Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
SOLUBLE PROTEIN ZTMPO-1
BACKGROUND OF THE INVENTION
There is a growing family of proteins which
share regions of sequence homology and localization to the
nucleus. These proteins include the thymopoietins,
(Zevin-Sonkin et al., Immuno. Letts. 31:301-10, 1992;
Harris et al., Proc. Natl. Acad. Sci. USA 91:6283-87,
1994; Harris et al., Genomics 28:198-205, 1995; Berger et
al., Genome Res. 6:361-70, 1996 and Ishijima et al.,
Biochem. Biophys. Res. Comm. 226:431-8, 1996), lamina
associated proteins, (Senior and Gerace, J. Cell Biol.
107:2029-36, 1988; Worman et al., J. Cell Biol. 111:1535-
42, 1990; Wozniak and Blobel J. Cell Biol. 119:1441-9,
1992; Foisner and Gerace, Cell 73:1267-79, 1993; Ye and
Worman, J. Biol. Chem. 269:11306-11, 1994 and Furukawa et
al., EMBO J. 14:1626-36, 1995) and emerin (Bione et al.,
Nat. Genet. 8:323-7, 1994; Manilal et al., Hum. Mol. Gen.
5:801-8, 1996 and Small et al., Mamm. Genom. 8:337-41,
1997) .
Emerin is a nuclear membrane protein responsible
for the X-linked recessive disorder Emery-Dreifuss
muscular dystrophy. Mouse, rat and human emerin sequences
have been reported .(Bione et al., Nat. Genet. 8:323-7,
1994; Manila et al., Hum. Mol. Genet. 5:801-8, 1996 and
Small et al., Mammal. Genom. 8:337-41, 1997). The mouse,
rat and human emerin share 73-95% nucleotide and amino
acid identity. All share some structural homology with
the thymopoietins and LAP2, in particular within portions
of the conserved N-terminal region and the hydrophobic
putative transmembrane domain of thymopoietin. Like the
thymopoietins and LAP2, emerin is ubiquitous expressed and
it is predicted that emerin has the same inner nuclear
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membrane organization as do thymopoietin and LAP2 (Manilal
et al., ibid.). Antisera raised against emerin peptides
localized expression of the protein to the nuclear
membranes of normal skeletal and cardiac muscle cells, but
found it to be absent in those cells of patients with
muscular dystrophy. It is unclear how a deficiency of a
nuclear protein results in the disease (Nagano et al.,
Nat. Genet. 12:254-9, 1996 and Small et al., ibid.).
The present invention provides associated
polypeptides for these and other uses that should be
apparent to those skilled in the art from the teachings
herein.
SUMMARY OF THE INVENTION
Within one aspect the invention provides an
isolated polypeptide comprising a sequence of amino acid
residues that is at least 80% identical in amino acid
sequence to residues 1 through 876 of SEQ ID N0:2. Within
one embodiment the sequence of amino acid residues is at
least 90% identical. Within another embodiment any
differences between said polypeptide and residues 1
through 876 of SEQ ID N0:2 are due to conservative amino
acid substitutions. Within another embodiment the
polypeptide specifically binds with an antibody that
specifically binds with a polypeptide consisting of the
amino acid sequence of SEQ ID N0:2. Within a further
embodiment the polypeptide is covalently linked to a
moiety selected from the group consisting of affinity
tags, radionucleotides, enzymes and fluorophores. Within
a related embodiment the moiety is an affinity tag
selected from the group consisting of polyhistidine, FLAG,
Glu-Glu, glutathione S transferase and an immunoglobulin
heavy chain constant region.
Also provided is an isolated polypeptide
comprising the amino acid sequence of SEQ ID N0:2.
Within another aspect the invention provides a
fusion protein consisting essentially of a first portion
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and a second portion joined by a peptide bond, said first
portion consisting of a polypeptide comprising a sequence
of amino acid residues that is at least 80o identical in
amino acid sequence to residues 1 through 876 of SEQ ID
N0:2; and said second portion comprising another
polypeptide.
Within yet another aspect the invention provides
a pharmaceutical composition comprising a polypeptide as
described above, in combination with a pharmaceutically
acceptable vehicle.
Within still another aspect is provided an
antibody or antibody fragment that specifically binds to a
polypeptide as described above. Within one embodiment the
antibody is selected from the group consisting of: a)
polyclonal antibody; b) murine monoclonal antibody; c)
humanized antibody derived from b); and d) human
monoclonal antibody. Within another embodiment the
antibody fragment is selected from the group consisting of
F(ab'), F(ab), Fab', Fab, Fv, scFv, and minimal
recognition unit. Within still another embodiment is
provided an anti-idiotype antibody that specifically binds
to the antibody described above.
Also provided is a binding protein that
specifically binds to an epitope of a polypeptide as
described above.
Within another aspect of the invention is
provided an isolated polynucleotide selected from the
group consisting of: a) a polynucleotide encoding a
polypeptide comprising a sequence of amino. acid residues
that is at least 80% identical in amino acid sequence to
residues 1 through 876 of SEQ ID N0:2; b) a polynucleotide
~ comprising the nucleotide sequence of SEQ ID N0:5; c) a
polynucleotide that remains hybridized following stringent
wash conditions to a polynucleotide consisting of the
nucleotide sequence of SEQ ID NO:1, or the complement of
SEQ ID NO:1. Within one embodiment the sequence of amino
acid residues is at least 90% identical. Within another
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embodiment any difference between the amino acid sequence
encoded by the polynucleotide and the corresponding amino
acid sequence of SEQ ID N0:2 is due to a conservative
amino acid substitution. Within yet another embodiment
the polynucleotide comprises nucleotide 127 to nucleotide
2754 of SEQ ID NO:1. Within still another embodiment the
polynucleotide is DNA.
Within another aspect the invention provides an
expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment
consisting of a polynucleotide as described above; and a
transcriptional terminator. Within one embodiment the
sequence of amino acid residues is at least 90% identical.
Within another embodiment any difference between the amino
acid sequence encoded by the polynucleotide and the
corresponding amino acid sequence of SEQ ID N0:2 is due to
a conservative amino acid substitution. Within another
embodiment the DNA segment encodes a polypeptide
covalently linked to an affinity tag selected from the
group consisting of polyhistidine, Glu-Glu, glutathione S
transferase and an immunoglobulin heavy chain constant
region. Within yet another embodiment the expression
vector further comprises a secretory signal sequence
operably linked to said DNA segment.
Also provided is a cultured cell into which has
been introduced an expression vector as described above,
wherein the cell expresses the polypeptide encoded by the
DNA segment.
Within a further aspect the invention provide a
method of producing a ZTMPO-1 polypeptide comprising:
culturing a cell into which has been introduced an
expression vector as described above, whereby the cell
expresses the polypeptide encoded by the DNA segment; and
recovering the expressed polypeptide.
Also provided by the invention is a method for
detecting a genetic abnormality in a patient, comprising:
obtaining a genetic sample from a patient; incubating the
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genetic sample with a polynucleotide comprising at least
14 contiguous nucleotides of SEQ ID NO:1 or the complement
of SEQ ID NO: l, under conditions wherein said
polynucleotide will hybridize to complementary
5 polynucleotide sequence, to produce a first reaction
product; comparing said first reaction product to a
control reaction product, wherein a difference between
said first reaction product and said control reaction
product is indicative of a genetic abnormality in the
patient.
These and other. aspects of the invention will
become evident upon reference to the following detailed
description of the invention and attached drawing.
BRIEF DESCRIPTION OF THE DRAWINGS
The figure shows a multiple amino acid sequence
alignment for ZTMPO-I (SEQ ID N0:2), human emerin (EMD HU)
Bione et al., Nat. Genet. 8:323-27, 1994 (SEQ ID N0:3),
human thymopoietin a (PIR A5) Harris et al., Proc. Natl.
Acad. Sci. USA 91: 6283-7, 1994 (SEQ ID N0:4), human
thymopoietin (3 (PIR B5) Harris et al., ibid. (SEQ ID
N0:30) and human thymopoietin y (PIR-C5) Harris et al.,
ibid. (SEQ ID N0:31).
DETAILED DESCRIPTION OF THE INVENTION
Prior to setting forth the invention in detail,
it may be helpful to the understanding thereof to define
the following terms:
The term "affinity tag" is used herein to denote
a polypeptide segment that can be attached to a second
polypeptide to provide for purification of the second
polypeptide or provide sites for attachment of the second
polypeptide to a substrate. In principal, any peptide or
protein for which an antibody or other specific binding
agent is available can be used as an affinity tag.
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Affinity tags include a poly-histidine tract, protein A
(Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,
Methods Enzymol. 198:3, 1991), glutathione S transferase
(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity
tag, substance P, FlagTM peptide (Hopp et al.,
BiotechnoloQV 6:1204-10, 1988), streptavidin binding
peptide, or other antigenic epitope or binding domain.
See, in general, Ford et al., Protein Expression and
Purification 2: 95-107, 1991. DNAs encoding affinity tags
are available from commercial suppliers (e. g., Pharmacies
Biotech, Piscataway, NJ).
The term "allelic variant" is used herein to
denote any of two or more alternative forms of a gene
occupying the same chromosomal locus. Allelic variation
arises naturally through mutation, and may result in
phenotypic polymorphism within populations. Gene
mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered
amino acid sequence. The term allelic variant is also
used herein to denote a protein encoded by an allelic
variant of a gene.
The terms "amino-terminal" and "carboxyl-
terminal" are used herein to denote positions within
polypeptides. Where the context allows, these terms are
used with reference to a particular sequence or portion of
a polypeptide to denote proximity or relative position.
For example, a certain sequence positioned carboxyl-
terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus
of the complete polypeptide.
The term "complements of a polynucleotide
molecule" is a polynucleotide molecule having a
complementary base sequence and reverse orientation as
compared to a reference sequence. For example, the
sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT
3'.
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The term "contig" denotes a polynucleotide that
has a contiguous stretch of identical or complementary
sequence to another polynucleotide. Contiguous sequences
are said to "overlap" a given stretch of polynucleotide
sequence either in their entirety or alone a partial
stretch of the polynucleotide. For example,
representative contigs to the polynucleotide sequence 5'-
ATGGCTTAGCTT-3' are 5'-TAGCTTgagtct-3' and 3'-
gtcgacTACCGA-5'.
The term "degenerate nucleotide sequence"
denotes a sequence of nucleotides that includes one or
more degenerate codons (as compared to a reference
polynucleotide molecule that encodes a polypeptide).
Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e.,
GAU and GAC triplets each encode Asp).
The term "expression vector" is used to denote a
DNA molecule, linear or circular, that comprises a segment
encoding a polypeptide of interest operably linked to
additional segments that provide for its transcription.
Such additional segments include promoter and terminator
sequences, and may also include one or more origins of
replication, one or more selectable markers, an enhancer,
a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may
contain elements of both.
The term "isolated", when applied to a
polynucleotide, denotes that the polynucleotide has been
removed from its natural genetic milieu and is thus free
of other extraneous or unwanted coding sequences, and is
in a form suitable for use within genetically engineered
protein production systems. Such isolated molecules are
those that are separated from their natural environment
and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes
with which they are ordinarily associated, but may include
naturally occurring 5' and 3' untranslated regions such as
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promoters and terminators. The identification of
associated regions will be evident to one of ordinary
skill in the art (see for example, Dynan and Tijan, Nature
316:774-78, 1985).
An "isolated" polypeptide or protein is a
polypeptide or protein that is found in a condition other
than its native environment, such as apart from blood and
animal tissue. In a preferred form, the isolated
polypeptide is substantially free of other polypeptides,
particularly other polypeptides of animal origin. It is
preferred to provide the polypeptides in a highly purified
form, i.e. greater than 95% pure, more preferably greater
than 99% pure. When used in this context, the term
"isolated" does not exclude the presence of the same
polypeptide in alternative physical forms, such as dimers
or alternatively glycosylated or derivatized forms.
The term "operably linked", when referring to
DNA segments, indicates that the segments are arranged so
that they function in concert for their intended purposes,
e.g., transcription initiates in the promoter and proceeds
through the coding segment to the terminator.
The term "ortholog" denotes a polypeptide or
protein obtained from one species that is the functional
counterpart of a polypeptide or protein from a different
species. Sequence differences among orthologs are the
result of speciation.
A "polynucleotide" is a single- or double-
stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end. Polynucleotides
include RNA and DNA, and may be isolated from natural
sources, synthesized in vitro, or prepared from a
combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated
"bp"), nucleotides ("nt"), or kilobases ("kb"). Where the
context allows, the latter two terms may describe
polynucleotides that are single-stranded or double-
stranded. When the term is applied to double-stranded
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molecules it is used to denote overall length and will be
understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the
two strands of a double-stranded polynucleotide may differ
slightly in length and that the ends thereof may be
staggered as a result of enzymatic cleavage; thus all
nucleotides within a double-stranded polynucleotide
molecule may not be paired. Such unpaired ends will in
general not exceed 20 nt in length.
A "polypeptide" is a polymer of amino acid
residues joined by peptide bonds, whether produced
naturally or synthetically. Polypeptides of less than
about 10 amino acid residues are commonly referred to as
"peptides".
"Probes and/or primers" as used herein can be
RNA or DNA. DNA can be either cDNA or genomic DNA.
Polynucleotide probes and primers are single or double-
stranded DNA or RNA, generally synthetic oligonucleotides,
but may be generated from cloned cDNA or genomic sequences
or its complements. Analytical probes will generally be
at least 20 nucleotides in length, although somewhat
shorter probes (14-17 nucleotides) can be used. PCR
primers are at least 5 nucleotides in length, preferably
15 or more nt, more preferably 20-30 nt. Short
polynucleotides can be used when a small region of the
gene is targeted for analysis. For gross analysis of
genes, a polynucleotide probe may comprise an entire exon
or more. Probes can be labeled to provide a detectable
signal, such as with an enzyme, biotin, a radionuclide,
fluorophore, chemiluminescer, paramagnetic particle and
the like, which are commercially available from many
sources, such as Molecular Probes, Inc., Eugene, OR, and
Amersham Corp., Arlington Heights, IL, using techniques
that are well known in the art. Examples of ZTMPO-1
probes and primers include, but are not limited to, the
sequences disclosed herein as SEQ ID NOs:6-29.
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The term "promoter" is used herein for its art-
recognized meaning to denote a portion of a gene
containing DNA sequences that provide for the binding of
RNA polymerase and initiation of transcription. Promoter
5 sequences are commonly, but not always, found in the 5'
non-coding regions of genes.
A "protein" is a macromolecule comprising one or
more polypeptide chains. A protein may also comprise non-
peptidic components, such as carbohydrate groups.
10 Carbohydrates and other non-peptidic substituents may be
added to a protein by the cell in which the protein is
produced, and will vary with the type of cell. Proteins
are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are
generally not specified, but may be present nonetheless.
The term "receptor" denotes a cell-associated
protein that binds to a bioactive molecule (i.e., a
ligand) and mediates the effect of the ligand on the cell.
Membrane-bound receptors are characterized by a multi-
domain structure comprising an extracellular ligand-
binding domain and an intracellular effector domain that
is typically involved in signal transduction. Binding of
ligand to receptor results in a conformational change in
the receptor that causes an interaction between the
effector domain and other molecules) in the cell. This
interaction in turn leads to an alteration in the
metabolism of the cell. Metabolic events that are linked
to receptor-ligand interactions include gene
transcription, phosphorylation, dephosphorylation,
increases in cyclic AMP production, mobilization of
cellular calcium, mobilization of membrane lipids, cell
adhesion, hydrolysis of inositol lipids and hydrolysis of
phospholipids. In general, receptors can be membrane
bound, cytosolic or nuclear; monomeric (e. g., thyroid
stimulating hormone receptor, beta-adrenergic receptor) or
multimeric (e. g., PDGF receptor, growth hormone receptor,
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IL-3 receptor, GM-CSF receptor, G-CSF receptor,
erythropoietin receptor and IL-6 receptor).
The term "secretory signal sequence" denotes a
DNA sequence that encodes a polypeptide (a "secretory
peptide") that, as a component of a larger polypeptide,
directs the larger polypeptide through a secretory pathway
of a cell in which it is synthesized. The larger
polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
The term "splice variant" is used herein to
denote alternative forms of RNA transcribed from a gene.
Splice variation arises naturally through use of
alternative splicing sites within a transcribed RNA
molecule, or less commonly between separately transcribed
RNA molecules, and may result in several mRNAs transcribed
from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term
splice variant is also used herein to denote a protein
encoded by a splice variant of an mRNA transcribed from a
gene.
Molecular weights and lengths of polymers
determined by imprecise analytical methods (e.g., gel
electrophoresis) will be understood to be approximate
values. When such a value is expressed as "about" X or
"approximately" X, the stated value of X will be
understood to be accurate to t10%.
The present invention is based in part upon the
discovery of a novel protein having regions of homology to
members of the thymopoietin-emerin family of nuclear
3o membrane proteins. This protein has been designated
"ZTMPO-1". The human ZTMPO-1 nucleotide sequence is
represented in SEQ ID NO:1 and the deduced amino acid
sequence in SEQ ID N0:2. The ZTMPO-1 proteins and
polypeptides encoded by polynucleotides of the present
invention were initially identified by querying an EST
(Expressed Sequence Tag) database for sequences homologous
to conserved motifs within the thymopoietin family.
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ZTMPO-1 as represented in SEQ ID NO:1 is a 2,754 by
polynucleotide which has an open reading frame encoding an
876 amino acid residue protein. Sequence analysis of the
deduced amino acid sequence as represented in SEQ ID N0:2
does not indicate the presence of a secretion signal
sequence or transmembrane domain. There is a putative
ankyrin-like region, amino acid residues 333-385 of SEQ ID
N0:2, having an ankyrin repeat (residues 347-379 of SEQ ID
N0:2) which may indicate that ZTMPO-1 is retained in the
plasma membrane. Ankyrin repeats have been described as a
33 amino acid motif, usually found in tandem arrays of
four to seven copies, that mediate protein interactions
(Michaely and Bennett, J. Biol. Chem. 268:22703-9, 1993).
Ankyrin repeats have been reported in numerous proteins in
species from bacteria to man (Sentenac et al., Science
256:663-5, 1992; Zhang et al., Plant Cell 4:1575-88, 1992;
Gustine et al., Plant Physiol. 108:1748, 1995; Andrews and
Herskowitz, Nature 342:830-3, 1989; Warton et al., Cell
43:567-81, 1995 and Yochem and Greenwald, Cell 58:53-63,
1989. Ankyrin repeats have been proposed as a generalized
protein binding motif, one function of ankyrin repeats is
to serve as adaptors, associating with the spectrin-based
cytoplasmic skeleton and membrane proteins. Ankyrin is
used as a membrane attachment site in neurons and may
provide a transport mechanism through secretory vesicles.
At the C-terminal end of ZTMPO-1 is a calcium
binding protein-like region having two potential calcium
binding sites (residues 678-692 and residues 719-731 pf
SEQ ID N0:2) similar to that seen in the sea urchin
calcium binding protein LPS1-beta (Xiang et al., J. Biol.
Chem. 16:10524-33, 1991).
The ZTMPO-1 polynucleotide of SEQ ID N0:1
encodes an 876 amino acid residue protein which is much
larger than other members of the thymopoietin/emerin
family. Human thymopoietin a, is a 693 amino acid residue
protein, human emerin is a 254 amino acid residue protein
and rat LAP2 is a 452 amino acid residue protein.
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Like emerin, the amino acid sequence of ZTMPO-1
does not contain the 42 amino acid thymopoietin peptide
originally identified by Goldstein (Nature 247:11-14,
1974) but shares discrete regions of homology with the
human thymopoietins a, (3 and y (Harris et al., ibid.,
Genbank Accession Nos. a (U09086), (3 (U09087) and
(U09088)) and the mouse thymopoietins a, , ,
~3 y, s, 8 and
(Berger et al., ibid., Genbank Accession Nos. a (U39078),
U39074, y (U39077), s (U39074), 8 (U39076) and
(U39073)). In particular, over the region defined by
amino acid residues 13 to 44 of SEQ ID N0:2, ZTMPO-1
shares 50% amino acid identity with the corresponding
regions of the mouse and human thymopoietins and 30% with
human emerin. In particular, the region defined by amino
acid residues 30-44 of SEQ ID N0:2 is highly conserved
between the proteins, see Figure.
As would be expected, ZTMPO-1 also shares
discrete regions of homology with rat lamina associated
protein 2, LAP2, (Furukawa et al., ibid., Genbank
Accession No. U18314). These regions correspond to many
of the same regions with which ZTMPO-1 shares identity
with the thymopoietins. ZTMPO-1 and rat LAP2 share 70%
amino acid identity over the region corresponding to amino
acid residues 13 to 44 of SEQ ID N0:2.
ZTMPO-1 also shares a limited degree of homology
to regions of the yeast transcription factor IIF alpha
subunit over the region corresponding to amino acid
residues 86 to 160 and amino acid residues 205 to 260 of
SEQ ID N0:2.
Additionally, ZTMPO-2 shares 27% amino acid
identity with Trypanosoma brucei ribonuclease H1 (Hesslein
and Campbell, Mol. Biochem. Parasitol. 86:221-6, 1997,
Genbank Accession No. U74470) over the region
corresponding to amino acid residues 156 to 203 of SEQ ID
N0:2. This homology, along with that shared with LAP2, as
well as the possible ankyrin repeat, suggests the
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possibility that ZTMPO-1 possesses chromatin or DNA
binding properties.
Those skilled in the art will recognize that
these domain boundaries are approximate, and are based on
alignments with known proteins and predictions of protein
folding.
Northern blot analysis of various human tissues
was performed using a 218 by human DNA probe (SEQ ID
N0:8). A 3.2 and a 5 kb transcript corresponding to
ZTMPO-1 were ubiquitously expressed with the highest level
being in testis tissue. Similar ubiquitous expression
patterns were also reported for the thymopoietins and
emerin (Harris et al., ibid. and Small et al., ibid.).
Chromosomal localization results show that
ZTMPO-1 maps 636.18 cR_3000 from the top of the human
chromosome 12 linkage group on the WICGR radiation hybrid
map. The proximal framework marker was D12S367. The use
of surrounding markers positions ZTMPO-1 in the 12q24.33
region on the integrated LDB chromosome 12 map. Among the
genes mapping around this region are insulin-like growth
factor 1 which is involved in growth and development;
melanin concentrating hormone, a neuropeptide associated
with goal-associated behaviors and general arousal (Nahon
et al., Genomics 12: 846-8, 1992); spinal muscular atrophy
a nonprogressive muscular atrophy involving mainly the
lower extremities (van Ravenswaaij, et al., Am. J. Hum.
Genet. 61 (suppl.): A299, 1997); spinal muscular atrophy 4
(Timmerman, et al., Hum. Molec. Genet. 5: 1065-9, 1996)
and myosin regulatory light chain which is involved in
regulation of myosin ATPase activity in smooth muscle
(Macera, et al., Genomics 13: 829-31, 1992). Thymopoietin
maps to chromosome 12q22 (Harris et al., ibid.).
The nucleotide sequences encoding regions of
conserved amino acid residues between ZTMPO-1 and nuclear
proteins such as the thymopoietins, LAP2 and emerin, for
example, the region between nucleotides 163 and 258 of SEQ
ID N0:1, in particular the region between nucleotides 214
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and 258 of SEQ ID NO:1, can be used as a tool to identify
new family members. For instance, reverse transcription-
polymerase chain reaction (RT-PCR) can be used to amplify
sequences encoding these conserved regions from RNA
5 obtained from a variety of tissue sources or cell lines.
In particular, highly degenerate primers designed from the
ZTMPO-1 sequences are useful for this purpose.
The present invention also provides
polynucleotide molecules, including DNA and RNA molecules,
10 that encode the ZTMPO-1 polypeptides disclosed herein.
Those skilled in the art will readily recognize that, in
view of the degeneracy of the genetic code, considerable
sequence variation is possible among these polynucleotide
molecules. SEQ ID N0:5 is a degenerate DNA sequence that
15 encompasses all DNAs that encode the ZTMPO-1 polypeptide
of SEQ ID N0:2. Those skilled in the art will recognize
that the degenerate sequence of SEQ ID N0:5 also provides
all RNA sequences encoding SEQ ID N0:2 by substituting U
(uracil) for T (thymine). Thus, ZTMPO-1 polypeptide-
encoding polynucleotides comprising nucleotide 1 to
nucleotide 2628 of SEQ ID N0:5 and their RNA equivalents
are contemplated by the present invention. Table 1 sets
forth the one-letter codes used within SEQ ID N0:5 to
denote degenerate nucleotide positions. "Resolutions" are
the nucleotides denoted by a code letter. "Nucleotide
Complement" indicates the code for the complementary
nucleotide(s). For example, the code Y denotes either C
(cytosine) or T, and its complement R denotes A
(adenosine) or G (guanine), A being complementary to T,
and G being complementary to C.
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TABLE 1
Nucleotide
Base Code Resolutions Base Code Complement
A A T T
C C G G
G G C C
T T A A
R A~G Y CST
Y CST R A~G
M ABC K GET
K GET M ABC
S CMG S CMG
W ACT W ACT
H A~C~T D A~G~T
B C~G~T V A~C~G
V A~C~G B C~G~T
D A~G~T H A~C~T
N A~C~G~T N A~C~G~T
The degenerate codons used in SEQ ID N0:5,
encompassing all possible codons for a given amino acid,
are set forth in Table 2.
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TABLE 2
Three One
Letter Letter Degenerate
Code Code Synonymous Colon
Colons
Cys C TGC TGT TGY
Ser S AGC AGTTCA TCC TCG TCT WSN
Thr T ACA ACCACG ACT ACN
Pro P CCA CCCCCG CCT CCN
Ala A GCA GCCGCG GCT GCN
Gly G GGA GGCGGG GGT GGN
Asn N AAC AAT qAY
Asp D~ GAC GAT GAY
Glu E GAA GAG GAR
Gln Q CAA CAG CAR
His H CAC CAT CAY
Arg R AGA AGGCGA CGC CGG CGT MGN
Lys K AAA AAG AAR
Met M ATG ATG
Ile I ATA ATCATT ATH
Leu L CTA CTCCTG CTT TTA TTG YTN
Val V GTA GTCGTG GTT GTN
Phe F TTC TTT ~Y
Tyr Y TAC TAT TAY
Trp W TGG TGG
Ter . TAA TAGTGA TRR
Asn~Asp B RAY
GIuJGIn Z SAR
Any X NNN
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18
One of ordinary skill in the art will appreciate
that some ambiguity is introduced in determining a
degenerate codon, representative of all possible codons
encoding each amino acid. For example, the degenerate
codon for serine (WSN) can, in some circumstances, encode
arginine (AGR), and the degenerate codon for arginine
(MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding
phenylalanine and leucine. Thus, some polynucleotides
encompassed by the degenerate sequence may encode variant
amino acid sequences, but one of ordinary skill in the art
can easily identify such variant sequences by reference to
the amino acid sequence of SEQ ID N0:2. Variant sequences
can be readily tested for functionality as described
herein.
One of ordinary skill in the art will also
appreciate that different species can exhibit
"preferential codon usage." In general, see, Grantham, et
al., Nuc. Acids Res., 8_:1893-912, 1980; Haas, et al. Curr.
Biol., 6:315-24, 1996; Wain-Hobson, et al., Gene, 13:355-
64, 1981; Grosjean and Fiers, Gene, 18:199-209, 1982;
Holm, Nuc. Acids Res., 14:3075-87, 1986; Ikemura, J. Mol.
Biol., 158:573-97, 1982. As used herein, the term
"preferential codon usage" or "preferential codons" is a
term of art referring to protein translation codons that
are most frequently used in cells of a certain species,
thus favoring one or a few representatives of the possible
codons encoding each amino acid (See Table 2). For
example, the amino acid threonine (Thr) may be encoded by
ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the
most commonly used codon; in other species, for example,
insect cells, yeast, viruses or bacteria, different Thr
codons may be preferential. Preferential codons for a
particular species can be introduced into the
polynucleotides of the present invention by a variety of
methods known in the art. Introduction of preferential
codon sequences into recombinant DNA can, for example,
CA 02325822 2000-10-18
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19
enhance production of the protein by making protein
translation more efficient within a particular cell type
or species. Therefore, the degenerate codon sequence
disclosed in SEQ ID N0:5 serves as a template for
optimizing expression of polynucleotides in various cell
types and species commonly used in the art and disclosed
herein. Sequences containing preferential codons can be
tested and optimized for expression in various species,
and tested for functionality as disclosed herein.
The present invention also provides polypeptide
fragments or peptides comprising an epitope-bearing
portion of an ZTMPO-1 polypeptide described herein. Such
fragments or peptides may comprise an "immunogenic
epitope," which is a part of a protein that elicits an
antibody response when the entire protein is used as an
immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example,
Geysen et al., Proc. Nat. Acad. Sci. USA 81:3998, 1983).
In contrast, polypeptide fragments or peptides
may comprise an "antigenic epitope," which is a region of
a protein molecule to which an antibody can specifically
bind. Certain epitopes consist of a linear or contiguous
stretch of amino acids, and the antigenicity of such an
epitope is not disrupted by denaturing agents. It is known
in the art that relatively short synthetic peptides that
can mimic epitopes of a protein can be used to stimulate
the production of antibodies against the protein (see, for
example, Sutcliffe et al., Science 219:660, 1983).
Accordingly, antigenic epitope-bearing peptides and
polypeptides of the present invention are useful to raise
antibodies that bind with the polypeptides described
herein .
Antigenic epitope-bearing peptides and
polypeptides preferably contain at least four to ten amino
acids, at least ten to fifteen amino acids, or about 15 to
about 30 amino acids of SEQ ID N0:2. Such epitope-bearing
peptides and polypeptides can be produced by fragmenting a
CA 02325822 2000-10-18
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ZTMPO-1 polypeptide, or by chemical peptide synthesis, as
described herein. Moreover, epitopes can be selected by
phage display of random peptide libraries (see, for
example, Lane and Stephen, Curr. Opin. Immunol. 5:268,
5 1993, and Cortese et al., Curr. Opin. Biotechnol. 7:616,
1996). Standard methods for identifying epitopes and
producing antibodies from small peptides that comprise an
epitope are described, for example, by Mole, "Epitope
Mapping," in Methods in Molecular Biologv, Vol. 10, Manson
10 (ed.), pages 105-116 (The Humana Press, Inc. 1992), Price,
"Production and Characterization of Synthetic Peptide-
Derived Antibodies," in Monoclonal Antibodies: Production,
Engineering, and Clinical Application, Ritter and Ladyman
(eds.), pages 60-84 (Cambridge University Press 1995), and
15 Coligan et al. (eds.), Current Protocols in ImmunolocTV,
pages 9.3.1 - 9.3.5 and pages 9.4.1 - 9.4.11 (John Wiley &
Sons 1997).
Potential antigenic sites in ZTMPO-1 can be
identified using the Jameson-Wolf method (Jameson and
20 Wolf, CABIOS 4:181, 1988), as implemented by the PROTEAN
program (version 3.14) of LASERGENE (DNASTAR; Madison,
WI). The Jameson-Wolf method predicts potential antigenic
determinants by combining six major subroutines for
protein structural prediction. Briefly, the Hopp-Woods
method (Hopp et al., Proc. Nat. Acad. Sci. USA 78:3824,
1981), is first used to identify amino acid sequences
representing areas of greatest local hydrophilicity
(parameter: seven residues averaged). In the second step,
Emini's method (Emini et al., J. Virolocry 55:836, 1985),
is used to calculate surface probabilities (parameter:
surface decision .threshold (0.6) - 1). Third, the
Karplus-Schultz method, (Karplus and Schultz;
Naturwissenschaften 72:212, 1985), is used to predict
backbone chain flexibility (parameter: flexibility
threshold (0.2) - 1). In the fourth and fifth steps of
the analysis, secondary structure predictions are applied
to the data using the methods of Chou-Fasman, Chou,
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21
"Prediction of Protein Structural Classes from Amino Acid
Composition," in Prediction of Protein Structure and the
Princit~les of Protein Conformation, Fasman (ed.), pages
549-586 (Plenum Press 1990), and Gamier-Robson, Gamier
et al., J. Mol. Biol. 120:97, 1978 (Chou-Fasman
parameters: conformation table - 64 proteins; a region
threshold - 103; b region threshold - 105; Garnier-Robson
parameters: a and b decision constants - 0). In the sixth
subroutine, flexibility parameters and hydropathy/solvent
accessibility factors are combined to determine a surface
contour value, designated as the "antigenic index."
Finally, a peak broadening function is applied to the
antigenic index, which broadens major surface peaks by
adding 20, 40, 60, or 80% of the respective peak value to
account for additional free energy derived from the
mobility of surface regions relative to interior regions.
Regardless of the particular nucleotide sequence
of a variant ZTMPO-1 gene, the gene encodes a polypeptide
that is characterized by its glycoprotein synthesis or
cell-cell interaction activity, or by the ability to bind
specifically to an anti-ZTMPO-1 antibody. More
specifically, variant ZTMPO-1 genes encode polypeptides
which exhibit at least 50%, and preferably, greater than
70, 80, or 90°s, of the activity of polypeptide encoded by
the human ZTMPO-1 gene described herein.
For any ZTMPO-1 polypeptide, including variants
and fusion proteins, one of ordinary skill in the art can
readily generate a fully degenerate polynucleotide
sequence encoding that variant using the information set
forth in Tables 1 and 2 above. Moreover, those of skill
in the art can use standard software to devise ZTMPO-1
variants based upon the nucleotide and amino acid
sequences described herein. Accordingly, the present
invention includes a computer-readable medium encoded with
a data structure that provides at least one of the
following sequences: SEQ ID NO:1, SEQ ID N0:2 or SEQ ID
N0:5. Suitable forms of computer-readable media include
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22
magnetic media and optically-readable media. Examples of
magnetic media include a hard or fixed drive, a random
access memory (RAM) chip, a floppy disk, digital linear
tape (DLT), a disk cache, and a ZIP disk. Optically
S readable media are exemplified by compact discs (e.g., CD-
read only memory (ROM), CD-rewritable (RW), and CD-
recordable), and digital versatile/video discs (DVD)
(e. g., DVD-ROM, DVD-RAM, and DVD+RW).
Within preferred embodiments of the invention,
the isolated polynucleotides can hybridize under stringent
conditions to polynucleotides having the nucleotide
sequence of SEQ ID NO:1 or to nucleic acid molecules
having a nucleotide sequence complementary to SEQ ID NO:1.
In general, stringent conditions are selected to be about
5°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength and pH. The
Tm is the temperature (under defined ionic strength and
pH) at which 50% of the target sequence hybridizes to a
perfectly matched probe.
A pair of nucleic acid molecules, such as DNA-
DNA, RNA-RNA and DNA-RNA, can hybridize if the nucleotide
sequences have some degree of complementarity. Hybrids
can tolerate mismatched base pairs in the double helix,
but the stability of the hybrid is influenced by the
degree of mismatch. The Tm of the mismatched hybrid
decreases by 1°C for every 1-1.5% base pair mismatch.
Varying the stringency of the hybridization conditions
allows control over the degree of mismatch that will be
present in the hybrid. The degree of stringency increases
as the hybridization temperature increases and the ionic
strength of the hybridization buffer decreases. Stringent
hybridization conditions encompass temperatures of about
5-25°C below the Tm of the hybrid and a hybridization
buffer having up to 1 M Na'. Higher degrees of stringency
at lower temperatures can be achieved with the addition of
formamide which reduces the Tm of the hybrid about 1°C for
CA 02325822 2000-10-18
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23
each 1% formamide in the buffer solution. Generally, such
stringent conditions include temperatures of 20-70°C and a
hybridization buffer containing up to 6xSSC and 0-500
formamide. A higher degree of stringency can be achieved
at temperatures of from 40-70°C with a hybridization
buffer having up to 4xSSC and from 0-50o formamide.
Highly stringent conditions typically encompass
temperatures of 42-70°C with a hybridization buffer having
up to lxSSC and 0-50% formamide. Different degrees of
stringency can be used during hybridization and washing to
achieve maximum specific binding to the target sequence.
Typically, the washes following hybridization are
performed at increasing degrees of stringency to remove
non-hybridized polynucleotide probes from hybridized
complexes.
The above conditions are meant to serve as a
guide and it is well within the abilities of one skilled
in the art to adapt these conditions for use with a
particular polypeptide hybrid. The Tm for a specific
target sequence is the temperature (under defined
conditions) at which 50% of the target sequence will
hybridize to a perfectly matched probe sequence. Those
conditions which influence the Tm include, the size and
base pair content of the polynucleotide probe, the ionic
strength of the hybridization solution, and the presence
of destabilizing agents in the hybridization solution.
Numerous equations for calculating Tm are known in the
art, and are specific for DNA, RNA and DNA-RNA hybrids and
polynucleotide probe sequences of varying length (see, for
example, Sambrook et al., Molecular Cloning: A Laboratory
Manual, Second Edition (Cold Spring Harbor Press 1989);
Ausubel et al., (eds.), Current Protocols in Molecular
Bioloc~v (John Wiley and Sons, Inc. 1987); Berger and
Kimmel (eds.), Guide to Molecular Cloning Technigues,
(Academic Press, Inc. 1987); and Wetmur, Crit. Rev.
Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis
CA 02325822 2000-10-18
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24
software, such as OLIGO 6.0 (LSR; Long Lake, MN) and
Primer Premier 4.0 (Premier Biosoft International; Palo
Alto, CA), as well as sites on the Internet, are available
tools for analyzing a given sequence and calculating Tm
based on user defined criteria. Such programs can also
analyze a given sequence under defined conditions and
identify suitable probe sequences. Typically,
hybridization of longer polynucleotide sequences, >50 base
pairs, is performed at temperatures of about 20-25°C below
the calculated Tm. For smaller probes, <50 base pairs,
hybridization is typically carried out at the Tm or 5-10°C
below. This allows for the maximum rate of hybridization
for DNA-DNA and DNA-RNA hybrids.
The length of the polynucleotide sequence
influences the rate and stability of hybrid formation.
Smaller probe sequences, <50 base pairs, reach equilibrium
with complementary sequences rapidly, but may form less
stable hybrids. Incubation times of anywhere from minutes
to hours can be used to achieve hybrid formation. Longer
probe sequences come to equilibrium more slowly, but form
more stable complexes even at lower temperatures.
Incubations are typically allowed to proceed overnight or
longer. Generally, incubations are carried out for a
period equal to three times the calculated Cot time. Cot
time, the time it takes for the polynucleotide sequences
to reassociate, can be calculated for a particular
sequence by methods known in the art.
The base pair composition of polynucleotide
sequence will effect the thermal stability of the hybrid
complex, thereby influencing the choice of hybridization
temperature and the ionic strength of the hybridization
buffer. A-T pairs are less stable than G-C pairs in
aqueous solutions containing sodium chloride. Therefore,
the higher the G-C content, the more stable the hybrid.
Even distribution of G and C residues within the sequence
also contribute positively to hybrid stability. In
addition, the base pair composition can be manipulated to
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
alter the Tm of a given sequence. For example, 5-
methyldeoxycytidine can be substituted for deoxycytidine
and S-bromodeoxuridine can be substituted for thymidine to
increase the Tm, whereas 7-deazz-2'-deoxyguanosine can be
5 substituted for guanosine to reduce dependence on Tm.
The ionic concentration of the hybridization
buffer also affects the stability of the hybrid.
Hybridization buffers generally contain blocking agents
such as Denhardt's solution (Sigma Chemical Co., St.
10 Louis, Mo.), denatured salmon sperm DNA, tRNA, milk
powders (BLOTTO), heparin or SDS, and a Na' source, such as
SSC (lx SSC: 0.15 M sodium chloride, 15 mM sodium citrate)
or SSPE ( lx SSPE : 1 . 8 M NaCl , 10 mM NaH2P0, , 1 mM EDTA, pH
7.7). By decreasing the ionic concentration of the
15 buffer, the stability of the hybrid is increased.
Typically, hybridization buffers contain from between 10
mM - 1 M Na'. The addition of destabilizing or denaturing
agents such as formamide, tetralkylammonium salts,
guanidinium cations or thiocyanate cations to the
20 hybridization solution will alter the Tm of a hybrid.
Typically, formamide is used at a concentration of up to
SO% to allow incubations to be carried out at more
convenient and lower temperatures. Formamide also acts to
reduce non-specific background when using RNA probes.
25 As an illustration, a polynucleotide encoding a
variant ZTMPO-1 polypeptide can be hybridized with a
polynucleotide having the nucleotide sequence of SEQ ID
NO:1 (or its complement) at 42°C overnight in a solution
comprising SOo formamide, SxSSC (lxSSC: 0.15 M sodium
chloride and 15 mM sodium citrate), 50 mM sodium phosphate
(pH 7.6), 5x Denhardt's solution (100x Denhardt's
solution: 2% (w/v) Ficoll 400, 2% (w/v)
polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin),
loo dextran sulfate, and 20 ~g/ml denatured, sheared
salmon sperm DNA. One of skill in the art can devise
variations of these hybridization conditions. For
example, the hybridization mixture can be incubated at a
CA 02325822 2000-10-18
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26
higher or lower temperature, such a~ about 65°C, in a
solution that does not contain formamide. Moreover,
premixed hybridization solutions are available (e. g.,
EXPRESSHYB Hybridization Solution from CLONTECH
Laboratories, Inc.), and hybridization can be performed
according to the manufacturer's instructions.
Following hybridization, the nucleic acid
molecules can be washed to remove non-hybridized nucleic
acid molecules under stringent conditions, or under highly
stringent conditions. Typical stringent washing
conditions include washing in a solution of 0.5x-2x SSC
with 0.1% sodium dodecyl sulfate (SDS) at 55-65°C. That
is, nucleic acid molecules encoding a variant ZTMPO-1
polypeptide hybridize with a nucleic acid molecule having
the nucleotide sequence of SEQ ID NO:1 (or its complement)
under stringent washing conditions, in which the wash
stringency is equivalent to 0.5x-2x SSC with 0.1% SDS at
50-65°C, including 0.5x SSC with O.la SDS at 55°C, or 2x
SSC with 0.1% SDS at 65°C. One of skill in the art can
readily devise equivalent conditions, for example, by
substituting SSPE for SSC in the wash solution.
Typical highly stringent washing conditions
include washing in a solution of O.lx-0.2x SSC with 0.1%
sodium dodecyl sulfate (SDS) at 50-65°C. In other words,
polynucleotides encoding a variant ZTMPO-1 polypeptide
hybridize with a polynucleotide having the nucleotide
sequence of SEQ ID NO:1 (or its complement) under highly
stringent washing conditions, in which the wash stringency
is equivalent to O.lx-0.2x SSC with 0.1% SDS at 50-65°C,
including O.lx SSC with 0.1% SDS at 50°C, or 0.2x SSC with
O.lo SDS at 65°C.
The present invention also contemplates ZTMPO-1
variant polypeptides that can be identified using two
criteria: a determination of the similarity between the
encoded polypeptide with the amino acid sequence of SEQ ID
N0:2, and a hybridization assay, as described above. Such
ZTMPO-1 variants include nucleic acid molecules (1) that
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27
hybridize with a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:1 (or its complement)
under stringent washing conditions, in which the wash
stringency is equivalent to 0.5x-2x SSC with O.lo SDS at
50-65°C, and (2) that encode a polypeptide having at least
80%, at least 900, at least 95% or greater than 95%
sequence identity to the amino acid sequence of SEQ ID
N0:2. Alternatively, ZTMPO-1 variants can be
characterized as nucleic acid molecules (1) that hybridize
with a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:1 (or its complement) under highly
stringent washing conditions, in which the wash stringency
is equivalent to O.lx-0.2x SSC with 0.1% SDS at 50-65°C,
and ( 2 ) that encode a polypeptide having at least 80 0 , at
least 90%, at least 95% or greater than 95% sequence
identity to the amino acid sequence of SEQ ID N0:2.
As previously noted, the isolated
polynucleotides of the present invention include DNA and
RNA. Methods for preparing DNA and RNA are well known in
the art. In general, RNA is isolated from a tissue or
cell that produces large amounts of ZTMPO-1 RNA. Such
tissues and cells are identified by Northern blotting
(Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), an
exemplary source being human testis tissue. Total RNA can
be prepared using guanidine HC1 extraction followed by
isolation by centrifugation in a CsCl gradient (Chirgwin
et al., Biochemistrv 18:52-94, 1979). Poly (A)+ RNA is
prepared from total RNA using the method of Aviv and Leder
(Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).
Complementary DNA (cDNA) is prepared from poly(A)+ RNA
using known methods. In the alternative, genomic DNA can
be isolated. Polynucleotides encoding ZTMPO-1
polypeptides are then identified and isolated by, for
example, hybridization or PCR.
The polynucleotides of the present invention can
also be synthesized using techniques widely known in the
art. See, for example, Glick and Pasternak, Molecular
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
28
Biotechnology, Principles & Applications of Recombinant
DNA, (ASM Press, Washington, D.C. 1994); Itakura et al.,
Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al.,
Proc. Natl. Acad. Sci. USA 87:633-7, 1990.
The present invention further provides
counterpart polypeptides and polynucleotides from other
species (orthologs). These species include, but are not
limited to mammalian, avian, amphibian, reptile, fish,
insect and other vertebrate and invertebrate species. Of
particular interest are ZTMPO-1 polypeptides from other
mammalian species, including murine, porcine, ovine,
bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human ZTMPO-1 can be cloned
using information and compositions provided by the present
invention in combination with conventional cloning
techniques. For example, a cDNA can be cloned using mRNA
obtained from a tissue or cell type that expresses ZTMPO-1
as disclosed herein. Suitable sources of mRNA can be
identified by probing Northern blots with probes designed
from the sequences disclosed herein. A library is then
prepared from mRNA of a positive tissue or cell line. A
ZTMPO-1-encoding cDNA can then be isolated by a variety of
methods, such as by probing with a complete or partial
human cDNA or with one or more sets of degenerate probes
based on the disclosed sequences. A cDNA can also be
cloned using the polymerase chain reaction, or PCR
(Mullis, U.S. Patent No. 4,683,202), using primers
designed from the representative human ZTMPO-1 sequence
disclosed herein. Within an additional method, the cDNA
library can be used to transform or transfect host cells,
and expression of the cDNA of interest can be detected
with an antibody to ZTMPO-1 polypeptide. Similar
techniques can also be applied to the isolation of genomic
clones.
Those skilled in the art will recognize that the
sequence disclosed in SEQ ID NO:1 represents a single
allele of human ZTMPO-1 and that allelic variation and
CA 02325822 2000-10-18
WO 99154468 PCT/US99/08601
29
alternative splicing are expected to occur. Allelic
variants of this sequence can be cloned by probing cDNA or
genomic libraries from different individuals according to
standard procedures. Allelic variants of the DNA
sequence shown in SEQ ID N0:2, including those containing
silent mutations and those in which mutations result in
amino acid sequence changes, are within the scope of the
present invention, as are proteins which are allelic
variants of SEQ ID N0:2. cDNAs generated from
alternatively spliced mRNAs, which retain the properties
of the ZTMPO-1 polypeptide are included within the scope
of the present invention, as are polypeptides encoded by
such cDNAs and mRNAs. Allelic variants and splice variants
of these sequences can be cloned by probing cDNA or
genomic libraries from different individuals or tissues
according to standard procedures known in the art.
The present invention also provides isolated
ZTMPO-1 polypeptides that are substantially homologous to
the polypeptides of SEQ ID N0:2 and their orthologs. The
term "substantially homologous" is used herein to denote
polypeptides having 50%, preferably 60%, more preferably
at least 80%, sequence identity to the sequences shown in
SEQ ID N0:2 or their orthologs. Such polypeptides will
more preferably be at least 90% identical, and most
preferably 95a or more identical to SEQ ID N0:2 or its
orthologs). Percent sequence identity is determined by
conventional methods. See, for example, Altschul et al.,
Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and
Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9, 1992. The
present invention further includes nucleic acid molecules
that encode such polypeptides. Methods for determining
percent identity are described below.
Briefly, two amino acid sequences are aligned
to optimize the alignment scores using a gap opening
penalty of 10, a gap extension penalty of 1, and the
"blosum 62" scoring matrix of Henikoff and Henikoff
(ibid.) as shown in Table 3 (amino acids are indicated by
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
the standard one-letter codes). The percent identity is
then calculated as:
Total number of identical matches
x 100
5 (length of the longer sequence plus the
number of gaps introduced into the longer
sequence in order to align the two sequences]
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
31
i
H N M
i
111N N O
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r1M N N
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l0 d~N N r-IM rl
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CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
32
Those skilled in the art appreciate that there
are many established algorithms available to align two
amino acid sequences. The "FASTA" similarity search
algorithm of Pearson and Lipman is a suitable protein
alignment method for examining the level of identity
shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative variant ZTMPO-1. The
FASTA algorithm is described by Pearson and Lipman, Proc.
Nat. Acad. Sci. USA 85:2444, 1988, and by Pearson, Meth.
Enzymol. 183:63, 1990.
Briefly, FASTA first characterizes sequence
similarity by identifying regions shared by the query
sequence (e. g., SEQ ID N0:2) and a test sequence that have
either the highest density of identities (if the ktup
variable is 1) or pairs of identities (if ktup=2), without
considering conservative amino acid substitutions,
insertions, or deletions. The ten regions with the
highest density of identities are then re-scored by
comparing the similarity of all paired amino acids using
an amino acid substitution matrix, and the ends of the
regions are "trimmed" to include only those residues that
contribute to the highest score. If there are several
regions with scores greater than the "cutoff" value
(calculated by a predetermined formula based upon the
length of the sequence and the ktup value), then the
trimmed initial regions are examined to determine whether
the regions can be joined to form an approximate alignment
with gaps. Finally, the highest scoring regions of the
two amino acid sequences are aligned using a modification
of the Needleman-Wunsch-Sellers algorithm (Needleman and
Wunsch, J. Mol. Biol. 48:444, 1970; Sellers, SIAM J. Appl.
Math. 26:787, 1974), which allows for amino acid
insertions and deletions. Illustrative parameters for
FASTA analysis are: ktup=1, gap opening penalty=10, gap
extension penalty=1, and substitution matrix=BLOSUM62.
These parameters can be introduced into a FASTA program by
modifying the scoring matrix file ("SMATRIX"), as
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33
explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63,
1990.
FASTA can also be used to determine the sequence
identity of nucleic acid molecules using a ratio as
disclosed above. For nucleotide sequence comparisons, the
ktup value can range between one to six, preferably from
four to six.
Substantially homologous proteins and
polypeptides are characterized as having one or more amino
acid substitutions, deletions or additions. These changes
are preferably of a minor nature, that is conservative
amino acid substitutions and other substitutions that do
not significantly affect the folding or activity of the
protein or polypeptide; small deletions, typically of one
to about 30 amino acids; and small amino- or carboxyl-
terminal extensions, such as an amino-terminal methionine
residue, a small linker peptide of up to about 20-25
residues, or an affinity tag. Polypeptides comprising
affinity tags can further comprise a proteolytic cleavage
site between the zsig37 polypeptide and the affinity tag.
Preferred such sites include thrombin cleavage sites and
factor Xa cleavage sites.
The present invention includes nucleic acid
molecules that encode a polypeptide having one or more
"conservative amino acid substitutions," compared with the
amino acid sequence of SEQ ID N0:2. Conservative amino
acid substitutions can be based upon the chemical
properties of the amino acids. That is, variants can be
obtained that contain one or more amino acid substitutions
of SEQ ID N0:2, in which an alkyl amino acid is
substituted for an alkyl amino acid in a ZTMPO-1 amino
acid sequence, an aromatic amino acid is substituted for
an aromatic amino acid in a ZTMPO-1 amino acid sequence, a
sulfur-containing amino acid is substituted for a sulfur-
containing amino acid in a ZTMPO-1 amino acid sequence, a
hydroxy-containing amino acid is substituted for a
hydroxy-containing amino acid in a ZTMPO-1 amino acid
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34
sequence, an acidic amino acid is substituted for an
acidic amino acid in a ZTMPO-1 amino acid sequence, a
basic amino acid is substituted for a basic amino acid in
a ZTMPO-1 amino acid sequence, or a dibasic monocarboxylic
amino acid is substituted for a dibasic monocarboxylic
amino acid in a ZTMPO-1 amino acid sequence.
Among the common amino acids, for example, a
"conservative amino acid substitution" is illustrated by a
substitution among amino acids within each of the
following groups: (1) glycine, alanine, valine, leucine,
and isoleucine, (2) phenylalanine, tyrosine, and
tryptophan, (3) serine and threonine, (4) aspartate and
glutamate, (5) glutamine and asparagine, and (6) lysine,
arginine and histidine. Other conservative amino acid
substitutions are provided in Table 4.
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Table 4
Conservative amino acid substitutions
Basic: arginine
lysine
histidine
Acidic: glutamic acid
aspartic acid
Polar: glutamine
asparagine
10 Hydrophobic: leucine
isoleucine
valine
Aromatic: phenylalanine
tryptophan
15 tyrosine
Small: glycine
alanine
serine
threonine
20 methionine
The BLOSUM62 table is an amino acid substitution
matrix derived from about 2,000 local multiple alignments
of protein sequence segments, representing highly
conserved regions of more than 500 groups of related
25 proteins (Henikoff and Henikoff, Proc. Natl. Acad. Sci
USA 89:10915, 1992). Accordingly, the BLOSUM62
substitution frequencies can be used to define
conservative amino acid substitutions that may be
introduced into the amino acid sequences of the present
30 invention. Although it is possible to design amino acid
substitutions based solely upon chemical properties (as
discussed above), the language "conservative amino acid
substitution" preferably refers to a substitution
represented by a BLOSUM62 value of greater than -1. For
35 example, an amino acid substitution is conservative if the
substitution is characterized by a BLOSUM62 value of 0,
1, 2, or 3. According to this system, preferred
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36
conservative amino acid substitutions are characterized by
a BLOSUM62 value of at least 1 (e. g., 1, 2 or 3), while
more preferred conservative amino acid substitutions are
characterized by a BLOSUM62 value of at least 2 (e.g., 2
or 3 ) .
Conservative amino acid changes in a ZTMPO-1
gene can be introduced by substituting nucleotides for the
nucleotides recited in SEQ ID NO:1. Such "conservative
amino acid" variants can be obtained, for example, by
oligonucleotide-directed mutagenesis, linker-scanning
mutagenesis, mutagenesis using the polymerase chain
reaction, and the like (see Ausubel (1995) at pages 8-10
to 8-22; and McPherson (ed.), Directed MutaQenesis: A
Practical Approach (IRL Press 1991)). The ability of such
variants to promote proliferation and cardiac functions as
will as other properties of the wild-type protein can be
determined using a standard methods, such as the assays
described herein. Alternatively, a variant ZTMPO-1
polypeptide can be identified by the ability to
specifically bind anti-ZTMPO-1 antibodies.
The proteins of the present invention can also
comprise non-naturally occurring amino acid residues.
Non-naturally occurring amino acids include, without
limitation, traps-3-methylproline, 2,4-methanoproline,
cis-4-hydroxyproline, traps-4-hydroxyproline, N-methyl-
glycine, allo-threonine, methylthreonine, hydroxy-
ethylcysteine, hydroxyethylhomocysteine, nitro-glutamine,
homoglutamine, pipecolic acid, thiazolidine carboxylic
acid, dehydroproline, 3- and 4-methylproline, 3,3-
dimethylproline, tert-leucine, norvaline, 2-azaphenyl-
alanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-
fluorophenylalanine. Several methods are known in the art
for incorporating non-naturally occurring amino acid
residues into proteins. For example, an in vitro system
can be employed wherein nonsense mutations are suppressed
using chemically aminoacylated suppressor tRNAs. Methods
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37
for synthesizing amino acids and aminoacylating tRNA are
known in the art. Transcription and translation of
plasmids containing nonsense mutations is carried out in a
cell-free system comprising an E. coli S30 extract and
commercially available enzymes and other reagents.
Proteins are purified by chromatography. See, for
example, Robertson et al., J. Am. Chem. Soc. 113:2722,
1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung
et al., Science 259:806-9, 1993; and Chung et al., Proc.
Natl. Acad. Sci. USA 90:10145-9, 1993). In a second
method, translation is carried out in Xenopus oocytes by
microinjection of mutated mRNA and chemically
aminoacylated suppressor tRNAs (Turcatti et al., J. Biol.
Chem. 271:19991-8, 1996). Within a third method, E. coli
cells are cultured in the absence of a natural amino acid
that is to be replaced (e.g., phenylalanine) and in the
presence of the desired non-naturally occurring amino
acids) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-
azaphenylalanine, or 4-fluorophenylalanine). The non-
naturally occurring amino acid is incorporated into the
protein in place of its natural counterpart. See, Koide
et al., Biochem. 33:7470-6, 1994. Naturally occurring
amino acid residues can be converted to non-naturally
occurring species by in vitro chemical modification.
Chemical modification can be combined with site-directed
mutagenesis to further expand the range of substitutions
(Wynn and Richards, Protein Sci. 2:395-403, 1993).
A limited number of non-conservative amino
acids, amino acids that are not encoded by the genetic
code, non-naturally occurring amino~acids, and unnatural
amino acids may be substituted for ZTMPO-1 amino acid
residues.
Essential amino acids in the polypeptides of the
present invention can be identified according to
procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham
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38
and Wells, Science 244: 1081-5, 1989; Bass et al., Proc.
Natl. Acad. Sci. USA 88:4498-502, 1991). In the latter
technique, single alanine mutations are introduced at
every residue in the molecule, and the resultant mutant
molecules are tested for biological activity as disclosed
below to identify amino acid residues that are critical to
the activity of the molecule. See also, Hilton et al. , ,T.
Biol. Chem. 271:4699-708, 1996. Sites of ligand-receptor
interaction can also be determined by physical analysis of
structure, as determined by such techniques as nuclear
magnetic resonance, crystallography, electron diffraction
or photoaffinity labeling, in conjunction with mutation of
putative contact site amino acids. See, for example, de
Vos et al., Science 255:306-12, 1992; Smith et al., J.
Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett.
309:59-64, 1992. The identities of essential amino acids
can also be inferred from analysis of homologies with
related nuclear membrane bound proteins.
Multiple amino acid substitutions can be made
and tested using known methods of mutagenesis and
screening, such as those disclosed by Reidhaar-Olson and
Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc.
Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these
authors disclose methods for simultaneously randomizing
two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the
mutagenized polypeptides to determine the spectrum of
allowable substitutions at each position. Other methods
that can be used include phage display (e.g., Lowman et
al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent
No. 5,223,409; Huse, WIPO Publication WO 92/06204) and
region-directed mutagenesis (Derbyshire et al., Gene
46:145, 1986; Ner et al., DNA 7:127, 1988).
Variants of the disclosed ZTMPO-1 DNA and
polypeptide sequences can be generated through DNA
shuffling as disclosed by Stemmer, Nature 370:389-91, 1994
and Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994.
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39
Briefly, variant DNAs are generated by in vitro homologous
recombination by random fragmentation of a parent DNA
followed by reassembly using PCR, resulting in randomly
introduced point mutations. This technique can be
modified by using a family of parent DNAs, such as allelic
variants or genes from different species, to introduce
additional variability into the process. Selection or
screening for the desired activity, followed by additional
iterations of mutagenesis and assay provides for rapid
"evolution" of sequences by selecting for desirable
mutations while simultaneously selecting against
detrimental changes.
Mutagenesis methods as disclosed herein can be
combined with high-throughput, automated screening methods
to detect activity of cloned, mutagenized polypeptides in
host cells. Preferred assays in this regard include cell
proliferation assays and biosensor-based ligand-binding
assays, which are described below. Mutagenized DNA
molecules that encode active polypeptides can be recovered
from the host cells and rapidly sequenced using modern
equipment. These methods allow the rapid determination of
the importance of individual amino acid residues in a
polypeptide of interest, and can be applied to
polypeptides of unknown structure.
Using the methods discussed herein, one of
ordinary skill in the art can identify and/or prepare a
variety of polypeptide fragments or variants of SEQ ID
N0:2 or that retain the receptor binding properties of the
wild-type ZTMPO-1 protein. Such polypeptides may also
include additional polypeptide segments as generally
disclosed herein.
For any ZTMPO-1 polypeptide, including variants
and fusion proteins, one of ordinary skill in the art can
readily generate a fully degenerate polynucleotide
sequence encoding that variant using the information set
forth in Tables 1 and 2 above.
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As used herein a fusion protein consists
essentially of a first portion and a second portion joined
by a peptide bond. In one embodiment the first portion
consists of a polypeptide comprising a sequence of amino
5 acid residues that is at least 80% identical in amino acid
sequence to residues 1 through 876 of SEQ ID N0:2 and the
second portion is any other polypetide. The other
polypeptide may be alternative or additional domains from
other members of the thymopoietin or emerin family, a
10 signal peptide to facilitate secretion of the fusion
protein, affinity tags, Ig domains or the like.
The ZTMPO-1 polypeptides of the present
invention, including full-length polypeptides,
biologically active fragments, and fusion polypeptides,
15 can be produced in genetically engineered host cells
according to conventional techniques. Suitable host cells
are those cell types that can be transformed or
transfected with exogenous DNA and grown in culture, and
include bacteria, fungal cells, and cultured higher
20 eukaryotic cells. Eukaryotic cells, particularly cultured
cells of multicellular organisms, are preferred.
Techniques for manipulating cloned DNA molecules and
introducing exogenous DNA into a variety of host cells are
disclosed by Sambrook et al., Molecular Cloning: A
25 Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, 1989, and Ausubel et al.,
eds., Current Protocols in Molecular Bioloav, John Wiley
and Sons, Inc., NY, 1987.
In general, a DNA sequence encoding a ZTMPO-1
30 polypeptide is operably linked to other genetic elements
required for its expression, generally including a
transcription promoter and terminator, within an
expression vector. The vector will also commonly contain
one or more selectable markers and one or more origins of
35 replication, although those skilled in the art will
recognize that within certain systems selectable markers
may be provided on separate vectors, and replication of
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41
the exogenous DNA may be provided by integration into the
host cell genome. Selection of promoters, terminators,
selectable markers, vectors and other elements is a matter
of routine design within the level of ordinary skill in
the art. Many such elements are described in the
literature and are available through commercial suppliers.
To direct a ZTMPO-1 polypeptide into the
secretory pathway of a host cell, a secretory signal
sequence (also known as a leader sequence, signal
sequence, prepro sequence or pre sequence) is provided in
the expression vector. The secretory signal sequence may
be derived from another secreted protein (e.g., t-PA) or
synthesized de novo. The secretory signal sequence is
operably linked to the ZTMPO-1 DNA sequence, i.e., the two
sequences are joined in the correct reading frame and
positioned to direct the newly synthesized polypeptide
into the secretory pathway of the host cell. Secretory
signal sequences are commonly positioned 5' to the DNA
sequence encoding the polypeptide of interest, although
certain secretory signal sequences may be positioned
elsewhere in the DNA sequence of interest (see, e.g.,
Welch et al., U.S. Patent No. 5,037,743; Holland et al.,
U.S. Patent No. 5,143,830).
Cultured mammalian cells are suitable hosts
within the present invention. Methods for introducing
exogenous DNA into mammalian host cells include calcium
phosphate-mediated transfection (Wigler et al., Cell
14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics
7:603, 1981: Graham and Van der Eb, ViroloQV 52:456,
1973), electroporation (Neumann et al., EMBO J. 1:841-5,
1982), DEAE-dextran mediated transfection (Ausubel et al.,
ibid.), and liposome-mediated transfection (Hawley-Nelson
et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,
1993, and viral vectors (Miller and Rosman, BioTechniaues
7:980-90, 1989; Wang and Finer, Nature Med. 2:714-6,
1996). The production of recombinant polypeptides in
cultured mammalian cells is disclosed, for example, by
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42
Levinson et al., U.S. Patent No. 4,713,339; Hagen et al.,
U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent
No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134.
Suitable cultured mammalian cells include the COS-1 (ATCC
No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No.
CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL
1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and
Chinese hamster ovary (e. g. CHO-K1; ATCC No. CCL 61) cell
lines. Additional suitable cell lines are known in the
art and available from public depositories such as the
American Type Culture Collection, Rockville, Maryland. In
general, strong transcription promoters are preferred,
such as promoters from SV-40 or cytomegalovirus. See,
e.g., U.S. Patent No. 4,956,288. Other suitable promoters
include those from metallothionein genes (U. S. Patent Nos.
4,579,821 and 4,601,978) and the adenovirus major late
promoter.
Drug selection is generally used to select for
cultured mammalian cells into which foreign DNA has been
inserted. Such cells are commonly referred to as
"transfectants". Cells that have been cultured in the
presence of the selective agent and are able to pass the
gene of interest to their progeny are referred to as
"stable transfectants." A preferred selectable marker is
a gene encoding resistance to the antibiotic neomycin.
Selection is carried out in the presence of a neomycin-
type drug, such as G-418 or the like. Selection systems
can also be used to increase the expression level of the
gene of interest, a process referred to as
"amplification." Amplification is carried out by
culturing transfectants in the presence of a low level of
the selective agent and then increasing the amount of
selective agent to select for cells that produce high
levels of the products of the introduced genes. A
preferred amplifiable selectable marker is dihydrofolate
reductase, which confers resistance to methotrexate.
Other drug resistance genes (e. g. hygromycin resistance,
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43
multi-drug, resistance, puromycin acetyltransferase) can
also be used. Alternative markers that introduce an
altered phenotype, such as green fluorescent protein, or
cell surface proteins such as CD4, CDB, Class I MHC,
placental alkaline phosphatase may be used to sort
transfected cells from untrarisfected cells by such means
as FACS sorting or magnetic bead separation technology.
Other higher eukaryotic cells can also be used
as hosts, including plant cells, insect cells and avian
cells. The use of Agrobacterium rhizogenes as a vector
for expressing genes in plant cells has been reviewed by
Sinkar et al., J. Biosci. (Banaalore) 11:47-58, 1987.
Transformation of insect cells and production of foreign
polypeptides therein is disclosed by Guarino et al., U.S.
Patent No. 5,162,222 and WIPO publication WO 94/06463.
Insect cells can be infected with recombinant baculovirus
vectors, which are commonly derived from Autographa
californica multiple nuclear polyhedrosis virus (AcMNPV).
DNA encoding the polypeptide of interest is inserted into
the viral genome in place of the polyhedrin gene coding
sequence by homologous recombination in cells infected
with intact, wild-type AcMNPV and transfected with a
transfer vector comprising the cloned gene operably linked
to polyhedrin gene promoter, terminator, and flanking
sequences. The resulting recombinant virus is used to
infect host cells, typically a cell line derived from the
fall armyworm, Spodoptera frugiperda. See, in general,
Glick and Pasternak, Molecular Biotechnolocry: Principles
and Applications of Recombinant DNA, ASM Press,
Washington, D.C., 1994.
Fungal cells, including yeast cells, can also be
used within the present invention. Yeast species of
particular interest in this regard include Saccharomyces
cerevisiae, Pichia pastoris, and Pichia methanolica.
Methods for transforming S. cerevisiae cells with
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44
exogenous DNA and producing recombinant polypeptides
therefrom are disclosed by, for example, Kawasaki, U.S.
Patent No. 4,599,311; Kawasaki et al., U.S. Patent No.
4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al.,
U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent
No. 4,845,075. Transformed cells are selected by
phenotype determined by the selectable marker, commonly
drug resistance or the ability to grow in the absence of a
particular nutrient (e. g., leucine). A preferred vector
system for use in Saccharomyces cerevisiae is the POTI
vector system disclosed by Kawasaki et al. (U. S. Patent
No. 4,931,373), which allows transformed cells to be
selected by growth in glucose-containing media. Suitable
promoters and terminators for use in yeast include those
from glycolytic enzyme genes (see, e.g., Kawasaki, U.S.
Patent No. 4,599,311; Kingsman et al., U.S. Patent No.
4,615,974; and Bitter, U.S. Patent No. 4,977,092) and
alcohol dehydrogenase genes. See also U.S. Patents Nos.
4,990,446; 5,063,154; 5,139,936 and 4,661,454.
Transformation systems for other yeasts, including
Hansenula polymorpha, Schizosaccharornyces pombe,
Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago
maydis, Pichia pastoris, Pichia methanolica, Pichia
guillermondii and Candida maltosa are known in the art.
See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279.
Aspergillus cells may be utilized according to the methods
of McKnight et al., U.S. Patent No. 4,935,349. Methods
for transforming Acremonium chrysogenum are disclosed by
Sumino et al., U.S. Patent No. 5,162,228. Methods for
transforming Neurospora are disclosed by Lambowitz, U.S.
Patent No. 4,486,533.
The use of Pichia methanolica as host for the
production of recombinant proteins is disclosed in WIPO
Publications WO 9717450 and W09717451. DNA molecules for
use in transforming P. methanolica will commonly be
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prepared as double-stranded, circular plasmids, which are
preferably linearized prior to transformation. For
polypeptide production in P. methanolica, it is preferred
that the promoter and terminator in the plasmid be that of
5 a P. methanolica gene, such as a P. methanolica alcohol
utilization gene (AUGI or AUG2) . Other useful nrnmntP,-
include those of the dihydroxyacetone synthase (DHAS),
formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host
10 chromosome, it is preferred to have the entire expression
segment of -the plasmid flanked at both ends by host DNA
sequences. A preferred selectable marker for use in
Pichia methanolica is a P. rnethanolica ADE2 gene, which
encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;
15 EC 4 . 1 .1. 21 ) , which allows ade2 host cells to grow in the
absence of adenine. For large-scale, industrial processes
where it is desirable to minimize the use of methanol, it
is preferred to use host cells in which both methanol
utilization genes (AUGI and AUG2) are deleted. For
20 production of secreted proteins, host cells deficient in
vacuolar protease genes (PEP4 and PRBI) are preferred.
Electroporation is used to facilitate the introduction of
a plasmid containing DNA encoding a polypeptide of
interest into P. methanolica cells. It is preferred to
25 transform P. methanolica cells by electroporation using
an exponentially decaying, pulsed electric field having a
field strength of from 2.5 to 4.5 kV/cm, preferably about
3.75 kV/cm, and a time constant (t) of from 1 to 40
milliseconds, most preferably about 20 milliseconds.
30 Prokaryotic host cells, including strains of the
bacteria Escherichia coli, Bacillus and other genera are
also useful as host cells within the present invention.
Techniques for transforming these hosts and expressing
foreign DNA sequences cloned therein are well known in the
35 art (see, e.g., Sambrook et al., ibid.). When expressing
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46
a ZTMPO-1 polypeptide in bacteria such as E. coli, the
polypeptide may be retained in the cytoplasm, typically as
insoluble granules, or may be directed to the periplasmic
space by a bacterial secretion sequence. In the former
case, the cells are lysed, and the granules are recovered
and denatured using, for example, guanidine isothiocyanate
or urea. The denatured polypeptide can then be refolded
and dimerized by diluting the denaturant, such as by
dialysis against a solution of urea and a combination of
reduced and oxidized glutathione, followed by dialysis
against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic
space in a soluble and functional form by disrupting the
cells (by, for example, sonication or osmotic shock) to
I5 release the contents of the periplasmic space and
recovering the protein, thereby obviating the need for
denaturation and refolding.
Transformed or transfected host cells are
cultured according to conventional procedures in a culture
medium containing nutrients and other components required
for the growth of the chosen host cells. A variety of
suitable media, including defined media and complex media,
are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins
and minerals. Media may also contain such components as
growth factors or serum, as required. The growth medium
will generally select for cells containing the exogenously
added DNA by, for example, drug selection or deficiency in
an essential nutrient which is complemented by the
selectable marker carried on the expression vector or co-
transfected into the host cell. P. methanolica cells are
cultured in a medium comprising adequate sources of
carbon, nitrogen and trace nutrients at a temperature of
about 25°C to 35°C. Liquid cultures are provided with
sufficient aeration by conventional means, such as shaking
of small flasks or sparging of fermentors. A preferred
culture medium for P. methanolica is YEPD (2% D-glucose,
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47
2o BactoTM Peptone (Difco Laboratories, Detroit, MI), to
BactoT"' yeast extract (Difco Laboratories), 0.004% adenine
and 0.0060 L-leucine).
It is preferred to purify the polypeptides of
the present invention to >_80% purity, more preferably to
>_90o purity, even more preferably ?95% purity, and
particularly preferred is a pharmaceutically pure state,
that is greater than 99.9% pure with respect to
contaminating macromolecules, particularly other proteins
and nucleic acids, and free of infectious and pyrogenic
agents. Preferably, a purified polypeptide is
substantially free of other polypeptides, particularly
other polypeptides of animal origin.
Expressed recombinant ZTMPO-1 polypeptides (or
fusion or chimeric ZTMPO-1 polypeptides) can be purified
using fractionation and/or conventional purification
methods and media. Ammonium sulfate precipitation and
acid or chaotrope extraction may be used for fractionation
of samples. Exemplary purification steps may include
hydroxyapatite, size exclusion, FPLC and reverse-phase
high performance liquid chromatography. Suitable
chromatographic media include derivatized dextrans,
agarose, cellulose, polyacrylamide, specialty silicas, and
the like. PEI, DEAE, QAE and Q derivatives are preferred.
Exemplary chromatographic media include those media
derivatized with phenyl, butyl, or octyl groups, such as
Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso
Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia)
and the like; or polyacrylic resins, such as Amberchrom CG
71 (Toso Haas) and the like. Suitable solid supports
include glass beads, silica-based resins, cellulosic
resins, agarose beads, cross-linked agarose beads,
polystyrene beads, cross-linked polyacrylamide resins and
the like that are insoluble under the conditions in which
they are to be used. These supports may be modified with
reactive groups that allow attachment of proteins by amino
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48
groups, carboxyl groups, sulfhydryl groups, hydroxyl
groups and/or carbohydrate moieties. Examples of coupling
chemistries include cyanogen bromide activation, N-
hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl
and amino derivatives for carbodiimide coupling
chemistries. These and other solid media are well known
and widely used in the art, and are available from
commercial suppliers. Methods for binding receptor
polypeptides to support media are well known in the art.
Selection of a particular method is a matter of routine
design and is determined in part by the properties of the
chosen support. See, for example, Affinitv
Chromatoaraphv: Principles & Methods, Pharmacia LKB
Biotechnology, Uppsala, Sweden, 1988.
The polypeptides of the present invention can be
isolated by exploitation of their binding properties. For
example, immobilized metal ion adsorption (IMAC)
chromatography can be used to purify histidine-rich
proteins, including those comprising polyhistidine tags.
Briefly, a gel is first charged with divalent metal ions
to form a chelate (Sulkowski, Trends in Biochem. 3:1-7,
1985). Histidine-rich proteins will be adsorbed to this
matrix with differing affinities, depending upon the metal
ion used, and will be eluted by competitive elution,
lowering the pH, or use of strong chelating agents. Other
methods of purification include purification of
glycosylated proteins by lectin affinity chromatography
and ion exchange chromatography (Methods in Enzymol., Vol.
182, "Guide to Protein Purification", M. Deutscher, (ed.),
Acad. Press, San Diego, 1990, pp.529-39). Within
additional embodiments of the invention, a fusion of the
polypeptide of interest and an affinity tag (e.g., Glu-Glu
tag) may be constructed to facilitate purification.
ZTMPO-1 polypeptides or fragments thereof may
also be prepared through chemical synthesis according to
methods known in the art, including exclusive solid phase
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49
synthesis, partial solid phase methods, fragment
condensation or classical solution synthesis. See, for
example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963.
Using methods known in the art, ZTMPO-1
S polypeptides may be prepared as monomers or multimers;
glycosylated or non-glycosylated; pegylated or non
pegylated; and may or may not include an initial
methionine amino acid residue.
An in vivo approach for assaying proteins of the
present invention involves viral delivery systems.
Exemplary viruses for this purpose include adenovirus,
herpesvirus, vaccinia virus and adeno-associated virus
(AAV). Adenovirus, a double-stranded DNA virus, is
currently the best studied gene transfer vector for
delivery of heterologous nucleic acid (for a review, see
Becker et al., Meth. Cell Biol. 43:161-89, 1994; and
Douglas and Curiel, Science & Medicine 4:44-53). The
adenovirus system offers several advantages: adenovirus
can (i) accommodate relatively large DNA inserts; (ii) be
grown to high-titer; (iii) infect a broad range of
mammalian cell types; and (iv) be used with a large number
of available vectors containing different promoters.
Also, because adenoviruses are stable in the bloodstream,
they can be administered by intravenous injection.
By deleting portions of the adenovirus genome,
larger inserts (up to 7 kb) of heterologous DNA can be
accommodated. These inserts may be incorporated into the
viral DNA by direct ligation or by homologous
recombination with a co-transfected plasmid. In an
exemplary system, the essential E1 gene has been deleted
from the viral vector, and the virus will not replicate
unless the E1 gene is provided by the host cell (the human
293 cell line is exemplary). When intravenously
administered to intact animals, adenovirus primarily
targets the liver. If the adenoviral delivery system has
an E1 gene deletion, the virus cannot replicate in the
host cells. However, the host's tissue (e. g., liver) will
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express and process (and, if a secretory signal sequence
is present, secrete) the heterologous protein. Secreted
proteins will enter the circulation in the highly
vascularized liver, and effects on the infected animal can
5 be determined.
The adenovirus system can also be used for
protein production in vitro. By culturing adenovirus-
infected non-293 cells under conditions where the cells
are not rapidly dividing, the cells can produce proteins
10 for extended periods of time. For instance, BHK cells are
grown to confluence in cell factories, then exposed to the
adenoviral vector encoding the secreted protein of
interest. The cells are then grown under serum-free
conditions, which allows infected cells to survive for
15 several weeks without significant cell division.
Alternatively, adenovirus vector infected 2935 cells can
be grown in suspension culture at relatively high cell
density to produce significant amounts of protein (see
Gamier et al., Cytotechnol. 15:145-55, 1994). With
20 either protocol, an expressed, secreted heterologous
protein can be repeatedly isolated from the cell culture
supernatant. Within the infected 293S cell production
protocol, non-secreted proteins may also be effectively
obtained.
25 The broad tissue distribution of ZTMPO-1
suggests it may play a critical role in biological
processes of an organism and as such altered expression of
ZTMPO-1 is likely involved in numerous pathologies
associated with genetic and other human disease states, in
30 particular those related to immunological, reproductive,
cardiac and muscle pathologies, such as diabetes, muscular
dystrophys, hematopoietic disorders, immune disorders,
leukemias, hypertension and cardiac disorders and
diseases. ZTMPO-1 polypeptides, agonists and antagonists
35 have potential in both in vitro and in vivo applications.
ZTMPO-1 is expressed ubiquitously, many of those
tissues are characterized by a high rate of cellular
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51
proliferation. ZTMPO-1 polypeptides would find use as
regulators of cellular proliferation and/or
differentiation. Proliferation and differentiation can be
measured using cultured cells or in vivo by administering
molecules of the present invention to the appropriate
animal model. Suitable cultured cells, include but are
not limited to, testicular, muscle, lymphatic and tumor
cell lines which are all readily available to one skilled
in the art from such sources as American Type Culture
Collection, Rockville, MD. In particular, proliferation
can be measured using cultured cardiac cells or in vivo by
administering molecules of the present invention to the
appropriate animal model. Generally, proliferative
effects are seen as an increase in cell number, and may
include inhibition of apoptosis as well as stimulation of
mitogenesis. Cultured cells for use in these assays
include cardiac fibroblasts, cardiac myocytes, skeletal
myocytes, and human umbilical vein endothelial cells from
primary cultures. Suitable established cell lines
include: NIH 3T3 fibroblasts (ATCC No. CRL-1658), CHH-1
chum heart cells (ATCC No. CRL-1680), H9c2 rat heart
myoblasts (ATCC No. CRL-1446), Shionogi mammary carcinoma
cells (Tanaka et al., Proc. Natl. Acad. Sci. 89:8928-32,
1992), and LNCap.FGC adenocarcinoma cells (ATCC No. CRL-
1740). Cultured testicular cells include dolphin DBl.Tes
cells (ATCC No. CRL-6258); mouse GC-1 spg cells (ATCC No.
CRL-2053); TM3 cells (ATCC No. CRL-1714); TM4 cells (ATCC
No. CRL-1715); and pig ST cells (ATCC No. CRL-1746).
Mouse skeletal muscle (ATCC No. CRL-2174), human muscle
(ATCC No. CRL-7522) and Raji, (Burkitt's human lymphoma,
ATCC No. CCL86), Ramos (Burkitt's lymphoma cell line, ATCC
No. CRL-1596), Daudi (Burkitt's human lymphoma, ATCC No.
CCL213) and RPMI 1788 (a B lymphocyte cell line, CCL-156)
all available from American Type Culture Collection, 10801
University Boulevard, Manassas, VA 20110-2209. Cultured
Assays measuring cell proliferation are well known in the
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52
art. For example, assays measuring proliferation include
chemosensitivity to neutral red dye (Cavanaugh et al.,
Investictational New Drugs 8:347-54, 1990), incorporation
of radiolabelled nucleotides (Cook et al., Analytical
Biochem. 179:1-7, 1989), incorporation of 5-bromo-2'-
deoxyuridine (BrdU) in the DNA of proliferating cells
(Porstmann et al., J. Immunol. Methods 82:169-79, 1985),
and use of tetrazolium salts (Mosmann, J. Immunol. Methods
65:55-63, 1983; Alley et al., Cancer Res. 48:589-601,
1988; Marshall et al., Growth Reg. 5:69-84, 1995; and
Scudiero et al., Cancer Res. 48:4827-33, 1988).
Additional methods can be found in the art, for example,
Current Protocols in Molecular Biolocty, John Wiley and
Sons, Inc., NY, 1997.
Assays measuring differentiation include, for
example, measuring cell-surface markers associated with
stage-specific expression of a tissue, enzymatic activity,
functional activity or morphological changes (Watt, FASEB,
5:281-4, 1991; Francis, Differentiation 57:63-75, 1994;
Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-71,
1989). Bioassays and ELISAs are available to measure
cellular response to ZTMPO-1, in particular are those
which measure changes in cytokine production as a measure
of cellular response (see for example, Current Protocols
in Immunolocrv ed. John E. Coligan et al., NIH, 1996).
In vivo assays are available for evaluating
cardiac neogenesis or hyperplasia include treating
neonatal and mature rats with the molecules of the present
invention. The animals' cardiac function is measured as
heart rate, blood pressure, and cardiac output to
determine left ventricular function. Post-mortem methods
for assessing cardiac decline or improvement include:
increased or decreased cardiac weight, nuclei/cytoplasmic
volume, and staining of cardiac histology sections to
determine proliferating cell nuclear antigen (PCNA) vs.
cytoplasmic actin levels (Quaini et al., Circulation Res.
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53
75:1050-63, 1994 and Reiss et al., Proc. Natl. Acad Sci
93:8630-5, 1996.).
Cardiac defects related to conduction have been
reported in patients having a deleted emerin gene (Emery,
J. Med. Genet. 2-66:637-41, 1989). The resulting cardiac
conduction defect is life threatening in these patients.
Defects in the intrinsic conduction system can cause
irregularities in the heart rhythm, such as arrhythmia and
fibrillation. Tissue distribution and sequence
similarities between emerin and ZTMPO-1 suggest that
ZTMPO-1 may be involved in re-polarization of cardiac cell
membranes. Localization of emerin to the desmosomes and
fasciae adherentes suggests that association with the
connection between epithelial cells accounts for the
cardiac conduction defect when the gene is absent.
ZTMPO-1 polypeptides and antagonists may influence cell-
cell communication, either independently, or in
conjunction with other proteins, such as emerin, and may
regulate messages between cell membranes. To verify the
presence of this capability in ZTMPO-1 polypeptides,
agonists or antagonists of the present invention, such
ZTMPO-1 polypeptides, agonists or antagonists are
evaluated with respect to their ability to modulate
cardiac conductance according to procedures known in the
art. If desired, ZTMPO-1 polypeptide performance in this
regard can be compared to emerin and may be evaluated in
combination with emerin to identify synergistic effects.
With respect to cardiac conductance, a resulting increase
or decrease is measured by assessing voltage-dependent
conductance, sodium or calcium ion flux in an appropriate
assay system known in the art. Changes in the voltage
conductance or in indicator substrates reflect the
activities of ZTMPO-1 polypeptides on enhancing or
inhibition cardiac conductance relative to a control not
subjected to treatment. An electrocardiograph is used to
monitor the electrical currents generated and transmitted
through the heart. Changes in the electrocardiogram (ECG)
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54
tracing (wave pattern and/or timing) would indicate an
alteration in the heart's conduction system. Therefore a
return to a normal ECG pattern following ZTMPO-1
administration would indicate a re-establishment of a
regular heart rhythm.
The invention also provides isolated and
purified ZTMPO-1 polynucleotide probes or primers. Such
polynucleotide probes can be RNA or DNA. DNA can be
either cDNA or genomic DNA. Polynucleotide probes are
single or double-stranded DNA or RNA, generally synthetic
oligonucleotides, but may be generated from cloned cDNA or
genomic sequences and will generally comprise at least 16
nucleotides, more often from 17 nucleotides to 25 or more
nucleotides, sometimes 40 to 60 nucleotides, and in some
instances a substantial portion, domain or even the entire
ZTMPO-1 gene or cDNA. Probes and primers are generally
synthetic oligonucleotides, but may be generated from
cloned cDNA or genomic sequences or its complements.
Analytical probes will generally be at least 20
nucleotides in length, although somewhat shorter probes
(14-I7 nucleotides) can be used. PCR primers are at least
5 nucleotides in length, preferably 15 or more
nucleotides, more preferably 20-30 nucleotides. Short
polynucleotides can be used when a small region of the
gene is targeted for analysis. For gross analysis of
genes, a polynucleotide probe may comprise an entire exon
or more. Probes can be labeled to provide a detectable
signal, such as with an enzyme, biotin, a radionuclide,
fluorophore, chemiluminescer, paramagnetic particle and
the like, which are commercially available from many
sources, such as Molecular Probes, Inc., Eugene, OR, and
Amersham Corp., Arlington Heights, IL, using techniques
that are well known in the art. Preferred regions from
which to construct probes include regions of homology with
other thymopoietins and emerin as described herein, the
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ankyrin-like region, the calcium binding protein-like
region, the signal sequence, and the like. Techniques for
developing polynucleotide probes and hybridization
techniques are known in the art, see for example, Ausubel
5 et al., eds., Current Protocols in Molecular Biology, John
Wiley and Sons, Inc., NY, 1991.
ZTMPO-1 polypeptides may be used within
diagnostic systems to detect the presence of ZTMPO-1. The
information derived from such detection methods would
10 provide insight into the significance of ZTMPO-1
polypeptides in various diseases, and as a would serve as
diagnostic tools for diseases for which altered levels of
ZTMPO-1 are significant. Altered levels of ZTMPO-1
receptor polypeptides may be indicative of pathological
15 conditions including cancer, cardiac and autoimmune
disorders and infectious diseases.
In a basic assay, a single-stranded probe
molecule is incubated with RNA, isolated from a biological
sample, under conditions of temperature and ionic strength
20 that promote base pairing between the probe and target
ZTMPO-1 RNA species. After separating unbound probe from
hybridized molecules, the amount of hybrids is detected.
Well-established hybridization methods of RNA
detection include northern analysis and dot/slot blot
25 hybridization (see, for example, Ausubel ibid. and Wu et
al. (eds.), "Analysis of Gene Expression at the RNA
Level," in Methods in Gene Biotechnology, pages 225-239
(CRC Press, Inc. 1997)). Nucleic acid probes can be
detectably labeled with radioisotopes such as 32P or 355.
30 Alternatively, ZTMPO-1 RNA can be detected with a
nonradioactive hybridization method (see, for example,
Isaac (ed.), Protocols for Nucleic Acid Analysis by
Nonradioactive Probes, Humana Press, Inc., 1993).
Typically, nonradioactive detection is achieved by
35 enzymatic conversion of chromogenic or chemiluminescent
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56
substrates. Illustrative nonradioactive moieties include
biotin, fluorescein, and digoxigenin.
ZTMPO-1 oligonucleotide probes are also useful
for in vivo diagnosis. As an illustration, 18F-labeled
oligonucleotides can be administered to a subject and
visualized by positron emission tomography (Tavitian et
al., Nature Medicine 4:467, 1998).
Numerous diagnostic procedures take advantage of
the polymerase chain reaction (PCR) to increase
sensitivity of detection methods. Standard techniques for
performing PCR are well-known (see, generally, Mathew
(ed.), Protocols in Human Molecular Genetics (Humans
Press, Inc. 1991), White (ed.), PCR Protocols: Current
Methods and Applications {Humans Press, Inc. 1993), Cotter
(ed.), Molecular Diagnosis of Cancer (Humans Press, Inc.
1996), Hanausek and Walaszek (eds.), Tumor Marker
Protocols (Humans Press, Inc. 1998), Lo (ed.), Clinical
Applications of PCR (Humans Press, Inc. 1998), and Meltzer
(ed.), PCR in Bioanalysis (Humans Press, Inc. 1998)).
PCR primers can be designed to amplify a sequence encoding
a particular ZTMPO-1 domain or region of homology as
described herein.
One variation of PCR for diagnostic assays is
reverse transcriptase-PCR (RT-PCR). In the RT-PCR
technique, RNA is isolated from a biological sample,
reverse transcribed to cDNA, and the cDNA is incubated
with ZTMPO-1 primers (see, for example, Wu et al. (eds.),
"Rapid Isolation of Specific cDNAs or Genes by PCR," in
Methods in Gene Biotechnology, CRC Press, Inc., pages 15-
28, 1997). PCR is then performed and the products are
analyzed using standard techniques.
As an illustration, RNA is isolated from
biological sample using, for example, the guanidinium-
thiocyanate cell lysis procedure described above.
Alternatively, a solid-phase technique can be used to
isolate mRNA from a cell lysate. A reverse transcription
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57
reaction can be primed with the isolated RNA using random
oligonucleotides, short homopolymers of dT, or ZTMPO-1
anti-sense oligomers. Oligo-dT primers offer the
advantage that various mRNA nucleotide sequences are
amplified that can provide control target sequences.
ZTMPO-1 sequences are amplified by the polymerase chain
reaction using two flanking oligonucleotide primers that
are typically at least S bases in length.
PCR amplification products can be detected using
a variety of approaches, For example, PCR products can be
fractionated by gel electrophoresis, and visualized by
ethidium bromide staining. Alternatively, fractionated
PCR products can be transferred to a membrane, hybridized
with a detectably-labeled ZTMPO-1 probe, and examined by
autoradiography. Additional alternative approaches
include the use of digoxigenin-labeled deoxyribonucleic
acid triphosphates to provide chemiluminescence detection,
and the C-TRAK colorimetric assay.
Another approach is real time quantitative PCR
(Perkin-Elmer Cetus, Norwalk, Ct.). A fluorogenic probe,
consisting of an oligonucleotide with both a reporter and
a quencher dye attached, anneals specifically between the
forward and reverse primers. Using the 5' endonuclease
activity of Taq DNA polymerase, the reporter dye is
separated from the quencher dye and a sequence-specific
signal is generated and increases as amplification
increases. The fluorescence intensity can be continuously
monitored and quantified during the PCR reaction.
Another approach for detection of ZTMPO-1
expression is cycling probe technology (CPT), in which a
single-stranded DNA target binds with an excess of DNA
RNA-DNA chimeric probe to form a complex, the RNA portion
is cleaved with RNase H, and the presence of cleaved
chimeric probe is detected (see, for example, Beggs et
al., J. Clin. Microbiol. 34:2985, 1996 and Bekkaoui et
al., Biotechniques 20:240, 1996). Alternative methods for
detection of ZTMPO-1 sequences can utilize approaches such
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58
as nucleic acid sequence-based amplification (NASBA),
cooperative amplification of templates by cross-
hybridization (CATCH), and the ligase chain reaction (LCR)
(see, for example, Marshall et al., U.S. Patent No.
5,686,272 (1997), Dyer et al., J. Virol. Methods 60:161,
1996; Ehricht et al., Eur. J. Biochem. 243:358, 1997 and
Chadwick et al., J. Virol. Methods 70:59, 1998). Other
standard methods are known to those of ski-11 in the art.
ZTMPO-1 probes and primers can also be used to
detect and to localize ZTMPO-1 gene expression in tissue
samples. Methods for such in situ hybridization are well
known to those of skill in the art (see, for example, Choo
(ed.), In Situ Hybridization Protocols, Humana Press, Inc.,
1999; Wu et al. (eds.), "Analysis of Cellular DNA or
Abundance of mRNA by Radioactive In Situ Hybridization
IRISH)," in Methods in Gene Biotechnology, CRC Press, Inc.,
pages 259-278, 1997 and Wu et al. (eds.), "Localization of
DNA or Abundance of mRNA by Fluorescence In Situ
Hybridization IRISH)," in Methods in Gene Biotechnology,
CRC Press, Inc., pages 279-289, 1997).
Various additional diagnostic approaches are
well-known to those of skill in the art (see, for example,
Mathew (ed.), Protocols in Human Molecular Genetics Humana
Press, Inc., 1991; Coleman and Tsongalis, Molecular
Diagnostics, Humana Press, Inc., 1996 and Elles, Molecular
Diagnosis of Genetic Diseases, Humana Press, Inc., 1996).
The invention also provides antagonists or
inhibitors of ZTMPO-1 activity. Such antagonists would
include anti-ZTMPO-1 antibodies, soluble ZTMPO-1
receptors, as well as other peptidic and non-peptidic
agents (including ribozymes). Such antagonists would have
use as research reagents for characterizing sites of
ligand-receptor interaction. Antagonists would also find
use in modulating cellular proliferation and
differentiation such as in tumor growth and development.
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High levels of expression of ZTMPO-1 in testis tissue
suggest a role in spermatogenesis. These ZTMPO-1
antagonists would be useful for inhibiting spermatogenesis
and sperm activation. Such ZTMPO-1 antagonists can be
used for contraception in humans and animals, and in
particular, domestic and zoological animals and livestock,
where they would act to prevent fertilization of an egg.
Such ZTMPO-1 antagonists could be used, for instance, in
place of surgical forms of contraception (such as spaying
and neutering), and would allow for the possibility of
future breeding of treated animals if desired. ZTMPO-1
antagonists could also be used to mediate immune response,
for instance by boosting the humoral response in
individuals at risk for an infectious disease or as a
supplement to vaccination.
ZTMPO-1 can be used to identify inhibitors
(antagonists) of its activity. Test compounds are
transfected into cells or possibly added to the assays
disclosed herein to identify compounds that inhibit the
activity of ZTMPO-1. In addition to those assays
disclosed herein, samples can be tested for inhibition of
ZTMPO-1 activity within a variety of assays designed to
measure receptor binding or the stimulation/inhibition of
ZTMPO-1-dependent cellular responses. For example, ZTMPO-
1-responsive cell lines can be transfected with a reporter
gene construct that is responsive to a ZTMPO-1-stimulated
cellular pathway. Reporter gene constructs of this type
are known in the art, and will generally comprise a ZTMPO-
1-DNA response element operably linked to a gene encoding
an assayable protein, such as luciferase. DNA response
elements can include, but are not limited to, cyclic AMP
response elements (CRE), hormone response elements (HRE)
insulin response element (IRE) (Nasrin et al., Proc. Natl.
Acad. Sci. USA 87:5273-7, 1990) and serum response
elements (SRE) (Shaw et al. Cell 56: 563-72, 1989).
Cyclic AMP response elements are reviewed in Roestler et
al., J. Biol. Chem. 263: 9063-6; 1988 and Habener, Molec.
CA 02325822 2000-10-18
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Endocrinol. 4:1087-94; 1990. Hormone response elements
are reviewed in Beato, Cell 56:335-44; 1989. Candidate
compounds, solutions, mixtures or extracts are tested for
the ability to inhibit the activity of ZTMPO-1 on the
5 target cells as evidenced by a decrease in ZTMPO-1
stimulation of reporter gene expression. Assays of this
type will detect compounds that directly block ZTMPO-1
binding to cell-surface receptors, as well as compounds
that block processes in the cellular pathway subsequent to
10 receptor-ligand binding. In the alternative, compounds or
other samples can be tested for direct blocking of ZTMPO-1
binding to receptor using ZTMPO-1 tagged with a detectable
label (e. g., 'ZSI, biotin, horseradish peroxidase, FITC,
and the like). Within assays of this type, the ability of
15 a test sample to inhibit the binding of labeled ZTMPO-1 to
the receptor is indicative of inhibitory activity, which
can be confirmed through secondary assays. Receptors used
within binding assays may be cellular receptors or
isolated, immobilized receptors.
20 ZTMPO-1 polypeptides can also be used to prepare
antibodies that specifically bind to ZTMPO-1 epitopes,
peptides or polypeptides. The ZTMPO-1 polypeptide or a
fragment thereof serves as an antigen (immunogen) to
inoculate an animal and elicit an immune response.
25 Suitable antigens would be the ZTMPO-1 polypeptide encoded
by SEQ ID N0:2 from amino acid number 1 to amino acid
number 876, or contiguous 9 to 25 amino acid residue
fragments thereof. Antibodies generated from this immune
response can be isolated and purified as described herein.
30 Methods for preparing and isolating polyclonal and
monoclonal antibodies are well known in the art. See, for
example, Current Protocols in Immunolocrv, Cooligan, et al.
(eds.), National Institutes of Health, John Wiley and
Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A
35 Laboratory Manual, Second Edition, Cold Spring Harbor, NY,
1989; and Hurrell, (Ed.), Monoclonal Hybridoma Antibodies:
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61
Techniques and Applications, CRC Press, Inc., Boca Raton,
FL, 1982 .
As would be evident to one of ordinary skill in
the art, polyclonal antibodies can be generated from
inoculating a variety of warm-blooded animals such as
horses, cows, goats, sheep, dogs, chickens, rabbits, mice,
and rats with a ZTMPO-1 polypeptide or a fragment thereof.
The immunogenicity of a ZTMPO-1 polypeptide may be
increased through the use of an adjuvant, such as alum
(aluminum hydroxide) or Freund's complete or incomplete
adjuvant. Polypeptides useful for immunization also
include fusion polypeptides, such as fusions of ZTMPO-1 or
a portion thereof with an immunoglobulin polypeptide or
with maltose binding protein. The polypeptide immunogen
may be a full-length molecule or a portion thereof. If
the polypeptide portion is "hapten-like", such portion may
be advantageously joined or linked to a macromolecular
carrier (such as keyhole limpet hemocyanin (FCLH), bovine
serum albumin (BSA) or tetanus toxoid) for immunization.
As used herein, the term "antibodies" includes
polyclonal antibodies, affinity-purified polyclonal
antibodies, monoclonal antibodies, and antigen-binding
fragments, such as F(ab')2 and Fab proteolytic fragments.
Genetically engineered intact antibodies or fragments,
such as chimeric antibodies, Fv fragments, single chain
antibodies and the like, as well as synthetic antigen-
binding peptides and polypeptides, are also included.
Non-human antibodies may be humanized by grafting non-
human CDRs onto human framework and constant regions, cr
by incorporating the entire non-human variable domains
(optionally "cloaking" them with a human-like surface by
replacement of exposed residues, wherein the result is a
"veneered" antibody). In some instances, humanized
antibodies may retain non-human residues within the human
variable region framework domains to enhance proper
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62
binding characteristics. Through humanizing antibodies,
biological half-life may be increased, and the potential
for adverse immune reactions upon administration to humans
is reduced.
Alternative techniques for generating or
selecting antibodies useful herein include in vitro
exposure of lymphocytes to ZTMPO-1 protein or peptide, and
selection of antibody display libraries in phage or
similar vectors (for instance, through use of immobilized
or labeled ZTMPO-1 protein or peptide). Genes encoding
polypeptides having potential ZTMPO-1 polypeptide binding
domains can be obtained by screening random peptide
libraries displayed on phage (phage display) or on
bacteria, such as E. coli. Nucleotide sequences encoding
the polypeptides can be obtained in a number of ways, such
as through random mutagenesis and random polynucleotide
synthesis. These random peptide display libraries can be
used to screen for peptides which interact with a known
target which can be a protein or polypeptide, such as a
ligand or receptor, a biological or synthetic
macromolecule, or organic or inorganic substances.
Techniques for creating and screening such random peptide
display libraries are known in the art (Ladner et al., US
Patent NO. 5,223,409; Ladner et al., US Patent NO.
4,946,778; Ladner et al., US Patent NO. 5,403,484 and
Ladner et al., US Patent N0. 5,571,698) and random peptide
display libraries and kits for screening such libraries
are available commercially, for instance from Clontech
(Palo Alto, CA), Invitrogen Inca (San Diego, CA), New
England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB
Biotechnology Inc. (Piscataway, NJ). Random peptide
display libraries can be screened using the ZTMPO-1
sequences disclosed herein to identify proteins which bind
to ZTMPO-1. These "binding proteins" which interact with
ZTMPO-1 polypeptides can be used for tagging cells; for
isolating homolog polypeptides by affinity purification;
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they can be directly or indirectly conjugated to drugs,
toxins, radionuclides and the like. These binding
proteins can also be used in analytical methods such as
for screening expression libraries and neutralizing
activity. The binding proteins can also be used for
diagnostic assays for determining circulating levels of
polypeptides; for detecting or quantitating soluble
polypeptides as marker of underlying pathology or disease.
These binding proteins can also act as ZTMPO-1
"antagonists" to block ZTMPO-1 binding and signal
transduction in vitro and in vivo. These anti-ZTMPO-1
binding proteins would be useful for inhibiting binding.
Antibodies are determined to be specifically
binding if: 1) they exhibit a threshold level of binding
activity, and/or 2) they do not significantly cross-react
with related polypeptide molecules. First, antibodies
herein specifically bind if they bind to a ZTMPO-1
polypeptide, peptide or epitope with a binding affinity
(Ka) of 106 M 1 or greater, preferably 10~ M 1 or greater,
more preferably 108 M 1 or greater, and most preferably
109 M 1 or greater. The binding affinity of an antibody
can be readily determined by one of ordinary skill in the
art, for example, by Scatchard analysis (Scatchard, Ann.
NY Acad. Sci. 51: 660-72, 1949).
Second, antibodies are determined to
specifically bind if they do not significantly cross-react
with related polypeptides. Antibodies do not significantly
cross-react with related polypeptide molecules, for
example, if they detect ZTMPO-1 but not known related
polypeptides using a standard Western blot analysis
(Ausubel et al., ibid.). Examples of known related
polypeptides are those disclosed in the prior art, such as
known orthologs, and paralogs, and similar known members
of a protein family. Moreover, antibodies may be
CA 02325822 2000-10-18
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64
"screened against" known related polypeptides, such as
non-human ZTMPO-l, and ZTMPO-1 mutant polypeptides, to
isolate a population that specifically binds to the
inventive polypeptides. For example, antibodies raised to
ZTMPO-1 are adsorbed to related polypeptides adhered to
insoluble matrix; antibodies specific to ZTMPO-1 will flow
through the matrix under the proper buffer conditions.
Such screening allows isolation of polyclonal and
monoclonal antibodies non-crossreactive to closely related
polypeptides (Antibodies: A Laboratory Manual, Harlow and
Lane (eds.), Cold Spring Harbor Laboratory Press, 1988;
Current Protocols in Immunology, Cooligan, et al. (eds.),
National Institutes of Health, John Wiley and Sons, Inc.,
1995). Screening and isolation of specific antibodies is
well known in the art. See, Fundamental Immunology, Paul
(eds.), Raven Press, 1993: Getzoff et al., Adv. in
Immunol. 43: 1-98, 1988; Monoclonal Antibodies:
Principles and Practice, Goding, J.W. (eds.), Academic
Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2:
67-101, 1984.
A variety of assays known to those skilled in
the art can be utilized to detect antibodies and binding
proteins which specifically bind to ZTMPO-1 proteins or
peptides. Exemplary assays are described in detail in
Antibodies: A Laboratory Manual, Harlow and Lane (Eds.),
Cold Spring Harbor Laboratory Press, 1988. Representative
examples of such assays include: concurrent
immunoelectrophoresis, radioimmunoassay, radioimmuno-
precipitation, enzyme-linked immunosorbent assay (ELISA),
dot blot or Western blot assay, inhibition or competition
assay, and sandwich assay. In addition, antibodies can be
screened for binding to wild-type versus mutant ZTMPO-1
protein or polypeptide.
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Antibodies to ZTMPO-1 may be used for tagging
cells that express ZTMPO-1; for isolating ZTMPO-1 by
affinity purification; for diagnostic assays for
determining circulating levels of ZTMPO-1 polypeptides;
S for detecting or quantitating soluble ZTMPO-1 as marker of
underlying pathology or disease; in analytical methods
employing FACS; for screening expression libraries; for
generating anti-idiotypic antibodies; and as neutralizing
antibodies or as antagonists to block ZTMPO-1 binding in
10 vitro and in vivo. Suitable direct tags or labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent markers, chemiluminescent markers, magnetic
particles and the like; indirect tags or labels may
feature use of biotin-avidin or other complement/anti-
15 complement pairs as intermediates. Antibodies herein may
also be directly or indirectly conjugated to drugs,
toxins, radionuclides and the like, and these conjugates
used for in vivo diagnostic or therapeutic applications.
Moreover, antibodies to ZTMPO-1 or fragments thereof may
20 be used in vitro to detect denatured ZTMPO-1 or fragments
thereof in assays, for example, Western Blots or other
assays known in the art.
Antibodies or polypeptides herein may also be
directly or indirectly conjugated to drugs, toxins,
25 radionuclides and the like, and these conjugates used for
in vivo diagnostic or therapeutic applications. For
instance, polypeptides or antibodies of the present
invention may be used to identify or treat tissues or
organs that express a corresponding anti-complementary
30 molecule (receptor or antigen, respectively, for
instance). More specifically, ZTMPO-1 polypeptides or
anti-ZTMPO-1 antibodies, or bioactive fragments or
portions thereof, can be coupled to detectable or
cytotoxic molecules and delivered to a mammal having
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66
cells, tissues or organs that express the anti-
complementary molecule.
Suitable detectable molecules may be directly or
indirectly attached to the polypeptide or antibody, and
include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent markers, chemiluminescent markers,
magnetic particles and the like. Suitable cytotoxic
molecules may be directly or indirectly attached to the
polypeptide or antibody, and include bacterial or plant
toxins (for instance, diphtheria toxin, Pseudomonas
exotoxin, ricin, abrin and the like), as well as
therapeutic radionuclides, such as iodine-131, rhenium-188
or yttrium-90 (either directly attached to the polypeptide
or antibody, or indirectly attached through means of a
chelating moiety, fox instance). Polypeptides or
antibodies may also be conjugated to cytotoxic drugs, such
as adriamycin. For indirect attachment of a detectable or
cytotoxic molecule, the detectable or cytotoxic molecule
may be conjugated with a member of a complementary/
anticomplementary pair, where the other member is bound to
the polypeptide or antibody portion. For these purposes,
biotin/streptavidin is an exemplary complementary/
anticomplementary pair.
Molecules of the present invention can be used
to identify and isolate receptors involved in ZTMPO-1
binding. For example, proteins and peptides of the
present invention can be immobilized on a column and
membrane preparations run over the column (Immobilized
Affinity Ligand Techniques, Hermanson et al., eds.,
Academic Press, San Diego, CA, 1992, pp.195-202).
Proteins and peptides can also be radiolabeled (Methods in
Enzymol., vol. 182, "Guide to Protein Purification", M.
Deutscher, ed., Acad. Press, San Diego, 1990, 721-37) or
photoaffinity labeled (Brunner et al., Ann. Rev. Biochem.
62:483-514, 1993 and Fedan et al., Biochem. Pharmacol.
33:1167-80, 1984) and specific cell-surface proteins can
be identified.
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The molecules of the present invention will be
useful regulators in multiple cellular organisms. The
molecules of the present invention may used to modulate
cellular proliferation and differentiation, for example
spermatogenesis. In particular, certain proliferative
disorders such as cancers may be amenable to such
diagnosis, treatment or prevention. ZTMPO-1 would be
useful in modulating the cell cycle such as during
differentiation or in rapidly proliferating cells such as
in tumor tissues. ZTMPO-1 would find application in a
diverse array of tissues as testis, skeletal muscle,
thyroid and adrenal gland for example.
Polynucleotides encoding ZTMPO-1 polypeptides
are useful within gene therapy applications where it is
desired to increase or inhibit ZTMPO-1 activity. If a
mammal has a mutated or absent ZTMPO-1 gene, the ZTMPO-1
gene can be introduced into the cells of the mammal. In
one embodiment, a gene encoding a ZTMPO-1 polypeptide is
introduced in vivo in a viral vector. Such vectors
include an attenuated or defective DNA virus, such as, but
not limited to, herpes simplex virus (HSV),
papillomavirus, Epstein Barr virus (EBV), adenovirus,
adeno-associated virus (AAV), and the like. Defective
viruses, which entirely or almost entirely lack viral
genes, are preferred. A defective virus is not infective
after introduction into a cell. Use of defective viral
vectors allows for administration to cells in a specific,
localized area, without concern that the vector can infect
other cells. Examples of particular vectors include, but
are not limited to, a defective herpes simplex virus 1
(HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.
2:320-30, 1991); an attenuated adenovirus vector, such as
the vector described by Stratford-Perricaudet et al., J.
Clin. Invest. 90:626-30, 1992; and a defective adeno-
associated virus vector (Sarnulski et al., J. Virol.
61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8,
1989) .
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68
In another embodiment, a ZTMPO-1 gene can be
introduced in a retroviral vector, e.g., as described in
Anderson et al., U.S. Patent No. 5,399,346; Mann et al.
Cell 33:153, 1983; Temin et al., U.S. Patent No.
4,650,764; Temin et al., U.S. Patent No. 4,980,289;
Markowit2 et al., J. Virol. 62:1120, 1988; Temin et al.,
U.S. Patent No. 5,124,263; International Patent
Publication No. WO 95/07358, published March 16, 1995 by
Dougherty et al.; and Kuo et al., Blood 82:845, 1993.
Alternatively, the vector can be introduced by lipofection
in vivo using liposomes. Synthetic cationic lipids can be
used to prepare liposomes for in vivo transfection of a
gene encoding a marker (Felgner et al., Proc. Natl. Acad.
Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad.
Sci. USA 85:8027-31, 1988). The use of lipofection to
introduce exogenous genes into specific organs in vivo has
certain practical advantages. Molecular targeting of
liposomes to specific cells represents one area of
benefit. More particularly, directing transfection to
particular cells represents one area of benefit. For
instance, directing transfection to particular cell types
would be particularly advantageous in a tissue with
cellular heterogeneity, such as the pancreas, liver,
kidney, and brain. Lipids may be chemically coupled to
other molecules for the purpose of targeting. Targeted
peptides (e. g., hormones or neurotransmitters), proteins
such as antibodies, or non-peptide molecules can be
coupled to liposomes chemically.
It is possible to remove the target cells from
the body; to introduce the vector as a naked DNA plasmid;
and then to re-implant the transformed cells into the
body. Naked DNA vectors for gene therapy can be
introduced into the desired host cells by methods known in
the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAF dextran,
calcium phosphate precipitation, use of a gene gun or use
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69
of a DNA vector transporter. See, e.g., Wu et al., J.
Biol. Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem.
263:14621-4, 1988.
The present invention also provides reagents for
use in diagnostic applications. For example, the ZTMPO-1
gene, a probe comprising ZTMPO-1 DNA or RNA, or a
subsequence thereof can be used to determine if the ZTMPO
1 gene is present on chromosome 12 or if a mutation has
occurred. The emerin gene is not detected in samples from
patients with Emery-Dreifuss muscular dystrophy, and is
present in normal patients (Bione et al., Nat. Genet.
8:323-7, 1994 and Nagano et al., Nat. Genet. 12:254-9,
1996) and thus serves as a marker for the disease.
Detectable chromosomal aberrations at the ZTMPO-1 gene
locus include, but are not limited to, aneuploidy, gene
copy number changes, insertions, deletions, restriction
site changes and rearrangements. These aberrations can
occur within the coding sequence, within introns, or
within flanking sequences, including upstream promoter and
regulatory regions, and may be manifested as physical
alterations within a coding sequence or changes in gene
expression level.
In general, these diagnostic methods comprise
the steps of (a) obtaining a genetic sample from a
patient; (b) incubating the genetic sample with a
polynucleotide probe or primer as disclosed above, under
conditions wherein the polynucleotide will hybridize to
complementary polynucleotide sequence, to produce a first
reaction product; and (iii) comparing the first reaction
product to a control reaction product. A difference
between the first reaction product and the control
reaction product is indicative of a genetic abnormality in
the patient. Genetic samples for use within the present
invention include genomic DNA, cDNA, and RNA. The
polynucleotide probe or primer can be RNA or DNA, and will
comprise a portion of SEQ ID NO:1, the complement of SEQ
ID NO: l, or an RNA equivalent thereof. Suitable assay
CA 02325822 2000-10-18
W O 99/54468 PCT/US99/08601
methods in this regard include molecular genetic
techniques known to those in the art, such as restriction
fragment length polymorphism (RFLP) analysis, short tandem
repeat (STR) analysis employing PCR techniques, ligation
5 chain reaction (Barany, PCR Methods and Applications 1:5-
16, 1991), ribonuclease protection assays, and other
genetic linkage analysis techniques known in the art
(Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian,
Chest 108:255-65, 1995). Ribonuclease protection assays
10 (see, e.g., Ausubel et al., ibid., ch. 4) comprise the
hybridization of an RNA probe to a patient RNA sample,
after which the reaction product (RNA-RNA hybrid) is
exposed to RNase. Hybridized regions of the RNA are
protected from digestion. Within PCR assays, a patient's
15 genetic sample is incubated with a pair of polynucleotide
primers, and the region between the primers is amplified
and recovered. Changes in size or amount of recovered
product are indicative of mutations in the patient.
Another PCR-based technique that can be employed is single
20 strand conformational polymorphism (SSCP) analysis
(Hayashi, PCR Methods and Applications 1:34-8, 1991).
Transgenic mice, engineered to express the
ZTMPO-1 gene, and mice that exhibit a complete absence of
ZTMPO-1 gene function, referred to as "knockout mice"
25 (Snouwaert et al., Science 257:1083, 1992), may also be
generated (Lowell et al., Nature 366:740-42, 1993). These
mice may be employed to study the ZTMPO-1 gene and the
protein encoded thereby in an in vivo system. Such mice
could be used, for example, in breeding studies to
30 determine the effect ZTMPO-1 has on spermatogenesis and
sperm function as well as on conductivity of the heart.
For pharmaceutical use, the proteins of the
present invention are formulated for parenteral,
particularly intravenous or subcutaneous, delivery
35 according to conventional methods. Intravenous
administration will be by bolus injection or infusion over
a typical period of one to several hours. In general,
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pharmaceutical formulations will include a ZTMPO-1 protein
in combination with a pharmaceutically acceptable vehicle,
such as saline, buffered saline, 5% dextrose in water or
the like. Formulations may further include one or more
excipients, preservatives, solubilizers, buffering agents,
albumin to prevent protein loss on vial surfaces, etc.
Methods of formulation are well known in the art and are
disclosed, for example, in Remington: The Science and
Practice of Pharmacv, Gennaro, ed., Mack Publishing Co.,
Easton, PA, 19th ed., 1995. Determination of dose is
within the level of ordinary skill in the art. The
proteins may be administered for acute treatment, over one
week or less, often over a period of one to three days or
may be used in chronic treatment, over several months or
years. Evaluation of therapeutic effect of ZTMPO-1 for
cardiac applications can be done by looking for changes in
ECG. Decreases in creatine kinase levels and a decrease
in weakness would serve as indicators for changes in
muscle wasting associated with muscular dystrophy.
The invention is further illustrated by the
following non-limiting examples.
EXAMPLES
Example 1
Isolation of ZTMPO-1
Novel ZTMPO-1 encoding polynucleotides and
polypeptides of the present invention were initially
identified by querying an EST database. To identify the
corresponding cDNA, two clones from which an identified
EST was derived that were considered likely to contain the
entire human ZTMPO-1 sequence were used for sequencing.
Using a QIAwell 8 plasmid kit (Qiagen, Inc., Chatsworth,
CA) according to manufacturer's instructions, a 5 ml
overnight culture in LB + 50 ~g/ml ampicillin was
prepared. The templates were sequenced on an Applied
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72
BiosystemsT"' model 377 DNA sequences (Perkin-Elmer Cetus,
Norwalk, Ct.) using the ABI PRISMT"' Dye Terminator Cycle
Sequencing Ready Reaction Kit (Perkin-Elmer Corp.)
according to the manufacturer's instructions.
Oligonucleotides ZC694 (SEQ ID N0:9), ZC976 (SEQ ID N0:10)
and ZC447 (SEQ ID N0:14) were used as sequencing primers.
Oligonucleotides ZC15976 (SEQ ID NO:11), ZC15485 (SEQ ID
N0:12), ZC15526 (SEQ ID N0:13), 215620 (SEQ ID N0:15) and
ZC15823 (SEQ ID N0:16) were used to complete the sequence
from the clones.
Sequencing reactions were carried out in a
Hybaid OmniGene Temperature Cycling System (National
Labnet Co., Woodbridge, NY). SequencherTM 3.0 sequence
analysis software (Gene Codes Corporation, Ann Arbor, MI)
was used for data analysis. The sequences from the two
clones overlapped by 740 by and contained the 3' end of
the gene and the poly A tail. A third clone prepared as
described above was sequenced resulting in the remaining
5' sequence. Oligonucleotides ZC447 (SEQ ID N0:14), ZC976
(SEQ ID NO:10), ZC16162 (SEQ ID N0:17), ZC16038 (SEQ ID
N0:18), ZC16249 (SEQ ID N0:19), ZC16164 (SEQ ID N0:20),
ZC16163 (SEQ ID N0:21), ZC16165 (SEQ ID N0:22) and ZC16037
(SEQ ID N0:23) were used in sequencing. Differences
between the original EST sequences and the final sequence
of ZTMPO-1 were detected. The lack of identity arose from
ambiguity in the original EST sequences.
To confirm that the polynucleotide sequence
encoding the initial methionine had been identified, ,a
nested 5'RACE (rapid amplification of cDNA ends) was
performed. Several Marathon's'"' cDNA libraries (human
prostate, spleen, testis and uterus) were prepared using a
Marathon cDNA kit (Clontech) according the manufacturer's
instructions. For the first round PCR oligonucleotides
AP1 (SEQ ID N0:24, supplied with the kit or synthesized)
and ZC15527 (SEQ ID N0:25) were used as primers and the 5°
RACE reaction was carried out at 94oC, for 2 minutes,
followed by 25 cycles at 94oC for 15 seconds, 6loC for 20
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73
seconds and 72oC for 30 seconds, followed by a 1 minute
extension at 72oC. The PCR products from the first round
reaction were diluted 1/100 and used as templates for a
second round of PCR using oligonucleotides AP2 (SEQ ID
N0:32, supplied with the Marathon Kit or synthesized) and
ZC15526 (SEQ ID N0:13) as primers. The PCR derived DNA
fragments were resolved by gel electrophoresis, excised
and ligated into the expression vector was the vector
pCR2.1 (TA Cloning Kit, Invitrogen Inc., San Diego, CA)
according to manufacturer's instructions. The sequence of
the inserts was confirmed by sequence analysis using
oligos ZC694 (SEQ ID N0:9) and ZC695 (SEQ ID N0:26) as
primers, as described above and confirmed that the Met
(amino acid residue 1 of SEQ ID N0:2) was indeed the start
methionine. The resulting 2,754 by polynucleotide (SEQ ID
NO:1) had an open reading frame encoding an 876 amino acid
residue protein sequence (SEQ ID N0:2) and was designated
ZTMPO-1.
Example 2
Northern Blot Analysis of ZTMPO-1
Human Multiple Tissue Northern Blots (MTN I, MTN
II and MTN III; Clontech) were probed to determine the
tissue distribution of human ZTMPO1 expression. An
approximately 218 by PCR derived probe (SEQ ID N0:8) was
amplified using EST clone EST934031 (SEQ ID N0:27) as a
template and oligonucleotide ZC15521 (SEQ ID N0:28) and
ZC15525 (SEQ ID N0:29) as primers. The amplification was
carried out as follows : 1 cycle at 94°C for 2 minutes, 30
cycles of 94°C for 15 seconds, 65°C 20 seconds and 72°C
30
seconds, followed by 1 cycle at 72°C for 1 minute. The PCR
product was gel purified using the QIAquick method
(Qiagen, Chatsworth, CA) and radioactively labeled using
the Rediprime DNA labeling kit (Amersham, Arlington
Heights, IL) both according to the manufacturer's
suggestion. The probe was purified using a NUCTRAP push
column (Stratagene). EXPRESSHYB (Clontech) solution was
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used for prehybridization and as a hybridizing solution
for the Northern blots. Hybridization took place
overnight at 65°C using 4 x 106 cpm/ml of labeled probe.
The blots were then washed in 2X SSC and 0.05% SDS at RT,
followed by washes in O.1X SSC and 0.1% SDS at 50°C twice
and at 55°C once. Two transcripts of approximately 3.2 kb
and 5 kb were seen in nearly all the tissues with the most
predominant expression being in testis.
Example 3
Chromosomal Assicrnment and Placement of ZTMPO-1
ZTMPO-1 was mapped to chromosome 12 using the
commercially available GeneBridge 4 Radiation Hybrid Panel
(Research Genetics, Inc., Huntsville, AL). The GeneBridge
4 Radiation Hybrid Panel contains PCRable DNAs from each
of 93 radiation hybrid clones, plus two control DNAs (the
HFL donor and the A23 recipient). A publicly available
WWW server (http://www-genome.wi.mit.edu/cgi-bin/contig/
rhmapper.pl) allows mapping relative to the Whitehead
Institute/MIT Center for Genome Research's radiation
hybrid map of the human genome (the "WICGR" radiation
hybrid map) which was constructed with the GeneBridge 4
Radiation Hybrid Panel.
For the mapping of ZTMPO-1 with the GeneBridge 4
RH Panel, 20 ~.1 reactions were set up in a 96-well
microtiter plate (Stratagene, La Jolla, CA) and used in a
RoboCycler Gradient 96 thermal cycler (Stratagene). Each
of the 95 PCR reactions consisted of 2 ul lOX KlenTaq PCR
reaction buffer (CLONTECH Laboratories, Inc., Palo Alto,
CA), 1.6 ~.1 dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster
City, CA) , 1 ~,1 sense primer, ZC15, 487 (SEQ ID NO: 6) , 1 ~.1
antisense primer, ZC 15486 (SEQ ID N0:7), 2 ~.1 RediLoad
(Research Genetics, Inc.), 0.4 ~1 50X Advantage KlenTaq
Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA
from an individual hybrid clone or control and ddH20 for a
total volume of 20 ~1. The reactions were overlaid with
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an equal amount of mineral oil and sealed. The PCR cycler
conditions were as follows: an initial 1 cycle 5 minute
denaturation at 95°C, 35 cycles of a 1 minute denaturation
at 95°C, 1 minute annealing at 62°C and 1.5 minute
5 extension at 72°C, followed by a final 1 cycle extension of _
7 minutes at 72°C. The reactions were separated by
electrophoresis on a 2o agarose gel (Life Technologies,
Gaithersburg, MD).
The results showed that ZTMPO-1 maps 636.18
10 cR_3000 from the top of the human chromosome 12 linkage
group on the WICGR radiation hybrid map. The proximal
framework marker was D12S367. This positions ZTMPO-1 in
the 12q24.33 region on the integrated LDB chromosome 12
map (The Genetic Location Database, University of
15 Southhampton, wWW server: http://cedar.genetics.soton.ac.
uk/public html/).
From the foregoing, it will be appreciated that,
although specific embodiments of the invention have been
described herein for purposes of illustration, various
20 modifications may be made without deviating from the
spirit and scope of the invention. Accordingly, the
invention is not limited except as by the appended claims.
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1
SEQUENCE LISTING
<110> ZyrnoGenetics. Inc.
1201 Eastlake Avenue East
Seattle. Washington 98102
United States of America
<120> SOLUBLE PROTEIN ZTMPO-1
<130> 97-67PC
<150> 60/082.513
<151> 1998-04-21
<160> 32
<170> FastSEQ for Windows Version 3.0
<210>1
<211>2884
<212>DNA
<213>Homo Sapiens
<220>
<221> CDS
<222> (127)...(2754)
<400> 1
aaagttttta atgaaagaaa cagaaactga tgccattata taatgaaccc tagtacccat 60
cacccagctt cagcaggtgt tagtattttg tgactctttg atttttttgt cttgggccta 120
ggtgaa atg aca atg gat get ctg ttg get cga ttg aaa ctt ctg aat 168
Met Thr Met Asp Ala Leu Leu Ala Arg Leu Lys Leu Leu Asn
1 5 lp '
cca gat gac ctt aga gaa gaa atc gtc aaa gcc gga ttg aaa tgt gga 216
Pro Asp Asp Leu Arg Glu Glu Ile Val Lys Ala Gly Leu Lys Cys Gly
15 20 25 30
ccc att aca tca act aca agg ttc att ttt gag aaa aaa ttg get cag 264
Pro Ile Thr Ser Thr Thr Arg Phe Ile Phe Glu Lys Lys Leu Ala Gln
35 40 45
get tta ctg gag caa gga gga agg ctg tct tct ttc tac cac cat gag 312
CA 02325822 2000-10-18
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2
Ala Leu Leu Glu Gln Gly Gly Arg Leu Ser Ser Phe Tyr His His Glu
50 55 60
gca ggt gtc aca get ctc agc cag gac cca caa agg att ttg aag cca 360
Ala Gly Val Thr Ala Leu Ser Gln Asp Pro Gln Arg Ile Leu Lys Pro
65 70 75
get gaa ggg aac cca act gat cag get ggt ttt tct gaa gac aga gat 408
Ala Glu Gly Asn Pro Thr Asp Gln Ala Gly Phe Ser Glu Asp Arg Asp
80 85 90
ttt ggt tac agt gtg ggc ctg aat cct cca gag gag gaa get gtg aca 456
Phe Gly Tyr Ser Val Gly Leu Asn Pro Pro Glu Glu Glu Ala Val Thr
95 100 105 110
tcc aag acc tgc tcg gtg ccc cct agt gac acc gac acc tac aga get 504
Ser Lys Thr Cys Ser Val Pro Pro Ser Asp Thr Asp Thr Tyr Arg Ala
115 120 125
gga gcg act gcg tct aag gag ccg ccc ctg tac tat ggg gtg tgt cca 552
Gly Ala Thr Ala Ser Lys Glu Pro Pro Leu Tyr Tyr Gly Val Cys Pro
130 135 140
gtg tat gag gac gtc cca gcg aga aat gaa agg atc tat gtt tat gaa 600
Ual Tyr Glu Asp Val Pro Ala Arg Asn Glu Arg Ile Tyr Val Tyr Glu
145 150 155
aat aaa aag gaa gca ttg caa get gtc aag atg atc aaa ggg tcc cga 648
Asn Lys Lys Glu Ala Leu Gln Ala Val Lys Met Ile Lys Gly Ser Arg
160 165 170
ttt aaa get ttt tct acc aga gaa gac get gag aaa ttt get aga gga 696
Phe Lys Ala Phe Ser Thr Arg Glu Asp Ala Glu Lys Phe Ala Arg Gly
175 180 185 190
att tgt gat tat ttc cct tct cca agc aaa acg tcc tta cca ctg tct 744
Ile Cys Asp Tyr Phe Pro Ser Pro Ser Lys Thr Ser Leu Pro Leu Ser
195 200 205
cct gtg aaa aca get cca ctc ttt agc aat gac agg ttg aaa gat ggt 792
Pro Ual Lys Thr Ala Pro Leu Phe Ser Asn Asp Arg Leu Lys Asp Gly
210 215 220
ttg tgc ttg tcg gaa tca gaa aca gtc aac aaa gag cga gcg aac agt 840
Leu Cys Leu Ser Glu Ser Glu Thr Val Asn Lys Glu Arg Ala Asn Ser
CA 02325822 2000-10-18
WO 99/54468 PCTNS99/08601
3
225 230 235
tac aaa aat ccc cgc acg cag gac ctc acc gcc aag ctt cgg aaa get 888
Tyr Lys Asn Pro Arg Thr Gln Asp Leu Thr Ala Lys Leu Arg Lys Ala
240 245 250
gtg gag aag gga gag gag gac acc ttt tct gac ctt atc tgg agc aac 936
Val Glu Lys Gly Glu Glu Asp Thr Phe Ser Asp Leu Ile Trp Ser Asn
255 260 265 270
ccc cgg tat ctg ata ggc tca gga gac aac ccc act atc gtg cag gaa 984
Pro Arg Tyr Leu Ile Gly Ser Gly Asp Asn Pro Thr Ile Val Gln Glu
275 280 285
ggg tgc agg tac aac gtg atg cat gtt get gcc aaa gag aac cag get 1032
Gly Cys Arg Tyr Asn Ual Met His Val Ala Ala Lys Glu Asn Gln Ala
290 295 300
tcc atc tgc cag ctg act ctg gac gtc ctg gag aac cct gac ttc atg 1080
Ser Ile Cys Gln Leu Thr Leu Asp Val Leu Glu Asn Pro Asp Phe Met
305 310 315
agg ctg atg tac cct gat gac gac gag gcc atg ctg cag aag cgt atc 1128
Arg Leu Met Tyr Pro Asp Asp Asp Glu Ala Met Leu Gln Lys Arg Ile
320 325 330
cgt tac gtg gtg gac ctg tac ctc aac acc ccc gac aag atg ggc tat 1176
Arg Tyr Val Val Asp Leu Tyr Leu Asn Thr Pro Asp Lys Met Gly Tyr
335 340 345 350
gac aca ccg ttg cat ttt get tgt aag ttt gga aat gca gat gta gtc 1224
Asp Thr Pro Leu His Phe Ala Cys Lys Phe Gly Asn Ala Asp Val Val
355 360 365
aac gtg ctt tcg tca cac cat ttg att gta aaa aac tca agg aat aaa 1272
Asn Val Leu Ser Ser His His Leu Ile Val Lys Asn Ser Arg Asn Lys
370 375 380
tat gat aaa aca cct gaa gat gta att tgt gaa aga agc aaa aat aaa 1320
Tyr Asp Lys Thr Pro Glu Asp Ual Ile Cys Glu Arg Ser Lys Asn Lys
385 390 395
tct gtg gaa ctg aag gag cgg atc aga gag tat tta aag ggc cac tac 1368
Ser Val Glu Leu Lys Glu Arg Ile Arg Glu Tyr Leu Lys Gly His Tyr
400 405 410
CA 02325822 2000-10-18
WO 99/54468 PCT/US99l08601
4
tac gtg ccc ctc ctg aga gcg gaa gag act tct tct cca gtc atc ggg 1416
Tyr Val Pro Leu Leu Arg Ala Glu Glu Thr Ser Ser Pro Val Ile Gly
415 420 425 430
gag ctg tgg tcc cca gac cag acg get gag gcc tct cac gtc agc cgc 1464
Glu Leu Trp Ser Pro Asp Gln Thr Ala Glu Ala Ser His Val Ser Arg
435 440 445
tat gga ggc agc ccc aga gac ccg gta ctg acc ctg aga gcc ttc gca 1512
Tyr Gly Gly Ser Pro Arg Asp Pro Val Leu Thr Leu Arg Ala Phe Ala
450 455 460
ggg ccc ctg agt cca gcc aag gca gaa gat ttt cgc aag ctc tgg aaa 1560
Gly Pro Leu Ser Pro Ala Lys Ala Glu Asp Phe Arg Lys Leu Trp Lys
465 470 475
act cca cct cga gag aaa gca ggc ttc ctt cac cac gtc aag aag tcg 1608
Thr Pro Pro Arg Glu Lys Ala Gly Phe Leu His His Ual Lys Lys Ser
480 485 490
gac ccg gaa aga ggc ttt gag aga gtg gga agg gag cta get cat gag 1656
Asp Pro Glu Arg Gly Phe Glu Arg Val Gly Arg Glu Leu Ala His Glu
495 500 505 510
ctg ggg tat ccc tgg gtt gaa tac tgg gaa ttt ctg ggc tgt ttt gtt 1704
Leu Gly Tyr Pro Trp Val Glu Tyr Trp Glu Phe Leu Gly Cys Phe Val
515 520 525
gat ctg tct tcc cag gaa ggc ctg caa aga cta gaa gaa tat ctc aca 1752
Asp Leu Ser Ser Gln Glu Gly Leu Gln Arg Leu Glu Glu Tyr Leu Thr
530 535 540
cag cag gaa ata ggc aaa aag get caa caa gaa aca gga gaa cgg gaa 1800
Gln Gln Glu Ile Gly Lys Lys Ala Gln Gln Glu Thr Gly Glu Arg Glu
545 550 555
gcc tcc tgc cga gat aaa gcc acc acg tct ggc agc aat tcc att tcc 1848
Ala Ser Cys Arg Asp Lys Ala Thr Thr Ser Gly Ser Asn Ser Ile Ser
560 565 570
gtg agg gcg ttt cta gat gaa gat gac atg agc ttg gaa gaa ata aaa 1896
Val Arg Ala Phe Leu Asp Glu Asp Asp Met Ser Leu Glu Glu Ile Lys
575 580 585 590
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
aat cgg caa aat gca get cga aat aac agc ccg ccc aca gtc ggt get 1944
Asn Arg Gln Asn Ala Ala Arg Asn Asn Ser Pro Pro Thr Val Gly Ala
595 600 605
ttt gga cat acg agg tgc agc gcc ttc ccc ttg gag cag gag gca gac 1992
Phe Gly His Thr Arg Cys Ser Ala Phe Pro Leu Glu Gln Glu Ala Asp
610 615 620
ctc ata gaa gcc gcc gag ccg gga ggt cca cac agc agc aga aat ggg 2040
Leu Ile Glu Ala Ala Glu Pro Gly Gly Pro His Ser Ser Arg Asn Gly
625 630 635
ctc tgc cat cct ctg aat cac agc agg acc ctg gcg ggc aag aga cca 2088
Leu Cys His Pro Leu Asn His Ser Arg Thr Leu Ala Gly Lys Arg Pro
640 645 650
aag gcc ccc cat ggg gag gaa gcc cat ctg cca cct gtc tcg gat ttg 2136
Lys Ala Pro His Gly Glu Glu Ala His Leu Pro Pro Val Ser Asp Leu
655 660 665 670
act gtt gag ttt gat aaa ctg aat ttg caa aat ata gga cgt agc gtt 2184
Thr Ual Glu Phe Asp Lys Leu Asn Leu Gln Asn Ile Gly Arg Ser Val
675 680 685
tcc aag aca cca gat gaa agt aca aaa act aaa gat cag atc ctg act 2232
Ser Lys Thr Pro Asp Glu Ser Thr Lys Thr Lys Asp Gln Ile Leu Thr
690 695 700
tca aga atc aat gca gta gaa aga gac ttg tta gag cct tct ccc gca 2280
Ser Arg Ile Asn Ala Val Glu Arg Asp Leu Leu Glu Pro Ser Pro Ala
705 710 715
gac caa ctc ggg aat ggc cac agg agg aca gaa agt gaa atg tca gcc 2328
Asp Gln Leu Gly Asn Gly His Arg Arg Thr Glu Ser Glu Met Ser Ala
720 725 130
agg atc get aaa atg tcc ttg agt ccc agc agc ccc agg cac gag gat 2376
Arg Ile Ala Lys Met Ser Leu Ser Pro Ser Ser Pro Arg His Glu Asp
735 740 745 750
cag ctc gag gtc acc agg gaa ccg gcc agg cgg ctc ttc ctt ttt gga 2424
Gln Leu Glu Val Thr Arg Glu Pro Ala Arg Arg Leu Phe Leu Phe Gly
755 760 765
gag gag cca tca aaa ctc gat cag gat gtt ttg gcc get ctt gaa tgt 2472
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
6
Glu Glu Pro Ser Lys Leu Asp Gln Asp Val Leu Ala Ala Leu Glu Cys
770 775 780
gca gac gtc gac ccc cat cag ttc ccg gcc gtg cac aga tgg aag agt 2520
Ala Asp Ual Asp Pro His Gln Phe Pro Ala Val His Arg Trp Lys Ser
785 790 795
get gtc ctg tgc tac tca ccc tcg gac aga cag agt tgg ccc agt ccc 2568
Ala Val Leu Cys Tyr Ser Pro Ser Asp Arg Gln Ser Trp Pro Ser Pro
800 805 810
gcg gtg aaa gga agg ttc aag tct cag ctg cca gat ctc agt ggc cct 2616
Ala Val Lys Gly Arg Phe Lys Ser Gln Leu Pro Asp Leu Ser Gly Pro
815 820 825 830
cac agc tac agt ccg ggg aga aac agc gtg get gga agc aac ccc gca 2664
His Ser Tyr Ser Pro Gly Arg Asn Ser Val Ala Gly Ser Asn Pro Ala
835 840 845
aag cca ggc ctg ggc agt cct ggg cgc tac agc ccc gtg cac ggg agc 2712
Lys Pro Gly Leu Gly Ser Pro Gly Arg Tyr Ser Pro Val His Gly Ser
850 855 860
cag ctc cgc agg atg gcg cgc ctg get gag ctt gcc gcc ctg 2754
Gln Leu Arg Arg Met Ala Arg Leu Ala Glu Leu Ala Ala Leu
865 870 875
taggcttggc gctgggctct cggtttgttc ttcattttta aagaaggaag ggtcatatgt 2814
ttattgctaa actgtcaaaa aggaatatat tctgattaaa ttattactcc tcaaaaaaaa 2874
aaaaaaaaaa 2884
<210>2
<211>876
<212>PRT
<213>Homo sapiens
<400> 2
Met Thr Met Asp Ala Leu Leu Ala Arg Leu Lys Leu Leu Asn Pro Asp
1 5 10 15
Asp Leu Arg Glu Glu Ile Val Lys Ala Gly Leu Lys Cys Gly Pro Ile
20 25 30
Thr Ser Thr Thr Arg Phe Ile Phe Glu Lys Lys Leu Ala Gln Ala Leu
35 40 45
Leu Glu Gln Gly Gly Arg Leu Ser Ser Phe Tyr His His Glu Ala Gly
50 55 60
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
7
Val Thr Ala Leu Ser Gln Asp Pro Gln Arg Ile Leu Lys Pro Ala Glu
65 70 75 80
Gly Asn Pro Thr Asp Gln Ala Gly Phe Ser Glu Asp Arg Asp Phe Gly
85 90 95
Tyr Ser Val Gly Leu Asn Pro Pro Glu Glu Glu Ala Val Thr Ser Lys
100 105 110
Thr Cys Ser Val Pro Pro Ser Asp Thr Asp Thr Tyr Arg Ala Gly Ala
115 120 125
Thr Ala Ser Lys Glu Pro Pro Leu Tyr Tyr Gly Ual Cys Pro Val Tyr
130 135 140
Glu Asp Val Pro Ala Arg Asn Glu Arg Ile Tyr Val Tyr Glu Asn Lys
145 150 155 160
Lys Glu Ala Leu Gln Ala Val Lys Met Ile Lys Gly Ser Arg Phe Lys
165 170 175
Ala Phe Ser Thr Arg Glu Asp Ala Glu Lys Phe Ala Arg Gly Ile Cys
180 185 190
Asp Tyr Phe Pro Ser Pro Ser Lys Thr Ser Leu Pro Leu Ser Pro Val
195 200 205
Lys Thr Ala Pro Leu Phe Ser Asn Asp Arg Leu Lys Asp Gly Leu Cys
210 215 220
Leu Ser Glu Ser Glu Thr Val Asn Lys Glu Arg Ala Asn Ser Tyr Lys
225 230 235 240
Asn Pro Arg Thr Gln Asp Leu Thr Ala Lys Leu Arg Lys Ala Val Glu
245 250 255
Lys Gly Glu Glu Asp Thr Phe Ser Asp Leu Ile Trp Ser Asn Pro Arg
260 265 270
Tyr Leu Ile Gly Ser Gly Asp Asn Pro Thr Ile Val Gln Glu Gly Cys
275 280 285
Arg Tyr Asn Val Met His Val Ala Ala Lys Glu Asn Gln Ala Ser Ile
290 295 300
Cys Gln Leu Thr Leu Asp Val Leu Glu Asn Pro Asp Phe Met Arg Leu
305 310 315 320
Met Tyr Pro Asp Asp Asp Glu Ala Met Leu Gln Lys Arg Ile Arg Tyr
325 330 335
Val Val Asp Leu Tyr Leu Asn Thr Pro Asp Lys Met Gly Tyr Asp Thr
340 345 350
Pro Leu His Phe Ala Cys Lys Phe Gly Asn Ala Asp Val Val Asn Val
355 360 365
Leu Ser Ser His His Leu Ile Val Lys Asn Ser Arg Asn Lys Tyr Asp
370 375 380
Lys Thr Pro Glu Asp Val Ile Cys Glu Arg Ser Lys Asn Lys Ser Val
385 390 395 400
Glu Leu Lys Glu Arg Ile Arg Glu Tyr Leu Lys Gly His Tyr Tyr Val
405 410 415
Pro Leu Leu Arg Ala Glu Glu Thr Ser Ser Pro Val Ile Gly Glu Leu
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
8
420 425 430
Trp Ser Pro Asp Gln Thr Ala Glu Ala Ser His Val Ser Arg Tyr Gly
435 440 445
Gly Ser Pro Arg Asp Pro Val Leu Thr Leu Arg Ala Phe Ala Gly Pro
450 455 460
Leu Ser Pro Ala Lys Ala Glu Asp Phe Arg Lys Leu Trp Lys Thr Pro
465 470 475 480
Pro Arg Glu Lys Ala Gly Phe Leu His His Val Lys Lys Ser Asp Pro
485 490 495
Glu Arg Gly Phe Glu Arg Val Gly Arg Glu Leu Ala His Glu Leu Gly
500 505 510
Tyr Pro Trp Val Glu Tyr Trp Glu Phe Leu Gly Cys Phe Val Asp Leu
515 520 525
Ser Ser Gln Glu Gly Leu Gln Arg Leu Glu Glu Tyr Leu Thr Gln Gln
530 535 540
Glu Ile Gly Lys Lys Ala Gln Gln Glu Thr Gly Glu Arg Glu Ala Ser
545 550 555 560
Cys Arg Asp Lys Ala Thr Thr Ser Gly Ser Asn Ser Ile Ser Val Arg
565 570 575
Ala Phe Leu Asp Glu Asp Asp Met Ser Leu Glu Glu Ile Lys Asn Arg
580 585 590
Gln Asn Ala Ala Arg Asn Asn Ser Pro Pro Thr Val Gly Ala Phe Gly
595 600 605
His Thr Arg Cys Ser Ala Phe Pro Leu Glu Gln Glu Ala Asp Leu Ile
610 615 620
Glu Ala Ala Glu Pro Gly Gly Pro His Ser Ser Arg Asn Gly Leu Cys
625 630 635 640
His Pro Leu Asn His Ser Arg Thr Leu Ala Gly Lys Arg Pro Lys Ala
645 650 655
Pro His Gly Glu Glu Ala His Leu Pro Pro Val Ser Asp Leu Thr Val
660 665 670
Glu Phe Asp Lys Leu Asn Leu Gln Asn Ile Gly Arg Ser Ual Ser Lys
675 680 685
Thr Pro Asp Glu Ser Thr Lys Thr Lys Asp Gln Ile Leu Thr Ser Arg
690 695 700
Ile Asn Ala Val Glu Arg Asp Leu Leu Glu Pro Ser Pro Ala Asp Gln
705 710 715 720
Leu Gly Asn Gly His Arg Arg Thr Glu Ser Glu Met Ser Ala Arg Ile
725 730 735
Ala Lys Met Ser Leu Ser Pro Ser Ser Pro Arg His Glu Asp Gln Leu
740 745 750
Glu Val Thr Arg Glu Pro Ala Arg Arg Leu Phe Leu Phe Gly Glu Glu
755 760 765
Pro Ser Lys Leu Asp Gln Asp Val Leu Ala Ala Leu Glu Cys Ala Asp
770 775 780
CA 02325822 2000-10-18
WO 99/544b8 PCT/US99/08601
9
Ual Asp Pro His Gln Phe Pro Ala Ual His Arg Trp Lys Ser Ala Val
785 790 795 800
Leu Cys Tyr Ser Pro Ser Asp Arg Gln Ser Trp Pro Ser Pro Ala Val
805 810 815
Lys Gly Arg Phe Lys Ser Gln Leu Pro Asp Leu Ser Gly Pro His Ser
820 825 830
Tyr Ser Pro Gly Arg Asn Ser Val Ala Gly Ser Asn Pro Ala Lys Pro
835 840 845
Gly Leu Gly Ser Pro Gly Arg Tyr Ser Pro Val His Gly Ser Gln Leu
850 855 860
Arg Arg Met Ala Arg Leu Ala Glu Leu Ala Ala Leu
865 870 875
<210> 3
<211> 254
<212> PRT
<213> Homo sapiens
<400> 3
Met Asp Asn Tyr Ala Asp Leu Ser Asp Thr Glu Leu Thr Thr Leu Leu
1 5 10 15
Arg Arg Tyr Asn Ile Pro His Gly Pro Val Val Gly Ser Thr Arg Arg
20 25 30
Leu Tyr Glu Lys Lys Ile Phe Glu Tyr Glu Thr Gln Arg Arg Arg Leu
35 40 45
Ser Pro Pro Ser Ser Ser Ala Ala Ser Ser Tyr Ser Phe Ser Asp Leu
50 55 60
Asn Ser Thr Arg Gly Asp Ala Asp Met Tyr Asp Leu Pro Lys Lys Glu
65 70 75 80
Asp Ala Leu Leu Tyr Gln Ser Lys Gly Tyr Asn Asp Asp Tyr Tyr Glu
85 90 95
Glu Ser Tyr Phe Thr Thr Arg Thr Tyr Gly Glu Pro Glu Ser Ala Gly
100 105 110
Pro Ser Arg Ala Ual Arg Gln Ser Val Thr Ser Phe Pro Asp Ala Asp
115 120 125
Ala Phe His His Gln Val His Asp Asp Asp Leu Leu Ser Ser Ser Glu
130 135 140
Glu Glu Cys Lys Asp Arg Glu Arg Pro Met Tyr Gly Arg Asp Ser Ala
145 150 155 160
Tyr Gln Ser Ile Thr His Tyr Arg Pro Val Ser Ala Ser Arg Ser Ser
165 170 175
Leu Asp Leu Ser Tyr Tyr Pro Thr Ser Ser Ser Thr Ser Phe Met Ser
180 185 190
Ser Ser Ser Ser Ser Ser Ser Trp Leu Thr Arg Arg Ala Ile Arg Pro
195 200 205
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
Glu Asn Arg Ala Pro Gly Ala Gly Leu Gly Gln Asp Arg Gln Val Pro
210 215 220
Leu Trp Gly Gln Leu Leu Leu Phe Leu Val Phe Val Ile Val Leu Phe
225 230 235 240
Phe Ile Tyr His Phe Met Gln Ala Glu Glu Gly Asn Pro Phe
245 250
<210>4
<211>694
<212>PRT
<213>Homo Sapiens
<400> 4
Met Pro Glu Phe Leu Glu Asp Pro Ser Val Leu Thr Lys Asp Lys Leu
1 5 10 15
Lys Ser Glu Leu Val Ala Asn Asn Val Thr Leu Pro Ala Gly Glu Gln
25 30
Arg Lys Asp Val Tyr Val Gln Leu Tyr Leu Gln His Leu Thr Ala Arg
35 40 45
Asn Arg Pro Pro Leu Pro Ala Gly Thr Asn Ser Lys Gly Pro Pro Asp
50 55 60
Phe Ser Ser Asp Glu Glu Arg Glu Pro Thr Pro Val Leu Gly Ser Gly
65 70 75 80
Ala Ala Ala Ala Gly Arg Ser Arg Ala Ala Val Gly Arg Lys Ala Thr
85 90 95
Lys Lys Thr Asp Lys Pro Arg Gln Glu Asp Lys Asp Asp Leu Asp Val
100 105 110
Thr Glu Leu Thr Asn Glu Asp Leu Leu Asp Gln Leu Ual Lys Tyr Gly
115 120 125
Val Asn Pro Gly Pro Ile Val Gly Thr Thr Arg Lys Leu Tyr Glu Lys
130 135 140
Lys Leu Leu Lys Leu Arg Glu Gln Gly Thr Glu Ser Arg Ser Ser Thr
145 150 155 160
Pro Leu Pro Thr Ile Ser Ser Ser Ala Glu Asn Thr Arg Gln Asn Gly
165 170 175
Ser Asn Asp Ser Asp Arg Tyr Ser Asp Asn Glu Glu Gly Lys Lys Lys
180 185 190
Glu His Lys Lys Val Lys Ser Thr Arg Asp Ile Val Pro Phe Ser Glu
195 200 205
Leu Gly Thr Thr Pro Ser Gly Gly Gly Phe Phe Gln Gly Ile Ser Phe
210 215 220
Pro Glu Ile Ser Thr Arg Pro Pro Leu Gly Ser Thr Glu Leu Gln Ala
225 230 235 240
Ala Lys Lys Val His Thr Ser Lys Gly Asp Leu Pro Arg Glu Pro Leu
245 250 255
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
11
Val Ala Thr Asn Leu Pro Gly Arg Gly Gln Leu Gln Lys Leu Ala Ser
260 265 270
Glu Arg Asn Leu Phe Ile Ser Cys Lys Ser Ser His Asp Arg Cys Leu
275 280 285
Glu Lys Ser Ser Ser Ser Ser Ser Gln Pro Glu His Ser Ala Met Leu
290 295 300
Val Ser Thr Ala Ala Ser Pro Ser Leu Ile Lys Glu Thr Thr Thr Gly
305 310 315 320
Tyr Tyr Lys Asp Ile Val Glu Asn Ile Cys Gly Arg Glu Lys Ser Gly
325 330 335
Ile Gln Pro Leu Cys Pro Glu Arg Ser His Ile Ser Asp Gln Ser Pro
340 345 350
Leu Ser Ser Lys Arg Lys Ala Leu Glu Glu Ser Glu Ser Ser Gln Leu
355 360 365
Ile Ser Pro Pro Leu Ala Gln Ala Ile Arg Asp Tyr Val Asn Ser Leu
370 375 380
Leu Val Gln Gly Gly Ual Gly Ser Leu Pro Gly Thr Ser Asn Ser Met
385 390 395 400
Pro Pro Leu Asp Val Glu Asn Ile Gln Lys Arg Ile Asp Gln Ser Lys
405 410 415
Phe Gln Glu Thr G1u Phe Leu Ser Pro Pro Arg Lys Ual Pro Arg Leu
420 425 430
Ser Glu Lys Ser Val Glu Glu Arg Asp Ser Gly Ser Phe Val Ala Phe
435 440 445
Gln Asn Ile Pro Gly Ser Glu Leu Met Ser Ser Phe Ala Lys Thr Val
450 455 460
Val Ser His Ser Leu Thr Thr Leu Gly Leu Glu Ual Ala Lys Gln Ser
465 470 475 480
Gln His Asp Lys Ile Asp Ala Ser Glu Leu Ser Phe Pro Phe His Glu
485 490 495
Ser Ile Leu Lys Val Ile Glu Glu Glu Trp Gln Gln Val Asp Arg Gln
500 505 510
Leu Pro Ser Leu Ala Cys Lys Tyr Pro Val Ser Ser Arg Glu Ala Thr
515 520 525
Gln Ile Leu Ser Ual Pro Lys Val Asp Asp Glu Ile Leu Gly Phe Ile
530 535 540
Ser Glu Ala Thr Pro Leu Gly Gly Ile Gln Ala Ala Ser Thr Glu Ser
545 550 555 560
Cys Asn Gln Gln Leu Asp Leu Ala Leu Cys Arg Ala Tyr Glu Ala Ala
565 570 575
Ala Ser Ala Leu Gln Ile Ala Thr His Thr Ala Phe Ual Ala Lys Ala
580 585 590
Met Gln Ala Asp Ile Ser Gln Ala A1a Gln Ile Leu Ser Ser Asp Pro
595 600 605
Ser Arg Thr His Gln Ala Leu Gly Ile Leu Ser Lys Thr Tyr Asp Ala
CA 02325822 2000-10-18
W O 99/54468 PCT/US99/08601
12
610 615 620
Ala Ser Tyr Ile Cys Glu Ala Ala Phe Asp Glu Val Lys Met Ala Ala
625 630 635 640
His Thr Met Gly Asn Ala Thr Val Gly Arg Arg Tyr Leu Trp Leu Lys
645 650 655
Asp Cys Lys Ile Asn Leu Ala Ser Lys Asn Lys Leu Ala Ser Thr Pro
660 665 670
Phe Lys Giy Gly Thr Leu Phe Gly Gly Glu Val Cys Lys Val Ile Lys
675 680 685
Lys Arg Gly Asn Lys His
690
<210> 5
<211> 2628
<212> DNA
<213> Artificial Sequence
<220>
<223> Degenerate nucleotide sequence encoding the
polypeptide of SEQ ID N0:2
<221> variation
<222> (1)...(2628)
<223> Each N is independently any one of A, T, G or C.
<400> 5
atgacnatgg aygcnytnytngcnmgnytnaarytnytnaayccngaygayytnmgngar 60
garathgtna argcnggnytnaartgyggnccnathacnwsnacnacnmgnttyathtty 120
garaaraary tngcncargcnytnytngarcarggnggnmgnytnwsnwsnttytaycay 180
caygargcng gngtnacngcnytnwsncargayccncarmgnathytnaarccngcngar 240
ggnaayccna cngaycargcnggnttywsngargaymgngayttyggntaywsngtnggn 300
ytnaayccnc cngargargargcngtnacnwsnaaracntgywsngtnccnccnwsngay 360
acngayacnt aymgngcnggngcnacngcnwsnaargarccnccnytntaytayggngtn 420
tgyccngtnt aygargaygtnccngcnmgnaaygarmgnathtaygtntaygaraayaar 480
aargargcny tncargcngtnaaratgathaarggnwsnmgnttyaargcnttywsnacn 540
mgngargayg cngaraarttygcnmgnggnathtgygaytayttyccnwsnccnwsnaar 600
acnwsnytnc cnytnwsnccngtnaaracngcnccnytnttywsnaaygaymgnytnaar 660
gayggnytnt gyytnwsngarwsngaracngtnaayaargarmgngcnaaywsntayaar 720
aayccnmgna cncargayytnacngcnaarytnmgnaargcngtngaraarggngargar 780
gayacnttyw sngayytnathtggwsnaayccnmgntayytnathggnwsnggngayaay 840
ccnacnathg tncargarggntgymgntayaaygtnatgcaygtngcngcnaargaraay 900
cargcnwsna thtgycarytnacnytngaygtnytngaraayccngayttyatgmgnytn 960
atgtayccng aygaygaygargcnatgytncaraarmgnathmgntaygtngtngayytn 1020
tayytnaaya cnccngayaaratgggntaygayacnccnytncayttygcntgyaartty 1080
ggnaaygcng aygtngtnaaygtnytnwsnwsncaycayytnathgtnaaraaywsnmgn 1140
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
13
aayaartaygayaaracnccngargaygtnathtgygarmgnwsnaaraayaarwsngtn 1200
garytnaargarmgnathmgngartayytnaarggncaytaytaygtnccnytnytnmgn 1260
gcngargaracnwsnwsnccngtnathggngarytntggwsnccngaycaracngcngar 1320
gcnwsncaygtnwsnmgntayggnggnwsnccnmgngayccngtnytnacnytnmgngcn 1380
ttygcnggnccnytnwsnccngcnaargcngargayttymgnaarytntggaaracnccn 1440
ccnmgngaraargcnggnttyytncaycaygtnaaraarwsngayccngarmgnggntty 1500
garmgngtnggnmgngarytngcncaygarytnggntayccntgggtngartaytgggar 1560
ttyytnggntgyttygtngayytnwsnwsncargarggnytncarmgnytngargartay 1620
ytnacncarcargarathggnaaraargcncarcargaracnggngarmgngargcnwsn 1680
tgymgngayaargcnacnacnwsnggnwsnaaywsnathwsngtnmgngcnttyytngay 1740
gargaygayatgwsnytngargarathaaraaymgncaraaygcngcnmgnaayaaywsn 1800
ccnccnacngtnggngcnttyggncayacnmgntgywsngcnttyccnytngarcargar 1860
gcngayytnathgargcngcngarccnggnggnccncaywsnwsnmgnaayggnytntgy 1920
cayccnytnaaycaywsnmgnacnytngcnggnaarmgnccnaargcnccncayggngar 1980
gargcncayytnccnccngtnwsngayytnacngtngarttygayaarytnaayytncar 2040
aayathggnmgnwsngtnwsnaaracnccngaygarwsnacnaaracnaargaycarath 2100
ytnacnwsnmgnathaaygcngtngarmgngayytnytngarccnwsnccngcngaycar 2160
ytnggnaayggncaymgnmgnacngarwsngaratgwsngcnmgnathgcnaaratgwsn 2220
ytnwsnccnwsnwsnccnmgncaygargaycarytngargtnacnmgngarccngcnmgn 2280
mgnytnttyytnttyggngargarccnwsnaarytngaycargaygtnytngcngcnytn 2340
gartgygcngaygtngayccncaycarttyccngcngtncaymgntggaarwsngcngtn 2400
ytntgytaywsnccnwsngaymgncarwsntggccnwsnccngcngtnaarggnmgntty 2460
aarwsncarytnccngayytnwsnggnccncaywsntaywsnccnggnmgnaaywsngtn 2520
gcnggnwsnaayccngcnaarccnggnytnggnwsnccnggnmgntaywsnccngtncay 2580
ggnwsncarytnmgnmgnatggcnmgnytngcngarytngcngcnytn 2628
<210> 6
<211> 18
<2I2> DNA
<2I3> Artificial Sequence
<220>
<223> Oligonucleotide ZC15487
<400> 6
ggacccatta catcaact Ig
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC15486
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
14
<400> 7
cctccttgct ccagtaaa lg
<210> 8
<211> 218
<212> DNA
<213> Artificial Sequence
<220>
<223> Northern Blot probe
<400> 8
ctcaggcttt actggagcaa ggaggaaggc tgtcttcttt ctaccaccat gaggcaggtg 60
tcacagctct cagccaggac ccacaaagga ttttgaagcc agctgaaggg aacccaactg 120
atcaggctgg tttttctgaa gacagagatt ttggttacag tgtgggcctg aatcctccag 180
aggaggaagc tgtgacatcc aagacctgct cggtgccc 218
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> ZC694
<400> 9
taatacgact cactatag lg
<210> 10
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC976
<400> 10
cgttgtaaaa cgacggcc 18
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
<223> Oligonucleotide ZC15976
<400> 11
cagctctgta ggtgtcggtg tc 22
<210> 12
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC15485
<400> 12
caccgacacc tacagagc 18
<210> 13
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> ZC15526
<400> 13
tgctccagta aagcctgagc caatt 25
<210> 14
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC447
<400> 14
taacaatttc acacagg 17
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC15620
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
16-
<400> 15
acagagctgg agcgactgcg 20
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC15823
<400> 16
tctctttggc agcaacatgc 20
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC16162
<400> 17
gtgcaggtac aacgtgatgc 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC16035
<400> 18 '
ctgacttcat gaggctgatg 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC16249
CA 02325822 2000-10-18
WO 99154468 PCT/US99/08601
17
<400> 19
cagggtacat cagcctcatg 20
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC16164
<400> 20
tctgtcttcc caggaaggcc 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC16163
<400> 21
ggaattgctg ccagacgtgg 20
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC16165
a
<400> 22
agagccttct cccgcagacc 20
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide 16037
<400> 23
CA 02325822 2000-10-18
WO 99/54468 PCTNS99/08601
18
ggctgctggg actcaaggac 20
<210> 24
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide AP1
<400> 24
ccatcctaat acgactcact atagggc 27
<210> 25
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC15527
<400> 25
ctcatggtgg tagaaagaag acagc 25
<210> 26
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC695
<400> 26
gatttaggtg acactatag lg
<210> 27
<211> 424
<212> DNA
<213> Artificial Sequence
<220>
<223> EST934031
<400> 27
gctcgattga aacttctgaa tccagatgac cttagagaag aaatcgtcaa agccggattg 60
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
19
aaatgtggacccattacatcaactacaaggttcatttttgagaaaaaattggctcaggct 120
ttactggagcaaggaggaaggctgtcttctttctaccaccatgaggcaggtgtcacagct 180
ctcagccaggacccacaaaggattttgaagccagctgaagggaacccaactgatcaggct 240
ggtttttctgaagacagagattttggttacagtgtgggcctgaatcctccagaggaggaa 300
gctgtgacatccaagacctgctcggtgccccctagtgacaccgacacctacagagctgga 360
gcgactgcgtctataggagccgccccctgtactatgngggtgtgtccagttgtatgagga 420
cgtc 424
<210> 28
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC15521
<400> 28
gggcaccgag caggtcttgg atgt 24
<210> 29
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide ZC15525
<400> 29
ctcaggcttt actggagcaa ggagg 25
<210>30
<211>454
<212>PRT
<213>Homo Sapiens
<400> 30
Met Pro Glu Phe Leu Glu Asp Pro Ser Val Leu Thr Lys Asp Lys Leu
1 5 10 15
Lys Ser Glu Leu Val Ala Asn Asn Val Thr Leu Pro Ala Gly Glu Gln
20 25 30
Arg Lys Asp Val Tyr Val Gln Leu Tyr Leu Gln His Leu Thr Ala Arg
35 40 45
Asn Arg Pro Pro Leu Pro Ala Gly Thr Asn Ser Lys Gly Pro Pro Asp
50 55 60
Phe Ser Ser Asp Glu Glu Arg Glu Pro Thr Pro Val Leu Giy Ser Gly
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
65 70 75 80
Ala Ala AlaAiaGly ArgSerArg AlaAla UalGlyArg LysAlaThr
85 90 95
Lys Lys ThrAspLys ProArgGln GluAsp LysAspAsp LeuAspVal
100 105 110
Thr Glu LeuThrAsn GluAspLeu LeuAsp GlnLeuVal LysTyrGly
115 120 125
Val Asn ProGlyPro IleValGly ThrThr ArgLysLeu TyrGluLys
130 135 140
Lys Leu LeuLysLeu ArgGluGln GlyThr GluSerArg SerSerThr
145 150 155 160
Pro Leu ProThrIle SerSerSer AlaGlu AsnThrArg GlnAsnGly
165 170 175
Ser Asn AspSerAsp ArgTyrSer AspAsn GluGluAsp SerLysIle
180 185 190
Glu Leu LysLeuGlu LysArgGlu ProLeu LysGlyArg AlaLysThr
195 200 205
Pro Val ThrLeuLys GlnArgArg ValGlu HisAsnGln SerTyrSer
210 215 220
Gln Ala GlyIleThr GluThrGlu TrpThr SerGlySer SerLysGly
225 230 235 240
Gly Pro LeuGlnAla LeuThrArg GluSer ThrArgGly SerArgArg
245 250 255
Thr Pro ArgLysArg ValGluThr SerGlu HisPheArg IleAspGly
260 265 270
Pro Val IleSerGlu SerThrPro IleAla GluThrIle MetAlaSer
275 280 285
Ser Asn GluSerLeu ValValAsn ArgVal ThrGlyAsn PheLysHis
290 295 300
Ala Ser ProIleLeu ProIleThr GluPhe SerAspIle ProArgArg
305 310 315 320
Ala Pro LysLysPro LeuThrArg AlaGlu UalGlyGlu LysThrGlu
325 330 335
Glu Arg ArgUalGlu ArgAspIle LeuLys GluMetPhe ProTyrGlu
340 345 350
Ala Ser ThrProThr GlyIleSer AlaSer CysArgArg ProIleLys
355 360 365
Gly Ala AlaGlyArg ProLeuGlu LeuSer AspPheArg MetGluGlu
370 375 380
Ser Phe SerSerLys TyrValPro LysTyr ValProLeu AlaAspUai
385 390 395 400
Lys Ser GluLysThr LysLysGly ArgSer IleProVal TrpIleLys
405 410 415
Ile Leu LeuPheVal ValValAla ValPhe LeuPheLeu ValTyrGln
420 425 430
CA 02325822 2000-10-18
WO 99/54468 PCT/US99/08601
21
Ala Met Glu Thr Asn Gln Val Asn Pro Phe Ser Asn Phe Leu His Val
435 440 445
Asp Pro Arg Lys Ser Asn
450
<210> 31
<211> 345
<212> PRT
<213> Homo sapiens
<400> 31
Met Pro Glu Phe Leu Glu Asp Pro Ser Val Leu Thr Lys Asp Lys Leu
1 5 10 15
Lys Ser Glu Leu Val Ala Asn Asn Val Thr Leu Pro Ala Gly Glu Gln
20 25 30
Arg Lys Asp Val Tyr Val Gln Leu Tyr Leu Gln His Leu Thr Ala Arg
35 40 45
Asn Arg Pro Pro Leu Pro Ala Gly Thr Asn Ser Lys Gly Pro Pro Asp
50 55 60
Phe Ser Ser Asp Glu Glu Arg Glu Pro Thr Pro Val Leu Gly Ser Gly
65 70 75 g0
Ala Ala Ala Ala Gly Arg Ser Arg Ala Ala Val Gly Arg Lys Ala Thr
85 90 95
Lys Lys Thr Asp Lys Pro Arg Gln Glu Asp Lys Asp Asp Leu Asp Val
100 105 110
Thr Glu Leu Thr Asn Glu Asp Leu Leu Asp Gln Leu Val Lys Tyr Gly
115 120 125
Val Asn Pro Gly Pro Ile Val Gly Thr Thr Arg Lys Leu Tyr Glu Lys
130 135 140
Lys Leu Leu Lys Leu Arg Glu Gln Gly Thr Glu Ser Arg Ser Ser Thr
145 150 155 160
Pro Leu Pro Thr Ile Ser Ser Ser Ala Glu Asn Thr Arg Gln Asn Gly
165 170 175
Ser Asn Asp Ser Asp Arg Tyr Ser Asp Asn Glu Glu Asp Ser Lys Ile
180 185 190 '
Glu Leu Lys Leu Glu Lys Arg Glu Pro Leu Lys Gly Arg Ala Lys Thr
195 200 205
Pro Val Thr Leu Lys Gln Arg Arg Val Glu His Asn Gln Val Gly Glu
210 215 220
Lys Thr Glu Glu Arg Arg Ual Glu Arg Asp Ile Leu Lys Glu Met Phe
225 230 235 240
Pro Tyr Glu Ala Ser Thr Pro Thr Gly Ile Ser Ala Ser Cys Arg Arg
245 250 255
Pro Ile Lys Gly Ala Ala Gly Arg Pro Leu Glu Leu Ser Asp Phe Arg
260 265 270
CA 02325822 2000-10-18
WO 99/54468
PCT/US99/08601
22
Met Glu Glu Ser Phe Ser Ser Lys Tyr Val Pro Lys Tyr Val Pro Leu
275 280 285
Ala Asp Val Lys Ser Glu Lys Thr Lys Lys Gly Arg Ser Ile Pro Val
290 295 300
Trp Ile Lys Ile Leu Leu Phe Val Val Val Ala Val Phe Leu Phe Leu
305 310 315 320
Val Tyr Gln Ala Met Glu Thr Asn Gln Val Asn Pro Phe Ser Asn Phe
325 330 335
Leu His Val Asp Pro Arg Lys Ser Asn
340 345
<210> 32
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide AP2
<400> 32
actcactata gggctcgagc ggc 23
