Note: Descriptions are shown in the official language in which they were submitted.
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
CYCLIC NUCLEOTIDE-ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of cyclic
nucleotide-
associated proteins and to the use of these sequences in the diagnosis,
treatment, and prevention of
cell proliferative, autoimmune/inflammatory, neurological, vision,
reproductive, and smooth
muscle disorders.
BACKGROUND OF THE INVENTION
Cyclic nucleotides include adenosine 3',5'-cyclic monophosphate (CAMP) and
guanosine
3'S'-cyclic monophosphate (cGMP). cAMP and cGMP, which serve as second
messengers in
many intracellular signaling pathways, are generated by the enzymes adenylyl
(adenylate) cyclase
and guanylyl (guanylate) cyclase. The cyclases are themselves regulated by
extracellular
signaling pathways.
cAMP has various tissue-specific effects. Increased levels of cAMP lead to an
increase in
triglyceride hydrolysis and a decrease in amino acid uptake in adipose tissue;
an increase in
conversion of glycogen to glucose, an inhibition of glycogen synthesis, and an
increase in
gluconeogenesis in liver; an increase in the synthesis of estrogen and
progesterone in ovarian
follicle; an increase in the synthesis of aldosterone and cortisol in adrenal
cortex; an increase in the
contraction rate in cardiac muscle cells; conversion of glycogen to glucose in
skeletal muscle;
secretion of thyroxine in thyroid; an increase in resorption of calcium from
bone in bone cells;
fluid secretion in intestine; resorption of water in kidney; and an inhibition
of aggregation and
secretion in blood platelets. cAMP activates cAMP-dependent protein kinases
which modify the
activities of specific enzymes in various cell types. (See Lodish, H. et ai. (
1995) Molecular Cell
Bi Scientific American Books, New York, NY pp. 871; 879-886.) In olfactory
epithelium
cAMP regulates a cation channel that functions in odorant signal transduction
(Prosite
PDOC00691 ).
cGMP plays a role in vascular and non-vascular smooth muscle contraction and
relaxation, peripheral and central neurotransmission, platelet reactivity, and
retinal
phototransduction (Hobbs, A.J. (1997) Trends Pharmacol. Sci. 18:484-491). cGMP
may also be
involved in immunomodulation (Chhajlani, V. et al. (1991) FEBS Lett. 290:157-
158). cGMP
activates cGMP-dependent protein kinases. cGMP binding to a retinal rod cell
sodium channel
leads to opening of the channel and depolarization of rod photoreceptors
(Prosite, ~uara; Lodish,
supra, pp. 975-976).
-1-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
Proteins associated with cyclic nucleotides therefore include adenyfyl
cyclases, guanylyl
cyclases, cAMP and cGMP-dependent protein kinases, and cyclic-nucleotide-gated
cation
channeis. Cyclic nucleotide binding domain motifs are found in cAMP- and cGMP-
dependent
kinases and in cyclic nucleotide-gated cation channels (Prosite, suara). These
motifs, around 120
residues in length, contain 6 invariant amino acids which seem to be important
for the
maintenance of the structural integrity of the domain. The kinases contain two
tandem copies of
the motif.
Adenylyl cyclase catalyzes the formation of cAMP and pyrophosphate from ATP.
Adenylyl cyclase, which has both membrane-associated and soluble forms, is
found in many
1o eukaryotes including mammals and yeasts (Lodish, su ra, pp.879-886; Mittal,
C.K. (1986)
Methods Enzymol. 132:422-428). The Saccharomvces cerevisiae adenylyl cyclase
has multiple
domains including an N-terminal domain of unknown function, a central domain
containing
multiple amphipathic leucine-rich repeats, which is required for activation of
adenylyl cyclase by
Ras, and a C-terminal catalytic domain (Young, D. et al. (1991) Gene 102:129-
132; Masuelli, L.
IS and M.L. Cutler (1996) Mol. Cell. Biol. 16:5466-5476). The leucine-rich
repeats, thought to
function in protein-protein interaction and cellular adhesion, are found in
many proteins. These
proteins include membrane-associated Schizosaccharomyces pombe and
Saccharomyces kluweri
adenylyl cyclases; the mammalian protein Rsu-I, which suppresses
transformation of mammalian
cells by v-Ras; flightless-I, a protein involved in Drosophila gastrulation
and muscle degeneration;
20 the Drosophila adhesion proteins Toll and Chaoptin; and von Willebrand
receptor factor proteins
(Masuelli, su ra; Claudianos, C. and H.D. Campbell (1995) Mol. Biol. Evol.
12:405-414).
Guanylyl cyclase catalyzes the formation of cGMP and pyrophosphate from GTP.
There
exist both membrane-bound and soluble guanylyl cyclases, which share a
conserved domain
probably important for catalytic activity. The soluble guanyiyl cyclases are
cytoplasmic
25 heterodimers of highly related subunits called alpha and beta. Multiple
forms of the alpha and
beta subunits, apparently tissue-specific, have been characterized (Chhajlani,
s" uora; Giuili, G. et
al. (1992) FEBS Lett. 304:83-88). Soluble guanylyl cyclase is associated with
heme and activated
by nitric oxide. The nitric oxide-soluble guanylyl cyclase-cGMP pathway is
widespread in
mammalian tissues and important in mediating numerous physiological processes
including
30 vascular and non-vascular smooth muscle relaxation, peripheral and central
neurotransmission,
platelet reactivity, and phototransduction. Overactivity of the nitric oxide-
soluble guanylyl
cyclase-cGMP pathway may be associated with septic shock and migraine, while
underactivity of
the pathway may be associated with impotence, hypertension, and asthma (Hobbs,
s_ uara).
The discovery of new cyclic nucleotide-associated proteins and the
polynucleotides
-2-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
encoding them satisfies a need in the art by providing new compositions which
are useful in the
diagnosis, prevention, and treatment of cell proliferative,
autoimmune/inflammatory, neurological,
vision, reproductive, and smooth muscle disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, cyclic nucleotide-
associated
proteins, referred to collectively as "CNAP" and individually as "CNAP-1,"
"CNAP-2," and
"CNAP-3". In one aspect, the invention provides a substantially purified
polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ
ID N0:2, and
SEQ ID N0:3 (SEQ ID NO:1-3), and fragments thereof.
The invention further provides a substantially purified variant having at
least 90% amino
acid identity to at least one of the amino acid sequences selected from the
group consisting of SEQ
ID NO:1-3, 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:1-3, and fragments thereof. The invention also
includes an
isolated and purified polynucleotide variant having at least 70%
poiynucleotide sequence identity
to the polynucleotide encoding the polypeptide comprising an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-3, 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:1-3,
and fragments
thereof. The invention also provides an isolated and purified polynucleotide
having a sequence
which is complementary to the palynucleotide encoding the polypeptide
comprising the amino
acid sequence selected from the group consisting of SEQ iD NO: l-3, and
fragments thereof.
The invention also provides an isolated and purified polynucleotide comprising
a
polynucleotide sequence selected from the group consisting of SEQ ID N0:4, SEQ
ID NO:S, and
SEQ ID N0:6 (SEQ ID N0:4-6), and fragments thereof. The invention further
provides an
isolated and purified polynucleotide variant having at least 70%
polynucleotide sequence identity
to the polynucleotide sequence selected from the group consisting of SEQ ID
N0:4-6, 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 N0:4-6, 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
-3-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
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. 1n one
aspect, the method further comprises amplifying the polynucleotide prior to
hybridization.
The invention further provides an expression vector containing at least a
fragment of the
polynucleotide encoding the poiypeptide comprising an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-3, and fragments thereof. 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 at least a
fragment of a polynucleotide under conditions suitable for the expression of
the polypeptide; and
(b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a
substantially
purified polypeptide having the amino acid sequence selected from the group
consisting of SEQ
ID NO: i-3, 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:1-3, 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 CNAP, the method comprising administering
to a subject in
need of such treatment an effective amount of a pharmaceutical composition
comprising a
substantially purified polypeptide having the amino acid sequence selected
from the group
consisting of SEQ ID NO:1-3, 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 CNAP, 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:1-3, and fragments
thereof.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows nucleotide and polypeptide sequence identification numbers (SEQ
ID NO),
clone identification numbers (clone ID), cDNA libraries, and cDNA fragments
used to assemble
full-length sequences encoding CNAP.
Table 2 shows features of each polypeptide sequence including potential
motifs,
homologous sequences, and methods and algorithms used for identification of
CNAP.
_q_
CA 02349210 2001-03-02
WO 00114248 PCT/US99/20287
Table 3 shows the tissue-specific expression patterns of each nucleic acid
sequence as
determined by northern analysis, diseases or disorders associated with these
tissues, and the vector
into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
Incyte
cDNA clones encoding CNAP were isolated.
Table 5 shows the programs, their descriptions, references, and threshold
parameters used
to analyze CNAP.
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 tines, 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
constrned as an admission that the invention is not entitled to antedate such
disclosure by virtue of
prior invention.
DEFINITIONS
"CNAP" refers to the amino acid sequences of substantially purified CNAP
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, murine, equine,
and preferably the human species, from any source, whether natural, synthetic,
semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which, when bound to CNAP, increases
or
-5-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
prolongs the duration of the effect of CNAP. Agonists may include proteins,
nucleic acids,
carbohydrates, or any other molecules which bind to and modulate the effect of
CNAP.
An "allelic variant" is an alternative form of the gene encoding CNAP. Allelic
variants
may result from at least one mutation in the nucleic acid sequence and may
result in altered
mIZNAs or in polypeptides whose structure or function may or may not be
altered. Any given
natural or recombinant gene may have none, one, or many allelic forms. 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 CNAP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polynucleotide the same as
CNAP or a polypeptide with at least one functional characteristic of CNAP.
Included within this
definition are polymorphisms which may or may not be readily detectable using
a particular
oligonucleotide probe of the polynucleotide encoding CNAP, and improper or
unexpected
hybridization to allelic variants, with a locus other than the normal
chromosomal locus for the
polynucleotide sequence encoding CNAP. The encoded protein may also be
"altered," and may
contain deletions, insertions, or substitutions of amino acid residues which
produce a silent change
and result in a functionally equivalent CNAP. 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 tong as the biological or immunological
activity of CNAP is
retained. For example, negatively charged amino acids may include aspartic
acid and glutamic
acid, positively charged amino acids may include lysine and arginine, and
amino acids with
uncharged polar head groups having similar hydrophilicity values may include
leucine, isoleucine,
and valine; glycine and alanine; asparagine and glutamine; serine and
threonine; and
phenylalanine and tyrosine.
The terms "amino acid" or "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or
synthetic molecules. In this context, "fragments," "immunogenic fragments," or
"antigenic
fragments" refer to fragments of CNAP which are preferably at least 5 to about
15 amino acids in
length, most preferably at least 14 amino acids, and which retain some
biological activity or
immunological activity of CNAP. 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.
-6-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerise chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which, when bound to CNAP,
decreases the
amount or the duration of the effect of the biological or immunological
activity of CNAP.
Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or
any other molecules
which decrease the effect of CNAP.
The term "antibody" refers to intact molecules as well as to fragments
thereof, such as
Fab, F(ab')2, and Fv fragments, which are capable of binding the epitopic
determinant. Antibodies
that bind CNAP polypeptides can be prepared using intact poiypeptides 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,
1 S and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to
immunize the animal.
The term "antigenic determinant" refers to that fragment of a molecule (i.e.,
an epitope)
that makes contact with a particular antibody. When a protein or a fragment of
a protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (given regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid
sequence which
is complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules
may be produced by any method including synthesis or transcription. Once
introduced into a cell,
the complementary nucleotides combine with natural sequences produced by the
cell to form
duplexes and to block either transcription or translation. The designation
"negative" can refer to
the antisense strand, and the designation "positive" can refer to the sense
strand.
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 CNAP, or of
any oligopeptide
thereof, to induce a specific immune response in appropriate animals or cells
and to bind with
specific antibodies.
The terms "complementary" or "complementarity" refer to the natural binding of
polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the
CA 02349210 2001-03-02
WO 00/14248 PCT/US991202$7
complementary sequence "3' T-C-A 5'." Complementarily between two single-
stranded molecules
may be "partial," such that only some of the nucleic acids bind, or it may be
"complete," such that
total complementarily exists between the single stranded molecules. The degree
of
complementarily between nucleic acid strands has significant effects on the
efficiency and strength
of the hybridization between the nucleic acid strands. This is of particular
importance in
amplification reactions, which depend upon binding between nucleic acids
strands, and in the
design and use of peptide nucleic acid (PNA) molecules.
A "composition comprising a given polynucleotide sequence" or a "composition
comprising a given amino acid sequence" 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
CNAP or
fragments of CNAP 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 XL-PCR kit (Perkin-Elmer, Norwatk CT)
in the 5' and/or
the 3' direction, and resequenced, or which has been assembled from the
overlapping sequences of
more than one Incyte Clone 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.
The term "correlates with expression of a polynucleotide" indicates that the
detection of
the presence of nucleic acids, the same or related to a nucleic acid sequence
encoding CNAP, by
northern analysis is indicative of the presence of nucleic acids encoding CNAP
in a sample, and
thereby correlates with expression of the transcript from the polynucleotide
encoding CNAP.
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, 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
-8-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
from which it was derived.
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" or "% identity" refer to the percentage of
sequence
similarity found in a comparison of two or more amino acid or nucleic acid
sequences. Percent
identity can be determined electronically, e.g., by using the MEGALIGN program
(DNASTAR,
Madison WI). The MEGALIGN program can create alignments between two or more
sequences
according to different methods, e.g., the clustal method. (See, e.g., Higgins,
D.G. and P.M. Sharp
( 1988) Gene 73:237-244.) The ciustal algorithm groups sequences into clusters
by examining the
distances between ail pairs. The clusters are aligned pairwise and then in
groups. The percentage
similarity between two amino acid sequences, e.g., sequence A and sequence B,
is calculated by
dividing the length of sequence A, minus the number of gap residues in
sequence A, minus the
number of gap residues in sequence B, into the sum of the residue matches
between sequence A
and sequence B, times one hundred. Gaps of low or of no similarity between the
two amino acid
sequences are not included in determining percentage similarity. Percent
identity between nucleic
acid sequences can also be counted or calculated by other methods known in the
art, e.g., the Jotun
Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.)
Identity between
sequences can also be determined by other methods known in the art, e.g., by
varying
hybridization conditions.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of
the elements
-9-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
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 any process by which a strand of nucleic acid binds
with a
complementary strand through base pairing.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution {e.g., Cot or Ttot analysis)
or.formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a
solid support (e.g., paper, membranes, filters, chips, pins or glass slides,
or any other appropriate
substrate to which cells or their nucleic acids have been fixed).
The words "insertion" or "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively,
I S to the sequence found in the naturally occurring molecule.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by
expression of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which
may affect cellular and systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on
a substrate.
The terms "element" or "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 CNAP. For example,
modulation may cause an increase or a decrease in protein activity, binding
characteristics, or any
other biological, functional, or immunological properties of CHAP.
The phrases "nucleic acid" or "nucleic acid sequence," as used herein, refer
to a
nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These
phrases also refer to
DNA or RNA of genomic or synthetic origin which may be single-stranded or
double-stranded
and may represent the sense or the antisense strand, to peptide nucleic acid
(PNA), or to any
DNA-like or RNA-like material. In this context, "fragments" refers to those
nucleic acid
sequences which comprise a region of unique polynucleotide sequence that
specifically identifies
SEQ ID N0:4-6, for example, as distinct from any other sequence in the same
genome. For
example, a fragment of SEQ ID N0:4-6 is useful in hybridization and
amplification technologies
and in analogous methods that distinguish SEQ ID N0:4-6 from related
polynucleotide sequences.
-10-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
A fragment of SEQ ID N0:4-6 is at least about 1 S-20 nucleotides in length.
The precise length of
the fragment of SEQ ID N0:4-6 and the region of SEQ ID N0:4-6 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment. In some cases, a fragment, when translated, would
produce
polypeptides retaining some functional characteristic, e.g., antigenicity, or
structural domain
characteristic, e.g., ATP-binding site, of the full-length polypeptide.
The terms "operably associated" or "operably linked" refer to functionally
related nucleic
acid sequences. A promoter is operably associated or operably linked with a
coding sequence if
the promoter controls the translation of the encoded polypeptide. While
operably associated or
operably linked nucleic acid sequences can be contiguous and in the same
reading frame, certain
genetic elements, e.g., repressor genes, are not contiguously linked to the
sequence encoding the
polypeptide but still bind to operator sequences that control expression of
the polypeptide.
The term "oligonucleotide" refers to a nucleic acid sequence of at least about
6
nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most
preferably about 20
to 25 nucleotides, which can be used in PCR amplification or in a
hybridization assay or
microarray. "Oligonucleotide" is substantially equivalent to the terms
"amplimer," "primer,"
"oligomer," and "probe," as these terms are commonly defined in the art.
"Peptide nucleic acid" (PNA) 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.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding CNAP, or fragments thereof, or CNAP 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" or "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, or an antagonist. The
interaction is dependent upon
the presence of a particular structure of the protein, e.g., the antigenic
determinant or epitope,
recognized by the binding molecule. For example, if an antibody is specific
for epitope "A," the
presence of a polypeptide containing the epitope A, or the presence of free
unlabeled A, in a
reaction containing free labeled A and the antibody will reduce the amount of
labeled A that binds
to the antibody.
The term "stringent conditions" refers to conditions which permit
hybridization between
-11-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
polynucleotides and the claimed polynucleotides. Stringent conditions can be
defined by salt
concentration, the concentration of organic solvent, e.g., formamide,
temperature, and other
conditions well known in the art. In particular, stringency can be increased
by reducing the
concentration of salt, increasing the concentration of formamide, or raising
the hybridization
temperature.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least about 60%
free, preferably about 75% free, and most preferably about 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or
nucleotides by
different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
"Transformation" describes a process by which exogenous DNA enters and changes
a
recipient cell. Transformation may 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 CNAP polypeptides refers to an amino acid sequence that is
altered by one
or more amino acid residues. The variant may have "conservative" changes,
wherein a substituted
amino acid has similar structural or chemical properties (e.g., replacement of
leucine with
isoleucine). More rarely, a variant may have "nonconservative" changes (e.g.,
replacement of
glycine with tryptophan). Analogous minor variations may also include amino
acid deletions or
insertions, or both. Guidance in determining which amino acid residues may be
substituted,
inserted, or deleted without abolishing biological or immunologica) activity
may be found using
computer programs well known in the art, for example, LASERGENE software
(DNASTAR).
The term "variant," when used in the context of a polynucleotide sequence, may
-12-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
encompass a polynucleotide sequence related to CNAP. This definition may also
include, for
example, "allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice
variant may have significant identity to a reference molecule, but will
generally have a greater or
lesser number of polynucleotides due to alternate splicing of exons during
mRNA processing. The
corresponding polypeptide may possess additional functional domains or an
absence of domains.
Species variants are poiynucleotide sequences that vary from one species to
another. The resulting
polypeptides generally will have significant amino acid identity relative to
each other. A
polymorphic variant is a variation in the polynucleotide sequence of a
particular gene between
individuals of a given species. Polymorphic variants also may encompass
"single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one base.
The presence
of SNPs may be indicative of, for example, a certain population, a disease
state, or a propensity for
a disease state.
THE INVENTION
The invention is based on the discovery of new human cyclic nucleotide-
associated
proteins (CNAP), the poiynucleotides encoding CNAP, and the use of these
compositions for the
diagnosis, treatment, or prevention of cell proliferative,
autoimmune/inflammatory, neurological,
vision, reproductive, and smooth muscle disorders.
Table 1 lists the Incyte Clones used to derive full length nucleotide
sequences encoding
CNAP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NO) of
the amino
acid and nucleic acid sequences, respectively. Column 3 shows the Clone ID of
the Incyte Clone
in which nucleic acids encoding each CNAP were identified, and column 4, the
cDNA libraries
from which these clones were isolated. Column 5 shows Incyte clones, their
corresponding cDNA
libraries, and shotgun sequences useful as fragments in hybridization
technologies, and which are
part of the consensus nucleotide sequence of each CNAP.
The columns of Table 2 show various properties of the polypeptides of the
invention:
column I references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3, potential phosphorylation sites; column 4, potential
glycosylation sites;
column 5, the amino acid residues comprising signature sequences and motifs;
column 6, the
identity of each protein; and column 7, analytical methods used to identify
each protein through
sequence homology and protein motifs.
CNAP-1 has chemical and structural similarity with Saccharom ces kluyveri
adenylyl
cyclase (GI 117793). In particular, CNAP-1 and Saccharom,~es kluyveri adenylyl
cyclase (GI
117793) share 15% identity. PRINTS analysis identifies sixteen potential
leucine-rich repeats in
CNAP-1.
-13-
CA 02349210 2001-03-02
WO 00/14248
PCT/US99/20287
CNAP-2 has chemical and structural similarity with Aquifex p ~~rophilus
esterase 28LC
(fallen, W. et al. ( 1997) PCT Application W09730160-A 1 ). In particular,
CNAP-2 and A uifex
pyro~hilus esterase 28LC (fallen, supra) share 24% identity. PFAM analysis
indicates that
CNAP-2 has two potential cNMP-binding domains from N 144 through Q269 and from
5573
through H696. Within these potential cNMP-binding domains CHAP-2 contains
conserved
residues at G 178, G 190, 8233, 6612, 6624, 6650, and 8660. BLOCKS analysis
indicates that
CNAP-2 resembles cyclic-nucleotide binding domain proteins from 6605 through
5628 and from
Y643 through P676.
CNAP-3 has chemical and structural similarity with the human soluble guanylate
cyclase
large subunit (GI 31684). In particular, CNAP-3 and human soluble guanylate
cyclase large
subunit (GI 31684) share 89% identity. Motifs analysis indicates that CNAP-3
has a guanylate
cyclase signature sequence from 6585 through E608. PFAM analysis indicates
that CNAP-3
resembles guanylate cyclase from V472 through Q661. ProfileScan indicates that
CNAP-3
resembles guanylate cyclase from P566 through D628. BLOCKS analysis indicates
that CNAP-3
resembles guanylate cyclase from V472 through D514, from V522 through L538,
and from M573
through 5619.
The columns of Table 3 show the tissue-specificity and disease-association of
nucleotide
sequences encoding CNAP. The first column of Table 3 lists the polynucleotide
sequence
identifiers. The second column lists tissue categories which express CNAP as a
fraction of tots!
tissue categories expressing CNAP. The third column lists the disease classes
associated with
those tissues expressing CNAP. The fourth column lists the vectors used to
subclone the cDNA
library. Of particular note is the expression of CNAP in cancerous or
proliferating, inflamed,
nervous, and reproductive tissues.
The following fragments of the nucleotide sequences encoding CNAP are useful
in
hybridization or amplification technologies to identify SEQ ID N0:4-6 and to
distinguish between
SEQ ID N0:4-6 and related polynucleotide sequences. The useful fragments are
the fragment of
SEQ ID N0:4 from about nucleotide 414 to about nucleotide 446; the fragment of
SEQ ID NO:S
from about nucleotide 136 to about nucleotide 165; and the fragment of SEQ ID
N0:6 from about
nucleotide 863 to about nucleotide 892.
The invention also encompasses CNAP variants. A preferred CNAP variant is one
which
has at least about 80%, more preferably at least about 90%, and most
preferably at least about 95%
amino acid sequence identity to the CNAP amino acid sequence, and which
contains at least one
functional or structural characteristic of CNAP.
The invention also encompasses polynucleotides which encode CNAP. In a
particular
-19-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence
selected from the group consisting of SEQ ID N0:4-6, which encodes CNAP.
The invention also encompasses a variant of a polynucleotide sequence encoding
CNAP.
In particular, such a variant polynucleotide sequence will have at least about
70%, more preferably
at least about 85%, and most preferably at least about 95% polynucleotide
sequence identity to the
polynucleotide sequence encoding CNAP. A particular aspect of the invention
encompasses a
variant of a polynucleotide sequence comprising a sequence selected from the
group consisting of
SEQ ID N0:4-6 which has at least about 70%, more preferably at least about
85%, and most
preferably at least about 95% polynucleotide sequence identity to a nucleic
acid sequence selected
from the group consisting of SEQ ID N0:4-6. Any one of the polynucleotide
variants described
above can encode an amino acid sequence which contains at least one functional
or structural
characteristic of CNAP.
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 CNAP, 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 CNAP, and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode CNAP and its variants are
preferably
capable of hybridizing to the nucleotide sequence of the naturally occurring
CNAP under
appropriately selected conditions of stringency, it may be advantageous to
produce nucleotide
sequences encoding CNAP 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 CNAP and its
derivatives without altering
the encoded amino acid sequences include the production of RNA transcripts
having more
desirable properties, such as a greater half life, than transcripts produced
from the naturally
occurring sequence.
The invention also encompasses production of DNA sequences which encode CNAP
and
CNAP 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
-15-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
systems using reagents well known in the art. Moreover, synthetic chemistry
may be used to
introduce mutations into a sequence encoding CNAP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucieotide sequences, and, in particular, to
those shown in SEQ ID
S N0:4-6 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-S 11.) For example, stringent salt concentration will ordinarily be
less than about 7S0 mM
NaCI and 75 mM trisodium citrate, preferably less than about S00 mM NaCI and
SO mM trisodium
citrate, and most preferably less than about 250 mM NaCI and 25 mM trisodium
citrate. Low
stringency hybridization can be obtained in the absence of organic solvent,
e.g., formamide, while
high stringency hybridization can be obtained in the presence of at least
about 3S% formamide,
and most preferably at least about SO% formamide. Stringent temperature
conditions will
ordinarily include temperatures of at least about 30°C, more preferably
of at least about 37°C, and
most preferably of at least about 42°C. Varying additional parameters,
such as hybridization time,
IS the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the
inclusion or exclusion
of carrier DNA, are well known to those skilled in the art. Various levels of
stringency are
accomplished by combining these various conditions as needed. In a preferred
embodiment,
hybridization will occur at 30°C in 750 mM NaCI, 7S mM trisodium
citrate, and I% SDS. In a
more preferred embodiment, hybridization will occur at 37°C in 500 mM
NaCI, 50 mM trisodium
citrate, I% SDS, 35% formamide, and 100 ~g/ml denatured salmon sperm DNA
(ssDNA). In a
most preferred embodiment, hybridization will occur at 42°C in 2S0 mM
NaCI, 2S mM trisodium
citrate, I% SDS, SO % formamide, and 200 ~cg/ml ssDNA. Useful variations on
these conditions
will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash
stringency conditions can be defined by salt concentration and by temperature.
As above, wash
stringency can be increased by decreasing salt concentration or by increasing
temperature. For
example, stringent salt concentration for the wash steps will preferably be
less than about 30 mM
NaCI and 3 mM trisodium citrate, and most preferably less than about 1 S mM
NaCI and 1.S mM
trisodium citrate. Stringent temperature conditions for the wash steps will
ordinarily include
temperature of at least about 2S°C, more preferably of at least about
42°C, and most preferably of
at least about 68°C. In a preferred embodiment, wash steps will occur
at 25°C in 30 mM NaCI, 3
mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps
will occur at
42°C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1 % SDS. In a most
preferred embodiment,
wash steps will occur at 68°C in 15 mM NaCI, 1.5 mM trisodium citrate,
and 0.1 % SDS.
-16-
CA 02349210 2001-03-02
WO 00/1x248 PCT/US99/20287
Additional variations on these conditions will be readily apparent to those
skilled in the art.
Methods for DNA sequencing are well known in the art and may be used to
practice any
of the embodiments of the invention. The methods may employ such enzymes as
the Klenow
fragment of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq
polymerise
(Perkin-Eimer), 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 Hamilton MICROLAB 2200
(Hamilton, Reno
NV), Peltier Thermal Cycler 200 (PTC200; MJ Research, Watertown MA) and the
ABI
CATALYST 800 (Perkin-Elmer). Sequencing is then carried out using either ABI
373 or 377
DNA Sequencing Systems (Perkin-Elmer) or the MEGABACE capillary
electrophoresis system
(Molecular Dynamics, Sunnyvale CA). 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
Biology and Biotechnolo~v, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding CNAP may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream
sequences, such as promoters and regulatory elements. For example, one method
which may be
employed, restriction-site PCR, uses universal and nested primers to amplify
unknown sequence
from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR
Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend in divergent
directions to
amplify unknown sequence from a circularized template. The template is derived
from restriction
fragments comprising a known genomic locus and surrounding sequences. (See,
e.g., Triglia, T. et
al. ( 1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves
PCR
amplification of DNA fragments adjacent to known sequences in human and yeast
artificial
chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic.
1:1 I I-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
-17-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
another appropriate program, to be about 22 to 30 nucleotides in length, to
have a GC content of
about SO% 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. Tn particular,
capillary sequencing may employ flowable polymers for eiectrophoretic
separation, four different
nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled
device camera for
detection of the emitted wavelengths. Output/light intensity may be converted
to electrical signal
using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-
Elmer),
and the entire process from loading of samples to computer analysis and
electronic data display
may be computer controlled. Capillary electrophoresis is especially preferable
for sequencing
small DNA fragments which may be present in limited amounts in a particular
sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode CNAP may be cloned in recombinant DNA molecules that direct
expression of
CNAP, 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
CNAP.
The nucleotide sequences of the present invention can be engineered using
methods
generally known in the art in order to alter CNAP-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 CNAP may be synthesized, in whole or
in
part, using chemical methods well known in the art. (See, e.g., Caruthers,
M.H. et al. (1980)
Nucleic Acids Symp. Ser. 7:21 S-223; Horn, T. et al. ( 1980) Nucleic Acids
Symp. Ser. 7:225-232.)
Alternatively, CNAP itself or a fragment thereof may be synthesized using
chemical methods. For
-18-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
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 43 I A Peptide Synthesizer (Perkin-Elmer). Additionally, the amino
acid sequence of
CNAP, 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
Enrymol. 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 CNAP, the nucleotide sequences
encoding CNAP
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 CNAP. Such elements may vary in their
strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding CNAP. Such signals include the ATG initiation codon and
adjacent
sequences, e.g. the Kozak sequence. In cases where sequences encoding CNAP and
its initiation
codon and upstream regulatory sequences are inserted into the appropriate
expression vector, no
additional transcriptional or translational control signals may be needed.
However, in cases where
only coding sequence, or a fragment thereof, is inserted, exogenous
translational control signals
including an in-frame ATG initiation codon should be provided by the vector.
Exogenous
translational elements and initiation codons may be of various origins, both
natural and synthetic.
The efficiency of expression may be enhanced by the inclusion of enhancers
appropriate for the
particular host cell system used. (See, e.g., Scharf, D. et al. ( 1994)
Results Probi. 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 CNAP and appropriate
transcriptional and
translational control elements. These methods include in vitro recombinant DNA
techniques,
synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook,
J. et al. ( 1989)
Molecular Clonine. A Laboratory Manual, Cold Spring Harbor Press, Plainview
NY, ch. 4, 8, and
16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biology,
John Wiley & Sons,
New York NY, ch. 9, 13, and 16.)
-19-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
A variety of expression vector/host systems may be utilized to contain and
express
sequences encoding CNAP. 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 CNAP.
For example,
routine cloning, subcloning, and propagation of polynucleotide sequences
encoding CNAP can be
achieved using a muliifunctiona) E. coli vector such as PBLUESCRIPT
{Stratagene, La Jolla CA)
or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding CNAP
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 CNAP are
needed, e.g. for the production of antibodies, vectors which direct high level
expression of CNAP
may be used. For example, vectors containing the strong, inducible TS or T7
bacteriophage
promoter may be used.
Yeast expression systems may be used for production of CNAP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH,
may be used in the yeast Saccharomvces cerevisiae or Pichia pastoris. In
addition, such vectors
direct either the secretion or intracellular retention of expressed proteins
and enable integration of
foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Bitter, G.A. et al. ( 1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. ( 1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of CNAP. Transcription of
sequences
encoding CNAP 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. 6:307-3 I 1 ). 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; Winter, J. et al. ( 1991 )
Results Probl. Cell Differ.
-20-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
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
Technolosv (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 CNAP
may be ligated
into an adenovirus transcription/translation complex consisting of the late
promoter and tripartite
leader sequence. Insertion in a non-essential E1 or E3 region ofthe viral
genome may be used to
obtain infective virus which expresses CNAP 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 piasmid. HACs of about 6
kb to 10 Mb
are constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat.
Genet. 15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of CNAP in cell lines is preferred. For example, sequences encoding
CNAP 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 or apr cells, respectively.
(See, e.g., Wigler, M. et
al. ( 1977) Cell 11:223-232; Lowy, I. et al. ( 1980) Cell 22:817-823.) Also,
antimetabolite,
antibiotic, or herbicide resistance can be used as the basis for selection.
For example, dhfr confers
resistance to methotrexate; neo confers resistance to the aminoglycosides
neomycin and G-418;
and als or pat confer resistance to chlorsulfuron and phosphinotricin
acetyltransferase,
respectively. (See, e.g., Wigler, M, et al. (1980) Proc. Natl. Acad. Sci. USA
77:3567-3570;
Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14) Additional
selectable genes have been
-21-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/2028'7
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 conflrnned. For
example, if the sequence encoding CNAP is inserted within a marker gene
sequence, transformed
cells containing sequences encoding CNAP can be identified by the absence of
marker gene
function. Alternatively, a marker gene can be placed in tandem with a sequence
encoding CNAP
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 CNAP
and that
express CNAP 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 CNAP using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on CNAP is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art.
(See, e.g., Hampton, R. et al. (1990) Serological Methods. a Laboratory
Manual, APS Press, St.
Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in
Immunolosv, Greene Pub.
Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998)
Immunochemical
Protocols, Humana Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art
and may be used in various nucleic acid and amino acid assays. Means for
producing labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding CNAP
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled
-22-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
nucleotide. Alternatively, the sequences encoding CNAP, 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 !tits, 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, chemiIuminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors,
magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding CNAP 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 CNAP may be designed to
contain signal
sequences which direct secretion of CNAP through a prokaryotic or eukaryotic
cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications
of the polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro"
form of the protein may also be used to specify protein targeting, folding,
and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are
available from
the American Type Culture Collection (ATCC, 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 CNAP 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 CNAP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody
may facilitate the screening of peptide libraries for inhibitors of CNAP
activity. Heterologous
protein and peptide moieties may also facilitate purification of fusion
proteins using commercially
available affinity matrices. Such moieties include, but are not limited to,
glutathione S-transferase
(GST), maltose binding protein (MBP), thioredoxin (Trx), caimodulin binding
peptide {CBP), 6-
His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification
of their cognate fusion proteins on immobilized glutathione, maltose,
phenylarsine oxide,
-23-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and
hemagglutinin (1-iA) 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 CNAP
encoding sequence
and the heterologous protein sequence, so that CNAP may be cleaved away from
the heteroiogous
moiety following purification. Methods for fusion protein expression and
purification are
discussed in Ausubel ( 1995, su ra, 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 CNAP may
be
achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ
extract systems
(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, preferably 'SS-methionine.
Fragments of CNAP may be produced not only by recombinant production, but also
by
IS direct peptide synthesis using solid-phase techniques. (See, e.g.,
Creighton, su ra pp. 55-60.)
Protein synthesis may be performed by manual techniques or by automation.
Automated synthesis
may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin-
Elmer). Various
fragments of CNAP 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 CNAP and cyclic nucleotide-associated proteins. In addition, CNAP is
expressed in
cancerous or proliferating, inflamed, nervous, and reproductive tissues.
Therefore, in cell
proliferative, autoimmune/inflammatory, neurological, vision, reproductive,
and smooth muscle
disorders associated with decreased expression or activity of CNAP, it is
desirable to increase the
expression of CNAP. In cell proliferative, autoimmune/inflammatory,
neurological, vision,
reproductive, and smooth muscle disorders associated with increased expression
or activity of
CNAP, it is desirable to decrease the expression of CNAP.
Therefore, in one embodiment, CNAP 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 CNAP. Examples of such disorders include, but are not limited to,
cell proliferative
disorders such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis,
mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria,
poiycythemia vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma,
-24-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
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;
autoimmune/inflammatory disorders such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis,
amyloidosis, anemia, asthma, atheroscierosis, autoimmune hemolytic anemia,
autoimmune
thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease,
atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel
syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial
inflammation,
osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's
syndrome, rheumatoid
arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic
lupus erythematosus,
systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis,
Werner syndrome,
complications of cancer, hemodialysis, and extracorporeal circulation, viral,
bacterial, fungal,
parasitic, protozoal, and helminthic infections, and trauma; neurological
disorders such as
epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms,
Alzheimer's disease,
Pick's disease, Huntington's disease, dementia, Parkinson's disease and other
extrapyramidal
disorders, amyotrophic lateral sclerosis and other motor neuron disorders,
progressive neural
muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis
and other
demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidural
abscess, suppurative intracrania) thrombophlebitis, myelitis and radiculitis,
viral central nervous
system disease; prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the
central nervous system, cerebral palsy, neuroskeletal disorders, autonomic
nervous system
disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy
and other
neuromuscular disorders, peripheral nervous system disorders, derrnatomyositis
and polymyositis;
inherited, metabolic, endocrine, and toxic myopathies; myasthenia gravis,
periodic paralysis;
mental disorders including mood, anxiety, and schizophrenic disorders;
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, and Tourette's disorder; vision disorders such as conjunctivitis,
keratoconjunctivitis
-25-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
sicca, keratitis, episcleritis, iritis, posterior uveitis, glaucoma, amaurosis
fugax, ischemic optic
neuropathy, optic neuritis, Leber's hereditary optic neuropathy, toxic optic
neuropathy, vitreous
detachment, retinal detachment, cataract, macular degeneration, central serous
chorioretinopathy,
retinitis pigmentosa, melanoma of the choroid, retrobulbar tumor, and chiasmal
tumor;
reproductive disorders such as disorders of prolactin production; infertility,
including tubal
disease, ovulatory defects, and endometriosis; disruptions of the estrous
cycle, disruptions of the
menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome,
endometrial and
ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies,
and teratogenesis;
cancer of the breast, fibrocystic breast disease, and galactorrhea;
disruptions of spermatogenesis,
abnormal sperm physiology, cancer of the testis, cancer of the prostate,
benign prostatic
hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male
breast, and
gynecomastia; and smooth muscle disorders such as any impairment or alteration
in the normal
action of smooth muscle and may include, but is not limited to, angina,
anaphylactic shock,
arrhythmias, asthma, cardiovascular shock, Cushing's syndrome, hypertension,
hypoglycemia,
IS myocardial infarction, migraine, and pheochromocytoma, and myopathies
including
cardiomyopathy, encephalopathy, epilepsy, Kearns-Sayre syndrome, lactic
acidosis, myoclonic
disorder, and ophthalmoplegia. Smooth muscle includes, but is not limited to,
that of the blood
vessels, gastrointestinal tract, heart, and uterus.
In another embodiment, a vector capable of expressing CNAP 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 CNAP including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially
purified CNAP 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 CNAP
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of CNAP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of CNAP including, but not limited to, those listed above.
In a further embodiment, an antagonist of CNAP may be administered to a
subject to treat
or prevent a disorder associated with increased expression or activity of
CNAP. Examples of such
disorders include, but are not limited to, the cell proiiferative,
autoimmune/inflammatory,
neurological, vision, reproductive, and smooth muscle disorders described
above. In one aspect,
an antibody which specifically binds CNAP may be used directly as an
antagonist or indirectly as
a targeting or delivery mechanism for bringing a pharmaceutical agent to cells
or tissue which
-26-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/ZOZ87
express CNAP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding CNAP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of CNAP 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 CNAP may be produced using methods which are generally known
in
the art. In particular, purified CNAP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind CNAP.
Antibodies to CNAP may
also be generated using methods that are well known in the art. Such
antibodies may include, but
are not limited to, polyclonal, monoclonal, chimeric, and single chain
antibodies, Fab fragments,
and fragments produced by a Fab expression library. Neutralizing antibodies
(i.e., those which
inhibit dimer formation) are especially preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice,
humans, and others may be immunized by injection with CNAP or with any
fragment or
oligopeptide thereof which has immunogenic properties. Depending on the host
species, various
adjuvants may be used to increase immunological response. Such adjuvants
include, but are not
limited to, Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, ICLH,
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
CNAP have an amino acid sequence consisting of at least about 5 amino acids,
and, more
preferably, of at least about 10 amino acids. 1t 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
CNAP amino acids may be fused with those of another protein, such as KLH, and
antibodies to the
chimeric molecule may be produced.
-27-
CA 02349210 2001-03-02
WO 00114248 PCT/US99/20287
Monoclonal antibodies to CNAP 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 a1. (1983) Proc. Natl.
Acad. Sci. USA
80:2026-2030; and Cole, S.P. et ai. (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;
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
CNAP-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 CNAP may also be
generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments
produced by
pepsin digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide
bridges ofthe 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
CNAP and its specific antibody. A two-site, monoclonal-based immunoassay
utilizing
monoclonal antibodies reactive to two non-interfering CNAP epitopes is
preferred, but a
competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
_28_
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
techniques may be used to assess the affinity of antibodies for CNAP. Affinity
is expressed as an
association constant, K" which is defined as the molar concentration of CNAP-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium
conditions. The K, determined for a preparation of polyclonal antibodies,
which are
heterogeneous in their affinities for multiple CNAP epitopes, represents the
average affinity, or
avidity, of the antibodies for CNAP. The K, determined for a preparation of
monoclonal
antibodies, which are monospecific for a particular CNAP epitope, represents a
true measure of
affinity. High-affinity antibody preparations with K, ranging from about 109
to 10'2 L/mole are
preferred for use in immunoassays in which the CNAP-antibody complex must
withstand rigorous
manipulations. Low-affinity antibody preparations with K, ranging from about
106 to 10' L/mole
are preferred for use in immunopurification and similar procedures which
ultimately require
dissociation of CNAP, preferably in active form, from the antibody (Catty, D.
( 1988) Antibodies.
Volume I: A Practical Approach, IRL Press, Washington DC; and Liddell, J.E.
and A. Cryer
( 1991 ) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New
York NY).
The titre 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 preferred for use in procedures
requiring precipitation
of CNAP-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, s, upra, and Coligan et al. suara.)
In another embodiment of the invention, the polynucleotides encoding CNAP, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, the
complement of the polynucleotide encoding CNAP 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 CNAP. Thus, complementary
molecules
or fragments may be used to modulate CNAP 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 CNAP.
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
-29-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
polynucleotides encoding CNAP. (See, e.g., Sambrook, supra; Ausubei, 1995,
supra.)
Genes encoding CNAP can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding CNAP. Such
constructs may be used to introduce untranslatable sense or antisense
sequences into a cell. Even
in the absence of integration into the DNA, such vectors may continue to
transcribe RNA
molecules until they are disabled by endogenous nucleases. Transient
expression may last for a
month or more with a non-replicating vector, and may last even longer if
appropriate replication
elements are part of the vector system.
As mentioned above, modifications of gene expression can be obtained by
designing
complementary sequences or antisense molecules (DNA, RNA, or PNA) to the
control, 5', or
regulatory regions of the gene encoding CNAP. Oligonucleotides derived from
the transcription
initiation site, e.g., between about positions -10 and +10 from the start
site, are preferred.
Similarly, inhibition can be achieved using triple helix base-pairing
methodology. Triple helix
pairing is useful because it causes inhibition of the ability of the double
helix to open sufficiently
for the binding of polymerises, 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-ig-c Approaches,
Future 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 CNAP.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences:
GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides, corresponding to the region of the target gene containing the
cleavage site, may
be evaluated for secondary structural features which may render the
oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing
accessibility to hybridization
with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared 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
-30-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
phosphoramidite chemical synthesis. Alternatively, RNA molecules may be
generated by in vitro
and in vivo transcription of DNA sequences encoding CNAP. 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 f 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. Biotech. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as dogs, cats, cows,
horses, rabbits,
monkeys, and most preferably, humans.
An additional embodiment of the invention relates to the administration of a
pharmaceutical or sterile composition, in conjunction with a pharmaceutically
acceptable carrier,
for any of the therapeutic effects discussed above. Such pharmaceutical
compositions may consist
of CNAP, antibodies to CNAP, and mimetics, agonists, antagonists, or
inhibitors of CNAP. 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, intro-arterial,
intramedullary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
-31-
CA 02349210 2001-03-02
WO 00/14248 PCTNS99/20287
In addition to the active ingredients, these pharmaceutical compositions may
contain
suitable pharmaceutically-acceptable carriers comprising excipients and
auxiliaries which
facilitate processing of the active compounds into preparations which can be
used
pharmaceutically. Further details on techniques for formulation and
administration may be found
in the latest edition of Remington's Pharmaceutical Sciences (Maack
Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically acceptable carriers well known in the art in dosages suitable
for oral
administration. Such carriers enable the pharmaceutical compositions to be
formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and
the like, for ingestion by
the patient.
Pharmaceutical preparations for oral use can be obtained through combining
active
compounds with solid excipient and processing the resultant mixture of
granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be
added, if desired. Suitable
excipients include carbohydrate or protein fillers, such as sugars, including
lactose, sucrose,
l5 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.
1f 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
-32-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
cellulose, sorbitol, or dextran. Additionally, suspensions of the active
compounds may be
prepared as appropriate oily injection suspensions. Suitable lipophilic
solvents or vehicles include
fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl
oleate, triglycerides, or
liposomes. Non-lipid polycationic amino polymers may also be used for
delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to increase the
solubility of the
compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art.
The pharmaceutical compositions of the present invention may be manufactured
in a
manner that is known in the art, e.g., by means of conventional mixing,
dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping, or
lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many
acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other protonic
solvents than are the
corresponding free base forms. In other cases, the preferred preparation may
be a lyophilized
powder which may contain any or all ofthe 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
CNAP, 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
CNAP or fragments thereof, antibodies of CNAP, and agonists, antagonists or
inhibitors of CNAP,
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be
determined by standard pharmaceutical procedures in cell cultures or with
experimental animals,
-33-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
such as by calculating the EDs° (the dose therapeutically effective in
50% of the population) or
LDSO (the dose lethal to SO% of the population) statistics. The dose ratio of
therapeutic to toxic
effects is the therapeutic index, and it can be expressed as the
ED~/LDS° ratio. Pharmaceutical
compositions which exhibit large therapeutic indices are preferred. The data
obtained from cell
culture assays and animal studies are used to formulate a range of dosage for
human use. The
dosage contained in such compositions is preferably within a range of
circulating concentrations
that includes the ED,° with little or no toxicity. The dosage varies
within this range depending
upon the dosage form employed, the sensitivity of the patient, and the route
of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of
the active moiety or to maintain the desired effect. Factors which may be
taken into account
include the severity of the disease state, the general health of the subject,
the age, weight, and
gender of the subject, time and frequency of administration, drug
combination(s), reaction
sensitivities, and response to therapy. Long-acting pharmaceutical
compositions may be
administered every 3 to 4 days, every week, or biweekly depending on the half
life and clearance
rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 fig, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular
cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind CNAP may be used for
the
diagnosis of cell proliferative, autoimmune/inflammatory, neurological,
vision, reproductive, and
smooth muscle disorders characterized by expression of CNAP, or in assays to
monitor patients
being treated with CNAP or agonists, antagonists, or inhibitors of CNAP.
Antibodies useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for CNAP include methods which utilize the antibody and a
label to detect
CNAP 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 CNAP, including ELISAs, RIAs, and FACS,
are
-34-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
known in the art and provide a basis for diagnosing altered or abnormal levels
of CNAP
expression. Normal or standard values for CNAP expression are established by
combining body
fluids or cell extracts taken from normal mammalian subjects, preferably
human, with antibody to
CNAP under conditions suitable for complex formation. The amount of standard
complex
formation may be quantitated by various methods, preferably by photometric
means. Quantities of
CNAP expressed in subject, control, and disease samples from biopsied tissues
are compared with
the standard values. Deviation between standard and subject values establishes
the parameters for
diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding CNAP may
be used
for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides
may be
used to detect and quantitate gene expression in biopsied tissues in which
expression of CNAP
may be correlated with disease. The diagnostic assay may be used to determine
absence,
presence, and excess expression of CNAP, and to monitor regulation of CNAP
levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide sequences, including genomic sequences, encoding CNAP or
closely related
molecules may be used to identify nucleic acid sequences which encode CNAP.
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 (maximal, high, intermediate, or low), will determine whether
the probe identifies
only naturally occurring sequences encoding CNAP, allelic variants, or related
sequences.
Probes may also be used for the detection of related sequences, and should
preferably
have at least 50% sequence identity to any of the CNAP encoding sequences. The
hybridization
probes of the subject invention may be DNA or RNA and may be derived from the
sequence of
SEQ ID N0:4-6 or from genomic sequences including promoters, enhancers, and
introns of the
CNAP gene.
Means for producing specific hybridization probes for DNAs encoding CNAP
include the
cloning of polynucleotide sequences encoding CNAP or CNAP 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'ZP or "S, or
by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
-35-
CA 02349210 2001-03-02
WO 00/14248 PCTNS99/20287
Polynucleotide sequences encoding CNAP may be used for the diagnosis of
disorders
associated with expression of CNAP. Examples of such disorders include, but
are not limited to,
cell proiiferative disorders such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,
paroxysmal nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas,
parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus;
autoimmune/inflammatory disorders such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune
thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease,
atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel
syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial
inflammation,
osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's
syndrome, rheumatoid
arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic
lupus erythematosus,
systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis,
Werner syndrome,
complications of cancer, hemodialysis, and extracorporeal circulation, viral,
bacterial, fungal,
parasitic, protozoal, and helminthic infections, and trauma; neurological
disorders such as
epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms,
Alzheimer's disease,
Pick's disease, Huntington's disease, dementia, Parkinson's disease and other
extrapyramidal
disorders, amyotrophic lateral sclerosis and other motor neuron disorders,
progressive neural
muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis
and other
demye(inating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidurai
abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis,
viral central nervous
system disease; prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developments!
disorders of the
central nervous system, cerebral palsy, neuroskeletal disorders, autonomic
nervous system
disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy
and other
-36-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis;
inherited, metabolic, endocrine, and toxic myopathies; myasthenia gravis,
periodic paralysis;
mental disorders including mood, anxiety, and schizophrenic disorders;
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, and Tourette's disorder; vision disorders such as conjunctivitis,
keratoconjunctivitis
sicca, keratitis, episcleritis, iritis, posterior uveitis, glaucoma, amaurosis
fugax, ischemic optic
neuropathy, optic neuritis, Leber's hereditary optic neuropathy, toxic optic
neuropathy, vitreous
detachment, retinal detachment, cataract, macular degeneration, central serous
chorioretinopathy,
retinitis pigmentosa, melanoma of the choroid, retrobulbar tumor, and chiasmal
tumor;
reproductive disorders such as disorders of prolactin production; infertility,
including tubal
disease, ovulatory defects, and endometriosis; disruptions of the estrous
cycle, disruptions of the
menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome,
endometriat and
ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies,
and teratogenesis;
cancer of the breast, fibrocystic breast disease, and galactorrhea;
disruptions of spermatogenesis,
abnormal sperm physiology, cancer of the testis, cancer of the prostate,
benign prostatic
hyperpiasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male
breast, and
gynecomastia; and smooth muscle disorders such as any impairment or alteration
in the normal
action of smooth muscle and may include, but is not limited to, angina,
anaphylactic shock,
arrhythmias, asthma, cardiovascular shock, Cushing's syndrome, hypertension,
hypoglycemia,
myocardial infarction, migraine, and pheochromocytoma, and myopathies
including
cardiomyopathy, encephalopathy, epilepsy, Kearns-Sayre syndrome, lactic
acidosis, myoclonic
disorder, and ophthalmoplegia. Smooth muscle includes, but is not limited to,
that of the blood
vessels, gastrointestinal tract, heart, and uterus. The polynucleotide
sequences encoding CNAP
may be used in Southern or northern analysis, dot blot, or other membrane-
based technologies; in
PCR technologies; in dipstick, pin, and ELISA assays; and in microarrays
utilizing fluids or
tissues from patients to detect altered CNAP expression. Such qualitative or
quantitative methods
are well known in the art.
In a particular aspect, the nucleotide sequences encoding CHAP may be useful
in assays
that detect the presence of associated disorders, particularly those mentioned
above. The
nucleotide sequences encoding CNAP may be labeled by standard methods and
added to a fluid or
tissue sample from a patient under conditions suitable for the formation of
hybridization
complexes. After a suitable incubation period, the sample is washed and the
signal is quantitated
and compared with a standard value. If the amount of signal in the patient
sample is significantly
altered in comparison to a control sample then the presence of altered levels
of nucleotide
-37-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
sequences encoding CNAP 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
CNAP, 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 CNAP, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from
normal subjects with values from an experiment in which a known amount of a
substantially
purified polynucleotide is used. Standard values obtained in this manner may
be compared with
values obtained from samples from patients who are symptomatic for a disorder.
Deviation from
standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in
the patient begins to approximate that which is observed in the normal
subject. The results
obtained from successive assays may be used to show the efficacy of treatment
over a period
ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the
appearance of actual clinical symptoms. A more definitive diagnosis of this
type may allow health
professionals to employ preventative measures or aggressive treatment earlier
thereby preventing
the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
CNAP may involve the use of PCR. These oligomers may be chemically
synthesized, generated
enzymaticaily, or produced in vitro. Oligomers will preferably contain a
fragment of a
polynucleotide encoding CNAP, or a fragment of a polynucleotide complementary
to the
polynucleotide encoding CNAP, and will be employed under optimized conditions
for
identification of a specific gene or condition. Oligomers may also be employed
under less
stringent conditions for detection or quantitation of closely related DNA or
RNA sequences.
Methods which may also be used to quantitate the expression of CNAP 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) 3. Immunol.
Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The
speed of
-38-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
quantitation of multiple samples may be acceierated by running the assay in an
ELISA format
where the oligomer of interest is presented in various dilutions and a
spectrophotometric or
colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as targets in a
microarray. The
microarray can be used to monitor the expression level of large numbers of
genes simultaneously
and to identify genetic variants, mutations, and polymorphisms. This
information may be used to
determine gene function, to understand the genetic basis of a disorder, to
diagnose a disorder, and
to develop and monitor the activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See,
e.g., Brennan, T.M. et al. ( 1995) U.S. Patent No. 5,474,796; Schena, M. et
al. ( 1996) Proc. Natl.
Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116;
Shalon, D. et ai. (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 CNAP
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- I 54. )
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich,
et al. ( 1995) in
Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in
various scientific
journals or at the Online Mendeiian Inheritance in Man (OMIM) site.
Correlation between the
location of the gene encoding CNAP on a physical chromosomal map and a
specific disorder, or a
predisposition to a specific disorder, may help define the region of DNA
associated with that
disorder. The nucleotide sequences of the invention may be used to detect
differences in gene
sequences among normal, carrier, and affected individuals.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such
as linkage analysis using established chromosomal markers, may be used for
extending genetic
maps. Often the placement of a gene on the chromosome of another mammalian
species, such as
mouse, may reveal associated markers even if the number or arm of a particular
human
-39-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
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
positiona) 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, CNAP, its catalytic or immunogenic
fragments,
or oligopeptides thereof can be used for screening libraries of compounds in
any of a variety of
drug screening techniques. The fragment employed in such screening may be free
in solution,
affixed to a solid support, borne on a cell surface, or located
intracellutarly. The formation of
binding complexes between CNAP 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 CNAP, or
fragments thereof, and washed. Bound CNAP is then detected by methods well
known in the art.
Purified CNAP 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 CNAP specifically compete with a
test compound for
binding CNAP. In this manner, antibodies can be used to detect the presence of
any peptide which
shares one or more antigenic determinants with CNAP.
In additional embodiments, the nucleotide sequences which encode CNAP may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely
on properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the
preceding description, utilize the present invention to its fullest extent.
The following preferred
specific embodiments are, therefore, to be construed as merely illustrative,
and not limitative of
the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below,
-4 0-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
in particular U.S. Ser. No. [Attorney Docket No. PF-0588 P, filed September 4,
1998], are hereby
expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some
tissues were homogenized and lysed in guanidinium isothiocyanate, while others
were
homogenized and lysed in phenol or in a suitable mixture of denaturants, such
as TRIZOL (Life
Technologies), a monophasic solution of phenol and guanidine isothiocyanate.
The resulting
i0 lysates were centrifuged over CsCI cushions or extracted with chloroform.
RNA was precipitated
from the lysates with either isopropanol or sodium acetate and ethanol, or by
other routine
methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was
isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles
(QIAGEN, Valencia CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively,
RNA was isolated directly from tissue lysates using other RNA isolation kits,
e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding
cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were
constructed with the
UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life
Technologies),
using the recommended procedures or similar methods known in the art. (See,
e.g., Ausubel,
1997, supra, units S.I-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 S1000, SEPHAROSE CL2B, or
SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or
preparative
agarose gel electrophoresis. cDNAs were ligated into compatible restriction
enzyme sites of the
polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene),
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, DHIOB, or ElectroMAX DHIOB from Life Technologies.
II. Isolation of cDNA Clones
Plasmids were recovered from host cells by in vivo excision, using the UNIZAP
vector
-41-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
system (Stratagene) or 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 REAL Prep 96
plasmid kit
from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of
distilled water
and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCB in
a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and
thermal cycling steps were carried out in a single reaction mixture. Samples
were processed and
stored in 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
The cDNAs were prepared for sequencing using the ABI CATALYST 800 (Perkin-
Elmer)
or the HYDRA microdispenser (Bobbins Scientific, Sunnyvale CA) or MICROLAB
2200
(Hamilton, Reno NV) systems in combination with the PTC-200 thermal cyclers
(MJ Research,
Watertown MA). The cDNAs were sequenced using the ABI PRISM 373 or 377
sequencing
systems (Perkin-Elmer) and standard ABI protocols, base calling software, and
kits. In one
alternative, cDNAs were sequenced using the MEGABACE 1000 capillary
electrophoresis
sequencing system (Molecular Dynamics, Sunnyvale CA). In another alternative,
the cDNAs
were amplified and sequenced using the ABI PRISM BIGDYE Terminator cycle
sequencing ready
reaction kit (Perkin-Elmer). In yet another alternative, cDNAs were sequenced
using solutions
and dyes from Amersham Pharmacia Biotech. Reading frames for the ESTs were
determined
using standard methods (reviewed in Ausubel, 1997, s-upra, unit 7.7). Some
ofthe cDNA
sequences were selected for extension using the techniques disclosed in
Example V.
The polynucleotide sequences derived from cDNA, extension, and shotgun
sequencing
were assembled and analyzed using a combination of software programs which
utilize algorithms
well known to those skilled in the art. Table 5 summarizes the software
programs, descriptions,
references, and threshold parameters used. The first column of Table 5 shows
the tools, programs,
and algorithms used, the second column provides a brief description thereof,
the third column
presents the references which are incorporated by reference herein, 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 probability the
greater the
homology). Sequences were analyzed using MACDNASIS PRO software (Hitachi
Software
-42-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
Engineering, S. San Francisco CA) and LASERGENE software (DNASTAR).
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 GenBank primate,
rodent, mammalian,
vertebrate, and eukaryote databases, and BLOCKS 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,
PFAM, and Prosite.
The programs described above for the assembly and analysis of full length
polynucleotide
I S and amino acid sequences were also used to identify polynucleotide
sequence fragments from
SEQ ID N0:4-6. Fragments from about 20 to about 4000 nt 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, ~uyra, 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 database
(Incyte
Pharmaceuticals). This analysis is much faster than multiple membrane-based
hybridizations. In
addition, the sensitivity of the computer search can be modified to determine
whether any
particular match is categorized as exact or similar. The basis of the search
is the product score,
which is defined as:
sequence identiy x % maximum BLAST score
100
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. For example, with a product score of 40, the
match will be exact
within a 1% to 2% error, and, with a product score of 70, the match will be
exact. Similar
molecules are usually identified by selecting those which show product scores
between I S and 40,
-43-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
although lower scores may identify related molecules.
The results of northern analyses are reported a percentage distribution of
libraries in which
the transcript encoding CNAP 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 categories included cancer,
inflammation/trauma, fetal,
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 expression are reported in Table 3.
V. Extension of CNAP Encoding Polynucleotides
The full length nucleic acid sequence of SEQ ID N0:4-6 was produced by
extension of an
appropriate fragment of the full length molecule using oligonucleotide primers
designed from this
fragment. One primer was synthesized to initiate 5' extension of the known
fragment, and the
other primer, to initiate 3' extension of the known fragment. The initial
primers were designed
using OLIGO 4.06 software (National Biosciences), or another appropriate
program, to be about
22 to 30 nucleotides in length, to have a GC content of about 50% or more, and
to anneal to the
target sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which
would result in hairpin structures and primer-primer dimerizations was
avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art.
PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ
Research, Inc.). The
reaction mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2',
(NH,)ZS04, and ~i-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia
Biotech),
ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase {Stratagene), with
the
following parameters for primer pair PCI A and PCI B: Step I : 94°C, 3
min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3,
and 4 repeated 20 times; Step 6:
68°C, 5 min; Step 7: storage at 4°C. In the alternative, the
parameters for primer pair T7 and SK+
were as follows: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step
3: 57°C, 1 min; Step 4: 68°C, 2
min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min;
Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 pl PICO
GREEN quantitation reagent (0.25% (v/v) PICO GREEN) (Molecular Probes, Eugene
OR)
dissolved in 1 X TE and 0.5 pl of undiluted PCR product into each well of an
opaque fluorimeter
plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The
plate was
-94-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
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 ~sl to 10 ~I aliquot of
the reaction mixture
was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which
reactions were
successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC I 8 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% dimethysuiphoxide (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 sequence of SEQ ID N0:4-6 is used to obtain 5'
regulatory
sequences using the procedure above, oligonucleotides designed for such
extension, and an
appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:4-6 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 uCi of
[y-'ZP] adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
-45-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-
based hybridization analysis of human genomic DNA digested with one of the
following
endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xbal, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.?% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under increasingly stringent conditions up to 0.1 x saline sodium citrate and
0.5% sodium dodecyl
sulfate. After XOMAT-AR film (Eastman Kodak, Rochester NY) is exposed to the
blots to film
for several hours, hybridization patterns are compared visually.
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, s_unra.)
An array analogous to a
dot or slot blot may also be used to arrange and link elements to the surface
of a substrate using
thermal, W, chemical, or mechanical bonding procedures. A typical array may be
produced by
hand or using available methods and machines and contain any appropriate
number of elements.
After hybridization, nonhybridized probes are removed and a scanner used to
determine the levels
and patterns of fluorescence. The degree of 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 CNAP-encoding sequences, or any parts thereof,
are
used to detect, decrease, or inhibit expression of naturally occurring CNAP.
Although use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
-9 6-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
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
CNAP. 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
CNAP-encoding transcript.
IX. Expression of CNAP
Expression and purification of CNAP is achieved using bacterial or virus-based
expression systems. For expression of CNAP in bacteria, cDNA is subcloned into
an appropriate
l0 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 CNAP upon induction
with isopropyl beta-
IS D-thiogalactopyranoside (IPTG). Expression of CNAP 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 CNAP by either homologous
recombination or
bacterial-mediated transposition involving transfer plasmid intermediates.
Viral infectivity is
20 maintained and the strong polyhedrin promoter drives high levels of cDNA
transcription.
Recombinant baculovirus is used to infect Spodoptera frugigerda {Sfl7) 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.)
25 In most expression systems, CNAP 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
30 antigenicity (Amersham Pharmacia Biotech). Following purification, the GST
moiety can be
proteolytically cleaved from CNAP 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
_q7_
CA 02349210 2001-03-02
WO 00/14248 PCTNS99/2028'7
and purification are discussed in Ausubel (1995, supra, ch 10 and 16).
Purified CNAP obtained by
these methods can be used directly in the following activity assay.
X. Demonstration of CNAP Activity
CNAP-1
CNAP-1 activity is demonstrated by the ability to convert ATP to cAMP (Mittal,
supra).
In this assay CNAP-1 is incubated with the substrate [a-'ZP]ATP, following
which the excess
substrate is separated from the product cyclic ['ZP] AMP. CNAP-1 activity is
determined in 12 x
75 mm disposable culture tubes containing 5 pl of 0.6 M Tris-HCI, pH 7.5, 5 pl
of 0.2 M MgClz, S
pl of 150 mM creatine phosphate containing 3 units of creatine phosphokinase,
5 pl of 4.0 mM 1-
methyl-3-isobutyixanthine, 5 pl of 20 mM cAMP, 5 pl 20 mM dithiothreitol, 5 pl
of 10 mM ATP,
10 pl [a-'zP]ATP (2-4 x 106 cpm), and water in a total volume of 100 pl. The
reaction mixture is
prewarmed to 30°C. The reaction is initiated by adding CNAP-1 to the
prewarmed reaction
mixture. After 10-15 minutes of incubation at 30°C, the reaction is
terminated by adding 25 pl of
30% ice-cold trichloroacetic acid (TCA). Zero-time incubations and reactions
incubated in the
absence of CNAP-I are used as negative controls. Products are separated by ion
exchange
chromatography, and cyclic ['ZP] AMP is quantified using a (3-radioisotope
counter. The CNAP-1
activity is proportional to the amount of cyclic ['ZP] AMP formed in the
reaction.
CNAP-22
CNAP-2 activity is measured by the ability to convert ethyl butyrate to
butyric acid
(Sigma On-line Catalog, Enzymatic Assay of Esterase). The reaction is
performed at 25 °C. A
0.88 g/ml solution of butyric acid ethyl ester is prepared (Reagent C). A 0.1%
(v/v) solution of
ethyl butyrate (Reagent B) is prepared by diluting Reagent C in 10 mM borate
buffer, pH 8Ø 25
ml of Reagent B is added to a titration vessel placed on a magnetic stirrer,
and the solution is
equilibrated to 25 °C. The solution pH is constantly measured using a
pH meter. 0.025 ml of
Reagent C is added. The solution pH is adjusted to pH 8.1 by adding 10 rnM
NaOH. 0.1 ml of
CNAP-2, diluted in 10 mM borate buffer, pH 8, is then added to the solution.
After the pH
reaches 8.0, the reaction is followed for 1-5 minutes. The pH of the reaction
is maintained at pH
8.0 by the addition of small volumes {0.050 ml) of 10 mM NaOH. The volume of
NaOH used to
maintain the pH at 8.0 as well as the time required to consume the added 10 mM
NaOH are
recorded. The activity of CNAP-2 is proportional to the amount of butyric acid
formed in the
assay.
CNAP-3
CNAP-3 activity is demonstrated by the ability to convert GTP to cGMP (Mittal,
su ra).
In this assay CNAP-3 is incubated with the substrate [a-'zP]GTP, following
which the excess
-48-
CA 02349210 2001-03-02
WO 00/I4248 PCT/US99/20287
substrate is separated from the product cyclic [3zP] GMP. A reaction mixture
contains S pl of I M
Tris-HC1, pH 7.5, S pl 80 mM MnClz or MgCI,, 25 pl of 40 mM theophylline or
2.0 mM I-
methyl-3-isobutylxanthine, S pl I SO mM creatine phosphate containing 20 pg
creatine
phosphokinase (120-135 units/mg protein), 5 pl 20 mM cGMP, 10 pl 10 mM GTP, 10
pl [a-''-P]
GTP (containing 2-4 x 106 cpm), and water in a total volume of 100 pl. The
reaction is initiated
by the addition of CNAP-3. After 10-15 minutes of incubation at 37°C,
the reaction is terminated
by adding 20 pl of 40% ice-cold trichloroacetic acid. Zero-time incubations
and reactions
incubated in the absence of CNAP-3 are used as negative controls. Products are
separated by ion
exchange chromatography, and cyclic ['ZP] GMP is quantified using a (3-
radioisotope counter. The
i0 CNAP-3 activity is proportional to the amount of cyclic ['ZP] GMP formed in
the reaction.
XI. Functional Assays
CNAP function is assessed by expressing the sequences encoding CNAP 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.
S-10 /cg of
recombinant vector are transiently transfected into a human cell line,
preferably of endothelial or
hematopoietic origin, using either liposome formulations or electroporation. 1-
2 ~g of an
additional plasmid containing sequences encoding a marker protein are co-
transfected. Expression
of a marker protein provides a means to distinguish transfected cells from
nontransfected cells and
is a reliable predictor of cDNA expression from the recombinant vector. Marker
proteins of
choice include, e.g., Green Fluorescent Protein (GFP; Clontech), 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 properties, for
example, their
apoptotic state. FCM detects and quantifies the uptake of fluorescent
molecules that diagnose
events preceding or coincident with cell death. These events include changes
in nuclear DNA
content as measured by staining of DNA with propidium iodide; changes in cell
size and
granularity as measured by forward light scatter and 90 degree side light
scatter; down-regulation
of DNA synthesis as measured by decrease in bromodeoxyuridine uptake;
alterations in
expression of cell surface and intracellular proteins as measured by
reactivity with specific
antibodies; and alterations in plasma membrane composition as measured by the
binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cvtometry, Oxford, New York NY.
The influence of CNAP on gene expression can be assessed using highly purified
-49-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
populations of cells transfected with sequences encoding CNAP 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 CNAP and other genes
of interest can
be analyzed by northern analysis or microarray techniques.
XII. Production of CNAP Specific Antibodies
CNAP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
t0 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 CNAP 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. I I.)
Typically, oligopeptides IS 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, su ra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide activity by, for example, binding the peptide to plastic, blocking
with 1 % BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
XIII. Purification of Naturally Occurring CNAP Using Specific Antibodies
Naturally occurring or recombinant CNAP is substantially purified by
immunoaffinity
chromatography using antibodies specific for CNAP. An immunoaffinity column is
constructed
by covalently coupling anti-CNAP antibody to an activated chromatographic
resin, such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing CNAP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of CNAP (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/CNAP 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 CNAP is collected.
-50-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
XIV. Identification of Molecules Which Interact with CNAP
CNAP, or biologically active fragments thereof, are labeled with'ZSI Bolton-
Hunter
reagent. (See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-
539.) Candidate
molecules previously arrayed in the wells of a mufti-well plate are incubated
with the labeled
CNAP, washed, and any wells with labeled CNAP complex are assayed. Data
obtained using
different concentrations of CNAP are used to calculate values for the number,
affinity, and
association of CNAP 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
specific preferred
embodiments, it should be understood that the invention as claimed should not
be unduly limited
to such specific embodiments. Indeed, various modifications of the described
modes for carrying
out the invention which are obvious to those skilled in molecular biology or
related fields are
intended to be within the scope of the following claims.
-51-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
~o . . . .
w
M . r--i '-i N N
.-I .-I M ~~
N
O ~~. O O . O
O O O O O
--. M Z H H ~ H
.1 O: H H Z
N
~vo ooxoox Nowo
o ~ H z H z o z
~n z H w z
H N 0 o H U H w
vo H ~ z c~
oc x z w ~n z o oc
N cn ~ a z
U N c~ H o w z o
x w ~
z o z x w a. a
. m ~ U
m ~n
a o _ _
~ p .. ... H
~. ~.
o ~ a
~
U w -- ~ ~ w mo ,-mo
o ,-~ ,
-- N H x w x v H
H x x H
N O .-i ~-1 O'1
m 01 .--I rl
O1 N
.-I ~', m N ~-1
2 r lD t~ M l0
.-1
pG . ~o .-i o~ x r
z ~ o~ m v~
m
l0 ~ 01 N l0 O N
~ N V' t0 M N
N N l0 a' M v' O~
U' O N ~O O 41
a~ O N r c~ m r O~
Z ~r N N 41
O H d" .~ N N .-1
''' N M C N M
r O N N
r z ~ . . . o~
~ .
~n x
. (~ . r .-I . N
M M a LO r M
~ H ~ O O N ~ O
H O M O C?
M p ~ H H H M H
r Z H H Z
O ~- O O N O O O
lfl O O O O
H o H z r~ z H z
z z z z
O .-t O H .-~ O ~
M C7 W C7 H CO
z U z cn W z 2 oG
N fx Z v1
2
c~ o, z x x a c~ o
o o cx
o
x ~ m H a ~ U
~ ~ Fc a m a
' ~
~ N z
' N
,- o -- .-mo m- ~
o ~ ~ ,.- .-,
,-~
o H x w x x r.4
~''' x ~ ~
~o ~r .-i M awo .-~
O c ~ o~
r v~
H ~ H .-~ ~n x M
Z ~ r m M M
O N m ~O .1 O r
f~ M W M N M
N N r O O~ .-~ m N
!1 tJW ~ ~-t
O
H m O 01 01 01 01
v~ 01 N N
O . 01 M M l0 N O1
O ,-i lO rl
~
.-1 V' .-~1 .-W
~ N N M M -i
v N M
tn N m tf7
O .-1 . . . .
.H x
O m .-. M M ~ M
H r-1 O1 M '-i
M Z N O O O r-i
tn O O O O
N
o U7 o H H H o H
0~ H H H H
H o: H O O x W O
r O O O D
O H W Z Z H Z 2
~ Z Z Z H
'Z 'J 5C Z U H H ~Y,,
N D pG
H
aC N f4 C~ Z W x
O ~ ~
W w w w ~ H
x m o cn
"'
(CS ~- .-a ~-
N V.
N O l0 v--I ~D lD
r-1 .-1 '-i n-i
e--I
~ x ~o rs. x ~ r~
H H x x x rx
x
x ~0 x m M o x o
0 o m ,-, u~
o,
.-a ~ M ~ a~ m r
M Z a M o N
Z
m av vo ~o ov c~ M
tn m ~ ~ u~
H ~
M M e-f l0 M N W
(Y, OZ N O ' v-1
C/) V'
N l0 ~ O~ r M O~ tT
H R: ~r M lfl
N
~r N M N .-t ~ M
O Ip c' O u7 O N
a N M .-i N .~ H
w' N M M N N
'~'
N C~ .-1
~, O O O
t-i H Z a1
rtf O O 2
z z
.o ~n w z
04 aG w
a H o ra
m ~ m
a~ 'n r r
6W O N
r o~
.--I lO v~
U N
N M
'L3
O
.i
Z
1~
O Q, ~ t0
G1
(~
H
H
Ud
~
w
z
cn
'
o
w
z
H N M
~a
'''
w
o
~
w
-52-
CA 02349210 2001-03-02
WO 00/14248 PCTNS99/20287
a
U .~ U
v1
b
H H H x rt H Cw
FC H ~ U7 ~ U U V7 H U
~ W
a x ~ w a ~ ~ ~ w a o ~ ~.
m w x cn m a. m x ~n m ~ m w
w
c
0
"'' tr~
o.
U U b N b
C N
~0 N -.n tn N r0 Q1
J~
.-i m S7 ~ .-i .-1
al -rl
i s-r .~ ~, N
~
LL N ~ O .--1
p ~
~ +~ .-i rtl
U .O
FC U U a
N tn CT U
7
.-i t~ O~ cr t~ O l0 l0 ~ v~
N ~
U " M O N u) r~ O c I~
U
N ~r ~mn uwmo vo ~o y
~
o " ~n a a H ~, r--, ~ x ~,
a a ~
a ro i ~ i i i i i i -~ i ~ ~ .
~
M l0
-
~
~
CJ' . rU O' N
W ~
f p
M O~ trr
47 OD
'"'~ o~ ,-~ c ~o 0o
M ui
N p ~
t1 a~
<T C u7 u7 ~ u7 l0 "'
l0
' ~ a ~ a a a U U a ~' .~ c r
~; n '~ ~; ~
N N 'O N 'O ~ U
1
UV'0 ~f31 ~S U''~
01NrOM01 SOD ~j 4~ O
OP tn O M X17 00 O . l0
l0 y,a V~ ~ .~ l0 y~ ~
M . ~
a u7 u7 y1) ~O l0 N N O O lD
,' (n H H H a H Cv ~ Ot '~ M ~ ~ a
'~ ~r ~ l0 S~-1
~ ~ i i i i ~ ~ i i
m. a
c
~, .,~ ro
a~ ~ N
mo vo ov c~ r O ~o .
o~ ~
U v~
1
~ ~ ,-,i
,i ~
~ f'~ c~ O~ ~ ~r ~9 "1
~ ~ v'
. O
N
~
., ~
. ~ . V'
~ t0
~ ~--1
U M v~ a if) Ln Ln ri
tn l0 rl
.-I
. N
N ~ ~ ~.
~ ~
~ a a a a a a H a ~
a z
~ ~
z
,~ ~
d, ro
a H o,
i vH-
~
~ c
n
c~ ~ d o.
o r,
N O cr
z z
..~ h o
ro
~n
v --i o ~n r.,
v
~ >, ,-, ~ z
~
N fn N Z M
.-1
.v z o~ oo z
o
~n
o U r m ~ N o
W ~'' 01 M 01 O N I'~ O
.1 01 cr C .W -1 N ~D
z z z z z z z
d.
O OD V' 1D CO N v~ '-1 V" I~ tn
Cn v~ O~ v' u7 O M CO O~ M 00 00 M
O~
+~ M ~ f~ c~ N N v~ r ,-~ r oo m
a~ cn oo oo ~
H ~ .-i CO v' N fn fn I~ cT ~ (n
-1 N cn N N H
. u7 ~-1 Y~ fn ri e-1 ~O f/) ~--i
C 61 M
n M e-I (n N O O ~ [--i H CO H
S M l0 M N
1
- l0 M O M u7 O 00 Cl1 O M N
O l0 N n-i ?i
1
lfl
~
) N c f'~ In lf7 tn
N t CO OD l0 .-i
V l0
1
c~
- c
H
n ~ .-i vW n .-i ~r M v)
e eo H o ~ H
H
H
O OD H N N N M H .-1 N
M O
~O O v-1 .a CO f~ fn
O .4J l0 07 01 N v~
1~
-1
1 M O O ~r-1 ,-I N 01
~ V1 H 01 .-1
h OD u7 '
t1~
tn
M l
u7 ~ M l0 H 1D l0 f~ ~
0 Cl) ~ M
N I
H Cn Cn (!) Cn tn tn tn H c M M r-1 H
U1 O [-i
M f~- N O Cn r-i
O f~ O M N ~ 1~ O v-i ~-f
N M V~ N H .-1 Cn
O
c~ N O1 ~O ~ M N u7 l9 r-i ~ .-W O
~ ~ M N
.-1 ~ ,-~ c~ l0 M t5> N M ~ ~ H CW .-1 M u7
O ~ C M
H v) H u7 v7 H .-~ H u1 v1 N r H ~
v) H v1
a~ v~ ~ N m o H H
r H H
J-~ M r ~o m r ~ o~
01 O er N O1 u) 01 o r o u7 01 N
~ OD N t17 a1 o'1 tn
~ N M v~ r-i
c lfl M ~a 01 l0 N N
r-1 c '-1 .-i vC) M M
l0 N cr ~I) 01
H cn ~ v7 cn cn Y~ t~ v1 W v7 t~ Ul H
c~ vJ H v7 c~ c~
N
'O
'O o r o
U .ri O N 01
r
a
N M
-53-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
H
w
.-1 H
O ~ ~
U
U
W
a
00
w
ro
+'
ro
o
,~ ~.. ,..,
E-,
U M u~
a .-~ ~-,
w
O ~n ~o
O
o o 0
~ --- r..
v V v
ro O M l0
.i
C
U u1 O O t'-) O N
. .~, r.,
o o +~ a o .u o
a
w ro ro -~- m ---
a a a
o w w o
w o
.r-, -~.,
+~ .-~ .-~ .-a +~
~
u w u
.~ ~ .~
o a
w .--i ~ w .~ w
a a~ v C ar ~
,~ H U U H U H
CW
O
ro
-ri
+-~
~ ~ ,n
O
E'~ N
H H
uw
C3, OD O O
O
X OD
W -1 tt) ~T tf7 .~
C
O .-. N N .-. -- M CO
~ ~
N O 00 v-1 07 .-1 a7 .-1
.-f ~.., C O
-- 00 ~ OD
H O ~ r-1 M O
U1 . O ~
ro --I
..
'rl . ... O .-
Z-1 . O H
ro O H .-,,
if7
H C -- ~1 '-i ~-- l0
C...,\ ~t1 N ~- LI
S-a
\
-.t ro U t~) .-~ ro
N .~-~ ro .-1
U
J.-~ N .-i N ~ N N .-i
-W -I .i ro
--1
(n ~ ~ 1-r ~ ~ ,7 ~
O 1-i 1~
41 -.-i O -.~ .-i O
U N O v U ~ U C
a ~ m .-1 _. ~ i-m--
-- ~ --I tn N
C U ro O U ro U ro
U O
-~ ~ D R. (n ~ ~ u~ 3
m -.~ ~ C). G2,
O a O O ~ a a ~ O
~ tn O O O
O --i ~ O O O O
O O --a
+~
+~ a zs 5 a a > a
ro 5 .-i -o a~
ro
~n a, a a n, p, ~.,
~ a o a ~ ~, p
ro a~ ro a~ a~ v v ro
a~ w a ro m
a~
~ c~ U x z x x z U
z ~ U x o
v
.
a
o
-~
z
+-
o
ra
a, u) ~o
N
H
r~
Ua
~w
z
cn
-54-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
ro a
o .-~ ~ tr
~ >..~I ro o ~ v
O H +.~ ~ s-i U
H O U f-I v w O .-I v7
w +~ ~ H ~n H m ro m
~ v
v1 1-I T7 T3 rtS -~-i
b U CL v N
T1 -~I ro v 3 v cx
+.~ +.r C
~
v x ~n a a
-~ ro
a N C U F: O >, N TJ
ro
~ ro N -
O
>~ G ro ro O
tr~ c ~
N N rl ~ H S-a ~ C
G O
t-i .i O 1.~ +~ N N
ro O -i --
+~ U U S-a N r1 v T7
O C7 TJ
N ro O 4a ~ ~ -- ro
U ~
u) ~n
v) H v~ v1 N N 'O
O
tn T7 cy ..i .C N
N t~ H .-I H
..-t ?, C: J-~ E-~
N U -.,~ ttf ttl w
+~ ~ 'O O O m ro
C +.~
~
o ~ ro ~a v ~n +.~ .-+
-.~ o H
N +ml U U C ro v ~ w
.-i ~
~ U U O ro ~ ~ O C
v)
--i N ~ i.~ .-i ~
.C U ~n O O .~
S-1 1-I C: O~ ~0 S.r
i U N T7 I
07 O ro N LI w r-i N
N f.l oo
~ a a v ~ +~ ro -c
.a
a v~ v c: ro Ts a
o -.~ --
v
a. -.~ v G ~o v o
~ ~ v
~.~Hmro a.vrowcaH
+~ o v H v v H v A, v o
s.r .~ Ts o x ro H
a~ b v v ~n
a
w ro H a ro v ~ a w
v v ro
G ~ 'O -.1 ~ ro N
~ O
'b -I v1 E o -i -b
~ ~ ~
U O
~ ~ tn O ~ w C N t-
U . N >, ~
N t~ -.i w O M ro
a .-i
-~ ~
O N
O ro -.-1 y O
~ ~ ~ ~
~ v
ro rn a >, .~ms .~ -.~
H ro I ro
H -r1 t ~ ~ rl
P ~ ~
~ C N -,-i
O ~ 'O ~
-~~ fs,' -r1 ltJ O O N Q,'
IL J..~ w
~
a z H v~ cn v~ .~ U z
~n ~-, o~ .~I
cx ~ -.~ ro 3 C rx
r-a .~
b a .C .-i 3 tn ,~ U
.-i o~
tr~ N O ~ G a~ ..~ ~
N N
G v ~ >, ~ r.~ v -.~
3 a.
~, .-I o z .-I ~n
H .-I
o a.
~n ro o H x ro ~ c v~
.~I .~ ro o
+~
b ~ ~
N W oWT U
b~
'b w 'U TS G C 'O 'O
Cu ~-.I .-i C ~-
N
"" ~ ' v
m
" . ~ .N +
a v ~ ~ ms
ro ~
U ro a U f-a v1 O U
~ l.i re; ~ 'd r-1
N
o -.~ ~n ~ ro ro a a
v~ o .~
a v~ ro H +~ U ~ . H
-.~ H -,~ -~
-~
-~ ro a +~ cn ~ O
v1 H -O -- s-a
of U N W ro 3 ~ ~n
-..~ v
~ a .-I c: U a~ .,~ G
-i a o C1 T1
o ro ro c> a. U o0 0
. ro .-r
U U S-I U H TS G cn U
G ?~ .-i I ~ O
ro .--I ro -~ -.-1 I
~ ~ O
v) D -.-I V> 1.a O m
~ ro U a~ ~I s-i
ro ~ ~ m .ca ms b ro
H ~ cn r +~ ro
3o roOro 3-~uvrorn 3v
I -o a rl ro N U >,
a a ~ N
>, H v >~ v -~ r~ >,
o ro o .~ I
S-1 ro ~.1 N ~ ~-I N
-'O ~-1 ..-~ ~ M
ro v ~ ro ~ I ro ro
a +~ -p ~o .~
H >, -, a v~ o ~ rl H
ro H v o, a ro
,L7 I U .t~ N N H ,Q
ro S-1 ~ ~ .f~
-.~ c~ -~I -.t o -~I
c w v ro ~ >. w
v
a <n -.~ a +~ ro ~ a
o .c U z -- -~ o
N a~ -i
ay O O O
H H z f~1
ro O O z
H z z.
W
ra H G D
d
0o c
m r
N ~ ~ N
G
O ~ uo
N
V N r"1
v
'O
O
'~
z
U
OI
a
w
z
~n
-55-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
r
><
~o h, ,~ ~~ :: '
~o = . x ? o '~ 00
I I 4 ~ ~~ ~~ J . ~ 7 ~ ? c n
'C ~ .. '~ 'o .. ~Y. r _ ~ v
G ~.
II z ~ ~= ; ~ ~,.-~~ n _~ ~,a
>. L 3 ~ ~ ~ s ' '~'. ~ a
.= s ~ a %, ~, ~ no n > ~ '
w?' ~",> > h. ~ > 70 .y ~
W (,~~ = L' GL7 ~ ~ _ jp ~_ ~,, v
L V ~ ~ ? ~ '~'3 v
n,~ll .~'a~N~~ll II~~ 117
V _n ~ ~ > > Ew ~ ~l ~ > 3 ~ ~ ~ 7 n ' >
G.- ~ '.~zt ~ :i '°> '.Hcwt a 3' W 'i ~ ;n c~f a . :n
H
U ci 3 'g < 5
o ~~', ~ n o a N ~ E ~ ~'
Op N ~0
CO a ~ ~ ~ . 7 N y~~ - a
y ~ a~ c0.'c a Z ~ E'_'
'x n ' ~ ~ a ~ oo ~ ~ y .~t v r j v
n n ~ ~ ~ -~ LL: a C. d' -' ~ ~ C C ch
;J O _ O vj ex1 ~ N ~ ~ at V W ~ ...1
CO C7 v CG . ~"~ 3 ~ r" ~ _',~ N ~ .,J N
a v ~ ~ t ~ 0 'Q' ~ Vf a0 ~ ~ p r
N
w U ~ U .. U o ~ oG N
Gt: < r7~
11 ~ >> T ~ T N ~ ~ ~ ~ ~ ~ H N v C ''J
wu WU wU o~~,= ~~E-~ ~~~~~ <~<
.=
tI~ " ~ a y ~ y ~ ~ a ~~
~O a'~ u' ~~ y~ ~ vp;u o
,.", a o.~. o»~. o.c~ <Nz ~.z..~< s_x~u ~-z
e~s
C ~ ~ wt .~ H ~ ~ ~ id
H
.= ~f'- H ~~~ .5~'
,a~ O L. a as
°- ~ ~ 8
~ .Y v ~ ~ ,~ w ~
~1 ~I '~ .o
'3 Y .g '~ G - ~ ~ ~~ :.~ a h
'~a~
.x E
~ 53 ~o $ ,~, .e a
H °~ ~ ~ ~ s ~ ~, :~ H
a ,~ ~.~~ °'
o ~ ~° t~ ~
;. ~ ~ ~ y
~a
< ~ < < Haas <~3 ~= < A~..v...
V
< u.~
.
Li, ~ < N ~ ~l.
< a a
-56-
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
a
v
3
v
y O O
a ~ h
a II a a
v r
a~: ;n ~ v
a
..
Z _ ~ '-.
~t~ x a
mc~~ o:~ ct-~'.'e 'r'~
u~a ~~ ~~ ~3 g
a
~~< . c~~a 3Nm- '~
3~.~_
da ~~ '_'~ ~ i~o~"~o~'e ., ~.-.a~,r '3 !~3
w a ~ N a v°a ~ ~ N ~ ,~
~~ o ~''~ ~ 0. a N a >
V ~ ~ °~~ my H~ ~ ~3 ~= ~o =°'~~
'~ V ~ ~
,r ~ c~C~ °~ wac~~ ~n~3~r~ ~o~c z°u aon~ ~~
.a
0O '~~' H
~._ ~,C ~' =°
a ~ ~ ,~~~z ~ ."
'O
ag
eo o '
.
a ~ Y
a
~~3a
~o~o,r. ~ ~~'~ ,~5~
A ._
~~ a <c~~ 5~ a < ~ <~
g
3
-57-
CA 02349210 2001-03-02
WO 00/14248 PCTNS99/20287
SEQUENCE LISTING
<120> INCYTE PHARMACEUTICALS, INC.
HILLMAN, Jennifer L.
YUE, Henry
GUEGLER, Karl J.
CORLEY, Neil C.
PATTERSON, Chandra
TANG, Y. Tom
<120> CYCLIC NUCLEOTIDE-ASSOCIATED PROTEINS
<130> PF-0588 PCT
<140> To Be Assigned
<141> Herewith
<150> 09/148,904; unassigned
<151> 1998-09-04; 1998-09-04
<160> 6
<170> PERL Program
<210> 1
<211> 708
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2267958CD1
<400> 1
Met Lys Gly Leu Lys Thr Asp Leu Asp Leu Gln Gln Tyr Ser Phe
1 5 10 15
Ile Asn Gln Met Cys Tyr Glu Arg Ala Leu His Trp Tyr Ala Lys
20 25 30
Tyr Phe Pro Tyr Leu Val Leu Ile His Thr Leu Val Phe Met Leu
35 40 45
Cys Ser Asn Phe Trp Phe Lys Phe Pro Gly Ser Ser Ser Lys Ile
50 55 60
Glu His Phe Ile Ser Ile Leu Gly Lys Cys Phe Asp Ser Pro Trp
65 70 75
Thr Thr Arg Ala Leu Ser Glu Val Ser Gly Glu Asp Ser Glu Glu
80 85 90
Lys Asp Asn Gly Lys Asn Asn Met Asn Arg Ser Asn Thr Ile Gln
9S 100 105
Ser Gly Pro Glu Gly Ser Leu Val Asn Ser Gln Ser Leu Lys Ser
110 115 120
Ile Pro Glu Lys Phe Val Val Asp Lys Ser Thr Ala Gly Ala Leu
125 130 135
Asp Lys Lys Glu Gly Glu Gln Ala Lys Ala Leu Phe Glu Lys Val
140 145 150
Lys Lys Phe Arg Leu His Val Glu Glu Gly Asp Ile Leu Tyr Ala
155 160 165
Met Tyr Val Arg Gln Thr Val Leu Lys Val Ile Lys Phe Leu Ile
1/ll
CA 02349210 2001-03-02
WO 00/14248 PCTNS99/20287
170 175 180
Ile Ile Ala Tyr Asn Ser Ala Leu Val Ser Lys Val Gln Phe Thr
185 190 195
Val Asp Cys Asn Val Asp Ile Gln Asp Met Thr Gly Tyr Lys Asn
200 205 210
Phe Ser Cys Asn His Thr Met Ala His Leu Phe Ser Lys Leu Ser
215 220 225
Phe Cys Tyr Leu Cys Phe Val Ser Ile Tyr Gly Leu Thr Cys Leu
230 235 240
Tyr Thr Leu Tyr Trp Leu Phe Tyr Arg Ser Leu Arg Glu Tyr Ser
245 250 255
Phe Glu Tyr Val Arg Gln Glu Thr Gly Ile Asp Asp Ile Pro Asp
260 265 270
Val Lys Asn Asp Phe Ala Phe Met Leu His Met Ile Asp Gln Tyr
275 280 285
Asp Pro Leu Tyr Ser Lys Arg Phe Ala Val Phe Leu Ser Glu Val
290 295 300
Ser Glu Asn Lys Leu Lys Gln Leu Asn Leu Asn Asn Glu Trp Thr
305 310 315
Pro Asp Lys Leu Arg Gln Lys Leu Gln Thr Asn Ala His Asn Arg
320 325 330
Leu Glu Leu Pro Leu Ile Met Leu Ser Gly Leu Pro Asp Thr Val
335 340 345
Phe Glu Ile Thr Glu Leu Gln Ser Leu Lys Leu Glu Ile Ile Lys
350 355 360
Asn Val Met Ile Pro Ala Thr Ile Ala Gln Leu Asp Asn Leu Gln
365 3?0 375
Glu Leu Ser Leu His Gln Cys Ser Val Lys Ile His Ser Ala Ala
380 385 390
Leu Ser Phe Leu Lys Glu Asn Leu Lys Val Leu Ser Val Lys Phe
395 400 405
Asp Asp Met Arg Glu Leu Pro Pro Trp Met Tyr Gly Leu Arg Asn
410 415 420
Leu Glu Glu Leu Tyr Leu Val Gly Ser Leu Ser His Asp Ile Ser
425 430 435
Arg Asn Val Thr Leu Glu Ser Leu Arg Asp Leu Lys Ser Leu Lys
440 445 450
Ile Leu Ser Ile Lys Ser Asn Val Ser Lys Ile Pro Gln Ala Val
455 460 465
Val Asp Val Ser Ser His Leu Gln Lys Met Cys Ile His Asn Asp
470 475 480
Gly Thr Lys Leu Val Met Leu Asn Asn Leu Lys Lys Met Thr Asn
485 490 495
Leu Thr Glu Leu Glu Leu Val His Cys Asp Leu Glu Arg Ile Pro
500 505 510
His Ala Val Phe Ser Leu Leu Ser Leu Gln Glu Leu Asp Leu Lys
515 520 525
Glu Asn Asn Leu Lys Ser Ile Glu Glu Ile Val Ser Phe Gln His
530 535 540
Leu Arg Lys Leu Thr Val Leu Lys Leu Trp His Asn Ser Ile Thr
545 550 555
Tyr Ile Pro Glu His Ile Lys Lys Leu Thr Ser Leu Glu Arg Leu
560 565 570
Ser Phe Ser His Asn Lys Ile Glu Val Leu Pro Ser His Leu Phe
575 580 585
Leu Cys Asn Lys Ile Arg Tyr Leu Asp Leu Ser Tyr Asn Asp Ile
590 595 600
2/11
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
Arg Phe Ile Pro Pro Glu Ile Gly Val Leu Gln Ser Leu Gln Tyr
605 610 615
Phe Ser Ile Thr Cys Asn Lys Val Glu Ser Leu Pro Asp Glu Leu
620 625 630
Tyr Phe Cys Lys Lys Leu Lys Thr Leu Lys Ile Gly Lys Asn Ser
635 640 645
Leu Ser Val Leu Ser Pro Lys Ile Gly Asn Leu Leu Phe Leu Ser
650 655 660
Tyr Leu Asp Val Lys Gly Asn His Phe Glu Ile Leu Pro Pro Glu
665 670 675
Leu Gly Asp Cys Arg Ala Leu Lys Arg Ala Gly Leu Val Val Glu
680 685 690
Asp Ala Leu Phe Glu Thr Leu Pro Ser Asp Val Arg Glu Gln Met
695 700 705
Lys Thr Glu
<210> 2
<211> 132?
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID Na: 3149674CD1
<400> 2
Met Glu Ala Pro Leu Gln Thr Gly Met Val Leu Gly Val Met Ile
1 5 10 15
Gly Ala Gly Val Ala Val Val Val Thr Ala Val Leu Ile Leu Leu
20 25 30
Val Val Arg Arg Leu Arg Val Pro Lys Thr Pro Ala Pro Asp Gly
35 40 45
Pro Arg Tyr Arg Phe Arg Lys Arg Asp Lys Val Leu Phe Tyr Gly
50 55 60
Arg Lys Ile Met Arg Lys Val Ser Gln Ser Thr Ser Ser Leu Val
65 70 75
Asp Thr Ser Val Ser Ala Thr Ser Arg Pro Arg Met Arg Lys Lys
80 85 90
Leu Lys Met Leu Asn Ile Ala Lys Lys Ile Leu Arg Ile Gln Lys
95 100 105
Glu Thr Pro Thr Leu Gln Arg Lys Glu Pro Pro Pro Ala Val Leu
110 115 120
Glu Ala Asp Leu Thr Glu Gly Asp Leu Ala Asn Ser His Leu Pro
125 130 135
Ser Glu Val Leu Tyr Met Leu Lys Asn Val Arg Val Leu Gly His
140 145 150
Phe Glu Lys Pro Leu Phe Leu Glu Leu Cys Arg His Met Val Phe
155 160 165
Gln Arg Leu Gly Gln Gly Asp Tyr Val Phe Arg Pro Gly Gln Pro
170 175 180
Asp Ala Ser Ile Tyr Val Val Gln Asp Gly Leu Leu Glu Leu Cys
185 190 195
Leu Pro Gly Pro Asp Gly Lys Glu Cys Val Val Lys Glu Val Val
200 205 210
Pro Gly Asp Ser Val Asn Ser Leu Leu Ser Ile Leu Asp Val Ile
215 220 225
3/11
CA 02349210 2001-03-02
WO 00/14248 PCTNS99/20287
Thr Gly His Gln His Pro Gln Arg Thr Val Ser Ala Arg Ala Ala
230 235 240
Arg Asp Ser Thr Val Leu Arg Leu Pro Val Glu Ala Phe Ser Ala
245 250 255
Val Phe Thr Lys Tyr Pro Glu Ser Leu Val Arg Val Val Gln Ile
260 265 270
Ile Met Val Arg Leu Gln Arg Val Thr Phe Leu Ala Leu His Asn
275 280 285
Tyr Leu Gly Leu Thr Asn Glu Leu Phe Ser His Glu Ile Gln Pro
290 295 300
Leu Arg Leu Phe Pro Ser Pro Gly Leu Pro Thr Arg Thr Ser Pro
305 310 315
Val Arg Gly Ser Lys Arg Met Val Ser Thr Ser Ala Thr Asp Glu
320 325 330
Pro Arg Glu Thr Pro Gly Arg Pro Pro Asp Pro Thr Gly Ala Pro
335 340 345
Leu Pro Gly Pro Thr Gly Asp Pro Val Lys Pro Thr Ser Leu Glu
350 355 360
Thr Pro Ser Ala Pro Leu Leu Ser Arg Cys Val Ser Met Pro Gly
365 370 375
Asp Ile Ser Gly Leu Gln Gly Gly Pro Arg Ser Asp Phe Asp Met
380 385 390
Ala Tyr Glu Arg Gly Arg Ile Ser Val Ser Leu Gln Glu Glu Ala
395 400 405
Ser Gly Gly Ser Leu Ala Ala Pro Ala Arg Thr Pro Thr Gln Glu
410 415 420
Pro Arg Glu Gln Pro Ala Gly Ala Cys Glu Tyr Ser Tyr Cys Glu
425 430 435
Asp Glu Ser Ala Thr Gly Gly Cys Pro Phe Gly Pro Tyr Gln Gly
440 445 450
Arg Gln Thr Ser Ser Ile Phe Glu Ala Ala Lys Gln Glu Leu Ala
455 460 465
Lys Leu Met Arg Ile Glu Asp Pro Ser Leu Leu Asn Ser Arg Val
470 475 480
Leu Leu His His Ala Lys Ala Gly Thr Ile Ile Ala Arg Gln Gly
485 490 495
Asp Gln Asp Val Ser Leu His Phe Val Leu Trp Gly Cys Leu His
500 505 510
Val Tyr Gln Arg Met Ile Asp Lys Ala Glu Asp Val Cys Leu Phe
515 520 525
Val Ala Gln Pro Gly Glu Leu Val Gly Gln Leu Ala Val Leu Thr
530 535 540
Gly Glu Pro Leu Ile Phe Thr Leu Arg Ala Gln Arg Asp Cys Thr
545 550 555
Phe Leu Arg Ile Ser Lys Ser Asp Phe Tyr Glu Ile Met Arg Ala
560 565 570
Gln Pro Ser Val Val Leu Ser Ala Ala His Thr Val Ala Ala Arg
575 580 585
Met Ser Pro Phe Val Arg Gln Met Asp Phe Ala Ile Asp Trp Thr
590 595 600
Ala Val Glu Ala Gly Arg Ala Leu Tyr Arg Gln Gly Asp Arg Ser
605 610 615
Asp Cys Thr Tyr Ile Val Leu Asn Gly Arg Leu Arg Ser Val Ile
620 625 630
Gln Arg Gly Ser Gly Lys Lys Glu Leu Val Gly Glu Tyr Gly Arg
635 640 645
Gly Asp Leu Ile Gly Val Val Glu Ala Leu Thr Arg Gln Pro Arg
4/11
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
650 655 660
Ala Thr Thr Val His Ala Val Arg Asp Thr Glu Leu Ala Lys Leu
665 670 675
Pro Glu Gly Thr Leu Gly His Ile Lys Arg Arg Tyr Pro Gln Val
680 685 690
Val Thr Arg Leu Ile His Leu Leu Ser Gln Lys Ile Leu Gly Asn
695 700 705
Leu Gln Gln Leu Gln Gly Pro Phe Pro Ala Gly Ser Gly Leu Gly
710 715 720
Val Pro Pro His Ser Glu Leu Thr Asn Pro Ala Ser Asn Leu Ala
725 730 735
Thr Val Ala Ile Leu Pro Val Cys Ala Glu Val Pro Met Val Ala
740 745 750
Phe Thr Leu Glu Leu Gln His Ala Leu Gln Ala Ile Gly Pro Thr
755 760 765
Leu Leu Leu Asn Ser Asp Ile Ile Arg Ala Arg Leu Gly Ala Ser
770 775 780
Ala Leu Asp Ser Ile Gln Glu Phe Arg Leu Ser Gly Trp Leu Ala
785 790 795
Gln Gln Glu Asp Ala His Arg Ile Val Leu Tyr Gln Thr Asp Ala
800 805 810
Ser Leu Thr Pro Trp Thr Val Arg Cys Leu Arg Gln Ala Asp Cys
815 820 825
Ile Leu Ile Val Gly Leu Gly Asp Gln Glu Pro Thr Leu Gly Gln
830 835 840
Leu Glu Gln Met Leu Glu Asn Thr Ala Val Arg Ala Leu Lys Gln
845 850 855
Leu Val Leu Leu His Arg Glu Glu Gly Ala Gly Pro Thr Arg Thr
860 865 870
Val Glu Trp Leu Asn Met Arg Ser Trp Cys Ser Gly His Leu His
875 880 885
Leu Arg Cys Pro Arg Arg Leu Phe Ser Arg Arg Ser Pro Ala Lys
B90 895 900
Leu His Glu Leu Tyr Glu Lys Val Phe Ser Arg Arg Ala Asp Arg
905 910 915
His Ser Asp Phe Ser Arg Leu Ala Arg Val Leu Thr Gly Asn Thr
920 925 930
Ile Ala Leu Val Leu Gly Gly Gly Gly Ala Arg Gly Cys Ser His
935 940 945
Ile Gly Val Leu Lys Ala Leu Glu Glu Ala Gly Val Pro Val Asp
950 955 960
Leu Val Gly Gly Thr Ser Ile Gly Ser Phe Ile Gly Ala Leu Tyr
965 970 975
Ala Glu Glu Arg Ser Ala Ser Arg Thr Lys Gln Arg Ala Arg Glu
980 985 990
Trp Ala Lys Ser Met Thr Ser Val Leu Glu Pro Val Leu Asp Leu
995 1000 1005
Thr Tyr Pro Val Thr Ser Met Phe Thr Gly Ser Ala Phe Asn Arg
1010 1015 1020
Ser Ile His Arg Val Phe Gln Asp Lys Gln Ile Glu Asp Leu Trp
1025 1030 1035
Leu Pro Tyr Phe Asn Val Thr Thr Asp Ile Thr Ala Ser Ala Met
1040 1045 1050
Arg Val His Lys Asp Gly Ser Leu Trp Arg Tyr Val Arg Ala Ser
1055 1060 1065
Met Thr Leu Ser Gly Tyr Leu Pro Pro Leu Cys Asp Pro Lys Asp
1070 1075 1080
5~ 1 1
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
Gly His Leu Leu Met Asp Gly Gly Tyr Ile Asn Asn Leu Pro Ala
1085 1090 1095
Asp Ile Ala Arg Ser Met G1y Ala Lys Thr Val Ile Ala Ile Asp
1100 1105 1110
Val Gly Ser Gln Asp Glu Thr Asp Leu Ser Thr Tyr Gly Asp Ser
1115 1120 1125
Leu Ser Gly Trp Trp Leu Leu Trp Lys Arg Leu Asn Pro Trp Ala
1130 1135 1140
Asp Lys Val Lys Val Pro Asp Met Ala Glu Ile Gln Ser Arg Leu
1145 1150 1155
Ala Tyr Val Ser Cys Val Arg Gln Leu Glu Val Val Lys Ser Ser
1160 1165 1170
Ser Tyr Cys Glu Tyr Leu Arg Pro Pro Ile Asp Cys Phe Lys Thr
1175 1180 1185
Met Asp Phe Gly Lys Phe Asp Gln Ile Tyr Asp Val Gly Tyr Gln
1190 1195 1200
Tyr Gly Lys Ala Val Phe Gly Gly Trp Ser Arg Gly Asn Val Ile
1205 1210 1215
Glu Lys Met Leu Thr Asp Arg Arg Ser Thr Asp Leu Asn Glu Ser
1220 1225 1230
Arg Arg Ala Asp Val Leu Ala Phe Pro Ser Ser Gly Phe Thr Asp
1235 1240 1245
Leu Ala Glu Ile Val Ser Arg Ile Glu Pro Pro Thr Ser Tyr Val
1250 1255 1260
Ser Asp Gly Cys Ala Asp Gly Glu Glu Ser Asp Cys Leu Thr Glu
1265 1270 1275
Tyr Glu Glu Asp Ala Gly Pro Asp Cys Ser Arg Asp Glu Gly Gly
1280 1285 1290
Ser Pro Glu Gly Ala Ser Pro Ser Thr Ala Ser Glu Met Glu Glu
1295 1300 1305
Glu Lys Ser Ile Leu Arg Gln Arg Arg Cys Leu Pro Gln Glu Pro
1310 1315 1320
Pro Gly Ser Ala Thr Asp Ala
1325
<210> 3
<211> 690
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 159278CD1
<400> 3
Met Phe Cys Thr Lys Leu Lys Asp Leu Lys Ile Thr Gly Glu Cys
1 5 10 15
Pro Phe Ser Leu Leu Ala Pro Gly Gln Val Pro Asn Glu Ser Ser
20 25 30
Glu Glu Ala Ala Gly Ser Ser Glu Ser Cys Lys Ala Thr Val Pro
35 40 45
Ile Cys Gln Asp Ile Pro Glu Lys Asn Ile Gln Glu Ser Leu Pro
50 55 60
Gln Arg Lys Thr Ser Arg Ser Arg Val Tyr Leu His Thr Leu Ala
65 70 75
Glu Ser Ile Cys Lys Leu Ile Phe Pro Glu Phe Glu Arg Leu Asn
6/11
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
80 85 90
Val Ala Leu Gln Arg Thr Leu Ala Lys His Lys Ile Lys Glu Ser
95 100 105
Arg Lys Ser Leu Glu Arg Glu Asp Phe Glu Lys Thr Ile Ala Glu
110 115 120
Gln Ala Val Ala Aia Gly Val Pro Val Glu Val Ile Lys Glu Ser
125 130 135
Leu Gly Glu Glu Val Phe Lys Ile Cys Tyr Glu Glu Asp Glu Asn
140 ~ 145 150
Ile Leu Gly Val Val Gly Gly Thr Leu Lys Asp Phe Leu Asn Ser
155 160 165
Phe Ser Thr Leu Leu Lys Gln Ser Ser His Cys Gln Glu Ala Gly
170 175 180
Lys Arg Gly Arg Leu Glu Asp Ala Ser Ile Leu Cys Leu Asp Lys
185 190 195
Glu Asp Asp Phe Leu His Val Tyr Tyr Phe Phe Pro Lys Arg Thr
200 205 210
Thr Ser Leu Ile Leu Pro Gly Ile Ile Lys Ala Ala Ala His Val
215 220 225
Leu Tyr Glu Thr Glu Val Glu Val Ser Leu Met Pro Pro Cys Phe
230 235 240
His Asn Asp Cys Ser Glu Phe Val Asn Gln Pro Tyr Leu Leu Tyr
245 250 255
Ser Val His Met Lys Ser Thr Lys Pro Ser Leu Ser Pro Ser Lys
260 265 270
Pro Gln Ser Ser Leu Val Ile Pro Thr Ser Leu Phe Cys Lys Thr
275 ~ 280 285
Phe Pro Phe His Phe Met Phe Asp Lys Asp Met Thr Ile Leu Gln
290 295 300
Phe Gly Asn Gly Ile Arg Arg Leu Met Asn Arg Arg Asp Phe Gln
305 310 315
Gly Lys Pro Asn Phe Glu Glu Tyr Phe Glu Ile Leu Thr Pro Lys
320 325 330
Ile Asn Gln Thr Phe Ser Gly Ile Met Thr Met Leu Asn Met Gln
335 340 345
Phe Val Val Arg Val Arg Arg Trp Asp Asn Ser Val Lys Lys Ser
350 355 360
Ser Arg Val Met Asp Leu Lys Gly Gln Met Ile Tyr Ile Val Glu
365 370 375
Ser Ser Ala Ile Leu Phe Leu Gly Ser Pro Cys Val Asp Arg Leu
380 385 390
Glu Asp Phe Thr Gly Arg Gly Leu Tyr Leu Ser Asp Ile Pro Ile
395 400 405
His Asn Ala Leu Arg Asp Val Val Leu Ile Gly Glu Gln Ala Arg.
410 415 420
Ala Gln Asp Gly Leu Lys Lys Arg Leu Gly Lys Leu Lys Ala Thr
425 430 435
Leu Glu Gln Ala His Gln Ala Leu Glu Glu Glu Lys Lys Lys Thr
440 445 450
Val Asp Leu Leu Cys Ser Ile Phe Pro Cys Glu Val Ala Gln Gln
455 460 46S
Leu Trp Gln Gly Gln Val Val Gln Ala Lys Lys Phe Ser Asn Val
470 475 480
Thr Met Leu Phe Ser Asp Ile Val Gly Phe Thr Ala Ile Cys Ser
485 490 495
Gln Cys Ser Pro Leu Gln Val Ile Thr Met Leu Asn Ala Leu Tyr
500 505 510
7/ 1 1
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
Thr Arg Phe Asp Gln Gln Cys Gly Glu Leu Asp Val Tyr Lys Val
515 520 525
Glu Thr Ile Gly Asp Ala Tyr Cys Val Ala Gly Gly Leu His Lys
530 535 540
Glu Ser Asp Thr His Ala Val Gln Ile Ala Leu Met Ala Leu Lys
545 550 555
Met Met Glu Leu Ser Asp Glu Val Met Ser Pro His Gly Glu Pro
560 565 570
Ile Lys Met Arg Ile Gly Leu His Ser Gly Ser Val Phe Ala Gly
575 580 585
Val Val Gly Val Lys Met Pro Arg Tyr Cys Leu Phe Gly Asn Asn
590 595 600
Val Thr Leu Ala Asn Lys Phe Glu Ser Cys Ser Val Pro Arg Lys
605 610 615
Ile Asn Val Ser Pro Thr Thr Tyr Arg Leu Leu Lys Asp Cys Pro
620 625 630
Gly Phe Val Phe Thr Pro Arg Ser Arg Glu Glu Leu Pro Pro Asn
635 640 645
Phe Pro Ser Glu Ile Pro Gly Ile Cys His Phe Leu Asp Ala Tyr
650 655 660
Gln Gln Gly Thr Asn Ser Lys Pro Cys Phe Gln Lys Lys Asp Val
665 670 675
Glu Asp Gly Asn Ala Asn Phe Leu Gly Lys Ala Ser Gly Ile Asp
680 685 690
<210> 4
<211> 2446
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2267958CB1
<400> 4
aagacaagat aatctgcctt ccgaaaagag tgcagcctgc tcagaaccac tcttcccttt 60
cgaatgtctc tcaagcagtt gccagtacca ctccactgcc tccacctaaa ccatctcctg 120
ctaaccccat cactgtggaa atgaaaggcc tgaagacaga tttggacctt cagcagtaca 180
gctttataaa tcagatgtgt tatgagcgag ccctccactg gtatgccaag tatttccctt 240
accttgtcct catccatacc ctggtcttta tgctctgcag taacttttgg ttcaaattcc 300
ctggttccag ctccaaaata gaacatttca tctccattct ggggaagtgt tttgactctc 360
cttggaccac acgggcttta tctgaagtgt ctggggagga ctcagaagaa aaggacaacg 420
ggaagaacaa catgaacagg tccaacacca tccaatctgg tccagaaggc agcctggtca 480
actctcagtc tttaaagtcc attcctgaga agtttgtagt tgataaatcc actgcagggg 540
ctctggataa aaaggaaggt gagcaggcta aggccttatt tgagaaggtg aagaagttca 600
ggctgcatgt ggaagaaggt gatattctat atgccatgta tgttcgccag actgtactta 660
aagttatcaa attcctaatc atcattgcat ataatagtgc tctggtttcc aaggtccagt 720
ttacagtgga ctgtaatgtg gacattcagg acatgactgg atataaaaac ttttcttgca 780
atcataccat ggcacacttg ttctcaaaac tgtccttttg ctatctgtgc tttgttagta 840
tctatggatt gacgtgcctt tataccttat actggctgtt ctaccgttct ctacgggaat 900
attcctttga gtatgtccgt caggagactg gaattgatga tattccagat gtgaaaaatg 960
actttgcttt tatgcttcat atgatagatc agtatgaccc tctctattcc aagagatttg 1020
cagtgttcct gtctgaagtc agtgaaaaca aattaaagca gctgaactta aataacgaat 1080
ggactcctga taaactgagg cagaagctac agacaaatgc ccataatcga ctggaattgc 1140
ctcttatcat gctctctggc cttccagaca ctgtttttga aatcacagag ttgcaatctc 1200
taaaacttga aatcattaag aacgtaatga taccagccac cattgcacag ctagacaatc 1260
8/11
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
ttcaagagct ctctctgcac cagtgttctg tcaaaatcca cagtgcggcg ctctctttcc 1320
tgaaggaaaa cctcaaggtc ttgagcgtca agtttgatga catgagggaa ctccccccct 1380
ggatgtatgg gctccgaaat ctggaagagc tgtacctagt tggctctcta agtcatgata 1440
tttccagaaa tgtcaccctt gagtctctgc gggatctcaa aagccttaaa attctctcta 1500
tcaaaagcaa cgtttccaaa atccctcagg cagtggttga tgtttccagc catctccaga 1560
agatgtgcat acataatgat ggcaccaagc tggtgatgct caacaactta aagaagatga 1620
ccaatctgac agagctggag ctggtccact gtgacctgga gcgtattcct catgctgtgt 1680
tcagcctact cagcctccag gaattggacc tgaaggaaaa caatctgaaa tctatagaag 1740
aaatcgttag ctttcagcac ttaagaaagt tgacagtgct aaaactgtgg cataacagca 1800
tcacctacat cccagagcat ataaagaaac tcaccagcct ggaacgcctg tcctttagtc 1860
acaataaaat agaggtgctg ccttcccacc tcttcctatg caacaagatc cgatacttgg 1920
acttatcgta caatgacatt cgatttatcc cccctgaaat tggagttcta caaagtttac 1980
agtatttttc catcacatgt aacaaagtgg aaagccttcc agatgaactc tacttctgca 2040
agaaacttaa aactctgaag attggaaaaa acagcctatc tgtactttca ccgaaaattg 2100
gaaatttgct atttctttcc tacttagatg taaaaggtaa tcactttgaa atcctccctc 2160
ctgaactggg tgactgtcgg gctctgaagc gagctggttt agttgtagaa gatgctctgt 2220
ttgaaactct gccttctgac gtccgggagc aaatgaaaac agaataactt atttttcgtt 2280
aaagtttgac tgaaacacgc ttctaccaaa tacagtataa ataattaggt agtcttaatg 2340
cctttcctat ttttttttcc ttttcacaca aaatgtacac aaagatcgcg taaggagtat 2400
gtatttttaa taaaaattta attgtatttt ttcaataaaa aaaaaa 2446
<210> 5
<211> 4228
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3149674CB1
<400> 5
ccagatcggc cgtccagctg gaatcaaccg atggaggctc cgctgcaaac tggaatggtg 60
cttggcgtga tgatcggggc cggagtggcg gtggtggtca cggccgtgct catcctcctg 120
gtggtgcgga ggctgcgagt gccaaaaacc ccagccccgg atggcccccg gtatcggttc 180
cggaagaggg acaaagtgct cttctatggc cggaagatta tgcggaaggt gtcacaatcc 240
acctcctccc tcgtggatac ctctgtctcc gccacctccc ggccacgcat gaggaagaaa 300
ctgaagatgc tcaacattgc caagaagatc ctgcgcatcc agaaagagac gcccacgctg 360
cagcggaagg agcccccgcc cgcagtgcta gaagctgacc tgaccgaggg cgacctggct 420
aactcccatc tgccctctga agtgctttat atgctcaaga acgtccgggt gctgggccac 480
ttcgagaagc cactcttcct ggagctctgc cgccacatgg tcttccagcg gctgggccag 540
ggtgactacg tcttccggcc gggccagcca gatgccagca tctacgtggt gcaggacggg 600
ctgctggagc tctgtctgcc agggcctgac gggaaggagt gtgtggtgaa ggaagtggtt 660
cctggggaca gcgtcaacag ccttctcagc atcctggatg tcatcaccgg tcaccagcat 720
ccccagcgga ccgtgtctgc ccgggcggcc cgggactcca cggtgctgcg cctgccggtg 780
gaagcattct ccgcggtctt caccaagtac ccggagagct tggtgcgggt cgtgcagatc 840
atcatggtgc ggctgcagcg agtcaccttc ctggcactgc acaactacct gggtctgacc 900
aatgagctct tcagccacga gatccagccc ctgcgtctgt tccccagccc cggcctccca 960
actcgcacca gccctgtgcg gggctccaag agaatggtca gcacctcagc tacagacgag 1020
cccagggaga ccccagggcg gccacccgat cccaccgggg ccccgctgcc tggacctaca 1080
ggggaccctg tgaagcccac atccctggaa accccctcgg cccctctgct gagccgctgc 1140
gtctccatgc caggggacat ctcaggcttg cagggtggcc cccgctccga cttcgacatg 1200
gcctatgagc gtggccggat ctccgtgtcc ctgcaggaag aggcctccgg ggggtccctg 1260
gcagcccccg ctcggacccc cactcaggag cctcgtgagc agccggcagg cgcctgtgaa 1320
tacagctact gtgaggatga gtcggccact ggtggctgcc ctttcgggcc ctaccagggc 1380
cgccagacca gcagcatctt cgaggcagca aagcaggagc tggccaagct gatgcggatt 1440
gaggacccct ccctcctgaa cagcagagtc ttgctgcacc acgccaaagc tggcaccatc 1500
attgcccgcc agggagacca ggacgtgagc ctgcacttcg tgctctgggg ctgcctgcac 1560
9/11
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
gtgtaccagc gcatgatcga caaggcggag gacgtgtgcc tgttcgtagc gcagcccggg 1620
gaactggtgg ggcagctggc ggtgctcact ggcgaacctc tcatcttcac actgcgagcc 1680
caacgcgact gcaccttcct gcggatctcc aagtccgact tctatgagat catgcgcgca 1740
cagcccagtg tggtgctgag tgcggcgcac acggtggcag ccaggatgtc gcccttcgtg 1800
cgccagatgg acttcgccat cgactggact gcagtggagg cgggacgcgc gctgtacagg 1860
cagggcgacc gctccgactg cacttacatc gtgctcaatg ggcggctgcg tagcgtgatc 1920
cagcgaggca gtggcaagaa ggagctggtg ggcgagtacg gccgcggcga cctcatcggc 1980
gtggtggagg cactgacccg gcagccgcga gccacgacgg tgcacgcggt gcgcgacacg 2040
gagctggcca agcttcccga gggcaccttg ggtcacatca aacgccggta cccgcaggtc 2100
gtgacccgcc ttatccacct actgagccag aaaattctag ggaatttgca gcagctgcaa 2160
ggacccttcc cagcaggctc tgggttgggt gtgcccccac actcggaact caccaaccca 2220
gccagcaacc tggcaactgt ggcaatcctg cctgtgtgtg ctgaggtccc catggtggcc 2280
ttcacgctgg agctgcagca cgccctgcag gccatcggtc cgacgctact ccttaacagt 2340
gacatcatcc gggcacgcct gggggcctcc gcactggata gcatccaaga gttccggctg 2400
tcagggtggc tggcccagca ggaggatgca caccgtatcg tactctacca gacggacgcc 2460
tcgctgacgc cctggaccgt gcgctgcctg cgacaggccg actgcatcct cattgtgggc 2520
ctgggggacc aggagcctac cctcggccag ctggagcaga tgctggagaa cacggctgtg 2580
cgcgccctta agcagctagt cctgctccac cgagaggagg gcgcgggccc cacgcgcacc 2640
gtggagtggc taaatatgcg cagctggtgc tcggggcacc tgcacctgcg ctgtccgcgc 2700
cgcctctttt cgcgccgcag ccctgccaag ctgcatgagc tctacgagaa ggttttctcc 2760
aggcgcgcgg accggcacag cgacttctcc cgcttggcga gggtgctcac ggggaacacc 2820
attgcccttg tgctaggcgg gggcggggcc aggggctgct cgcacatcgg agtactaaag 2880
gcattagagg aggcgggggt ccccgtggac ctggtgggcg gcacgtccat tggctctttc 2940
atcggagcgt tgtacgcgga ggagcgcagc gccagccgca cgaagcagcg ggcccgggag 3000
tgggccaaga gcatgacttc ggtgctggaa cctgtgttgg acctcacgta cccagtcacc 3060
tccatgttca ctgggtctgc ctttaaccgc agcatccatc gggtcttcca ggataagcag 3120
attgaggacc tgtggctgcc ttacttcaac gtgaccacag atatcaccgc ctcagccatg 3180
cgagtccaca aagatggctc cctgtggcgg tacgtgcgcg ccagcatgac gctgtcgggc 3240
tacctgcccc cgctgtgcga ccccaaggac gggcacctac tcatggatgg cggctacatc 3300
aacaatctgc cagcggacat cgcccgcagc atgggtgcca aaacggtcat cgccattgac 3360
gtggggagcc aggatgagac ggacctcagc acctacgggg acagcctgtc cggctggtgg 3420
ctgctgtgga agcggctgaa tccctgggct gacaaggtaa aggttccaga catggctgaa 3480
atccagtccc gcctggccta cgtgtcctgt gtgcggcagc tagaggttgt caagtccagc 3540
tcctactgcg agtacctgcg cccgcccatc gactgcttca agaccatgga ctttgggaag 3600
ttcgaccaga tctatgatgt gggctaccag tacgggaagg cggtgtttgg aggctggagc 3660
cgtggcaacg tcattgagaa aatgctcaca gaccggcggt ctacagacct taatgagagc 3720
cgccgtgcag acgtgcttgc cttcccaagc tctggcttca ctgacttggc agagattgtg 3780
tcccggattg agccccccac gagctatgtc tctgatggct gtgctgacgg agaggagtca 3840
gattgtctga cagagtatga ggaggacgcc ggacccgact gctcgaggga tgaagggggg 3900
tcccccgagg gcgcaagccc cagcactgcc tccgagatgg aggaggagaa gtcgattctc 3960
cggcaacgac gctgtctgcc ccaggagccg cccggctcag ccacagatgc ctgaggacct 4020
cgacaggggt caccccctcc ctcccacccc tggactgggc tgggggtggc cccgtggggg 4080
tagctcactc cccctcctgc tgctatgcct gtgacccccg cggcccacac actggactga 4140
cctgccctga gcggggatgc agtgttgcac tgatgacttg accagcccct cccccaataa 4200
actcgcctct tggaaaaaaa aaaaaaaa 4228
<210> 6
<211> 2715
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 159278CB1
<400> 6
gcaggtaccg gatcgcaagg atgactcgcc agcgcgtcgg ggagatgggg aggaggcggg 60
10/11
CA 02349210 2001-03-02
WO 00/14248 PCT/US99/20287
cagaggtctg aaaaaatcga atgccttaag gaaaggaact gcaagggttc ctttggggtg 120
atcaaagagg gagacacaga cacagagaga caaaggcaag gaggactgtc tgggagccac 180
gcgggcgata cagtttccga ggcacgccgc gtcccgccta gcctgttgaa caggtagaca 240
tgagcgaccc aagctgcgga tttgcgaggc gctctctgga gctgctagag atccggaagc 300
acagccccga ggtgtgcgaa gcaccaaaca tcccagttac cagtgtcctt gaattgatag 360
tggcttctgt ttgtcagtct catataagaa ctacagctca tcaggaggag atcgcagcag 420
ggtaagagac accaacacca tgttctgcac gaagctcaag gatctcaaga tcacaggaga 480
gtgtcctttc tccttactgg caccaggtca agttcctaac gagtcttcag aggaggcagc 540
aggaagctca gagagctgca aagcaaccgt gcccatctgt caagacattc ctgagaagaa 600
catacaagaa agtcttcctc aaagaaaaac cagtcggagc cgagtctatc ttcacacttt 660
ggcagagagt atttgcaaac tgattttccc agagtttgaa cggctgaatg ttgcacttca 720
gagaacattg gcaaagcaca aaataaaaga aagcaggaaa tctttggaaa gagaagactt 780
tgaaaaaaca attgcagagc aagcagttgc agcaggagtt ccagtggagg ttatcaaaga 840
atctcttggt gaagaggttt ttaaaatatg ttacgaggaa gatgaaaaca tccttggggt 900
ggttggaggc acccttaaag attttttaaa cagcttcagt acccttctga aacagagcag 960
ccattgccaa gaagcaggaa aaaggggcag gcttgaggac gcctccattc tatgcctgga 1020
taaggaggat gattttctac atgtttacta cttcttccct aagagaacca cctccctgat 1080
tcttcccggc atcataaagg cagctgctca cgtattatat gaaacggaag tggaagtgtc 1140
gttaatgcct ccctgcttcc ataatgattg cagcgagttt gtgaatcagc cctacttgtt 1200
gtactccgtt cacatgaaaa gcaccaagcc atccctgtcc cccagcaaac cccagtcctc 1260
gctggtgatt cccacatcgc tattctgcaa gacatttcca ttccatttca tgtttgacaa 1320
agatatgaca attctgcaat ttggcaatgg catcagaagg ctgatgaaca ggagagactt 1380
tcaaggaaag cctaattttg aagaatactt tgaaattctg actccaaaaa tcaaccagac 1440
gtttagcggg atcatgacta tgttgaatat gcagtttgtt gtacgagtga ggagatggga 1500
caactctgtg aagaaatctt caagggttat ggacctcaaa ggccaaatga tctacattgt 1560
tgaatccagt gcaatcttgt ttttggggtc accctgtgtg gacagattag aagattttac 1620
aggacgaggg ctctacctct cagacatccc aattcacaat gcactgaggg atgtggtctt 1680
aataggggaa caagcccgag ctcaagatgg cctgaagaag aggctgggga agctgaaggc 1740
tacccttgag caagcccacc aagccctgga ggaggagaag aaaaagacag tagaccttct 1800
gtgctccata tttccctgtg aggttgctca gcagctgtgg caagggcaag ttgtgcaagc 1860
caagaagttc agtaatgtca ccatgctctt ctcagacatc gttgggttca ctgccatctg 1920
ctcccagtgc tcaccgctgc aggtcatcac catgctcaat gcactgtaca ctcgcttcga 1980
ccagcagtgt ggagagctgg atgtctacaa ggtggagacc attggcgatg cctattgtgt 2040
agctggggga ttacacaaag agagtgatac tcatgctgtt cagatagcgc tgatggccct 2100
gaagatgatg gagctctctg atgaagttat gtctccccat ggagaaccta tcaagatgcg 2160
aattggactg cactctggat cagtttttgc tggcgtcgtt ggagttaaaa tgccccgtta 2220
ctgtcttttt ggaaacaatg tcactctggc taacaaattt gagtcctgca gtgtaccacg 2280
aaaaatcaat gtcagcccaa caacttacag attactcaaa gactgtcctg gtttcgtgtt 2340
tacccctcga tcaagggagg aacttccacc aaacttccct agtgaaatcc ccggaatctg 2400
ccattttctg gatgcttacc aacaaggaac aaactcaaaa ccatgcttcc aaaagaaaga 2460
tgtggaagat ggcaatgcca attttttagg caaagcatca ggaatagatt agcaacctat 2520
atacctattt ataagtcttt ggggtttgac tcattgaaga tgtgtagagc ctctgaaagc 2580
actttaggga ttgtagatgg ctaacaagca gtattaaaat ttcaggagcc aagtcacaat 2640
ctttctcctg tttaacatga caaaatgtat gtactcactt cagtacttca gctcttcaag 2700
aaaaaaaaaa aaaaa 2715
11/11
