used to
determine the levels and
patterns of fluorescence. The degree of complementarity and the relative
abundance of each probe
which hybridizes to an element on the microarray may be assessed through
analysis of the scanned
images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may
comprise
the elements of the microarray. Fragments suitable for hybridization can be
selected using software
well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs,
ESTs, or
fragments thereof corresponding to one of the nucleotide sequences of the
present invention, or
selected at random from a cDNA library relevant to the present invention, are
arranged on an
appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide
using, e.g., UV cross-linking
followed by thermal and chemical treatments and subsequent drying. (See, e.g.,
Schena, M. et al.
(1995) Science 270:467-470; Shalom D. et al. (1996) Genome Res. 6:639-645.)
Fluorescent probes
are prepared and used for hybridization to the elements on the substrate. The
substrate is analyzed by
procedures described above.
VIII. Complementary Polynucleotides
Sequences complementary to the LRSP-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring LRSP. Although
use of oligonucleotides
46
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
comprising from about 15 to 30 base pairs is described, essentially the same
procedure is used with
smaller or with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of LRSP. 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 LRSP-encoding transcript.
IX. Expression of LRSP
Expression and purification of LRSP is achieved using bacterial or virus-based
expression
systems. For expression of LRSP in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express LRSP upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of LRSP in eukaryotic cells is
achieved by infecting insect
or mammalian cell lines with recombinant Autographica californica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding LRSP by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, LRSP is synthesized as a fusion protein with,
e.g., glutathione
S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His,
permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-
kilodalton enzyme from Schistosoma japonicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
LRSP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity
purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues, enables
purification on metal-chelate
resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel (1995,
47
CA 02348046 2001-04-20
WO 00/24779 PCT/US99124046
aunts, ch. 10 and 16). Purified LRSP obtained by these methods can be used
directly in the following
activity assay.
X. Demonstration of LRSP Activity
The activity of LRSP may be demonstrated by messing the effect that LSRP has
upon
bacterial growth. Antifungal and antibacterial agents inhibit bacterial cell
growth (Mot, A. and P.
Nicolas ( 1994) J. Biol. Chem. 269:1934-1939). Bacterial cells, such as E.
coli (0.05 OD~o units), are
incubated with LB medium containing various amounts of LRSP (5 to 1000 pg/ml)
for 24 hours at -
30 °C in 96-well plates as described by Mor and Nicolas su ra). The
absorbance of the cells in each
well is then measured at 600 nm or 490 nm using a 96-well plate reader. The
absorbance of the cells
is compared to that of control, untreated cells. The decrease in absorbance is
proportional to the
amount of LRSP in the sample.
Alternatively, LRSP, or biologically active fragments thereof, are labeled
with ''-5I
Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-
539). Candidate
molecules previously arrayed in the wells of a mufti-well plate are incubated
with the labeled LRSP,
washed, and any wells with labeled LRSP complex are assayed. Data obtained
using different
concentrations of LRSP are used to calculate values for the number, affinity,
and association of LRSP
with the candidate molecules.
XI. Functional Assays
LRSP function is assessed by expressing the sequences encoding LRSP 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 ,ug of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. I-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-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
48
CA 02348046 2001-04-20
WO 00/Z4779 PCT/US99/24046
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 LRSP on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding LRSP and either CD64 or CD64-GFP.
CD64 and CD64-
GFP are expressed on the surface of transfected cells and bind to conserved
regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success
NY). mRNA can be purified from the cells using methods well known by those of
skill in the art.
Expression of mRNA encoding LRSP and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XII. Production of LRSP Specific Antibodies
LRSP 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 LRSP amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to ICLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with
the oligopeptide-
ICLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-
LRSP activity by, for example, binding the peptide or LRSP to a substrate,
blocking with 1% BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
JOII. Purification of Naturally Occurring LRSP Using Specific Antibodies
Naturally occurring or recombinant LRSP is substantially purified by
immunoaffinity
chromatography using antibodies specific for LRSP. An immunoaffinity column is
constructed by
covalently coupling anti-LRSP antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
49
CA 02348046 2001-04-20
WO 00/24779 PCT/US99124046
blocked and washed according to the manufacturer's instructions.
Media containing LRSP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of LRSP (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/LRSP 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 LRSP is collected.
XIV. Identification of Molecules Which Interact with LRSP
LRSP, or biologically active fragments thereof, are labeled with 'ZSI Bolton-
Hunter reagent.
(See, e.g., Bolton and Hunter, s, unra.) Candidate molecules previously
arrayed in the wells of a multi-
well plate are incubated with the labeled LRSP, washed, and any wells with
labeled LRSP complex
are assayed. Data obtained using different concentrations of LRSP are used to
calculate values for the
number, affinity, and association of LRSP 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 with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are
obvious to those skilled in molecular biology or related fields are intended
to be within the scope of
the following claims.
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/Z404b
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51
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
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52
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
TANG, Y. Tom
CORLEY, Neil C.
GUEGLER, Karl J.
PATTERSON, Chandra
<120> LYSINE-RICH STATHERIN PROTEIN
<130> PF-0620 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/155,209
<151> 1998-10-23
<160> 6
<170> PERL Program
<210> 1
<211> 95
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2820214CD1
<400> 1
Met Trp Phe His Lys Val Gly Arg Lys Gln His Phe Lys Va1 Thr
1 5 10 15
Phe Trp Glu Thr Asp Leu Ser Asn Asn Lys Thr Leu Val Ser Leu
20 25 30
Lys Lys Lys Lys Pro Phe His Leu Tyr Cys Val Ile Tyr Ile Pro
35 40 45
Leu Val Pro Lys Leu Ile Ile Leu Phe Leu Asp Ile Ala Phe Ile
50 55 60
Pro Lys Ser Leu Ile Ser Gln Phe Gln Asn Asn His Tyr Thr His
65 70 75
Asn His Thr Asn His Asn Thr Asn Asn Ile Arg Phe Asn Ile Ile
80 85 90
Ser Asn Cys Arg Thr
<210> 2
<211> 1331
<212> DNA
<213> Homo sapiens
1/4
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
<220>
<221> unsure
<222> 1242
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 2820214CB1
<400> 2
ctatgttttt agaatcaaag atgaaccggt aagctgtctc atgtaccaaa cgtgaaattt 60
acagtgttta caaatgtctg gaattttgca ctgccatagg gaatgttaag gttacttggc 120
tggaatttat cagacttgtg agtaaacaag ttgaagttta gcagatgagg gggaatattg 180
aggcccctaa ggctaaacaa aataatcagt atctgagata gtggctaatg tggctcecca 240
ggcctaattt gggaacagtt tttcctgatt gctttgagaa gtactttctt ttgacagaaa 300
ttttcattct gcttgccatt gctatattct ccctttatag gagccattgg atttctttcc 360
ttttgtggga aatgtcccat tagcattttc agatcttttg atgtgcacta atgccattat 420
tggtaatgcc gttattggtg aatacagcat agttaaataa actgttacag taaatctaca 480
cttggatttg ctgcacctct accaatagcc ttttgaatga ctgaaagtgt taacagagaa 540
agaggcatgt ctgcagaaag agatagctaa tattttttgg tactttat:ct gaaatccaag 600
atgctgcttc ccctgcaggt tgttttcctt cttacgatcc tcattgaatc ccctctggga 660
gcacaggaca gttagtagaa ctctccattt ctttgttttg ttttttaaga cagagactct 720
gtctcaaaaa aaaggacatt tatcattata acatcttatt agagccccta atttcttatc 7B0
tgaaggcact gttttttttt ttaaacagtt aagtactgat gtcaacagac aaatatttct 840
gatcagatag tcccctgtca acagtagcaa atgtggtttc ataaagtggg aagaaaacag 900
cattttaaag taactttttg ggagactgat ttgagtaata ataaaactct ggtctccctt 960
aagaaaaaaa aacccttcca cctttactgt gtcatttata tccccttagt tccaaagtta 1020
attatcttat ttctggatat tgcttttata ccaaagagcc ttatcagcca gttccagaac 1080
aaccactata cgcacaacca taccaaccac aataccaaca atatacgttt taatatcatc 1140
agtaactgca ggacatgatt attgaggctt gattggcaaa tacgacttct acatccatat 1200
tctcatcttt cataccatat cacactacta ccactttttg tnagatcatc taagagcaat 1260
gcgaatgtaa aaccctataa tttactggat actctttggt tccagatact tgccttttcc 1320
aatgtcactt g 1331
<210> 3
<211> 62
<212> PRT
<213> Homo sapiens
<300> misc_feature
<308> GenBank ID No: 8338508
<400> 3
Met Lys Phe Leu Val Phe Ala Phe Ile Leu Ala Leu Met Val Ser
1 5 10 15
Met Ile Gly Ala Asp Ser Ser Glu Glu Lys Phe Leu Arg Arg Ile
20 25 30
Gly Arg Phe Gly Tyr Gly Tyr Gly Pro Tyr Gln Pro Val Pro Glu
35 40 45
Gln Pro Leu Tyr Pro Gln Pro Tyr Gln Pro Gln Tyr Gln Gln Tyr
50 55 60
Thr Phe
2/4
CA 02348046 2001-04-20
WO 00/24779 PCT/US99/24046
<210> 4
<211> 51
<212> PRT
<213> Homo sapiens
<300> misc_feature
<308> GenBank ID No: g179466
<400> 4
Met Lys Phe Phe Val Phe Ala Leu Ile Leu Ala Leu Met Leu Ser
1 5 10 15
Met Thr Gly Ala Asp Ser His Ala Lys Arg His His Gly Tyr Lys
20 25 30
Arg Lys Phe His Glu Lys His His Ser His Arg Gly Tyr Arg Ser
35 40 45
Asn Tyr Leu Tyr Asp Asn
<210> 5
<211> 552
<212> DNA
<213> Homo sapiens
<300> misc_feature
<308> GenBank ID No: 8338507
<400> 5
atctcttgaa gcttcacttc aacttcacta cttctgtagt ctcatcttga gtaaaagaga 60
acccagccaa ctatgaagtt ccttgtcttt gccttcatct tggctctcat ggtttccatg 120
attggagctg attcatctga agagaaattt ttgcgtagaa ttggaagatt cggttatggg 180
tatggccctt atcagccagt tccagaacaa ccactatacc cacaaccata ccaaccacaa 240
taccaacaat atacctttta atatcatcag taactgcagg acatgattat tgaggcttga 300
ttggcaaata cgacttctac atccatattc tcatctttca taccatatca cactactacc 360
actttttgaa gaatcatcaa agagcaatgc aaatgaaaaa cactataatt tactgtatac 420
tctttgtttc aggatacttg ccttttcaat tgtcacttga tgatataatt gcaatttaaa 480
ctgttaagct gtgttcagta ctgtttctga ataatagaaa tcacttctct aaaagcaata 540
aatttcaagc cc 552
<210> 6
<211> 491
<212> DNA
<213> Homo sapiens
3/4
CA 02348046 2001-04-20
WO 00/24779 PCT/U899/24046
<300> misc_feature
<308> GenBank ID No: 8179465
<400> 6
aggacgccta ccaggaggac ctgggattca accaactatg aagttttttg tttttgcttt 60
aatcttggct ctcatgcttt ccatgactgg agctgattca catgcaaaga gacatcatgg 120
gtataaaaga aaattccatg aaaagcatca ttcacatcga ggctatagat caaattatct 180
gtatgacaat tgatatcttc agtaatcatg gggcatgatt atggaggttt gactggcaaa 240
ttcgctttgg actcgtgtat tctcatttgt cataccgcat cacactacta ctgctttttg 300
aaggaattat cataaggcaa tgcagaataa aagaaatacc atgatttagt gaattctgtg 360
tttcaggata cttcccttcc taattatcat ttgattagat acttgcaatt taaatgttaa 420
gctgttttca ctgctgtttc tgagtaatag aaattcattc ctctccaaaa gcaataaaat 480
tcaagcacat t 491
4/4
