U.S. patent application number 09/880578 was filed with the patent office on 2002-04-18 for mammalian cytokine-like receptor 5.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Adams, Robyn L., Foster, Donald C., Gilbert, Teresa, Jelmberg, Anna C., Lehner, Joyce M., Lok, Si, Presnell, Scott R., Whitmore, Theodore E..
Application Number | 20020045733 09/880578 |
Document ID | / |
Family ID | 27366659 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020045733 |
Kind Code |
A1 |
Lok, Si ; et al. |
April 18, 2002 |
Mammalian cytokine-like receptor 5
Abstract
Novel receptor polypeptides, polynucleotides encoding the
polypeptides, and related compositions. The polypeptides of the
present invention can be used to down-regulate their natural
ligands. The polynucleotides and subsequences thereof can be used
as diagnostic probes to determine if chromosome 19 is mutated. The
antibodies which bind to the polypeptides can be used to purify the
receptors and to inhibit the binding of the ligands onto the
receptors.
Inventors: |
Lok, Si; (Seattle, WA)
; Presnell, Scott R.; (Seattle, WA) ; Jelmberg,
Anna C.; (Issaquah, WA) ; Gilbert, Teresa;
(Auburn, WA) ; Whitmore, Theodore E.; (Redmond,
WA) ; Foster, Donald C.; (Lake Forest Park, WA)
; Adams, Robyn L.; (Bellevue, WA) ; Lehner, Joyce
M.; (Seattle, WA) |
Correspondence
Address: |
Paul G. Lunn
Patent Department
ZymoGenetics Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
27366659 |
Appl. No.: |
09/880578 |
Filed: |
June 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09880578 |
Jun 13, 2001 |
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09071224 |
May 1, 1998 |
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6271343 |
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60074721 |
Feb 13, 1998 |
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60045287 |
May 1, 1997 |
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Current U.S.
Class: |
530/350 ;
435/320.1; 435/325; 435/69.5; 530/351; 536/23.5 |
Current CPC
Class: |
C07K 14/715
20130101 |
Class at
Publication: |
530/350 ;
536/23.5; 530/351; 435/69.5; 435/325; 435/320.1 |
International
Class: |
C07K 014/52; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
We claim:
1. An isolated polynucleotide which encodes a mammalian
polypeptide, said polypeptide being comprised of an amino acid
sequence selected from the group consisting of SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, and SEQ ID NOs: 17-31.
2. The isolated polynucleotide of claim 1 wherein said
polynucleotide is a DNA sequence.
3. The isolated polynucleotide of claim 1 wherein said
polynucleotide is an RNA sequence.
4. An expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment encoding a
mammalian polypeptide, said polypeptide being comprised of an amino
acid sequence selected from the group consisting of SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NOs: 17-37; and a transcription
terminator.
5. An isolated polypeptide said polypeptide being comprised of an
amino acid sequence selected from the group consisting of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NOs: 17-31.
6. A peptide or polypeptide which has the amino acid sequence of an
epitope-bearing portion of a Zcytor5 polypeptide.
7. A polypeptide of claim 6 wherein the polypeptide has amino acid
sequence of at least 15 amino acid residues.
8. The polypeptide of claim 7 wherein said polypeptide is selected
from the group of polypeptide consisting of the amino acid
sequences of SEQ ID NOs: 32-37.
9. An antibody which specifically binds to an epitope-binding
sequence of a Zcytor5 polypeptide.
10. An antibody of claim 9 wherein said antibody binds to a
polypeptide comprised of an amino acid sequence selected from the
group consisting of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO:6, and SEQ
ID NOs: 17-37.
11. An anti-idiotypic antibody of an antibody of claims 9 or
10.
12. A method for producing an antibody which binds to a Zcytor5
polypeptide comprising inoculating an animal with an
epitope-bearing amino acid sequence of Zcytor5 polypeptide under
conditions wherein said animal produces antibodies which bind to
the Zcytor5 polypeptide; and isolating said antibodies.
Description
BACKGROUND OF THE INVENTION
[0001] Proliferation and differentiation of cells of multicellular
organisms are controlled by hormones and polypeptide growth
factors. These diffusable molecules allow cells to communicate with
each other and act in concert to form cells and organs, and to
repair and regenerate damaged tissue. Examples of hormones and
growth factors include the steroid hormones (e.g. estrogen,
testosterone), parathyroid hormone, follicle stimulating hormone,
the interleukins, platelet derived growth factor (PDGF), epidermal
growth factor (EGF), granulocyte-macrophage colony stimulating
factor (GM-CSF), erythropoietin (EPO) and calcitonin.
[0002] Hormones and growth factors influence cellular metabolism by
binding to receptors. Receptors may be integral membrane proteins
that are linked to signaling pathways within the cell, such as
second messenger systems. Other classes of receptors are soluble
molecules, such as the transcription factors.
[0003] Of particular interest are receptors for cytokines,
molecules that promote the proliferation and/or differentiation of
cells. Examples of cytokines include erythropoietin (EPO), which
stimulates the development of red blood cells; thrombopoietin
(TPO), which stimulates development of cells of the megakaryocyte
lineage; and granulocyte-colony stimulating factor (G-CSF), which
stimulates development of neutrophils. These cytokines are useful
in restoring normal blood cell levels in patients suffering from
anemia or receiving chemotherapy for cancer. The demonstrated in
vivo activities of these cytokines illustrates the enormous
clinical potential of, and need for, other cytokines, cytokine
agonists, and cytokine antagonists.
SUMMARY OF THE INVENTION
[0004] The present invention addresses this need by providing a
novel mammalian cytokine-like receptor called mammalian Zcytor5,
and related compositions and methods. Within one aspect, the
present invention provides an isolated human polynucleotide
encoding a ligand-binding human receptor polypeptide. The
polypeptide comprises a sequence of amino acids containing (a) the
amino acid residues of SEQ ID NO:17, residues 35 to 422 of SEQ ID
NO:2; (b) allelic variants of (a); and (c) sequences that are at
least 90%, 95% or 99% identical to (a) or (b). In an alternative
embodiment, the polypeptide is comprised of amino acid residues 30
to and including amino acid residue 422 of SEQ ID NO:2.
[0005] The present invention also provides for a polynucleotide
encoding another allelic variant of SEQ ID NO:2 which is a human
polypeptide receptor and is defined by SEQ ID NO:4 in particular
the polypeptide comprised of a sequence of amino acids containing
(a)the amino acid residues of SEQ ID NO:18, residues 34 to 425 of
SEQ ID NO:4; (b) allelic variants of (a); and (c) sequences that
are at least 90%, 95% or 99% identical to (a) or (b). In an
alternative embodiment, the polypeptide is comprised of amino acid
residues 29 to and including amino acid residue 425 of SEQ ID
NO:4.
[0006] Other polynucleotides of the present invention encode the
amino acid sequence of SEQ ID NO:21 which is a soluble receptor of
SEQ ID NO:17 that does not contain a C-terminus
phosphatidylinositol signal sequence; the amino acid sequence of
SEQ ID NO:20 is a Zcytor5 polypeptide of SEQ ID NO:2 having an
alternative N-terminus cleavage site; SEQ ID NO:22 which has an
alternative N-terminus cleavage site of the Zcytor5 polypeptide of
SEQ ID NO:4; SEQ ID NO:23 which is an amino acid of SEQ ID NO:18
that does not contain a C-terminus phosphatidylinositol signal
sequence and the amino acid sequences defined by SEQ ID NOs: 24-31
which are variants of the Zcytor5 polypeptide of SEQ ID NO:4.
[0007] Another embodiment of the present invention is a
polynucleotide which encodes rat Zcytor5. In particular, a
polynucleotide is claimed which encodes a rat polypeptide
containing (a)the amino acid sequence of SEQ ID NO:19 residues 41
to 425 of SEQ ID NO:6; (b) allelic variants of (a); and (c)
sequences that are at least 90%, 95% or 99% identical to (a) or
(b).
[0008] Within a second aspect of the invention there is provided an
expression vector comprising (a) a transcription promoter; (b) a
DNA segment encoding a secretory peptide and a ligand-binding
Zcyotor5 receptor polypeptide, containing an amino acid sequence as
described above.
[0009] 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
mammalian Zcytor5 receptor polypeptide encoded by the DNA
segment.
[0010] Within a fourth aspect of the invention there is provided an
isolated polypeptide. The polypeptide comprises a sequence of amino
acids containing (a) the amino acid sequence of SEQ ID NO:17,
residues 35 to 422 of SEQ ID NO:2; (b) allelic variants of (a); and
(c) sequences that are at least 90%, 95% or 99% identical to (a) or
(b). In an alternative embodiment, the polypeptide is comprised of
amino acid residues 30 to and including amino acid residue 422 of
SEQ ID NO:2.
[0011] The present invention also provides for another allelic
variant of SEQ ID NO:2 which is a human polypeptide receptor and is
defined by SEQ ID NO:4 in particular the polypeptide is comprised
of a sequence of amino acids containing (a) the amino acid sequence
of SEQ ID NO:18, residues 34 to 425 of SEQ ID NO:4; (b) allelic
variants of (a); and (c) sequences that are at least 90%, 95% or
99% identical to (a) or (b). In an alternative embodiment, the
polypeptide is comprised of residues 29 to 425 of SEQ ID NO:4.
[0012] Another embodiment of the present invention is a rat Zcytor5
polypeptide containing (a) the amino acid sequence of SEQ ID NO:19,
residues 41 to 425 of SEQ ID NO:6; (b) allelic variants of (a); and
(c) sequences that are at least 80% identical to (a) or (b).
[0013] 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 Zcytor5 receptor
polypeptide as described above. The invention also provides
expression vectors encoding the chimeric polypeptides and host
cells transfected to produce the chimeric polypeptides.
[0014] The invention also provides a method for detecting a ligand
within a test sample, comprising contacting a test sample with a
Zcytor5 polypeptide as disclosed above, and detecting binding of
the polypeptide to ligand in the sample. 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.
[0015] Within an additional aspect of the invention there is
provided an antibody that specifically binds to a polypeptide as
disclosed above and an anti-idiotypic antibody of an antibody which
specifically binds to a Zcytor5 antibody, also a method for
producing an antibody to Zcytor5.
[0016] An additional embodiment of the present invention relates to
a peptide or polypeptide which has the amino acid sequence of an
epitope-bearing portion of a Zcytor5 polypeptide having an amino
acid sequence described above. Peptides or polypeptides having the
amino acid sequence of an epitope-bearing portion of a Zcytor5
polypeptide of the present invention include portions of such
polypeptides with at least nine, preferably at least 15 and more
preferably at least 30 to 50 amino acids, although epitope-bearing
polypeptides of any length up to and including the entire amino
acid sequence of a polypeptide of the present invention described
above are also included in the present invention. Examples of said
polypeptides are defined by the amino acid sequences of SEQ ID NOs:
32-37.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The teachings of all of the references cited in the present
specification are incorporated in their entirety herein by
reference.
[0018] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection 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. 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 [Grussenmeyer
et al., Proc. Natl. Acad. Sci. USA 82:7952-4 (1985)], substance P,
FLAG.TM. peptide (Hopp et al., Biotechnology 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., Pharmacia Biotech,
Piscataway, N.J.).
[0019] The term "allelic variant" denotes 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.
[0020] 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.
[0021] 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.
[0022] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complem- ent pair preferably has a binding affinity
of <10.sup.9 M.+-..sup.1.
[0023] 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'.
[0024] 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'.
[0025] 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).
[0026] 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.
[0027] 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 promoters and
terminators. The identification of associated regions will be
evident-to one of ordinary skill in the art, for example, Dynan and
Tijan, Nature 316:774-78 (1985).
[0028] 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. "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.
[0029] The term "ortholog" (or "species homolog") denotes a
polypeptide or protein obtained from one species that has homology
to an analogous polypeptide or protein from a different
species.
[0030] The term "paralog" denotes a polypeptide or protein obtained
from a given species that has homology to a distinct polypeptide or
protein from that same species.
[0031] 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.
[0032] 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".
[0033] 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.
[0034] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. 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.
[0035] 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 molecule(s) 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, IL-3 receptor, GM-CSF
receptor, G-CSF receptor, erythropoietin receptor and IL-6
receptor).
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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. Analysis
of the tissue distribution of the mRNA corresponding to this novel
DNA showed that expression was present in highest amounts in
placenta, thyroid, heart and skeletal muscle with lower levels in
prostate and trachea.
[0040] 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 ma and Ad .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.
Hematopoietic receptors, for example, are characterized by the
presence of a domain containing conserved cysteine residues and the
WSXWS motif. 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. The
cytokine receptor superfamily is subdivided as shown in Table
1.
1TABLE 1 Cytokine Receptor Superfamily Immunoglobulin family CSF-1
receptor MGF receptor IL-1 receptor PDGF receptor Hematopoietin
family erythropoietin receptor G-CSF receptor IL-2 receptor
b-subunit IL-3 receptor IL-4 receptor IL-5 receptor IL-6 receptor
IL-7 receptor IL-9 receptor GM-CSF receptor a-subunit GM-CSF
receptor b-subunit Prolactin receptor CNTF receptor Oncostatin M
receptor Leukemia inhibitory factor receptor Growth hormone
receptor MPL Leptin receptor TNF receptor family TNF (p80) receptor
TNF (p60) receptor TNFR-RP CD27 CD30 CD40 4-1BB OX-40 Fas NGF
receptor Other IL-2 receptor .alpha.-subunit IL-15 receptor
.alpha.-subunit IFN-.gamma. receptor
[0041] 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.
[0042] The novel receptor of the present invention was initially
identified by the presence of the conserved WSXWS motif. Analysis
of a human cDNA clone encoding human Zcytor5 (SEQ ID NO:1) revealed
an open reading frame encoding 422 amino acids (SEQ ID NO:2) or an
allelic variant reveals an open reading of 425 amino acid residues,
SEQ ID NO:3 and SEQ ID NO:4.
[0043] Within preferred embodiments of the invention the isolated
polynucleotides will hybridize to similar sized regions of SEQ ID
NO:1, SEQ ID NO:3, or SEQ ID NO:5, 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 testis, including whole testis tissue extracts or
testicular cells, such as Sertoli cells, Leydig cells,
spermatogonia, or epididymis, although DNA 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
Zcytor5 polypeptides are then identified and isolated by, for
example, hybridization or PCR.
[0044] Those skilled in the art will recognize that the sequences
disclosed in SEQ ID NOS:1, 2, 3, 4 represent single alleles of the
human and SEQ ID NOs 5 and 6 of the rat Zcytor5 receptors. Allelic
variants of these sequences can be cloned by probing cDNA or
genomic libraries from different individuals according to standard
procedures.
[0045] The present invention further provides counterpart receptors
and polynucleotides from other species ("species orthologs"). Of
particular interest are Zcytor5 receptors from other mammalian
species, including murine, porcine, ovine, bovine, canine, feline,
equine, and other primate receptors. Species orthologs of the human
and macaque Zcytor5 receptors 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 or macaque 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.
[0046] The polynucleotides of the present invention can be
synthesized using DNA synthesizers. 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) 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.
[0047] 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 or staggered 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. 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).
[0048] Another embodiment of the present invention provides for a
peptide or polypeptide comprising an epitope-bearing portion of a
polypeptide of the invention. The epitope of the this polypeptide
portion is an immunogenic or antigenic epitope of a polypeptide of
the invention. A region of a protein to which an antibody can bind
is defined as an "antigenic epitope". See for instance, Geysen, H.
M. et al., Proc. Natl. Acad Sci. USA 81:3998-4002 (1984).
[0049] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in the
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See Sutcliffe, J.
G. et al. Science 219:660-666 (1983). Peptides capable of eliciting
protein-reactive sera are frequently represented in the primary
sequence of a protein, can be characterized by a set of simple
chemical rules, and are confined neither to immunodominant regions
of intact proteins (i.e., immunogenic epitopes) nor to the amino or
carboxyl terminals. Peptides that are extremely hydrophobic and
those of six or fewer residues generally are ineffective at
inducing antibodies that bind to the mimicked protein; longer
soluble peptides, especially those containing proline residues,
usually are effective.
[0050] Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention. Antigenic epitope-bearing peptides and polypeptides
of the present invention contain a sequence of at least nine,
preferably between 15 to about 30 amino acids contained within the
amino acid sequence of a polypeptide of the invention. However,
peptides or polypeptides comprising a larger portion of an amino
acid sequence of the invention, containing from 30 to 50 amino
acids, or any length up to and including the entire amino acid
sequence of a polypeptide of the invention, also are useful for
inducing antibodies that react with the protein. Preferably, the
amino acid sequence of the epitope-bearing peptide is selected to
provide substantial solubility in aqueous solvents (i.e., the
sequence includes relatively hydrophilic residues and hydrophobic
residues are preferably avoided); and sequences containing proline
residues are particularly preferred. All of the polypeptides shown
in the sequence listing contain antigenic epitopes to be used
according to the present invention, however, specifically designed
antigenic epitopes include the peptides defined by SEQ ID
NOs:32-37.
[0051] The present invention also provides isolated receptor
polypeptides that are substantially identical to the receptor
polypeptides of SEQ ID NOs: 2, 4 and 6 and their species orthologs.
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, 4, or 7 or their
species orthologs. Such polypeptides will more preferably be at
least 90% identical, and most preferably 95% or more identical to
SEQ ID NO:2, 4 or 6 or their species orthologs. 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 2 (amino acids are indicated by
the standard one-letter codes). The percent identity is then
calculated as: 1 Total number of identical matches [length of the
longer sequence plus the number of gaps introduced into the longer
sequence in order to align the two sequences] .times. 100
2 TABLE 2 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 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 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
[0052] Sequence identity of polynucleotide molecules is determined
by similar methods using a ratio as disclosed above.
[0053] 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 by their
respective codons as follows.
3 Alanine (Ala) is encoded by GCA, GCC, GCG or GCT; Cysteine (Cys)
is encoded by TGC or TGT; Aspartic acid (Asp) is encoded by GAC or
GAT; Glutamic acid (Glu) is encoded by GAA or GAG; Phenylalanine
(Phe) is encoded by TTC or TTT; Glycine (Gly) is encoded by GGA,
GGC, GGG or GGT; Histidine (His) is encoded by CAC or CAT;
Isoleucine (Ile) is encoded by ATA, ATC or ATT; Lysine (Lys) is
encoded by AAA, or AAG; Leucine (Leu) is encoded by TTA, TTG, CTA,
CTC, CTG or CTT; Methionine (Met) is encoded by ATG; Asparagine
(Asn) is encoded by AAC or AAT; Proline (Pro) is encoded by CCA,
CCC, CCG or CCT; Glutamine (Gln) is encoded by CAA or CAG; Arginine
(Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT; Serine (Ser) is
encoded by AGC, AGT, TCA, TCC, TCG or TCT; Threonine (Thr) is
encoded by ACA, ACC, ACG or ACT; Valine (Val) is encoded by GTA,
GTC, GTG or GTT; Tryptophan (Trp) is encoded by TGG; and Tyrosine
(Tyr) is encoded by TAC or TAT.
[0054] 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) which
encodes the polypeptides of the present invention, and which mRNA
is encoded by 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).
[0055] Substantially identical 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 3)
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 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.).
4TABLE 3 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
[0056] 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.
[0057] 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).
[0058] 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.
[0059] Using the methods discussed above, one of ordinary skill in
the art can prepare a variety of polypeptides that are
substantially homologous to residues SEQ ID NOs:2, 4, 6, 17, 18, or
19or allelic variants thereof and retain the ligand-binding
properties of the wild-type receptor.
[0060] 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.
[0061] In general, a DNA sequence encoding a Zcytor5 receptor
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 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.
[0062] To direct a Zcytor5 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 Zcytor5 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).
[0063] 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., N.Y., 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-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, 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.
[0064] 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.
[0065] 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, which
are incorporated herein by reference. 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).
[0066] 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. 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, 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. 132:3459-3465 (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.
[0067] 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.
[0068] 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.
[0069] Mammalian cells suitable for use in expressing Zcytor5
receptors and transducing a receptor-mediated signal include cells
that express a .beta.-subunit, such as the human .beta..sub.c
subunit. In this regard it is generally preferred to employ a cell
that is responsive to other cytokines that bind to receptors in the
same subfamily, such as IL-3 or GM-CSF, because such cells will
contain the requisite signal transduction pathway(s). 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 a .beta.-subunit (e.g., bc) 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 human bc subunit (also known as KH97) as well as a Zcytor5
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
Zcytor5 ligand.
[0070] 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 colorimetric
assay based on the metabolic breakdown of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
(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, e.g., Shaw et al., Cell 56:563-572 (1989). A preferred
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.
[0071] A natural ligand for the Zcytor5 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 Zcytor5 and human b.sub.c 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 Zcytor5 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.
[0072] An additional screening approach provided by the present
invention includes the use of hybrid receptor polypeptides. These
hybrid polypeptides fall into two general classes. 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 Zcytor5 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 Zcytor5.
[0073] 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.
[0074] 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 are thus useful in
specifically promoting the growth and/or development of
testis-derived cells in culture. Agonists and antagonists may also
prove useful in the study of spermatogenesis and infertility.
Antagonists are useful as research reagents for characterizing
sites of ligand-receptor interaction. In vivo, receptor agonists
may find application in the treatment of male infertility.
Antagonists of receptor function may be useful as male
contraceptive agents.
[0075] The proposed cytokine binding domain of Zcytor5 appears to
be closest to the Interleukin-6 .beta.-chain or gp130 (29%
identity). The ligand for Zcytor5 is probably a member of the
Interleukin-6 family of cytokines which at present includes:
Interleukins-6, -11, Leukemia Inhibitory Factor, Oncostatin M,
Cardiotropin-1 and Ciliary Neurotrophic Factor.
[0076] All Zcytor5 cDNAs isolated thus far do not encode a
transmembrane domain nor any recognizable cytoplasmic signaling
motifs characteristic of the Class I receptors. Structurally,
Zcytor5 bears close similarity to .alpha.-subunit of the Ciliary
neutrophil Factor receptor (CNTF-R.alpha.). It is quite possible
Zcytor5 does not have a transmembrane domain form and that the
native molecule is phosphatidyl-inositol linked to the cell
membrane in a manner similar to CNTF-R.alpha..
[0077] Rebledo et al. (J. Biol. Chem., 272: 4855-4863) provide
evidence for the existence of a third component of the
Cardiotropin-1 receptor (CT-1R). CT-1R is believed to have a
tripartite structure comprised of gp130, gp190 (LIV Receptor
.beta.) and an uncharacterized 45 kDa (CT-1R.alpha.) subunit that
appears to be linked to the cell surface through a
phosphatidyl-inositol linkage. CT-1R .alpha.appears to be important
for increased sensitivity and specificity of the receptor complex
to Cardiotropin-1. The data suggests that Zcytor5 is CT-1R.alpha..
Cardiotropin-1 is a member of the Interleukin-6 family in which
gp130 and gp190 are members of a tripartite complex is the Ciliary
neurotropic Factor receptor. In this receptor complex,
CNTF-R.alpha. comprises the third receptor subunit and it mediates
specificity and high affinity binding of the ligand complex. These
functions are similar to the proposed ones for CT-1R.alpha.. One
might then argue on the basis of "symmetry of nature" that CT-1R
.alpha. would physically resemble CNTF-R.alpha. and that the close
structural similarity of Zcytor5 to CNTF-R.alpha. would make
Zcytor5 a possible candidate for the third subunit of CT-1R.
Furthermore, the proposed 45 kDa molecular mass of CT-R.alpha.
agrees with that of Zcytor5 and the transcripts of CT-1 and Zcytor5
are found in similar tissues. In particular, both transcripts are
found in high levels in heart and in skeletal muscles, which is
consistent with the observation that ligand and their receptor
subunits are often co-expressed in the same tissue.
[0078] Cardiotropin-1 was originally cloned by function as a factor
involved in cardiac hypertrophy, an adaptive response of heart
muscle to an increased work load. Hypertrophy is characterized by
reactivation of genes expressed during fetal heart development and
by the accumulation of carsomeric proteins. If Zcytor5 proves to be
the subunit that is important in the binding and specificity of
Cardiotropin-1 to its receptor, Zcytor5 may prove to be a useful
therapeutic antagonist to counteract the hypertrophic response to
injury. Cardiotropin-1 has also been shown to promote survival of
rat dopaminergic neurons in vitro. An agonist-active soluble
receptor may potentially be useful in the treatment of neuronal
disorders such as Parkinson's disease.
[0079] Zcytor5 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
Zcytor5 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] Zcytor5 receptor polypeptides can be prepared by expressing
a DNA encoding a Zcytor5 polypeptide as described in SEQ ID NO:1, 3
and 5. 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. It is believed that amino acid 1-34 or in the
alternative amino acid residues 1-30 are secretory peptides of SEQ
ID NO:2. For SEQ ID NO:4, it is believed that residues 1-33 or in
the alternative 1-29 are secretory peptides. For the rat sequence,
it is believed that amino acid residues 1-40 define a secretory
peptide. These peptides are generally cleaved after secretion by a
mammalian cell. In the alternative, other secretory peptides could
be fused to the Zcytor5 polypeptide, such as the 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 Fc 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 Zcytor5-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. The chimeras may be used in vivo
to induce infertility. 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. Immonol
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,
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] Zcytor5 polypeptides can also be used to prepare antibodies
that specifically bind to Zcytor5 polypeptides. As used herein, the
term "antibodies" includes polyclonal antibodies, monoclonal
antibodies, 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 Zcytor5 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] Zcytor5 polypeptides can also be used to prepare antibodies
that specifically bind to Zcytor5 polypeptides. These antibodies
can then be used to manufacture anti-idiotypic antibodies. As used
herein, the term "antibodies" includes polyclonal antibodies,
monoclonal antibodies, 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 Zcytor5 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.).
[0087] 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
Zcytor5 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 Zcytor5 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.
[0088] As would be evident to one of ordinary skill in the art,
polyclonal antibodies can be generated by inoculating a variety of
warm-blooded animals such as horses, cows, goats, sheep, dogs,
chickens, rabbits, mice, hamsters, guinea pigs and rats with a
Zcytor5 polypeptide or a fragment thereof. The immunogenicity of a
Zcytor5 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 Zcytor5 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
(KLH), bovine serum albumin (BSA) or tetanus toxoid) for
immunization.
[0089] As used herein, the term "antibodies" includes polyclonal
antibodies, affinity-purified polyclonal antibodies, monoclonal
antibodies, and antigen-binding fragments, such as F(ab').sub.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, or 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 binding characteristics.
Through humanizing antibodies, biological half-life may be
increased, and the potential for adverse immune reactions upon
administration to humans is reduced.
[0090] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to Zcytor5 protein or peptide, and selection of antibody display
libraries in phage or similar vectors (for instance, through use of
immobilized or labeled Zcytor5 protein or peptide). Genes encoding
polypeptides having potential Zcytor5 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., U.S.
Pat. No. 5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner
et al., U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No.
5,571,698) and random peptide display libraries and kits for
screening such libraries are available commercially, for instance
from Clontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego,
Calif.), New England Biolabs, Inc. (Beverly, Mass.) and Pharmacia
LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the Zcytor5 sequences disclosed
herein to identify proteins which bind to Zcytor5. These "binding
proteins" which interact with Zcytor5 polypeptides can be used for
tagging cells; for isolating homolog polypeptides by affinity
purification; 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 Zcytor5 "antagonists" to
block Zcytor5 binding and signal transduction in vitro and in
vivo.
[0091] Antibodies can also be generated gene therapy. The animal is
administered the DNA or RNA which encodes Zcytor5 or an immunogenic
fragment thereof so that cells of the animals are transfected with
the nucleic acid and express the protein which in turn elicits an
immunogenic response. Antibodies which then are produced by the
animal are isolated in the form of polyclonal or monoclonal
antibodies.
[0092] Antibodies to Zcytor5 may be used for tagging cells that
express the protein, for affinity purification, within diagnostic
assays for determining circulating levels of soluble protein
polypeptides, and as antagonists to block ligand binding and signal
transduction in vitro and in vivo.
[0093] 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. Generally speaking,
antibodies against which bind to the claimed Zcytor5 polypeptides
can be raised by immunization of animals with a Zcytor5 polypeptide
or a fragment thereof. The immunogenicity of a Zcytor5 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 Zcytor5 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.
[0094] Antibodies to Zcytor5 are 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.
Uses
[0095] The tissue specificity of Zcytor5 expression suggests that
Zcytor5 may be a receptor for growth and/or maintenance factor in
the thyroid heart and skeletal muscle. Zcytor5 could therefor be
used to down regulate the effects of the factor by administering
soluble Zcytor5 to the patient. For example the soluble receptor
could be used to lessen the effect of cardiotrophin-1 on cardiac
pathologies. Thus preventing enlargement of the heart due to heart
disease. Zcytor5 could also be used as a diagnostic to test for the
presence of cardiotrophin-1 in the blood. Furthermore, Zcytor5 can
be used to discover other possible ligands which would bind to
Zcytor5.
[0096] The present invention also provides reagents which will find
use in diagnostic applications. For example, the Zcytor5 gene. A
probe comprising the Zcytor5 DNA or RNA or a subsequence thereof
can be used to determine if the Zcytor5 gene is present on
chromosome 1 or if a mutation has occurred.
[0097] Antibodies to Zcytor5 could be used to purify Zcytor5 and as
a therapeutic to modulate the effect of the Zcytor5 ligand. The
anti-idiotypic antibody to Zcytor5 could be used to purify the
ligand of Zcytor5 and the administration of the anti-idiotypic
antibody could be used to modulate the effect of the Zcytor5
ligand.
[0098] The invention is further illustrated by the following
non-limiting examples.
EXAMPLE 1
Cloning of Human Zcytor 5
[0099] Human Zcytor5 was identified from expressed sequence tag
(EST) 698365 (SEQ ID NO:7) identified in an EST database. The cDNA
containing EST 698365 was obtained from Incyte Pharmaceuticals,
Inc. as dried DNA. Upon reconstitution in water, the cDNA was
transfected into E. coli strain DH10B. The plasmid was designated
pSL8365. The EST in plasmid pSL8365 was sequenced, revealing an
insert of 952 bp.
[0100] The GENE TRAPPER.RTM. cDNA positive selection system (Life
Technologies, Gaithersburg, Md.) employing oligonucleotide ZC11,286
(SEQ ID NO:8) was used to isolate the plasmid Hzcytor5-9 from a
human lung cDNA library (obtained from Life Technologies Inc.,
Gaithersburg, Md.) in accordance with the manufacturer's
directions. Hzcytor5-9 extended the sequence of pSL8365 by 459 bp.
The sequence present in Hzcytor5-9 allowed the isolation of an
overlapping EST No. 485212 (SEQ ID NO:9), which extended the open
reading frame of Hzcytor5-9 by a further 33 codons.
[0101] A cDNA encoding full-length Zcytor5 was isolated from a
human testis cDNA library. (See Example 2 for the preparation of
the human cDNA testis library.) The library was comprised of eighty
pools of plasmid DNA, each pool comprised of 10,000 independent
recombinants. The presence of Zcytor5 cDNA in each library pool was
determined by PCR employing primers ZC11,663 (SEQ ID NO:10) and
ZC12,212 (SEQ ID NO:11). PCR was carried out using AMPLITAQ.RTM.
DNA polymerase (Perkin-Elmer) in buffer conditions recommended by
the supplier. The amplification was carried out at 94.degree. C.
for 1 minute followed by 30 cycles, each cycle consisting of 20
seconds at 94.degree. C., 1 minute at 66.degree. C. and 7 minutes
at 74.degree. C. Five cDNA pools were found to be positive for the
420 bp PCR product by agarose gel electrophoresis.
[0102] Plasmid DNA from one positive library pool was
electrophoresed into DH10B cells and plated. Colony lifts were
prepared using Hybond-N filters (Amersham; Arlington Heights, Ill.)
according to the procedure provided by the manufacturer. Following
denaturation and neutralization, DNA was cross-linked onto the
filters with 1,200 .mu.Joules of UV energy in a STRATALINKER.RTM.
(Stratagene Cloning Systems). Cell debris was removed-by several
washes in 0.25.times.standard sodium citrate (SSC), 0.25% sodium
dodecyl sulfate (SDS) and 1 mM EDTA at 65.degree. C. The filters
were then pre-hybridized overnight at 65.degree. C. in
EXPRESSHYB.RTM. solution (Clontech) with 1 mg/ml heat denatured
salmon sperm DNA. Colonies positive for Zcytor5 were identified by
hybridization with a probe that was generated from EST 484212 (SEQ
ID NO:9) cDNA employing PCR primers ZC11,663 (SEQ ID NO:10) and
ZC12,212 (SEQ ID NO:11). The PCR product probe was purified by
agarose gel electrophoresis. 100 ng of the probe was labeled with
.sup.32p dCTP using the MULTI-PRIME.RTM. DNA labeling system
(Amersham). Unincorporated label was removed with a NUCTRAP.RTM.
column (Stratagene). Probe hybridization was carried out overnight
at 65.degree. C. in EXPRESSHYB.RTM. solution at a probe
concentration of 1.times.10.sup.6 cpm/ml. The filters were washed
at 65.degree. C. in a wash buffer containing 0.25.times.SSC, 0.25
SDS and 1 mM EDTA.
[0103] Three positive signals were identified and were subjected to
colony purification via a second round of filter hybridization.
Sequence analysis of one positive clone, SEQ ID NO:3 was found to
be full length human Zcytor5. Sequencing of a several overlapping
clones revealed a second full-length sequence SEQ ID NO:1 which is
an allelic variant of SEQ ID NO:3.
EXAMPLE 2
Construction of the Human Testis cDNA Library
[0104] Fourteen .mu.l of poly d(T)--selected poly (A).sup.+human
testis mRNA (Clontech) at a concentration of 1.0 .mu.g/.mu.l was
mixed with 2 .mu.l of 20 pmole/.mu.l first strand primer ZC2938
(SEQ ID NO:12 ) containing an Sst I restriction site. The mixture
was heated at 65.degree. C. for 4 minutes and cooled by chilling on
ice. First strand cDNA synthesis was initiated by the addition of 8
.mu.l of 250 mM Tris-HCl, pH 8.3, 375 mM KC1, 15 mM MgCl.sub.2
(5.times.SUPERSCRIPT.TM. buffer; GIECO BRL), 4 .mu.l of 100 mM
dithiothreitol (DTT) and 2 .mu.l of a deoxynucleotide triphosphate
solution containing 10 mM each of DATP, dGTP, dTTP and
5-methyl-dCTP (Pharmacia LKB Biotechnology Inc.) to the RNA-primer
mixture. The reaction mixture was incubated at 45.degree. C. for 4
minutes followed by the addition of 10 .mu.l of 200 U/.mu.l RNase
H.sup.- reverse transcriptase (GIBCO BRL). The efficiency of the
first strand synthesis was analyzed in a parallel reaction by the
addition of 10 .mu.Ci of .sup.32P-.alpha.dCTP to a 10 .mu.l aliquot
of the reaction mixture to label the reaction for analysis. The
reactions were incubated at 45.degree. C. for 1 hour followed by an
incubation at 50.degree. C. for 15 minutes. Unincorporated
.sup.32P-.alpha.dCTP in the labeled reaction was removed by
chromatography on a 400 pore size gel filtration column (CHROMA
SPIN+TE-400.TM.; Clontech Laboratories Inc.). Unincorporated
nucleotides in the unlabeled first strand reaction were removed by
twice precipitating the cDNA in the presence of 10 .mu.g of
glycogen carrier, 2.5 M ammonium acetate and 2.5 volume ethanol.
The unlabeled cDNA was resuspended in 50 .mu.l water for use in
second strand synthesis. The length of the labeled first strand
cDNA was determined by agarose gel electrophoresis.
[0105] Second strand synthesis was performed on first strand cDNA
under conditions that promoted first strand priming of second
strand synthesis resulting in DNA hairpin formation. The reaction
mixture was assembled at room temperature and was comprised of 66
.mu.l of the unlabeled first strand cDNA, 20 .mu.l of 5.times.
polymerase I buffer (100 mM Tris: HCl, pH 7.4, 500 mM KCl, 25 mM
MgCl.sub.2, 50 mM (NH.sub.4).sub.2SO.sub.4), 1 .mu.l of 100 mM DTT,
1 .mu.l of a solution containing 20 mM of each deoxynucleotide
triphosphate, 3 .mu.l of 5 mM .beta.-NAD, 1 .mu.l of 4 U/.mu.l of
E. coli DNA ligase (New England Biolabs Inc., Beverly, Mass.) and 5
.mu.l of 10 U/.mu.l E. coli DNA polymerase I (New England Biolabs,
Inc.). The reaction was incubated at room temperature for 5 minutes
followed by the addition of 2 .mu.l of 2.2 U/.mu.l RNase H (GIBCO
BRL). A parallel reaction in which a 10 .mu.l aliquot of the second
strand synthesis mixture was labeled by the addition of 10 .mu.Ci
.sup.32P-.alpha.dCTP was used to monitor the efficiency of second
strand synthesis. The reactions were incubated at 15.degree. C. for
two hours followed by a 15 minute incubation at room temperature.
Unincorporated .sup.32 P-.alpha.dCTP in the labeled reaction was
removed by chromatography through a 400 pore size gel filtration
column (Clontech Laboratories, Inc.) before analysis by agarose gel
electrophoresis. The unlabeled reaction was terminated by two
extractions with phenol/chloroform and a chloroform extraction
followed by ethanol precipitation in the presence of 2.5 M ammonium
acetate.
[0106] The single-stranded DNA of the hairpin structure was cleaved
using mung bean nuclease. The reaction mixture contained 100 .mu.l
of second strand cDNA, 20 .mu.l of 10.times.mung bean nuclease
buffer (Stratagene Cloning Systems, La Jolla, Calif.), 16 .mu.l of
100 mM DTT, 51.5 .mu.l of water and 12.5 .mu.l of a 1:10 dilution
of mung bean nuclease (Promega Corp.; final concentration 10.5
U/.mu.l) in mung bean nuclease dilution buffer. The reaction was
incubated at 37.degree. C. for 15 minutes. The reaction was
terminated by the addition of 20 .mu.l of 1 M Tris: HCl, pH 8.0
followed by sequential phenol/chloroform and chloroform extractions
as described above. Following the extractions, the DNA was
precipitated in ethanol and resuspended in water.
[0107] The resuspended cDNA was blunt-ended with T4 DNA polymerase.
The cDNA, which was resuspended in 138 .mu.l of water, was mixed
with 40 .mu.l of 5.times.T4 DNA polymerase buffer (250 mM Tris:
HCl, PH 8.0, 250 mM KCl, 25 mM MgCl.sub.2), 3 .mu.l 0.1 M DTT, 5
.mu.l of a solution containing 10 mM of each deoxynucleotide
triphosphate and 4 .mu.l of 1 U/.mu.l T4 DNA polymerase (Boehringer
Mannheim Corp., Indianapolis, Ind.). After incubation of 1 hour at
10.degree. C., the reaction was terminated by the addition of 10
.mu.l of 0.5 M EDTA followed by serial phenol/chloroform and
chloroform extractions as described above. The DNA was
chromatographed through a 400 pore size gel filtration column
(Clontech Laboratories Inc. Palo Alto, Calif.) to remove trace
levels of protein and to remove short cDNAs less than about 400 bp
in length. The DNA was ethanol precipitated in the presence of 12
.mu.g glycogen carrier and 2.5 M ammonium acetate and was
resuspended in 10 .mu.l of water. Based on the incorporation of
.sup.32P-.alpha.dCTP, the yield of cDNA was estimated to be about 2
.mu.g from a starting template of 12.5 .mu.g.
[0108] Eco RI adapters were ligated onto the 5' ends of the cDNA to
enable cloning into a lambda phage vector. A 10 .mu.l aliquot of
cDNA (containing about 2 .mu.g of cDNA) and 11 .mu.l of 65
pmole/.mu.l of Eco RI adapter (Pharmacia LKB Biotechnology Inc.)
were mixed with 3 .mu.l 10.times. ligase buffer (Promega Corp.), 3
.mu.l 10 mM ATP and 3 .mu.l of 15 U/.mu.l T4 DNA ligase (Promega
Corp.). The reaction was incubated overnight (about 18 hours) at
12.5.degree. C. The reaction was terminated by the addition of 150
.mu.l of water and 10 .mu.l of 3 M Na acetate, followed by
incubation at 65.degree. C. for 30 minutes. After incubation, the
cDNA was extracted with phenol/chloroform and chloroform as
described above and precipitated in the presence of 2.5 M ammonium
acetate and 1.2 volume of isopropanol. Following centrifugation,
the cDNA pellet was washed with 70% ethanol, air dried and
resuspended in 89 .mu.l water.
[0109] To facilitate the directional cloning of the cDNA into a
lambda phage vector, the cDNA was digested with Sst-I resulting in
a cDNA having 5' Eco RI and 3' Sst-I cohesive ends. The Sst-I
restriction site at the 3' end of the cDNA had been previously
introduced through primer ZC2938 (SEQ ID NO:12). Restriction enzyme
digestion was carried out in a reaction containing 89 .mu.l of cDNA
described above, 10 .mu.l of 6 mM Tris: HCl, 6 mM MgCl.sub.2, 150
mM NaCl, 1 mM DTT (10 .times.D buffer; Promega Corp., Madison,
Wis.) and 1 .mu.l of 12 U/.mu.l Not I (Promega Corp.). Digestion
was carried out at 37.degree. C. for 1 hour. The reaction was
terminated by serial phenol/chloroform and chloroform extractions.
The cDNA was ethanol precipitated, washed with 70% ethanol, air
dried and resuspended in 20 .mu.l of 1.times. gel loading buffer
(10 mM Tris: HCl, pH 8.0, 1 mM EDTA, 5% glycerol and 0.125%
bromphenol blue).
[0110] The resuspended cDNA was heated to 65.degree. C. for 5
minutes, cooled on ice and electrophoresed on a 0.8% low melt
agarose gel ( SEA PLAQUE GTG.TM. low melt agarose; FMC Corp.).
Unincorporated adapters and cDNA below 1.6 kb in length were
excised from the gel. The electrodes were reversed, and the cDNA
was electrophoresed until concentrated near the lane of 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. A 300 .mu.l aliquot of water, approximately
three times the volume of the gel slice, was added to the tube. The
agarose was then melted by heating to 65.degree. C. for 15 minutes.
Following equilibration of the sample to 42.degree. C., 10 .mu.l of
1 U/.mu.l .beta.-agarose I ( New England Biolabs, Inc.) was added,
and the mixture was incubated for 90 minutes 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 in the
supernatant was ethanol precipitated, washed in 70% ethanol,
air-dried and resuspended in 37 .mu.l of water for the kinase
reaction to phosphorylate the ligated Eco RI adapters.
[0111] To the 37 .mu.l cDNA solution described above was added 10
.mu.l of 10.times.ligase buffer (Stratagene Cloning Systems), and
the mixture was heated to 65.degree. C. for 5 minutes. The mixture
was cooled on ice, and 5 .mu.l of 10 mM ATP and 3 .mu.l of 10
U/.mu.l of T4 polynucleotide kinase (Stratagene Cloning Systems)
were added. The reaction was incubated at 37.degree. C. for 45
minutes and was terminated by heating to 65.degree. C. for 10
minutes followed by serial extractions with phenol/chloroform and
chloroform. The phosphorylated cDNA was ethanol precipitated in the
presence of 2.5 M ammonium acetate, washed with 70%. ethanol, air
dried and resuspended in 12.5 .mu.l water. The concentration of the
phosphorylated cDNA was estimated to be about 40 fmole/.mu.l.
EXAMPLE 3
Northern Blot Analysis of Human Zcytor5
[0112] A 300 bp double stranded DNA probe for Northern analysis was
prepared from pSL1034 by PCR using oligonucleotide primers ZC
10,787 (SEQ ID NO:13) and ZC 11,097 (SEQ ID NO:14). The 300 bp PCR
fragment was gel-purified using a QIAQUICK.RTM. purification kit
(Qiagen Inc., Chatsworth, Calif.) and random-primer labeled using a
MULTIPRIME.RTM. kit (Amersham Corp.). Labeled cDNA was purified
from free counts using a- Stratagene push column. Human multiple
tissue Northern blots (Clontech Laboratories) and a human fetal
tissue Northern blot (Clontech Laboratories) were pre-hybridized
for three hours at 68.degree. C. using EXPRESSHYB hybridization
solution (Clontech Laboratories). The .sup.32 P-labeled cDNA probe
was then added to 10 mls of fresh hybridization solution at
10.sup.6cpm/ml overnight at 68.degree. C. The-blots were washed
several times at room temperature in wash solution containing
2.times.SSC, 0.05% SDS, then with continuous agitation for 40 min
at room temperature. The blots were then washed in 0.1.times.SSC,
0.1% SDS at 50.degree. C. for 40 min with one change of wash
solution.
[0113] A single transcript of .about.2.3 kb was detected after
exposure to film. In the multiple tissue blots (MTN, MTN II and MTN
III; Clontech Laboratories) the transcript was present in highest
abundance in placenta, thyroid, heart and skeletal muscle with
lower levels in prostate and trachea. Trace mRNA levels were found
in kidney, pancreas, testis, small intestine, colon, lymph node,
adrenal cortex and bone marrow.
EXAMPLE 4
Chromosomal Assignment and Placement of Human Zcytor-5
[0114] Zcytor5 was mapped to chromosome 19 using the commercially
available version of the "Stanford G3 Radiation Hybrid Mapping
Panel" (Research Genetics, Inc., Huntsville, Ala.). The "Stanford
G3 RH Panel" contains PCRable DNAs from each of 83 radiation hybrid
clones of the whole human genome, plus two control DNAs (the RM
donor and the A3 recipient). A publicly available WWW server
(http://shgc- www.stanford.edu) allows chromosomal localization of
markers.
[0115] For the mapping of Zcytor5 with the "Stanford G3 RH Panel",
20 .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 85 PCR
reactions consisted of 2 .mu.l 10.times.KlenTaq PCR reaction buffer
(CLONTECH Laboratories, Inc., Palo Alto, Calif.), 1.6 .mu.l dNTPs
mix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1 .mu.l sense
primer, (SEQ ID NO:13) 5' TAT GGC CAG GAC AAC ACA 3', 1 .mu.l
antisense primer, (SEQ ID NO:14), 5' ATA GGG CGT AAA GAG AGC 3', 2
.mu.l "RediLoad" (Research Genetics, Inc., Huntsville, Ala.), 0.4
.mu.l 50.times.Advantage KlenTaq Polymerase Mix (Clontech
Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone
or control and x .mu.l ddH20 for a total volume of 20 .mu.l. The
reactions were overlaid with an equal amount of mineral oil and
sealed. The PCR cycler conditions were as follows: an initial 1
cycle 5 minute denaturation at 95.degree. C., 35 cycles of a 1
minute denaturation at 95.degree. C., 1 minute annealing at
66.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 2% agarose gel
(Life Technologies, Gaithersburg, Md.).
[0116] The results showed linkage of Zcytor5 to the framework
marker WI-7289 with a LOD score of >10 and at a distance of
14.67 cR.sub.--10000 from the marker. The use of surrounding
markers positions Zcytor5 in the 19p13.1-p11 region on the
integrated LDB chromosome 19 map (The Genetic Location Database,
University of Southhampton, WWW server: http://cedar.genetics.
soton.ac.uk/public.sub.13 html/)
EXAMPLE 5
Cloning of the Rat Zcytor5 Gene
[0117] Rat Zcytor5 cDNA encoding Zcytor5 was isolated from an
amplified Rat testis cDNA library with a probe that was generated
by primers ZC12212 (SEQ ID NO: 11) and ZC10785 (SEQ ID NO:15) and
10 ng of plasmid pSL85212 as a template obtained from cDNA
containing EST 698365 as described in Example 1. The probe was
prepared by PCR by combining 1 .mu.l containing 10 ng of pSL85212,
1 .mu.l of ZC12212 having a concentration of 20 pmole/.mu.l, 1
.mu.l of ZC10785 having a concentration of 20 pmole/.mu.l, 0.5
.mu.l of dNTP having a concentration of 20 mM of dATP, dGTP, dCTP
and dTTP, 5 .mu.l of 10.times.Klentaq polymerase buffer (Clontech)
5 .mu.l Klentaq DNA polymerase (Clontech) and 39.5 .mu.l water. The
amplification was carried out at 94.degree. C. for 1 minute
followed by 30 cycles, each cycle consisting of 15 seconds at
95.degree. C., 20 seconds at 62.degree. C. and 1 minutes at
68.degree. C. The reaction had a final incubation at 68.degree. C.
for 10 minutes.
[0118] The resulting PCR product was diluted 1:100 with water. Four
.mu.l of the diluted PCR product was re-amplified using the
above-described conditions and the resultant PCR product was
further purified by electrophoresis on low-melt agarose gel. The
DNA probe was recovered from low-melt gel by digestion with
.beta.-Agarose I digestion. The rat Zcytor5 gene was then cloned
from a rat testis library which was constructed as described below
in Example 6.
[0119] In cloning the rat Zcytor5 gene, the library was first
amplified by plating 3.10.sup.6 plaque forming units (pfu) from the
previously constructed primary library onto 98 150 mm NZY plates.
Ten ml of serum medium was added to each plate and was incubated
for several hours at room temperature. Following incubation, the
phage lysates were collected and pooled to yield the amplified
phage library.
[0120] 1.5 million pfus from the amplified rat testis cDNA library
were plated onto 150 mm NZY plates at a density of 40,000 pfu/plate
on XL-1 Blue MRF' host cells. Following incubation at 37.degree. C.
overnight, filter lifts were made using HYBOND-N.TM. membranes
(Amersham), 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 8 minutes at room
temperature. The filters were neutralized in 0.5 M Tris: HCl, pH
7.2 for 5 minutes. Phage DNA was fixed onto the filters with 1,200
.mu.Joules of UV energy in a UV Cross-linker (Stratagene). The
filters were then washed with 0.25.times.SSC at 70.degree. C. to
remove excess cellular debris. Filter pre-hybridization was carried
out in a hybridization solution containing 5.times.SSC,
5.times.Denhardt solution, 0.2% SDS, 1 mM EDTA and heat denatured
sheared salmon-sperm DNA at a final concentration of 100 .mu.g/ml
for 72 hours at 60.degree. C.
[0121] 75 ng of probe DNA was labeled with .sup.32p-dCTP using a
MEGAPRIME.RTM. labeling kit (Amersham) and was purified with a
NUCTRAP.RTM. column (Stratagene). The labeled probe was
heat-denatured and added to fresh hybridization solution at a
concentration of 1.5.times.10.sup.6 cpm/ml. Into this solution were
also added the filters containing the phage particles.
Hybridization of the probes to the phage-containing filters was
completed overnight at 45.degree. C. Following hybridization, the
filters were washed in a solution containing 0.25.times.SSC, 0.25%
SDS and 1 mM EDTA at 50.degree. C. The washed filters were
autoradiographed for 72 hours at -70.degree. C. with intensifying
screens. Examination of the autoradiographs revealed multiple
regions that hybridized with the labeled probe. Agar plugs were
picked from 56 regions for plaque purification. Of the positive
signals, eleven produce positive phagemids following secondary and
tertiary hybridization screens. The plasmids within the positive
phagemids were recovered using the EXASSIT/SOLR.TM. system
according to the manufacturer's specifications. A clone designated
pSLRatR5-1 was sequenced and found to encode full length Rat
Zcytor5 (SEQ ID NO:5)
EXAMPLE 6
Production of Rat Testis cDNA library
[0122] The rat first strand cDNA reaction contained 10 .mu.l of rat
testis poly d(T)-selected poly (A).sup.+mRNA (Clontech, Palo Alto,
Calif.) at a concentration of 1.0 .mu.g/.mu.l, and 2 .mu.l of 20
pmole/.mu.L first strand primer ZC6091 (SEQ ID NO:16) containing an
Xho I restriction site. The mixture was heated at 70.degree. C. for
4 minutes and cooled by chilling on ice. First strand cDNA
synthesis was initiated by the addition of 8 .mu.l of first strand
buffer (5.times.SUPERSCRIPT.TM. buffer; Life Technologies,
Gaithersburg, Md.), 4 .mu.l of 100 mM dithiothreitol, and 2 .mu.l
of a deoxynucleotide triphosphate solution containing 10 mM each of
DATP, dGTP and 5-methyl-dCTP (Pharmacia LKB Biotechnology,
Piscataway, N.J.) to the RNA-primer mixture. The reaction mixture
was incubated at 45.degree. C. for 2 minutes, followed by the
addition of 10 .mu.l of 200 U/.mu.l RNase H- reverse transcriptase
(SUPERSCRIPT II.RTM.; Life Technologies). The efficiency of the
first strand synthesis was analyzed in a parallel reaction by the
addition of 10 .mu.Ci of .sup.32P-.alpha.dCTP 5 .mu.l aliquot from
one of the reaction mixtures to label he reaction for analysis. The
reactions were incubated at 45.degree. C. for 1 hour followed by an
incubation at 50.degree. C. for 10 minutes. Unincorporated
.sup.32P-.alpha.dCTP in the labeled reaction was removed by
chromatography on a 400 pore size gel filtration column (Clontech).
The unincorporated nucleotides and primers in the unlabeled first
strand retains were removed by chromatography on 400 pore size gel
filtration column (Clontech). The length of labeled first strand
cDNA was determined by agarose gel electrophoresis.
[0123] The second strand reaction contained 102 .mu.l of the
unlabeled first strand cDNA, 30 .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 .mu.l of 100 mM dithiothreitol, 3
.mu.l of a solution containing 10 mM of each deoxynucleotide
triphosphate, 5 .mu.l of 5 mM .beta.-NAD, 2 .mu.l of 3 U/.mu.l E.
coli DNA ligase (New England Biolabs), 5 .mu.l of 10 U/.mu.l E.
coli DNA polymerase I (New England Biolabs), and 1.5 .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 10 .mu.l T4 DNA
polymerase (10 U/.mu.l, Boerhinger Mannheim) 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 column
(Clontech) before analysis by agarose gel electrophoresis. The
unlabeled was terminated by the addition of 20 .mu.l 0.5 EDTA and
extraction with phenol/chloroform and chloroform followed by
ethanol precipitation in the presence of 2.5 M ammonium acetate.
The yield of cDNA was estimated to be approximately 2 .mu.g from
starting mRNA template of 10 .mu.g.
[0124] Eco RI adapters were ligated onto the 5' ends of the cDNA
described above to enable cloning into an expression vector. A 10.5
.mu.l aliquot of cDNA (.about.2 .mu.g) and 5 .mu.l of 65
pmole/.mu.l of Eco RI adapter (Pharmacia LKB Biotechnology Inc.)
were mixed with 2.5 .mu.l 10.times.ligase buffer 66 mM Tris-HCl pH
7.5, 10 mM MgCl.sub.2, 2.5 .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 overnight (.about.12 hours) at 12.degree. C. The
reaction was terminated by incubation at 70.degree. C. for 20
minutes. After incubation, the reaction was cooled to 37.degree. C.
To the reaction was added 2.5 .mu.l 10 mm ATP and 3 .mu.l 10
U/.mu.l T4 polynucleotide kinase (Life Technologies) to
phosphorylate the ligated Eco RI adapters.
[0125] 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 I 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:3).
Restriction enzyme digestion was carried out in a reaction mixture
containing 25 .mu.l of cDNA described above, 15 .mu.l of 10.times.H
Buffer (Boehringer Mannheim), 109 .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 65.degree. C for 10 minutes and chromatography
through a 400 pore size gel filtration column (Clontech).
[0126] The cDNA was ethanol precipitated, washed with 70% ethanol,
air dried and resuspended in 20 .mu.l of 1.times.gel loading buffer
(10 mM Tris:HCl, pH 8.0, 1 mM EDTA, 5% glycerol and 0.125%
bromphenol blue). The resuspended 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) was 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.
[0127] The resulting cDNA was cloned into the lambda phage vector
.lambda.ZapII that was predigested with Eco RI and Xho I and
dephosphorylated (Stratagene Cloning Systems, La Jolla, Calif.).
Ligation of the cDNA to the .lambda.ZapII vector was carried out in
a reaction mixture containing 1.0 .mu.l of prepared vector, 1.0
.mu.l of rat testis cDNA, 1.0 .mu.l 10.times.Ligase Buffer
(Promega), 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). The ligation mixture was
incubated at 5.degree. C.-15.degree. C. overnight in a temperature
gradient. After incubation, the ligation mixture was packaged into
phage using GIGPACK III GOLD packaging extract (Stratagene Cloning
Systems) and the resulting library was titered according to the
manufacturer's specifications.
Sequence CWU 1
1
* * * * *
References