U.S. patent application number 10/636716 was filed with the patent office on 2006-07-20 for polynucleotides encoding a class ii cytokine receptor.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Robyn L. Adams, Theresa M. Farrah, Anna C. Jelmberg, Choon J. Kho, Si Lok, Theodore E. Whitmore.
Application Number | 20060160091 10/636716 |
Document ID | / |
Family ID | 27122570 |
Filed Date | 2006-07-20 |
United States Patent
Application |
20060160091 |
Kind Code |
A9 |
Lok; Si ; et al. |
July 20, 2006 |
Polynucleotides encoding a class II cytokine receptor
Abstract
Novel receptor polypeptides, polynucleotides encoding the
polypeptides, and related compositions and methods are disclosed.
The polypeptides comprise an extracellular domain of a cell-surface
receptor that is expressed in kidneys, pancreas, prostate, adrenal
cortex and nervous tissue. The polypeptides may be used within
methods for detecting ligands that promote the proliferation and/or
differentiation of these organs.
Inventors: |
Lok; Si; (Seattle, WA)
; Kho; Choon J.; (Singapore, SG) ; Jelmberg; Anna
C.; (Issaquah, WA) ; Adams; Robyn L.;
(Bellevue, WA) ; Whitmore; Theodore E.; (Redmond,
WA) ; Farrah; Theresa M.; (Seattle, WA) |
Correspondence
Address: |
Shelby J. Walker;Patent Department
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Assignee: |
ZymoGenetics, Inc.
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20040072229 A1 US 20050244832 A9 |
November 3, 2005 |
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|
Family ID: |
27122570 |
Appl. No.: |
10/636716 |
Filed: |
August 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09861779 |
May 21, 2001 |
6686448 |
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10636716 |
Aug 7, 2003 |
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09275712 |
Mar 24, 1999 |
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09861779 |
May 21, 2001 |
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08943087 |
Oct 2, 1997 |
5945511 |
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09275712 |
Mar 24, 1999 |
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08803305 |
Feb 20, 1997 |
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08943087 |
Oct 2, 1997 |
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Current U.S.
Class: |
435/6.16 ;
435/193; 435/320.1; 435/325; 435/69.1; 530/350; 530/391.1;
536/23.5 |
Current CPC
Class: |
C07K 2319/02 20130101;
Y10S 930/12 20130101; C07K 14/715 20130101; Y10S 930/14
20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5; 435/193;
530/391.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12N 9/10 20060101
C12N009/10; C12P 21/02 20060101 C12P021/02; C12N 5/06 20060101
C12N005/06; C07K 14/705 20060101 C07K014/705 |
Claims
1. An isolated polynucleotide encoding a ligand-binding receptor
polypeptide, said polypeptide comprising a sequence of amino acids
selected from the group consisting of: (a) residues 30 to 250 of
SEQ ID NO:2; (b) allelic variants of (a); and (c) sequences that
are at least 80% identical to (a) or (b).
2. An isolated polynucleotide according to claim 1 wherein said
polypeptide further comprises a transmembrane domain.
3. An isolated polynucleotide according to claim 2 wherein said
transmembrane domain comprises residues 251 to 274 of SEQ ID NO:2,
or an allelic variant thereof.
4. An isolated polynucleotide according to claim 2 wherein said
polypeptide further comprises an intracellular domain.
5. An isolated polynucleotide according to claim 4 wherein said
intracellular domain comprises residues 275 to 553 of SEQ ID NO:2,
or an allelic variant thereof.
6. An isolated polynucleotide according to claim 1 which is a DNA
as shown in SEQ ID NO:1 from nucleotide 339 to nucleotide 1009.
7. An isolated polynucleotide according to claim 1 wherein said
polypeptide further comprises an affinity tag.
8. An isolated polynucleotide according to claim 7 wherein said
affinity tag is polyhistidine, protein A, glutathione S
transferase, substance P, or an immunoglobulin heavy chain constant
region.
9. An isolated polynucleotide according to claim 1 wherein said
polynucleotide is DNA.
10. An isolated polynucleotide encoding a polypeptide selected from
a group defined SEQ ID NO:2 consisting of residues 1 to 250,
residues 1 to 274, residues 1 to 553, residues 2 to 250, residues 2
to 274, residues 2 to 553, residues 251 to 274, residues 251 to 553
and residues 275 to 553.
11. An expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment encoding a
ligand-binding receptor polypeptide, said polypeptide comprising a
sequence of amino acids selected from the group consisting of: (a)
residues 30 to 250 of SEQ ID NO:2; (b) allelic variants of (a); and
(c) sequences that are at least 80% identical to (a) or (b); and a
transcription terminator.
12. An expression vector according to claim 11 wherein said
polypeptide further comprises a signal sequence.
13. An expression vector according to claim 11 wherein said
polypeptide further comprises a transmembrane domain.
14. An expression vector according to claim 11 wherein said
transmembrane domain comprises residues 251 to 274 of SEQ ID NO:2,
or an allelic variant thereof.
15. An expression vector according to claim 13 wherein said
polypeptide further comprises an intracellular domain.
16. An expression vector according to claim 15 wherein said
intracellular domain comprises residues 275 to 553 of SEQ ID NO:2,
or an allelic variant thereof.
17. An expression vector comprising the following operably linked
elements: (a) a transcription promoter; (b) a DNA segment encoding
a chimeric polypeptide, wherein said chimeric polypeptide is
comprised of a first portion and a second portion joined by a
peptide bond, said first portion consisting essentially of a ligand
binding domain of a receptor polypeptide selected from the group
consisting of: (i) a receptor polypeptide as shown in SEQ ID NO:2;
(ii) allelic variants of SEQ ID NO:2; and (iii) receptor
polypeptides that are at least 80% identical to (i) or (ii), and
said second portion consisting essentially of an affinity tag; and
(c) a transcription terminator.
18. An expression vector according to claim 17 wherein said
affinity tag is an immunoglobulin F.sub.c polypeptide.
19. A transformed or transfected cell into which has been
introduced an expression vector according to claim 11, wherein said
cell expresses a receptor polypeptide encoded by the DNA
segment.
20. The cell of claim 19 wherein said cell is prokaryotic.
21. The cell of claim 19 wherein said cell is eukaryotic.
Description
[0001] The present application is a divisional of U.S. patent
application Ser. No. 08/943,087, now U.S. Pat. No. 5,945,511, which
is a continuation-in-part of U.S. patent application Ser. No.
08/803,305 filed Feb. 20, 1997, which is now abandoned.
BACKGROUND OF THE INVENTION
[0002] Cytokines are soluble proteins that influence the growth and
differentiation of many cell types. Their receptors are composed of
one or more integral membrane proteins that bind the cytokine with
high affinity and transduce this binding event to the cell through
the cytoplasmic portions of the certain receptor subunits. Cytokine
receptors have been grouped into several classes on the basis of
similarities in their extracellular ligand binding domains. For
example, the receptor chains responsible for binding and/or
transducing the effect of interferons (IFNs) are members of the
type II cytokine receptor family (CRF2), based upon a
characteristic 200 residue extracellular domain. The demonstrated
in vivo activities of these interferons illustrate the enormous
clinical potential of, and need for, other cytokines, cytokine
agonists, and cytokine antagonists.
SUMMARY OF THE INVENTION
[0003] The present invention fills this need by providing novel
cytokine receptors and related compositions and methods. In
particular, the present invention provides for an extracellular
ligand-binding region of a mammalian Zcytor7 receptor,
alternatively also containing either a transmembrane domain or both
an intracellular domain and a transmembrane domain.
[0004] Within one aspect, the present invention provides an
isolated polynucleotide encoding a ligand-binding receptor
polypeptide. The polypeptide comprises a sequence of amino acids
selected from the group consisting of (a) residues 30 through 250
of SEQ ID NO:2; (b) allelic variants of (a); and (c) sequences that
are at least 80% identical to (a) or (b). Within one embodiment,
the polypeptide comprises residues 30 through 250 of SEQ ID NO:2.
Within another embodiment, the polypeptide encoded by the isolated
polynucleotide further comprises a transmembrane domain. The
transmembrane domain may comprise residues 251 through 274 of SEQ
ID NO:2, or an allelic variant thereof. Within another embodiment,
the polypeptide encoded by the isolated polynucleotide further
comprises an intracellular domain, such as an intracellular domain
comprising residues 275 through 553 of SEQ ID NO:2, or an allelic
variant thereof. Within further embodiments, the polynucleotide
encodes a polypeptide that comprises residues 1 through 553, 1
through 274, 1 through 250, 30 through 274 or 30 through 553 of SEQ
ID NO:2. Within an additional embodiment, the polypeptide further
comprises an affinity tag. Within a further embodiment, the
polynucleotide is DNA. Also claimed are the isolated polypeptides
encoded by these polynucleotides.
[0005] Within a second aspect of the invention there is provided an
expression vector comprising (a) a transcription promoter; (b) a
DNA segment encoding a ligand-binding receptor polypeptide, wherein
the ligand-binding receptor polypeptide comprises a sequence of
amino acids selected from the group consisting of: (i) residues 30
through 250 of SEQ ID NO:2; (ii) allelic variants of (i); and (iii)
sequences that are at least 80% identical to (i) or (ii); and (c) a
transcription terminator, wherein the promoter, DNA segment, and
terminator are operably linked. The ligand-binding receptor
polypeptide may further comprise a secretory peptide, a
transmembrane domain, a transmembrane domain and an intracellular
domain, or a secretory peptide, a transmembrane domain and an
intracellular domain.
[0006] Within a third aspect of the invention there is provided a
cultured eukaryotic cell into which has been introduced an
expression vector as disclosed above, wherein said cell expresses a
receptor polypeptide encoded by the DNA segment. Within one
embodiment, the cell further expresses a necessary receptor subunit
which forms a functional receptor complex. Within another
embodiment, the cell is dependent upon an exogenously supplied
hematopoietic growth factor for proliferation.
[0007] Within a fourth aspect of the invention there is provided an
isolated polypeptide comprising a sequence selected from the group
consisting of (a) residues 30, a valine, through residue 250, a
lysine, of SEQ ID NO:2; (b) allelic variants of (a); and (c)
sequences that are at least 80% identical to (a) or (b), wherein
said polypeptide is substantially free of transmembrane and
intracellular domains ordinarily associated with hematopoietic
receptors. Also claimed are polypeptides comprised of a sequence
defined by residues 30, a valine, through residue 274, a tyrosine;
and a polypeptide comprised of a sequence defined by residues 30, a
valine, through residue 553 an asparagine. Also claimed are the
polypeptides and polynucleotides defined by the sequences of SEQ ID
NOs: 13-60.
[0008] Within a further aspect of the invention there is provided a
chimeric polypeptide consisting essentially of a first portion and
a second portion joined by a peptide bond. The first portion of the
chimeric polypeptide consists essentially of a ligand binding
domain of a receptor polypeptide selected from the group consisting
of (a) a receptor polypeptide as shown in SEQ ID NO:2; (b) allelic
variants of SEQ ID NO:2; and (c) receptor polypeptides that are at
least 80% identical to (a) or (b). The second portion of the
chimeric polypeptide consists essentially of an affinity tag.
Within one embodiment the affinity tag is an immunoglobulin F.sub.c
polypeptide. The invention also provides expression vectors
encoding the chimeric polypeptides and host cells transfected to
produce the chimeric polypeptides.
[0009] The present invention also provides for an isolated
polynucleotide encoding a polypeptide selected from a group defined
SEQ ID NO:2 consisting of residues 1 through 250, residues 1
through 274, residues 1 through 553, residues 2 through 250,
residues 2 through 274, residues 2 through 553, residues 251
through 274, residues 251 through 553 and residues 275 through 553.
Also claimed are the isolated polypeptide expressed by these
polynucleotides.
[0010] The invention also provides a method for detecting a ligand
within a test sample, comprising contacting a test sample with a
polypeptide as disclosed above, and detecting binding of the
polypeptide to ligand in the sample. Within one embodiment the
polypeptide further comprises transmembrane and intracellular
domains. The polypeptide can be membrane bound within a cultured
cell, wherein the detecting step comprises measuring a biological
response in the cultured cell. Within another embodiment, the
polypeptide is immobilized on a solid support.
[0011] Within an additional aspect of the invention there is
provided an antibody that specifically binds to a polypeptide as
disclosed above, as well as an anti-idiotypic antibody which binds
to the antigen-binding region of an antibody to Zcytor7.
[0012] In still another aspect of the present invention,
polynucleotide primers and probes are provided which can detect
mutations in the Zcytor7 gene. The polynucleotide probe should at
least be 20-25 bases in length, preferably at least 50 bases in
length and most preferably about 80 to 100 bases in length. In
addition to the detection of mutations, these probes can be used to
discover the Zcytor7 gene in other mammalian species. The probes
can either be positive strand or anti-sense strands, and they can
be comprised of DNA or RNA.
[0013] These and other aspects of the invention will become evident
upon reference to the following detailed description and the
attached drawing.
DETAILED DESCRIPTION OF THE INVENTION
[0014] 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.
[0015] 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.
[0016] 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.
[0017] "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.
[0018] 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.
[0019] 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 sequences are commonly, but not always,
found in the 5' non-coding regions of genes.
[0020] The term "receptor" is used herein to denote a
cell-associated protein, or a polypeptide subunit of such a
protein, that binds to a bioactive molecule (the "ligand") and
mediates the effect of the ligand on the cell. Binding of ligand to
receptor results in a conformational change in the receptor (and,
in some cases, receptor multimerization, i.e., association of
identical or different receptor subunits) that causes interactions
between the effector domain(s) and other molecule(s) in the cell.
These interactions in turn lead to alterations in the metabolism of
the cell. Metabolic events that are linked to receptor-ligand
interactions include gene transcription, phosphorylation,
dephosphorylation, cell proliferation, increases in cyclic AMP
production, mobilization of cellular calcium, mobilization of
membrane lipids, cell adhesion, hydrolysis of inositol lipids and
hydrolysis of phospholipids. The term "receptor polypeptide" is
used to denote complete receptor polypeptide chains and portions
thereof, including isolated functional domains (e.g.,
ligand-binding domains).
[0021] A "secretory signal sequence" is 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.
[0022] A "soluble receptor" is a receptor polypeptide that is not
bound to a cell membrane. Soluble receptors are most commonly
ligand-binding receptor polypeptides that lack transmembrane and
cytoplasmic domains. Soluble receptors can comprise additional
amino acid residues, such as affinity tags that provide for
purification of the polypeptide or provide sites for attachment of
the polypeptide to a substrate, or immunoglobulin constant region
sequences. Many cell-surface receptors have naturally occurring,
soluble counterparts that are produced by proteolysis or translated
from alternatively spliced mRNAs. Receptor polypeptides are said to
be substantially free of transmembrane and intracellular
polypeptide segments when they lack sufficient portions of these
segments to provide membrane anchoring or signal transduction,
respectively.
[0023] The present invention is based in part upon the discovery of
a novel DNA sequence that encodes a protein having the structure of
a cytokine receptor, including the conserved WSXWS motif (SEQ ID
NO: 10). Analysis of the tissue distribution of the mRNA
corresponding to this novel DNA showed that mRNA level was highest
in pancreas, prostate, kidney and adrenal cortex followed by lower
levels in testis, stomach, adrenal medulla and thymus. The receptor
has been designated "Zcytor7".
[0024] Cytokine receptors subunits are characterized by a
multi-domain structure comprising a ligand-binding domain and an
effector domain that is typically involved in signal transduction.
Multimeric cytokine receptors include homodimers (e.g., PDGF
receptor .alpha..alpha. and .beta..beta. isoforms, erythropoietin
receptor, MPL [thrombopoietin receptor], and G-CSF receptor),
heterodimers whose subunits each have ligand-binding and effector
domains (e.g., PDGF receptor .alpha..beta. isoform), and multimers
having component subunits with disparate functions (e.g., IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSF receptors). Some receptor
subunits are common to a plurality of receptors. For example, the
AIC2B subunit, which cannot bind ligand on its own but includes an
intracellular signal transduction domain, is a component of IL-3
and GM-CSF receptors. Many cytokine receptors can be placed into
one of four related families on the basis of their structures and
functions. Class I hematopoietic receptors, for example, are
characterized by the presence of a domain containing conserved
cysteine residues and the WSXWS motif (SEQ ID NO: 10). Additional
domains, including protein kinase domains; fibronectin type III
domains; and immunoglobulin domains, which are characterized by
disulfide-bonded loops, are present in certain hematopoietic
receptors. Cytokine receptor structure has been reviewed by Urdal,
Ann. Reports Med. Chem. 26:221-228, 1991 and Cosman, Cytokine
5:95-106, 1993. It is generally believed that under selective
pressure for organisms to acquire new biological functions, new
receptor family members arose from duplication of existing receptor
genes leading to the existence of multi-gene families. Family
members thus contain vestiges of the ancestral gene, and these
characteristic features can be exploited in the isolation and
identification of additional family members.
[0025] Cell-surface cytokine receptors are further characterized by
the presence of additional domains. These receptors are anchored in
the cell membrane by a transmembrane domain characterized by a
sequence of hydrophobic amino acid residues (typically about 21-25
residues), which is commonly flanked by positively charged residues
(Lys or Arg). On the opposite end of the protein from the
extracellular domain and separated from it by the transmembrane
domain is an intracellular domain.
[0026] The novel receptor of the present invention, Zcytor7, is a
class II cytokine receptor. These receptors usually bind to
four-helix-bundle cytokines. Interleukin-10 and the interferons
have receptors in this class (e.g., interferon-gamma alpha and beta
chains and the interferon-alpha/beta receptor alpha and beta
chains). Class II cytokine receptors are characterized by the
presence of one or more cytokine receptor modules (CRM) in their
extracellular domains. The CRMs of class II cytokine receptors are
somewhat different than the better known CRMs of class I cytokine
receptors. While the class II CRMs contain two type-III
fibronectin-like domains, they differ in organization. In
particular, they contain two WSXWS (SEQ ID NO: 10) motifs, one in
each fibronectin III-like domain. These WSXWS (SEQ ID NO: 10)
motifs, however, are less conserved than those found in class I
CRMs.
[0027] Zcytor7, like all known class II receptors except
interferon-alpha/beta receptor alpha chain, has only a single class
II CRM in its extracellular domain. Zcytor7 appears to be a
receptor for a helical cytokine of the interferon/IL-10 class.
Using the Zcytor7 receptor we can identify ligands and additional
compounds which would be of significant therapeutic value.
Furthermore, the extracellular portion of Zcytor7 extending from
residue 30, a valine, through residue 250 of SEQ ID NO: 2 can be
expressed and used as a soluble receptor to down-regulate the
effects of the ligand of Zcytor7.
[0028] As was stated above, Zcytor7 was initially identified by the
overall homology to CRF2-4, an orphan Class II cytokine receptor.
See Lutfalla G. et al. Genomics, 16: 366-373 (1993). Analysis of a
human cDNA clone encoding Zcytor7 (SEQ ID NO: 1) revealed an open
reading frame encoding 553 amino acids (SEQ ID NO:2) comprising an
extracellular ligand-binding domain of approximately 221 amino acid
residues (residues 30-250 of SEQ ID NO:2), a transmembrane domain
of approximately 24 amino acid residues (residues 251-274 of SEQ ID
NO:2), and an intracellular domain of approximately 279 amino acid
residues (residues 275-553 of SEQ ID NO:2). 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. Deletion of residues from the ends of the domains
is possible.
[0029] Within preferred embodiments of the invention the isolated
polynucleotides will hybridize to similar sized regions of SEQ ID
NO: 1 or a sequence complementary thereto, under stringent
conditions. In general, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point (T.sub.m)
for the specific sequence at a defined ionic strength and pH. The
T.sub.m is the temperature (under defined ionic strength and pH) at
which 50% of the target sequence hybridizes to a perfectly matched
probe. Typical stringent conditions are those in which the salt
concentration is at least about 0.02 M at pH 7 and the temperature
is at least about 60.degree. C. As previously noted, the isolated
polynucleotides of the present invention include DNA and RNA.
Methods for isolating DNA and RNA are well known in the art. It is
generally preferred to isolate RNA from pancreas or prostate
tissues although cDNA can also be prepared using RNA from other
tissues or isolated as genomic DNA. Total RNA can be prepared using
guanidine HCl extraction followed by isolation by centrifugation in
a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979).
Poly (A).sup.+ RNA is prepared from total RNA using the method of
Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972).
Complementary DNA (cDNA) is prepared from poly(A).sup.+ RNA using
known methods. Polynucleotides encoding ZCytor7 polypeptides are
then identified and isolated by, for example, hybridization or
PCR.
[0030] Those skilled in the art will recognize that the sequences
disclosed in SEQ ID NOS:1 and 2 represent single alleles of the
human ZCytor7 receptor. Allelic variants of these sequences can be
cloned by probing cDNA or genomic libraries from different
individuals according to standard procedures. Other specific
embodiments include the polynucleotides and polypeptides defined by
SEQ ID NOs: 13-60.
[0031] The present invention further provides counterpart receptors
and polynucleotides from other species ("species orthologs"). Of
particular interest are ZCytor7 receptors from other mammalian
species, including murine, porcine, ovine, bovine, canine, feline,
equine, and other primate receptors. Species orthologs of the human
ZCytor7 receptor 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 the receptor.
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 receptor-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. Pat. No. 4,683,202), using
primers designed from the sequences 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 the receptor. Similar techniques can
also be applied to the isolation of genomic clones.
[0032] The present invention also provides isolated receptor
polypeptides that are substantially homologous to the receptor
polypeptide of SEQ ID NO: 2. By "isolated" is meant a protein or
polypeptide 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. 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
NO:2. Such polypeptides will more preferably be at least 90%
identical, and most preferably 95% or more identical to SEQ ID
NO:2. Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:
603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915-10919, 1992. 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 "blossom 62" scoring matrix
of Henikoff and Henikoff (ibid.) as shown in Table 1 (amino acids
are indicated by the standard one-letter codes). The percent
identity is then calculated as: Total .times. .times. number
.times. .times. of .times. .times. identical .times. .times.
matches [ length .times. .times. of .times. .times. the .times.
.times. longer .times. .times. sequence .times. .times. plus
.times. .times. the .times. .times. number .times. .times. of
.times. .times. gaps introduced .times. .times. into .times.
.times. the .times. .times. longer .times. .times. sequence .times.
.times. in .times. .times. order .times. .times. to .times. .times.
align .times. .times. the two .times. .times. sequences ] .times.
100 ##EQU1## TABLE-US-00001 TABLE 1 A R N D C Q E G H I L K M F P S
T W Y V A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0
-3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2
8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K
-1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5
F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2
-2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0
-1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W - -3 -4 -4 -2 -2 -3
-2 -2 -3 -2 -3 - 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2
-1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0
-3 -1 4
[0033] Sequence identity of polynucleotide molecules is determined
by similar methods using a ratio as disclosed above.
[0034] 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 (see Table 2)
and other substitutions that do not significantly affect the
folding or activity of the protein or polypeptide. Also claimedmall
deletions of SEQ ID NO:2, 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 a small extension that facilitates
purification (an affinity tag), such as 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), 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., Pharmacia Biotech,
Piscataway, N.J.). TABLE-US-00002 TABLE 2 Conservative amino acid
substitutions Basic: arginine lysine histidine Acidic: glutamic
acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine
isoleucine valine Aromatic: phenylalanine tryptophan tyrosine
Small: glycine alanine serine threonine methionine
[0035] Essential amino acids in the receptor 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 and Wells, Science 244, 1081-1085, 1989;
Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 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 (e.g., ligand binding and signal
transduction) to identify amino acid residues that are critical to
the activity of the molecule. Sites of ligand-receptor interaction
can also be determined by analysis of crystal structure as
determined by such techniques as nuclear magnetic resonance,
crystallography or photoaffinity labeling. See, for example, de Vos
et al., Science 255:306-312, 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 receptors.
[0036] 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 241:53-57, 1988) or
Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 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. 30:10832-10837, 1991; Ladner
et al., U.S. Pat. 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)
[0037] Mutagenesis methods as disclosed above can be combined with
high-throughput screening methods to detect activity of cloned,
mutagenized receptors 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 receptors or portions thereof (e.g.,
ligand-binding fragments) 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.
[0038] Using the methods discussed above, one of ordinary skill in
the art can prepare a variety of polypeptides that are
substantially homologous to residues 30 to 250 of SEQ ID NO:2 or
allelic variants thereof and retain the ligand-binding properties
of the wild-type receptor. Such polypeptides may include additional
amino acids from an extracellular ligand-binding domain of a
Zcytor7 receptor as well as part or all of the transmembrane and
intracellular domains. Such polypeptides may also include
additional polypeptide segments as generally disclosed above.
[0039] Polynucleotides, generally a cDNA sequence, of the present
invention encode the above-described polypeptides. A cDNA sequence
which encodes a polypeptide of the present invention is comprised
of a series of codons, each amino acid residue of the polypeptide
being encoded by a codon and each codon being comprised of three
nucleotides. The amino acid residues are encoded their respective
codons as follows. [0040] Alanine (Ala) is encoded by GCA, GCC, GCG
or GCT; [0041] Cysteine (Cys) is encoded by TGC or TGT; [0042]
Aspartic acid (Asp) is encoded by GAC or GAT; [0043] Glutamic acid
(Glu) is encoded by GAA or GAG; [0044] Phenylalanine (Phe) is
encoded by TTC or TTT; [0045] Glycine (Gly) is encoded by GGA, GGC,
GGG or GGT; [0046] Histidine (His) is encoded by CAC or CAT; [0047]
Isoleucine (Ile) is encoded by ATA, ATC or ATT; [0048] Lysine (Lys)
is encoded by AAA, or AAG; [0049] Leucine (Leu) is encoded by TTA,
TTG, CTA, CTC, CTG or CTT; [0050] Methionine (Met) is encoded by
ATG; [0051] Asparagine (Asn) is encoded by AAC or AAT; [0052]
Proline (Pro) is encoded by CCA, CCC, CCG or CCT; [0053] Glutamine
(Gin) is encoded by CAA or CAG; [0054] Arginine (Arg) is encoded by
AGA, AGG, CGA, CGC, CGG or CGT; [0055] Serine (Ser) is encoded by
AGC, AGT, TCA, TCC, TCG or TCT; [0056] Threonine (Thr) is encoded
by ACA, ACC, ACG or ACT; [0057] Valine (Val) is encoded by GTA,
GTC, GTG or GTT; [0058] Tryptophan (Trp) is encoded by TGG; and
[0059] Tyrosine (Tyr) is encoded by TAC or TAT.
[0060] It is to be recognized that according to the present
invention, when a cDNA is claimed as described above, it is
understood that what is claimed are both the sense strand, the
anti-sense strand, and the DNA as double-stranded having both the
sense and anti-sense strand annealed together by their respective
hydrogen bonds. Also claimed is the messenger RNA (mRNA) encodes
the polypeptides of the present invention, and which mRNA is
encoded by the above the above-described cDNA. A messenger RNA
(mRNA) will encode a polypeptide using the same codons as those
defined above, with the exception that each thymine nucleotide (T)
is replaced by a uracil nucleotide (U).
[0061] The receptor polypeptides of the present invention,
including full-length receptors, receptor fragments (e.g.
ligand-binding fragments), and fusion polypeptides 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 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 Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989, and Ausubel et al., ibid.
[0062] The polynucleotides of the present invention can be
synthesized using gene machines. Currently the method of choice is
the phosphoramidite method. If chemically synthesized double
stranded DNA is required for an application such as the synthesis
of a gene or a gene fragment, then each complementary strand is
made separately. The production of short genes (60 to 80 bp) is
technically straightforward and can be accomplished by synthesizing
the complementary strands and then annealing them. For the
production of longer genes (>300 bp), however, special
strategies must be invoked, because the coupling efficiency of each
cycle during chemical DNA synthesis is seldom 100%. To overcome
this problem, synthetic genes (double-stranded) are assembled in
modular form from single-stranded fragments that are from 20 to 100
nucleotides in length.
[0063] One method for building a synthetic gene requires the
initial production of a set of overlapping, complementary
oligonucleotides, each of which is between 20 to 60 nucleotides
long. The sequences of the strands are planned so that, after
annealing, the two end segments of the gene are aligned to give
blunt ends. Each internal section of the gene has complementary 3'
and 5' terminal extensions that are designed to base pair precisely
with an adjacent section. Thus, after the gene is assembled, the
only remaining requirement to complete the process is sealing the
nicks along the backbones of the two strands with T4 DNA ligase. In
addition to the protein coding sequence, synthetic genes can be
designed with terminal sequences that facilitate insertion into a
restriction endonuclease sites of a cloning vector and other
sequences should also be added that contain signals for the proper
initiation and termination of transcription and translation.
[0064] An alternative way to prepare a full-size gene is to
synthesize a specified set of overlapping oligonucleotides (40 to
100 nucleotides). After the 3' and 5' extensions (6 to 10
nucleotides) are annealed, large gaps still remain, but the
base-paired regions are both long enough and stable enough to hold
the structure together. The duplex is completed and the gaps filled
by enzymatic DNA synthesis with E. coli DNA polymerase I. This
enzyme uses the 3'-hydroxyl groups as replication initiation points
and the single-stranded regions as templates. After the enzymatic
synthesis is completed, the nicks are sealed with T4 DNA ligase.
For larger genes (.gtoreq.1,000 base pairs), the complete gene
sequence is usually assembled from double-stranded fragments that
are each put together by joining four to six overlapping
oligonucleotides (20 to 60 bp each). If there is a sufficient
amount of the double-stranded fragments after each synthesis and
annealing step, they are simply joined to one another. Otherwise,
each fragment is cloned into a vector to amplify the amount of DNA
available. In both cases, the double-stranded constructs are
sequentially linked to one another to form the entire gene
sequence. Because it is absolutely essential that a chemically
synthesized gene have the correct sequence of nucleotides, each
double-stranded fragment and then the complete sequence is
characterized by DNA sequence analysis. See Glick, Bernard R. and
Jack J. Pasternak, Molecular Biotechnology, Principles &
Applications of Recombinant DNA, (ASM Press, Washington, D.C.
1994), Itakura, K. et al. Synthesis and use of synthetic
oligonucleotides. Annu. Rev. Biochem. 53: 323-356 (1984), and
Climie, S. et al. Chemical synthesis of the thymidylate synthase
gene. Proc. Natl. Acad. Sci. USA 87:633-637 (1990).
[0065] A DNA sequence encoding a ZCytor7 receptor polypeptide can
then be 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 replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of 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.
[0066] To direct a Zcytor7 receptor polypeptide into the secretory
pathway of a host cell, a secretory signal sequence (also known as
a leader sequence, prepro sequence or pre sequence) is provided in
the expression vector. The secretory signal sequence may be that of
the receptor, or may be derived from another secreted protein
(e.g., t-PA) or synthesized de novo. The secretory signal sequence
is joined to the ZCytor7 DNA sequence in the correct reading frame.
Secretory signal sequences are commonly positioned 5' to the DNA
sequence encoding the polypeptide of interest, although certain
signal sequences may be positioned elsewhere in the DNA sequence of
interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland
et al., U.S. Pat. No. 5,143,830).
[0067] Cultured mammalian cells are preferred 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,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et
al., eds., Current Protocols in Molecular Biology, John Wiley and
Sons, Inc., NY, 1987), and liposome-mediated transfection
(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus
15:80, 1993. The production of recombinant polypeptides in cultured
mammalian cells is disclosed, for example, by Levinson et al., U.S.
Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter
et al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. 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-Ki; 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, Md. In
general, strong transcription promoters are preferred, such as
promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No.
4,956,288. Other suitable promoters include those from
metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and
the adenovirus major late promoter.
[0068] 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 may 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, multi-drug
resistance, puromycin acetyltransferase) can also be used.
[0069] Other higher eukaryotic cells can also be used as hosts,
including insect cells, plant cells and avian cells. Transformation
of insect cells and production of foreign polypeptides therein is
disclosed by Guarino et al., U.S. Pat. No. 5,162,222; Bang et al.,
U.S. Pat. No. 4,775,624; and WIPO publication WO 94/06463. The use
of Agrobacterium rhizogenes as a vector for expressing genes in
plant cells has been reviewed by Sinkar et al., J. Biosci.
(Bangalore) 11:47-58, 1987.
[0070] Fungal cells, including yeast cells, and particularly cells
of the genus Saccharomyces, can also be used within the present
invention, such as for producing receptor fragments or polypeptide
fusions. Methods for transforming yeast cells with exogenous DNA
and producing recombinant polypeptides therefrom are disclosed by,
for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al.,
U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et
al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. 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 yeast is the POT1 vector system
disclosed by Kawasaki et al. (U.S. Pat. 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. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No.
4,615,974; and Bitter, U.S. Pat. No. 4,977,092) and alcohol
dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154;
5,139,936 and 4,661,454. Transformation systems for other yeasts,
including Hansenula polymorpha, Schizosaccharomyces pombe,
Kluyveromyces lactis, Kluyveromycesfragilis, 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. 132:3459-3465, 1986 and Cregg, U.S. Pat.
No. 4,882,279. Aspergillus cells may be utilized according to the
methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods for
transforming Acremonium chrysogenum are disclosed by Sumino et al.,
U.S. Pat. No. 5,162,228. Methods for transforming Neurospora are
disclosed by Lambowitz, U.S. Pat. No. 4,486,533.
[0071] 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.
[0072] Within one aspect of the present invention, a novel receptor
is produced by a cultured cell, and the cell is used to screen for
ligands for the receptor, including the natural ligand, as well as
agonists and antagonists of the natural ligand. To summarize this
approach, a cDNA or gene encoding the receptor is combined with
other genetic elements required for its expression (e.g., a
transcription promoter), and the resulting expression vector is
inserted into a host cell. Cells that express the DNA and produce
functional receptor are selected and used within a variety of
screening systems.
[0073] Mammalian cells suitable for use in expressing ZCytor7
receptors and transducing a receptor-mediated signal include cells
that express other receptor subunits which may form a functional
complex with Zcytor7. These subunits may include those of the
interferon receptor family or of other class II or class I cytokine
receptors. It is also preferred to use a cell from the same species
as the receptor to be expressed. Within a preferred embodiment, the
cell is dependent upon an exogenously supplied hematopoietic growth
factor for its proliferation. Preferred cell lines of this type are
the human TF-1 cell line (ATCC number CRL-2003) and the AML-193
cell line (ATCC number CRL-9589), which are GM-CSF-dependent human
leukemic cell lines. In the alternative, suitable host cells can be
engineered to produce the necessary receptor subunit or other
cellular component needed for the desired cellular response. For
example, the murine cell line BaF3 (Palacios and Steinmetz, Cell
41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6:
4133-4135, 1986) or a baby hamster kidney (BHK) cell line can be
transfected to express the necessary .quadrature. subunit (also
known as KH97) as well as a ZCytor7 receptor. The latter approach
is advantageous because cell lines can be engineered to express
receptor subunits from any species, thereby overcoming potential
limitations arising from species specificity. In the alternative,
species orthologs of the human receptor cDNA can be cloned and used
within cell lines from the same species, such as a mouse cDNA in
the BaF3 cell line. Cell lines that are dependent upon one
hematopoietic growth factor, such as GM-CSF, can thus be engineered
to become dependent upon a ZCytor7 ligand.
[0074] Cells expressing functional receptor are used within
screening assays. A variety of suitable assays are known in the
art. These assays are based on the detection of a biological
response in a target cell. One such assay is a cell proliferation
assay. Cells are cultured in the presence or absence of a test
compound, and cell proliferation is detected by, for example,
measuring incorporation of tritiated thymidine or by calorimetric
assay based on the metabolic breakdown of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MT)
(Mosman, J. Immunol. Meth. 65: 55-63, 1983). An alternative assay
format uses cells that are further engineered to express a reporter
gene. The reporter gene is linked to a promoter element that is
responsive to the receptor-linked pathway, and the assay detects
activation of transcription of the reporter gene. A preferred
promoter element in this regard is a serum response element, or SRE
(see, e.g., Shaw et al., Cell 56:563-572, 1989). A preferred such
reporter gene is a luciferase gene (de Wet et al., Mol. Cell. Biol.
7:725, 1987). Expression of the luciferase gene is detected by
luminescence using methods known in the art (e.g., Baumgartner et
al., J. Biol. Chem. 269:29094-29101, 1994; Schenborn and Goiffin,
Promega Notes 41:11, 1993). Luciferase activity assay kits are
commercially available from, for example, Promega Corp., Madison,
Wis. Target cell lines of this type can be used to screen libraries
of chemicals, cell-conditioned culture media, fungal broths, soil
samples, water samples, and the like. For example, a bank of
cell-conditioned media samples can be assayed on a target cell to
identify cells that produce ligand. Positive cells are then used to
produce a cDNA library in a mammalian expression vector, which is
divided into pools, transfected into host cells, and expressed.
Media samples from the transfected cells are then assayed, with
subsequent division of pools, re-transfection, subculturing, and
re-assay of positive cells to isolate a cloned cDNA encoding the
ligand.
[0075] A natural ligand for the ZCytor7 receptor can also be
identified by mutagenizing a cell line expressing the receptor and
culturing it under conditions that select for autocrine growth. See
WIPO publication WO 95/21930. Within a typical procedure, BaF3
cells expressing ZCytor7 and the necessary additional subunits are
mutagenized, such as with 2-ethylmethanesulfonate (EMS). The cells
are then allowed to recover in the presence of IL-3, then
transferred to a culture medium lacking IL-3 and IL-4. Surviving
cells are screened for the production of a ZCytor7 ligand, such as
by adding soluble receptor to the culture medium or by assaying
conditioned media on wild-type BaF3 cells and BaF3 cells expressing
the receptor.
[0076] An additional screening approach provided by the present
invention includes the use of hybrid receptor polypeptides. These
hybrid polypeptides fall into two general classes. Within the first
class, the intracellular domain of Z-Cytor7, comprising
approximately residues 275 to 553 of SEQ ID NO:2, is joined to the
ligand-binding domain of a second receptor. It is preferred that
the second receptor be a hematopoietic cytokine receptor, such as
mpl receptor (Souyri et al., Cell 63: 1137-1147, 1990). The hybrid
receptor will further comprise a transmembrane domain, which may be
derived from either receptor. A DNA construct encoding the hybrid
receptor is then inserted into a host cell. Cells expressing the
hybrid receptor are cultured in the presence of a ligand for the
binding domain and assayed for a response. This system provides a
means for analyzing signal transduction mediated by ZCytor7 while
using readily available ligands. This system can also be used to
determine if particular cell lines are capable of responding to
signals transduced by ZCytor7. A second class of hybrid receptor
polypeptides comprise the extracellular (ligand-binding) domain of
ZCytor7 (approximately residues 30 to 250 of SEQ ID NO:2) with an
intracellular domain of a second receptor, preferably a
hematopoietic cytokine receptor, and a transmembrane domain. Hybrid
receptors of this second class are expressed in cells known to be
capable of responding to signals transduced by the second receptor.
Together, these two classes of hybrid receptors enable the
identification of a responsive cell type for the development of an
assay for detecting a Zcytor7 ligand.
[0077] Cells found to express the ligand are then used to prepare a
cDNA library from which the ligand-encoding cDNA can be isolated as
disclosed above. The present invention thus provides, in addition
to novel receptor polypeptides, methods for cloning polypeptide
ligands for the receptors.
[0078] The tissue specificity of Zcytor7 expression suggests a role
in the development of the kidney, pancreas, prostate or nervous
tissues. In view of the tissue specificity observed for this
receptor, agonists (including the natural ligand) and antagonists
have enormous potential in both in vitro and in vivo applications.
Compounds identified as receptor agonists are useful for
stimulating proliferation and development of target cells in vitro
and in vivo. For example, agonist compounds are useful as
components of defined cell culture media, and may be used alone or
in combination with other cytokines and hormones to replace serum
that is commonly used in cell culture. Agonists may be useful in
specifically promoting the growth and/or development of nervous,
pancreatic or prostate-derived cells in culture. Antagonists are
useful as research reagents for characterizing sites of
ligand-receptor interaction. In vivo, receptor agonists or
antagonists may find application in the treatment of renal, neural,
pancreatic or prostate diseases.
[0079] ZCytor7 may also be used within diagnostic systems for the
detection of circulating levels of ligand. Within a related
embodiment, antibodies or other agents that specifically bind to
ZCytor7 can be used to detect circulating receptor polypeptides.
Elevated or depressed levels of ligand or receptor polypeptides may
be indicative of pathological conditions, including cancer.
[0080] ZCytor7 receptor polypeptides can be prepared by expressing
a truncated DNA encoding the extracellular domain, for example, a
polypeptide which contains residues 30 through 250 of a human
ZCytor7 receptor (SEQ ID NO:2) or the corresponding region of a
non-human receptor. It is preferred that the extracellular domain
polypeptides be prepared in a form substantially free of
transmembrane and intracellular polypeptide segments. For example,
the C-terminus of the receptor polypeptide may be at residue 250 of
SEQ ID NO:2 or the corresponding region of an allelic variant or a
non-human receptor. To direct the export of the receptor domain
from the host cell, the receptor DNA is linked to a second DNA
segment encoding a secretory peptide, such as a t-PA secretory
peptide. To facilitate purification of the secreted receptor
domain, a C-terminal extension, such as a poly-histidine tag,
substance P, Flag.TM. peptide (Hopp et al., Biotechnology
6:1204-1210, 1988; available from Eastman Kodak Co., New Haven,
Conn.) or another polypeptide or protein for which an antibody or
other specific binding agent is available, can be fused to the
receptor polypeptide.
[0081] In an alternative approach, a receptor extracellular domain
can be expressed as a fusion with immunoglobulin heavy chain
constant regions, typically an F.sub.C fragment, which contains two
constant region domains and a hinge region but lacks the variable
region. Such fusions are typically secreted as multimeric molecules
wherein the F.sub.C portions are disulfide bonded to each other and
two receptor polypeptides are arrayed in closed proximity to each
other. Fusions of this type can be used to affinity purify the
cognate ligand from solution, as an in vitro assay tool, to block
signals in vitro by specifically titrating out ligand, and as
antagonists in vivo by administering them parenterally to bind
circulating ligand and clear it from the circulation. To purify
ligand, a ZCytor7-Ig chimera is added to a sample containing the
ligand (e.g., cell-conditioned culture media or tissue extracts)
under conditions that facilitate receptor-ligand binding (typically
near-physiological temperature, pH, and ionic strength). The
chimera-ligand complex is then separated by the mixture using
protein A, which is immobilized on a solid support (e.g., insoluble
resin beads). The ligand is then eluted using conventional chemical
techniques, such as with a salt or pH gradient. In the alternative,
the chimera itself can be bound to a solid support, with binding
and elution carried out as above. Chimeras with high binding
affinity are administered parenterally (e.g., by intramuscular,
subcutaneous or intravenous injection). Circulating molecules bind
ligand and are cleared from circulation by normal physiological
processes. For use in assays, the chimeras are bound to a support
via the F.sub.C region and used in an ELISA format.
[0082] A preferred assay system employing a ligand-binding receptor
fragment uses a commercially available biosensor instrument
(BIAcore.TM., Pharmacia Biosensor, Piscataway, N.J.), wherein the
receptor fragment is immobilized onto the surface of a receptor
chip. Use of this instrument is disclosed by Karlsson, J. Immunol.
Methods 145:229-240, 1991 and Cunningham and Wells, J. Mol. Biol.
234:554-563, 1993. A receptor fragment is covalently attached,
using amine or sulfhydryl chemistry, to dextran fibers that are
attached to gold film within the flow cell. A test sample is passed
through the cell. If ligand is present in the sample, it will bind
to the immobilized receptor polypeptide, causing a change in the
refractive index of the medium, which is detected as a change in
surface plasmon resonance of the gold film. This system allows the
determination of on- and off-rates, from which binding affinity can
be calculated, and assessment of stoichiometry of binding.
[0083] Ligand-binding receptor polypeptides can also be used within
other assay systems known in the art. Such systems include
Scatchard analysis for determination of binding affinity (see,
Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949) and calorimetric
assays (Cunningham et al., Science 253:545-548, 1991; Cunningham et
al., Science 254:821-825, 1991).
[0084] A receptor ligand-binding polypeptide can also be used for
purification of ligand. The receptor polypeptide is immobilized on
a solid support, such as beads of agarose, cross-linked agarose,
glass, cellulosic resins, silica-based resins, polystyrene,
cross-linked polyacrylamide, or like materials that are stable
under the conditions of use. Methods for linking polypeptides to
solid supports are known in the art, and include amine chemistry,
cyanogen bromide activation, N-hydroxysuccinimide activation,
epoxide activation, sulfhydryl activation, and hydrazide
activation. The resulting media will generally be configured in the
form of a column, and fluids containing ligand are passed through
the column one or more times to allow ligand to bind to the
receptor polypeptide. The ligand is then eluted using changes in
salt concentration or pH to disrupt ligand-receptor binding.
[0085] ZCytor7 polypeptides can also be used to prepare antibodies
that specifically bind to ZCytor7 polypeptides. As used herein, the
term "antibodies" includes polyclonal antibodies, monoclonal
antibodies, single-chain antibodies and antigen-binding fragments
thereof such as F(ab').sub.2 and Fab fragments, and the like,
including genetically engineered antibodies. Antibodies are defined
to be specifically binding if they bind to a ZCytor7 polypeptide
with a K.sub.a of greater than or equal to 10.sup.7/M. The affinity
of a monoclonal antibody can be readily determined by one of
ordinary skill in the art (see, for example, Scatchard, ibid.).
[0086] Methods for preparing polyclonal and monoclonal antibodies
are well known in the art (see for example, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor, N.Y., 1989; and Hurrell, J. G. R., Ed., Monoclonal
Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc.,
Boca Raton, Fla., 1982). As would be evident to one of ordinary
skill in the art, polyclonal antibodies can be generated from a
variety of warm-blooded animals such as horses, cows, goats, sheep,
dogs, chickens, rabbits, mice, and rats. The immunogenicity of a
ZCytor7 polypeptide may be increased through the use of an adjuvant
such as Freund's complete or incomplete adjuvant. A variety of
assays known to those skilled in the art can be utilized to detect
antibodies which specifically bind to ZCytor7 polypeptides.
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, radio-immunoassays,
radio-immunoprecipitations, enzyme-linked immunosorbent assays
(ELISA), dot blot assays, inhibition or competition assays, and
sandwich assays.
[0087] Antibodies to ZCytor7 may be used for tagging cells that
express the receptor, for affinity purification, within diagnostic
assays for determining circulating levels of soluble receptor
polypeptides, and as antagonists to block ligand binding and signal
transduction in vitro and in vivo.
[0088] Anti-idiotypic antibodies which bind to the antigenic
binding site of antibodies to Zcytor7 are also considered part of
the present invention. The antigenic binding region of the
anti-idiotypic antibody thus will mimic the ligand binding region
of Zcytor7. An anti-idiotypic antibody thus could be used to screen
for possible ligands of the Zcytor7 receptor. Thus neutralizing
antibodies to Zcytor7 can be used to produce anti-idiotypic
antibodies by methods well known in the art as is described in, for
example, Sambrook et al., Molecular Cloning: A Laboratory Manual,
Second Edition, (Cold Spring Harbor, N.Y., 1989); and Hurrell, J.
G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and
Applications, (CRC Press, Inc., Boca Raton, Fla., 1982).
[0089] Zcytor-7 maps 795.76 cR from the top of the human chromosome
6 linkage group on the WICGR radiation hybrid map. Relative to the
centromere, its nearest proximal marker was CHLC.GATA32B03 and its
nearest distal maker was SGC32063. The use of surrounding markers
also helped position zcytor-7 in the 6q22-q23 region on the CHLC
chromosome 6 version v8c7 integrated marker map. The locus where
Zcytor7 maps onto chromosome 6 is a common breakpoint area in ALL
(acute lymphoblastic leukemia) and NHL (non-Hodgkin lymphoma) as
well as in AML (acute myelogenous leukemia) and CML (chronic
myeloid leukemia). It is interesting to note that the MYB (avian
myeloblastosis viral oncogene homolog) gene, which encodes proteins
critical for hematopoetic. cell proliferation and development,
appears to be less than 800 kB from zcytor7. The 6q-deletion
breakpoints occur slightly distal to the MYB gene and although the
neoplasms show high levels of MYB mRNA, the gene itself appears to
be intact.
[0090] Thus Zcytor7 could be used to generate a probe that could
allow detection of an aberration of the Zctyor7 gene in the 6q
chromosome which may indicate the presence of a cancerous cell such
as leukemic cells which may still be present in after chemical or
radiation therapy. If the Zcytor7 gene is deleted by the
chromosomal abnormality, only one copy can be used to determine
whether one or two copies of the gene are present per nucleus, thus
indicating the percentage cancerous cells might be present relative
to normal cells. For further discussions on developing
polynucleotide probes and hybridization see Current Protocols in
Molecular Biology Ausubel, F. et al. Eds. (John Wiley & Sons
Inc. 1991).
Pharmaceutical Compositions
[0091] Pharmaceutical compositions can be formulated which contain
the soluble receptor, antibody or anti-idiotypic antibodies of the
present invention. Generally included in such protein therapeutic
compositions are buffers; surface adsorption inhibitors such as
surfactants and polyols; and isotonic amounts of a physiologically
acceptable salt. The composition may be formulated as an aqueous
solution or a lyophilized powder. The latter is reconstituted prior
to use with a pharmaceutically acceptable diluent such as sterile
water for injection.
[0092] Examples of buffers which can be used for the
above-described pharmaceutical compositions include low ionic
strength, physiologically acceptable buffers that are effective
within the pH range of 5.0-7.0. Such buffers include phosphate,
acetate, citrate, succinate and histidine buffers.
[0093] Examples of surface adsorption inhibitors which can be used
in the above-described pharmaceutical compositions include
non-ionic surfactants and polyols. Non-ionic surfactants include
polyoxyethylene sorbitan fatty acid esters, such as polysorbate 20
(polyoxyethylene sorbitan monolaurate), and the like. Other
non-ionic surfactants useful in this regard include polyethylene
oxides; sorbitan esters; polyoxyethylene alkyl ethers; and
glycerides of fatty acid, including glyceryl monooleate and
glyceryl monostearate. Polyols which can be used include
polyethylene glycol, e.g. PEG 3350, mannitol, xylitol, sorbitol,
inositol, and glycerol. In general, the surface adsorption
inhibitor will be included within the composition at a
concentration from 0.001% to 5%.
[0094] Physiologically acceptable salts are generally included in a
protein therapeutic composition generally in an amount isotonic to
human blood. Preferred salts in this regard include chloride salts
such as NaCl, KCl, CaCl.sub.2 and MgCl.sub.2.
[0095] Albumin may also be included in the above-described
pharmaceutical compositions. Human serum albumin is preferred for
inclusion in pharmaceutical compositions intended for human use.
Albumin is useful as an excipient in lyophilized compositions and
acts as a stabilizer when included at a concentration of 0.1-1.0%.
Albumin may useful as a surface adsorption inhibitor.
[0096] One or more preservatives may also be included in the
pharmaceutical compositions of the present invention. Common
preservative include methylparaben, propylparaben, benzyl alcohol,
m-cresol, ethylmercurithiosalycilate, phenol, thimerosol and the
like. Methods of formulation of pharmaceutical compositions are
well known in the art and are disclosed, for example, in
Remington's Pharmaceutical Sciences, Gennaro, ed., (Mack Publishing
Co., Easton, Pa., 1996) Dosages
[0097] Therapeutic doses of the protein compositions of the present
invention will generally be in the range of 0.1 to 100 .mu.g/kg of
patient per day with the exact dose determined by the
clinician.
[0098] The invention is further illustrated by the following
non-limiting examples.
EXAMPLE 1
Cloning of Zcytor7
[0099] Expressed sequence tag (EST) 277139 (SEQ ID NO:3) was
identified. The cDNA clone (ID No. 50416) was obtained from the
IMAGE consortium Lawrence Livermore National Laboratory through
Genome Systems, Inc. The cDNA was supplied as an agar stab
containing E. coli transfected with a plasmid having the cDNA of
interest. The E. coli was streaked on an agar plate. The plasmid
was designated pSL7139. The cDNA insert in plasmid pSL7139 was
sequenced. The insert was determined to be 1231 bp in length, but
was not a full length sequence.
[0100] A human testis cDNA template was made using a MARATHON.TM.
cDNA Amplification Kit (Clontech Laboratories, Inc., Palo Alto,
Calif.) according to the supplier s instructions. A 5 RACE reaction
was used to obtain a full-length cDNA. The RACE reaction was
carried out in two reactions employing two sets of primers.
Reaction I (outer nest), using primers ZC11,107 (SEQ ID NO:4) and
AP-1 (SEQ ID NO: 5) (Clontech Laboratories) was run for 35 cycles
at 98.degree. C. for 20 seconds, 45.degree. C. for 20 seconds;
68.degree. for 4 minutes and a final extension time of 10 minutes
at 68.degree. C. One .mu.l of a 1:100 dilution of the reaction
product was used as a template in reaction II (inner nest). Primers
were ZC11,108 (SEQ ID NO:6) and AP-2 (SEQ ID NO:7) (Clontech
Laboratories). The reaction was run at 98.degree. C. for 30
seconds, and 30 cycles each cycle being comprised of 98.degree. C.
for 28 seconds; 43.degree. C. for 20 seconds; and 68.degree. C. for
3.5 minutes with a final extension at 68.degree. C. for 10
minutes.
[0101] The product of the inner nest RACE reaction was subcloned
using a PCR-SCRIPT.TM. kit (Stratagene Cloning Systems, La Jolla,
Calif.) to prepare the plasmid pSLR7-1. Sequence analysis of this
plasmid indicated that the 5 RACE-generated sequence extended the
sequence of pSL7139 by 555 bp.
[0102] Full-length cDNA was obtained by screening a .lamda.ZAP.RTM.
human testis cDNA library using a probe that was generated by PCR
primers ZC11,526 (SEQ ID NO:9) and ZC11,108 (SEQ ID NO:6) and
pSLR7-1 as template and then re-amplified. The resulting probe was
purified through recovery from low-melt agarose gel electrophoresis
and was labeled with 32P-.alpha.-dCTP using a MEGAPRIME.TM.
labeling kit (Amersham Corp., Arlington, Heights, Ill.). The
labeled probe was purified on a push column (NUCTRAP.RTM. probe
purification column; Stratagene Cloning Systems).
[0103] The first strand cDNA reaction contained 15 .mu.l of human
testis twice poly d(T)-selected poly (A).sup.+ mRNA (Clontech
Laboratories) at a concentration of 1.0 .mu.g/.mu.l, and 3 .mu.l of
20 pmole/.mu.l first strand primer ZC6091 (SEQ ID NO:8) containing
an Xho I restriction site. The mixture was heated at 70.degree. C.
for 4 minutes and cooled by chilling on ice. First stand cDNA
synthesis was initiated by the addition of 12 .mu.l of first strand
buffer (5.times.SUPERSCRIP.TM. buffer; Life Technologies,
Gaithersburgh, Md.), 6 .mu.l of 100 mM dithiothreitol, 3 .mu.l of
deoxynucleotide triphosphate solution containing 10 mM each of
dTTP, dATP, dGTP, and 5-methyl-dCTP (Pharmacia LKB Biotechnology,
Piscataway, N.J.) to the RNA-primer mixture. The reaction mixture
was incubated at 37.degree. C. for 2 minutes, followed by the
addition of 15 .mu.l of 200 U/.mu.l Rnase H.sup.- reverse
transcriptase (SUPERSCRIPT II.sup.<<; Life Technologies). The
efficiency of the first strand synthesis was analyzed in a parallel
reaction by the addition of 5 .mu.Ci of .sup.32P-.alpha.dCTP to 5
.mu.l aliquot from one of the reaction mixtures to label the
reaction for analysis. The reactions were incubated at 37.degree.
C. for 10 minutes, 45.degree. C. for 1 hour, then incubated at
50.degree. C. for 10 minutes. Unincorporated .sup.32P-.alpha.dCTP
in the labeled reaction and the unincorporated nucleotides and
primers in the unlabeled first strand reactions were removed by
chromatography on a 400 pore size gel filtration column (Clontech
Laboratories). The length of labeled first strand cDNA was
determined by agarose gel electrophoresis.
[0104] The second strand reaction contained 120 .mu.l of the
unlabeled first strand cDNA, 36 .mu.l of 5.times.polymerase I
buffer (125 mM Tris: HCl, pH 7.5, 500 mM KCl, 25 mM MgCl.sub.2, 50
mM (NH.sub.4).sub.2SO.sub.4)), 2.4 .mu.l of 100 mM dithiothreitol,
3.6 .mu.l of a solution containing 10 mM of each deoxynucleotide
triphosphate, 6 .mu.l of 5 mM .beta.-NAD, 3.6 .mu.L of 3 U/.mu.l E.
coli DNA ligase (New England Biolabs), 9 .mu.l of 10 U/.mu.l E.
coli DNA polymerase I (New England Biolabs), and 1.8 .mu.l of 2
U/.mu.l RNase H (life Technologies). A 10 .mu.l aliquot from one of
the second strand synthesis reactions was labeled by the addition
of 10 .mu.Ci .sup.32P-.alpha.dCTP to monitor the efficiency of
second strand synthesis. The reactions were incubated at 16.degree.
C. for two hours, followed by the addition of 15 .mu.l T4 DNA
polymerase (10 U/.mu.l, Boerhinger Mannheim, Indianapolis, Ind.)
and incubated for an additional 5 minutes at 16.degree. C.
Unincorporated .sup.32P-.alpha.dCTP in the labeled reaction was
removed by chromatography through a 400 pore size gel filtration
(Clontech Laboratories) before analysis by agarose gel
electrophoresis. The unlabeled second strand reaction was
terminated by the addition of 20 .mu.l 0.5 M EDTA and extraction
with phenol/chloroform and chloroform followed by ethanol
precipitation in the presence of 2.5 M ammonium acetate and 4 .mu.g
of glycogen carrier. The yield of cDNA was estimated to be
approximately 3 .mu.g from starting mRNA template of 15 .mu.g.
[0105] Eco RI adapters were ligated onto the 5 ends of the cDNA
described above to enable cloning into an expression vector. A 10
.mu.l aliquot of cDNA (approximately 1.5 .mu.g) and 5 .mu.of 65
pmole/.mu.l of Eco RI adapter (Pharmacia LKB Biotechnology Inc.)
were mixed with 2 .mu.l 10.times. ligase buffer (660 mM Tris-HCl pH
7.5, 100 mM MgCl.sub.2), 2 .mu.l of 10 mM ATP and 1 .mu.l of
15.U/.mu.l T4 DNA ligase (Promega Corp., Madison, Wis.). The
reaction was incubated 2 hours at 5.degree. C., two hours at
7.5.degree. C., 2 hours at 10.degree. C, and 10 hours at
12.5.degree. C. The reaction was terminated by incubation at
70.degree. C. for 20 minutes.
[0106] To facilitate the directional cloning of the cDNA into an
expression vector, the cDNA was digested with Xho I, resulting in a
cDNA having a 5 Eco RI cohesive end and a 3 Xho cohesive end. The
Xho I restriction site at the 3 end of the cDNA had been previously
introduced using the ZC6091 primer (SEQ ID NO: 8). Restriction
enzyme digestion was carried out in a reaction mixture containing
20 .mu.l of cDNA as described above, 10 .mu.l of 10.times.H Buffer
Xho I (Boehringer Mannheim), 69 .mu.l H.sub.2O, and 1.0 .mu.l of 40
U/.mu.l Xho I (Boehringer Mannheim). Digestion was carried out at
37.degree. C. for 40 minutes. The reaction was terminated by
incubation at 70.degree. C. for 10 minutes and chromatography
through a 400 pore size gel filtration column (Clontech
Laboratories).
[0107] The cDNA was ethanol precipitated, washed with 70% ethanol,
air dried and resuspended in 14 .mu.l water, 2 .mu.l of ligase
buffer (Promega Corp., Madison, Wis.), 2 .mu.l T4 polynucleotide
kinase (10 U/.mu.l, Life Technologies). Following incubation at
37.degree. C. for 30 minutes, the cDNA was heated to 65.degree. C.
for 5 minutes, cooled on ice, and electrophoresed on a 0.8% low
melt agarose gel. The contaminating adapters and cDNA below 0.6 kb
in length were excised from the gel. The electrodes were reversed,
and the cDNA was electrophoresed until concentrated near the lane
origin. The area of the gel containing the concentrated cDNA was
excised and placed in a microfuge tube, and the approximate volume
of the gel slice was determined. An aliquot of water approximately
three times the volume of the gel slice (300 .mu.l) and 35 .mu.l
10.times..beta.-agarose I buffer (New England Biolabs) were added
to the tube, and the agarose was melted by heating to 65.degree. C.
for 15 minutes. Following equilibration of the sample to 45.degree.
C., 3 .mu.l of 1 U/.mu.l .beta.-agarose I (New England Biolabs) was
added, and the mixture was incubated for 60 minutes at 45.degree.
C. to digest the agarose. After incubation, 40 .mu.l of 3 M Na
acetate was added to the sample, and the mixture was incubated on
ice for 15 minutes. The sample was centrifuged at 14,000.times.g
for 15 minutes at room temperature to remove undigested agarose.
The cDNA was ethanol precipitated, washed in 70% ethanol, air-dried
and resuspended in 10 .mu.l water.
[0108] The resulting cDNA was cloned into the lambda phage vector
.lamda.Zap.sup.<< II (Stratagene Cloning Systems) that was
predigested with Eco RI and Xho I and dephosphorylated. Ligation of
the cDNA to the .lamda.Zap<< II vector was carried out in a
reaction mixture containing 1.0 .mu.l of prepared vector, 1.0 .mu.l
of human testis cDNA, 1.0 .mu.l 10.times. Ligase Buffer (Promega
Corp.), 1.0 .mu.l of 10 mM ATP, 5 .mu.l water, and 1.0 .mu.l of T4
DNA Ligase at 15 units/ml (Promega Corp.). The ligation mixture was
incubated at 5.degree.-15.degree. C. overnight in a temperature
gradient. After incubation, the ligation mixture was packaged into
phage using an in vitro packaging extract (Gigapack<< III
Gold packaging extract; Stratagene Cloning Systems), and the
resulting library was titered according to the manufacturer s
specifications.
[0109] The human testis .lamda.ZAP<< II library was used to
infect E. coli host cells (XL1-Blue MRF strain (Stratagene Cloning
Systems), and 1.5.times.10.sup.6 plaque forming units (pfu) were
plated onto 150-mm NZY plates at a density of about 50,000
pfu/plate. The inoculated plates were incubated overnight at
37.degree. C. Filter plaque lifts were made using nylon membranes
(Hybond.TM.-N; Amersham Corp., Arlington Heights, Ill.), according
to the procedures provided by the manufacturer. The filters were
processed by denaturation in solution containing 1.5 M NaCl and 0.5
M NaOH for 6 minutes at room temperature. The filters were blotted
briefly on filter paper to remove excess denaturation solution,
followed by neutralization for 6 minutes in 1 M Tris-HCl, pH 7.5,
and 1.5 M NaCl. Phage DNA was fixed onto the filters with 1,200
.mu.Joules of UV energy in a UV Crosslinker (Stratalinker<<;
Stratagene Cloning Systems). After fixing, the filters were first
pre-washed in an aqueous solution containing 0.25.times. standard
sodium citrate (SSC), 0.25% sodium dodecyl sulfate (SDS) and 1 mM
EDTA to remove cellular debris and then prehybridized in
hybridization solution (5.times.SSC, 5.times. Denhardts solution,
0.2% SDS and 1 mM EDTA). Heat-denatured, sheared salmon sperm DNA
at a final concentration of 100 .mu.g/ml was added. The filters
were prehybridized at 65.degree. C. overnight.
[0110] A probe was prepared as a PCR product by using
oligonucleotide primers designed to amplify the human Zcytor7
coding region. A PCR reaction mixture was prepared containing 2
.mu.l of ZC11526 (SEQ ID NO:9) 2 .mu.l of ZC11,108 (SEQ ID NO:6), 1
.mu.l of an overnight bacterial culture of pSLR7-1, 1 .mu.l of 10
mM dNTP, 10 .mu.l of 10.times. KlenTaq buffer (Clontech
Laboratories), 82 .mu.l water, and 2 .mu.l KlenTaq DNA polymerase
(Clontech laboratories). The PCR reaction was run as follows:
94.degree. C. for 1 minute; 30 cycles of 95.degree. C. for 20
seconds, 43.degree. C. for 20 seconds, 68.degree. C. for 1 minute;
then held at 68.degree. C. for 10 minutes. The PCR product was
re-amplified and gel purified on a 0.8% low melt agarose gel.
[0111] Fifty nanograms PCR product was radiolabeled with
32P-.alpha.-dCTP by random priming using the MEGAPRIME<< DNA
Labeling System (Amersham), according to the manufacturers
specifications. The prehybridization solution was replaced with
fresh hybridization solution containing 1.4.times.10.sup.6 cpm/ml
labeled probe and allowed to hybridize for 64 hours at 60.degree.
C. After hybridization, the hybridization solution was removed and
the filters were rinsed in a wash solution containing
0.25.times.SSC, 0.25% SDS and 1 mM EDTA at 65.degree. C. The
filters were placed on autoradiograph film and exposed at
-70.degree. C. with intensifying screens for 72 hours.
[0112] Examination of the autoradiographs revealed multiple regions
that hybridized with labeled probe. Agar plugs were picked from 12
regions for purification. Each agar plug was soaked 2 hours in 0.5
ml of SM solution containing 25 ml 4M NaCl, 10 ml 1M MgSO.sub.4, 25
ml 2M Tris HCl, 5 ml 2% gelatin and 935 ml H.sub.2O and 10% (v/v)
chloroform (Sambrook et al. Molecular Cloning: A Laboratory Manual,
2.sup.nd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989). After incubation, the phages from each plug
were diluted 1:1000 in SM. Aliquots of 50 .mu.l were plated on 100
mm plates containing 300 .mu.l of E. coli XL-1 Blue MRF cells. The
plates were incubated overnight at 37.degree. C., and filter lifts
were prepared, prehybridized overnight, hybridized overnight with a
hybridization solution containing 1.1.times.10.sup.6 cpm/ml labeled
probe, washed and autoradiographed. Examination of the resulting
autoradiographs revealed 10 positive signals. The positive plaques
were subjected to an additional round of purification.
[0113] The plasmids were excised using an ExASSIST/SOLR<<
system (Stratagene Cloning Systems), according to the manufacturers
specifications. These plasmid inserts were amplified by PCR for
size determination. A clone, designated pSLR7-2 was sequenced and
determined to have an insert of 3,532 bp in size.
EXAMPLE 2
Northern Blot Analysis
[0114] A 970 bp fragment of the Zcytor7 cDNA containing nucleotides
822-1791 was random primer labeled using a MULTIPRIME.TM. kit
(Amersham Corp.). Labeled cDNA was purified from free counts using
a push column (Stratagene Cloning Systems). A human RNA master dot
blot (Clontech Laboratories) for three hours at 65.degree. C., then
hybridized with 10.sup.6 cpm/ml of labeled cDNA probe. The
expression pattern for this blot, which contained RNA samples which
had been normalized to the mRNA expression levels of eight
different housekeeping genes, was highest in kidney, followed by
spinal cord, prostate, and cerebellum.
EXAMPLE 3
Expression of Human Zcytor7 mRNA in Human Tissues
[0115] Poly(A).sup.+ RNAs isolated from adrenal cortex, adrenal
medulla, brain, colon, heart, kidney, liver, lung, ovary, pancreas,
prostate, placenta, peripheral blood leukocytes, stomach, spleen,
skeletal muscle, small intestine, testis, thymus, thyroid, fetal
brain, fetal lung, fetal liver and fetal kidney were hybridized
under high stringency conditions with a radiolabeled DNA probe
containing nucleotides 822-1791 of (SEQ ID NO: 1). Membranes were
purchased from Clontech. The membrane were washed with
0.1.times.SSC, 0.1% SDS at 50.degree. C. and autoradiographed for
24 hours. The mRNA levels were highest in adrenal cortex, pancreas
and prostate with lower levels in testis, stomach, adrenal medulla
and thyroid.
EXAMPLE 4
Chromosomal Assignment and Placement of Zcytor-7
[0116] Zcytor-7 was mapped to chromosome 6 using the commercially
available version of the Whitehead Institute/MIT Center for Genome
Research's "GeneBridge 4 Radiation Hybrid Panel" (Research
Genetics, Inc., Huntsville, Ala.). The GeneBridge 4 Radiation
Hybrid Panel contains PCRable DNAs from each of 93 radiation hybrid
clones, plus two control DNAs (the BFL 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.
[0117] For the mapping of zcytor-7 with the, "GeneBridge 4 RH
Panel", 25 .mu.l reactions were set up in a PCRable 96-well
microtiter plate (Stratagene, La Jolla, Calif.) and used in a
"RoboCycler Gradient 96" thermal cycler (Stratagene). Each of the
95 PCR reactions consisted of 2.5 .mu.l 50.times. "Advantage
KlenTaq Polymerase Mix" (CLONTECH Laboratories, Inc., Palo Alto,
Calif.), 2=|1 dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,
Calif.), 1.25 .mu.l sense primer, ZC 11,131, (SEQ ID NO: 11), 1.25
.mu.l antisense primer, ZC 11,155, (SEQ ID NO: 12), 2.5 .mu.l
"RediLoad" (Research Genetics, Inc., Huntsville, Ala.), 0.5=|1
"Advantage KlenTaq Polymerase Mix" (Clontech Laboratories, Inc.),
25 ng of DNA from an individual hybrid clone or control and water
is added to bring up the total volume to 25=|1. The reactions were
overlaid with an equal amount of mineral oil and sealed. The PCR
cycler conditions were as follows: an initial 1 cycle 4 minute
denaturation at 94.degree. C., 35 cycles of a 1 minute denaturation
at 94.degree. C., 1.5 minute annealing at 63.degree. C. and 1.5
minute extension at 72.degree. C., followed by a final 1 cycle
extension of 7 minutes at 72.degree. C. The reactions were
separated by electrophoresis on a 3% NuSieve GTG agarose gel (FMC
Bioproducts, Rockland, Me.).
[0118] The results showed that zcytor-7 maps 795.76 cR from the top
of the human chromosome 6 linkage group on the WICGR radiation
hybrid map. Relative to the centromere, its nearest proximal marker
was CHLC.GATA32BO3 and its nearest distal maker was SGC32063. The
use of surrounding markers also helped position zcytor-7 in the
6q22-q23 region on the CHLC chromosome 6 version v8c7 integrated
marker map (The Cooperative Human Linkage Center, WWW server:
http://www.chlc.org/ChlclntegratedMaps.html) and to 6q22.33-q23.1
on the integrated LDB chromosome 6 map (The Genetic Location
Database, University of Southhampton, WWW
server:http://cedar.genetics.soton.ac.uk/public_html/).
[0119] This is a common breakpoint area in ALL (acute lymphoblastic
leukemia) and NHL (non-Hodgkin lymphoma) as well as in AML (acute
myelogenous leukemia) and CML (chronic myeloid leukemia). It is
interesting to note that the MYB (avian myeloblastosis viral
oncogene homolog) gene, which encodes proteins critical for
hematopoetic cell proliferation and development, appears to be less
than 800 kB from zcytor7. The 6q-deletion breakpoints occur
slightly distal to the MYB gene and although the neoplasms show
high levels of MYB mRNA, the gene itself appears to be intact.
Sequence CWU 1
1
* * * * *
References