Note: Descriptions are shown in the official language in which they were submitted.
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MAMMALIAN RECEPTOR PROTEINS;
RELATED REAGENTS AND METHODS
This filing claims priority to U.S. Patent Application
09/443,060, filed November 18, 1999, and U.S. Application
60/170,320, filed December 13, 1999, each of which is
incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to compositions and methods
for affecting mammalian physiology, including immune system
function. In particular, it provides methods to regulate
development and/or the immune system. Diagnostic and
therapeutic uses of these materials are also disclosed.
BACKGROUND OF THE INVENTION
Recombinant DNA technology refers generally to techniques
of integrating genetic information from a donor source into
vectors for subsequent processing, such as through introduction
into a host, whereby the transferred genetic information is
copied and/or expressed in the new environment. Commonly, the
genetic information exists in the form of complementary DNA
(cDNA) derived from messenger RNA (mRNA) coding for a desired
protein product. The carrier is frequently a plasmid having the
capacity to incorporate cDNA for later replication in a host
and, in some cases, actually to control expression of the cDNA
and thereby direct synthesis of the encoded product in the host.
See, e.g., Sambrook, et al. (1989) Molecular Cloning: A
Laboratory Manual, (2d ed.) vols.~ 1-3, CSH Press, NY.
For some time, it has been known that the mammalian immune
response is based on a series of complex cellular interactions,
called the "immune network". Recent research has provided new
insights into the inner workings of this network. While it
remains clear that much of the immune response does, in fact,
revolve around the network-like interactions of lymphocytes,
macrophages, granulocytes, and other cells, immunologists now
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generally hold the opinion that soluble proteins, known as
lymphokines, cytokines, or monokines, play critical roles in
controlling these cellular interactions. Thus, there is
considerable interest in the isolation, characterization, and
mechanisms of action of cell modulatory factors, an
understanding of which will lead to significant advancements in
the diagnosis and therapy of numerous medical abnormalities,
e.g., immune system disorders.
Lymphokines apparently mediate cellular activities in a
10~ variety of ways. See, e.g., Paul (ed. 1996) Fundamental
Immunoloav 3d ed., Raven Press, New York; and Thomson (ed. 1994)
The Cytokine Handbook 2d ed., Academic Press, San Diego. They
have been shown to support the proliferation, growth, and/or
differentiation of pluripotential hematopoietic stem cells into
vast numbers of progenitors comprising diverse cellular lineages
which make up a complex immune system. Proper and balanced
interactions between the cellular components are necessary for a
healthy immune response. The different cellular lineages often
respond in a different manner when lymphokines are administered
in conjunction with other agents.
Cell lineages especially important to the immune response
include two classes of lymphocytes: B-cells, which can produce
and secrete immunoglobulins (proteins with the capability of
recognizing and binding to foreign matter to effect its
removal), and T-cells of various subsets that secrete
lymphokines and induce or suppress the B-cells and various other
cells (including other T-cells) making up the immune network.
These lymphocytes interact with many other cell types.
Research to better understand and treat various immune
disorders has been hampered by the general inability to maintain
cells of the immune system in vitro. Immunologists have
discovered that culturing many of these cells can be
accomplished through the use of T-cell and other cell
supernatants, which contain various growth factors, including
many of the lymphokines.
Various growth and regulatory factors exist which modulate
morphogenetic development. Many receptors for cytokines are
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known. Often, there are at least two critical subunits in the
functional receptor. See, e.g., Gonda and D'Andrea (1997) Blood
89:355-369; Presky, et al. (1996) Proc. Nat'1 Acad. Sci..USA
93:14002-14007; Drachman and Kaushansky (1995) Curr. Opin.
Hematol. 2:22-28; Theze (1994) Eur. Cytokine Netw. 5:353-368;
and Lemmon and Schlessinger (1994) Trends Biochem. Sci. 19:459-
463.
From the foregoing, it is evident that the discovery and
development of new receptors, including ones similar to known
10~ receptors for lymphokines, should contribute to new therapies .
In particular, the discovery and understanding of novel
receptors for lymphokine-like molecules which enhance or
potentiate the beneficial activities of other lymphokines would
be highly advantageous. The present invention provides new
receptors for ligands exhibiting similarity to cytokine like
compositions and related compounds, and methods for their use.
SUMMARY OF THE INVENTION
The present invention is directed to novel receptors
related to cytokine receptors, e.g., primate, cytokine receptor
like molecular structures, designated DNAX Cytokine Receptor
Subunits (DCRS), and their biological activities. In
particular, it provides descriptions of subunits designated
DCRS3 (referring to two embodiments designated DCRS3.1 and
DCRS3.2) and DCRS4 (referring to three embodiments designated
DCRS4.1, DCRS4.2, and DCRS4.3). It includes nucleic acids
coding for the polypeptides themselves and methods for their
production and use. The nucleic acids of the invention are
characterized, in part, by their homology to cloned
complementary. DNA (cDNA) sequences enclosed herein.
The present invention provides a composition of matter
selected from: a substantially pure or recombinant: DCRS3
polypeptide comprising: at least three distinct nonoverlapping
segments of at least four amino acids identical to segments of
SEQ ID NO: 2 or 25; a substantially pure or recombinant DCRS3
polypeptide comprising at least two distinct nonoverlapping
segments of at least five amino acids identical to segments of
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SEQ ID NO: 2 or 25; a natural sequence DCRS3 comprising mature
SEQ ID NO: 2 or 25; a fusion polypeptide comprising DCRS3
sequence; or DCRS4 polypeptide comprising: at least three
distinct nonoverlapping segments of at least four amino acids
identical to segments of SEQ ID NO: 5, 28, or 31; a
substantially pure or recombinant DCRS4 polypeptide comprising
at least two distinct nonoverlapping segments of at least five
amino acids identical to segments of SEQ ID NO: 5, 28, or 31; a
natural sequence DCRS4 comprising mature SEQ ID NO: 5, 28, or
10~ 31; or a fusion polypeptide comprising DCRS4 sequence. In
certain embodiments, the invention embraces such a substantially
pure or isolated antigenic DCRS3 or DRS4 polypeptide, wherein
the distinct nonoverlapping segments of identity: include one of
at least eight amino acids; include one of at least four amino
acids and a second of at least five amino acids; include at
least three segments of at least four, five, and six amino
acids, or include one of at least twelve amino acids. Other
embodiments include wherein the: DCRS3 polypeptide: comprises a
mature sequence of Table 1; is an unglycosylated form of DCRS3;
is from a primate, such as a human; comprises at least seventeen
amino acids of SEQ ID NO: 2 or 25; exhibits at least four
nonoverlapping segments of at least seven amino acids of SEQ ID
NO: 2 or 25; comprises a sequence of at least 3 amino acids on
each side across an exon boundary; is a natural allelic variant
of DCRS3; has a length at least about 30 amino acids; exhibits
at least two non-overlapping epitopes which are specific for a
primate DCRS3; is glycosylated; has a molecular weight of at
least 30 kD with natural glycosylation; is a synthetic
polypeptide; is attached to a solid substrate; is conjugated to
another chemical moiety; is a 5-fold or less substitution from
natural sequence; or is a deletion or insertion variant from a
natural sequence; or DCRS4 polypeptide: comprises a mature
sequence of Table 3; is an unglycosylated form of DCRS4; is from
a primate, such as a human; comprises at least seventeen amino
acids of SEQ ID NO: 5; exhibits at least four nonoverlapping
segments of at least seven amino acids of SEQ ID NO: 5, 28, or
31; comprises a sequence of at least 3 amino acids on each side
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across an exon boundary; is a natural allelic variant of DCRSS;
has a length at least about 30 amino acids; exhibits at least
two non-overlapping epitopes which are specific for a primate
DCRS5; is glycosylated; has a molecular weight of at least 30 kD
5 with natural glycosylation; is a synthetic polypeptide; is
attached to a solid substrate; is conjugated to another chemical
moiety; is a 5-fold or less substitution from natural sequence;
or is a deletion or insertion variant from a natural sequence.
Still other embodiments include a composition comprising: a
10~ substantially pure DCRS3 and another cytokine receptor family
member; a sterile DCRS3 polypeptide; the DCRS3 polypeptide and a
carrier, wherein the carrier is: an aqueous compound, including
water, saline, and/or buffer; and/or formulated for oral,
rectal, nasal, topical, or parenteral administration; a
substantially pure DCRS4 and another cytokine receptor family
member; a sterile DCRS4 polypeptide; the DCRS4 polypeptide and a
carrier, wherein the carrier is: an aqueous compound, including
water, saline, and/or buffer; and/or formulated for oral,
rectal, nasal, topical, or parenteral administration. Fusion
polypeptide embodiments include those comprising: mature protein
sequence of Table 1 or 3; a detection or purification tag,
including a FLAG, His6, or Ig sequence; or sequence of another
interferon receptor protein. Kit embodiments include those
comprising such a polypeptide, and: a compartment comprising the
protein or polypeptide; or instructions for use or disposal of
reagents in the kit.
Binding compound embodiments include, e.g., a binding
compound comprising an antigen binding site from an antibody,
which specifically binds to a natural: DCRS3 polypeptide,
wherein: the binding compound is in a container; the DCRS3
polypeptide is from a human; the binding compound is an Fv, Fab,
or Fab2 fragment; the binding compound is conjugated to another
chemical moiety; or the antibody: is raised against a peptide
sequence of a mature polypeptide of Table 1; is raised against a
mature DCRS3; is raised to a purified human DCRS3; is
immunoselected; is a polyclonal antibody; binds to a denatured
DCRS3; exhibits a Kd to antigen of at least 30 ~M; is attached
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to a solid substrate, including a bead or plastic membrane; is
in a sterile composition; or is detectably labeled, including a
radioactive or fluorescent label; or DCRS4 polypeptide, wherein:
the binding compound is in a container; the DCRS4 polypeptide is
from a human; the binding compound is an Fv, Fab, or Fab2
fragment; the binding compound is conjugated to another chemical
moiety; or the antibody: is raised against a peptide sequence of
a mature polypeptide of Table 3; is raised against a mature
DCRS4; is raised to a purified human DCRS4; is immunoselected;
10~ is a polyclonal antibody; binds to a denatured DCRS4; exhibits a
Kd to antigen of at least 30 ~M; is attached to a solid
substrate, including a bead or plastic membrane; is in a sterile
composition; or is detectably labeled, including a radioactive
or fluorescent label. Kits include those comprising the binding
compound, and: a compartment comprising the binding compound; or
instructions for use or disposal of reagents in the kit.
Methods are provided, e.g., of producing an
antigen: antibody complex, comprising contacting under
appropriate conditions: a primate DCRS3 polypeptide with a
described antibody, thereby allowing the complex to form; or a
primate DCRS4 polypeptide with a described antibody, thereby
allowing the complex to form. This includes wherein: the
complex is purified from other cytokine receptors; the complex
is purified from other antibody; the contacting is with a sample
comprising another cytokine; the contacting allows quantitative
detection of the antigen; the contacting is with a sample
comprising the antibody; or the contacting allows quantitative
detection of the antibody.
Various related compositions are provided, e.g., a
composition comprising: a sterile binding compound, as
described, or the described binding compound and a carrier,
wherein the carrier is: an aqueous compound, including water,
saline, and/or buffer; and/or formulated for oral, rectal,
nasal, topical, or parenteral administration.
Nucleic acid embodiments include, e.g., an isolated or
recombinant nucleic acid encoding the DCRS3 polypeptide, wherein
the: DCRS3 is from a human; or the nucleic acid: encodes an
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antigenic peptide sequence of Table 1; encodes a plurality of
antigenic peptide sequences of Table 1; exhibits identity over
at least thirteen nucleotides to a natural cDNA encoding the
segment; is an expression vector; further comprises an origin of
replication; is from a natural source; comprises a detectable
label; comprises synthetic nucleotide sequence; is less than 6
kb, preferably less than 3 kb; is from a primate; comprises a
natural full length coding sequence; is a hybridization probe
for a gene encoding the DCRS3; or is a PCR primer, PCR product,
10~ or mutagenesis primer; or an isolated or recombinant nucleic
acid encoding the DCRS4 polypeptide, wherein the: DCRS4 is from
a human; or the nucleic acid: encodes an antigenic peptide
sequence of Table 3; encodes a plurality of antigenic peptide
sequences of Table 3; exhibits identity over at least thirteen
nucleotides to a natural cDNA encoding the segment; is an
expression vector; further comprises an origin of replication;
is from a natural source; comprises a detectable label;
comprises synthetic nucleotide sequence; is less than 6 kb,
preferably less than 3 kb; is from a primate; comprises a
natural full length coding sequence; is a hybridization probe
for a gene encoding the DCRS4; or is a PCR primer, PCR product,
or mutagenesis primer. Other embodiments of the invention
include a cell or tissue comprising the described recombinant
nucleic acid. Preferably, the cell is: a prokaryotic cell; a
eukaryotic cell; a bacterial cell; a yeast cell; an insect cell;
a mammalian cell; a mouse cell; a primate cell; or a human cell.
Kit embodiments include those comprising a described
nucleic acid, and: a compartment comprising the nucleic acid; a
compartment further comprising a primate DCRS3 or DCRS4
polypeptide; or instructions for use or disposal of reagents in
the kit.
Alternative nucleic acid embodiments include a nucleic acid
which: hybridizes under wash conditions of 30 minutes at 30° C
and less than 2M salt to the coding portion of: SEQ ID NO: 1,
24, 4, 27, or 30; or exhibits identity over a stretch of at
least about 30 nucleotides to a primate DCRS3 or DCRS4.
Preferred embodiments include those wherein: the wash conditions
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are at 45° C and/or 500 mM salt; the wash conditions are at 55°
C and/or 150 mM salt; the stretch is at least 55 nucleotides; or
the stretch is at least 75 nucleotides.
Other methods include those of modulating physiology or
development of a cell or tissue culture cells comprising
contacting the cell with an agonist or antagonist of a mammalian
DCRS3 or DCRS4. Preferably, the cell is transformed with a
nucleic acid encoding a DCRS3 or DCRS4 and another cytokine
receptor subunit.
10~
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
OUTLINE
I. General
II. Activities
III. Nucleic acids
A. encoding fragments, sequence, probes
B. mutations, chimeras, fusions
C. making nucleic acids
D. vectors, cells comprising
IV. Proteins, Peptides
A. fragments, sequence, immunogens, antigens
B. muteins
C. agonists/antagonists, functional equivalents
D. making proteins
V. Making nucleic acids, proteins
A. synthetic
B. recombinant
C. natural sources
VI. Antibodies
A. polyclonals
B. monoclonal
C . fragment s ; Kd
D, anti-idiotypic antibodies
E. hybridoma cell lines
VII. Kits and Methods to quantify DCRS
A. ELISA
B. assay mRNA encoding
C. qualitative/quantitative
D. kits
VIII. Therapeutic compositions, methods
A. combination compositions
B. unit dose
C. administration
IX. Screening
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X. Ligands
I. General
The present invention provides the amino acid sequences and
DNA sequences of mammalian, herein primate, cytokine receptor-
like subunit molecules, these designated DNAX Cytokine Receptor
Subunit 3 (DCRS3; 50R) and DNAX Cytokine Receptor Subunit 4
(DCRS4; cytor) having particular defined properties, both
structural and biological. Various cDNAs encoding these
10~ molecules were obtained from primate, e.g., human, cDNA sequence
libraries. Other primate or other mammalian counterparts would
also be desired.
Some of the standard methods applicable are described or
referenced, e.g., in Maniatis, et al. (1982) Molecular Cloning,
A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor Press; Sambrook, et al. (1989) Molecular Cloning: A
Laboratory Manual, (2d ed.), vols. 1-3, CSH Press, NY; Ausubel,
et al., Bioloay, Greene Publishing Associates, Brooklyn, NY; or
Ausubel, et al. (1987 and periodic supplements) Current
Protocols in Molecular Bioloav, Greene/Wiley, New York; each of
which is incorporated herein by reference.
Nucleotide (SEQ ID NO: 1) and corresponding amino acid
sequence (SEQ ID NO: 2) of a human DCRS3 coding segment are
shown in Table 1; likewise for the DCRS3.2 as SEQ ID NO: 24 and
25; comparison of DCRS3.1 and DCRS3.2 polypeptide sequences is
shown also in Table 1. Reverse translations based upon the
universal genetic code are provided in Table 2; comparison of
the encoding nucleic acid sequences is also presented in Table
2. The sequences are derived from genomic sequence at
chromosome location clones CIT987SK-582J2 HUAC004525 and CIT987-
SKA-67085 HUAC002303, at 16p12, and other cDNA sequences. The
predicted signal sequence is indicated, but may depend on cell
type, or may be a few residues in either direction. The
transmembrane segment (SEQ ID NO: 2) is predicted to run from
about 1eu248-ser264 (g1u242-his268). Predicted fibronectin
domain runs from about asn128-tyr220; cytokine receptor WS box
from about trp224-ser228; conserved disulfide motif between
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cys6-cys26; second conserved disulfide linkage at cys65-cys89;
five N glycosylation sites at Asn residues 61, 97, 121, 128, and
145; seven CAMP PK sites at lys4; 1ys68; 1ys184; arg191; arg201;
1ys202; and 1ys292; fourteen Ca phosphorylation sites at thr7l,
5 ser130, ser187, ser205, ser237, ser182, ser195, ser310, ser317,
thr323, ser374, ser385, ser403, and thr499; five myristoly sites
at 91y174, 91y303, 91y439, 91y449, and 91y466; four PKC
phosphorylation sites at ser7, ser147, ser180, and ser264; and
one tyrosine kinase site at 1ys163.
10~ Exon boundaries are predicted to be about between
nucleotides 949-c50, 9230-9231, 8284-9285, a484-8485, 9597-a598,
9775-a776, 8875-9876, and 8957-a958. Because the sequences have
been derived from genomic sequence, in which the introns have
not been spliced out, particularly important compositions will
be those which encode segments across the boundaries, e.9., both
nucleic acid sequence and amino acid sequence. The segments
will comprise, e.9., segments across the boundary which may
comprise 8, 9, 11, 13, 15, 17 20, 25, 30, 35, 50, or more
nucleotides on either or both sides adjacent to an exon
boundary, or 4, 5, 6, 7, or 8 amino acids on either or both
sides adjacent a boundary. The lengths on either side need not
be the same for purposes of novelty, e.9., three amino acids on
one side and 5 on the other side. Thus, e.9., compositions are
provided comprising, e.9, 15 contiguous nucleotides across a
boundary, of which at least 6 are from each side. Similarly,
compositions are provided, e.9., comprising at least 3 amino
acids from each side of the exon boundary, with a matching of at
least 8 amino acids across the boundary. Also provided are
compositions comprising a plurality of such segments across
multiple exon boundaries, which different segments need not have
the same length limitations. Thus, the invention provides a
nucleic acid comprising, e.9., at least 5 nucleotides in each
side across the exon 1/2 boundary, and at least 4 nucleotides on
either side of the the exon 3/4, 4/5, 5/6, and/or 6/7
boundaries. Natural sequence compositions would be preferred.
Nucleotide (SEQ ID NO: 4) and corresponding amino acid
sequence (SEQ.ID NO: 5) of a human DCRS4 coding segment are
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shown in Table 3; likewise for the DCRS4.2 as SEQ ID NO: 27 and
28 and the DCRS4.3 as SEQ ID N0: 30 and 31; comparison of DCRS4
polypeptide sequences is shown also in Table 3. Reverse
translations based upon the universal genetic code are provided
in Table 4; comparison of the encoding nucleic acid sequences is
also presented in Table 4. The sequence of DCRS4.1 is derived
from genomic sequence at chromosome location 6q24.1-25.2, within
some 50 kb of IFNyRl chain. The predicted DCRS4.1 signal
sequence is indicated, but may depend on cell type, or may be a
10~ few residues in either direction. This embodiment of the
receptor lacks a transmembrane segment, which is unusual, but
there is precedent for soluble forms of cytokine receptor
subunits. See, e.g, IL-l2Ra (p40 subunit) and the EBI3 receptor
subunit homolog. For the DCRS4.1, the predicted cytokine
receptor domain from prol0-arg49; conserved disulfide motif
between cys57-cys65; five N glycosylation sites at Asn residues
35, 131, 136, 157, and 174; four cAMP PK sites at arg30, 1ys98,
1ys106, and 1ys156; eight Ca phosphorylation sites at thr4,
thr60, ser64, thr68, thr7l, ser159, ser176, and ser220; three
myristoly sites at g1y89, g1y103, and g1y186; three PKC
phosphorylation sites at ser7, ser97, and ser217; one amidation
site at tyr79; one CAMP phosphorylation site at 1ys98; and two
CK2 phosphorylation sites at sera and ser159. Exon boundaries
are predicted to be about between nucleotides c59-a60; t197-
a198, 8206-a207, 8430-c431, and g601-a602. Alignment with the
other DCRS4 embodiments is provided. As described above,
compositions with sequence across the exon boundaries are
provided.
3 0 Table 1: Nucleotide and polypeptide sequences of DNAX Cytokine Receptor
Subunit like embodiments (DCRS3.1; 50R). Primate, e.g., human embodiment
(see SEQ ID NO: 1 and 2). Predicted signal sequence indicated, but may vary
by a few positions and depending upon cell type.
3 5 atg ccg cgt ggc tgg gcc gcc ccc ttg ctc ctg ctg ctg ctc cag gga 48
Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly
-20 -15 -10 -5
gcc ctc gag ggg atg gag agg aag ctc tgc agt ccc aag cca ccc ccc 96
4 0 Ala Leu Glu Gly Met Glu Arg Lys Leu Cys Ser Pro Lys Pro Pro Pro
-1 1 5 10
acc aag gcc tct ctc ccc act gac cct cca ggc tgg ggc tgc ccc gac 144
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Thr Lys Ala Ser Leu Pro Thr Asp Pro Pro Gly Trp Gly Cys Pro Asp
15 20 25
ctc gtc tgc tac acc gat tac ctc cag acg gtc atc tgc atc ctg gaa 192
Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys Ile Leu Glu
30 35 40
atg tgg aac ctc cac ccc agc acg ctc acc ctt acc tgg ata ctt tct 240
Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp Ile Leu Ser
45 50 55 60
15'
aat aat act ggg tgc tat atc aag gac aga aca ctg gac ctc agg caa 288
Asn Asn Thr Gly Cys Tyr Ile Lys Asp Arg Thr Leu Asp Leu Arg Gln
65 70 75
gac cag tat gaa gag ctg aag gac gag gcc acc tcc tgc agc ctc cac 336
Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu His
80 85 90
2 0 agg tcg gcc cac aat gcc acg cat gcc acc tac acc tgc cac atg gat 384
Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met Asp
95 100 105
gta ttc cac ttc atg gcc gac gac att ttc agt gtc aac atc aca gac 432
2 5 Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr Asp
110 115 120
cag tct ggc aac tac tcc cag gag tgt ggc agc ttt ctc ctg get gag 480
Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala Glu
3 0 125 130 135 140
agc aga cag tat aat atc tcc tgg cgc tca gat tac gaa gac cct gcc 528
Ser Arg Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp Pro Ala
145 150 155
ttc tac atg ctg aag ggc aag ctt cag tat gag ctg cag tac agg aac 576
Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn
160 165 170
4 0 cgg gga gac ccc tgg get gtg agt ccg agg aga aag ctg atc tca gtg 624
Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile Ser Val
175 180 185
gac tca aga agt gtc tcc ctc ctc ccc ctg gag ttc cgc aaa gac tcg 672
4 5 Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser
190 195 200
agc tat gag ctg cag gtg cgg gca ggg ccc atg cct ggc tcc tcc tac 720
Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr
5 0 205 210 215 220
cag ggg acc tgg agt gaa tgg agt gac ccg gtc atc ttt cag acc cag 768
Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln
225 230 235
tca gag gag tta aag gaa ggc tgg aac cct cac ctg ctg ctt ctc ctc 816
Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu Leu Leu Leu
240 245 250
ctg ctt gtc ata gtc ttc att cct gcc ttc tgg agc ctg aag acc cat 864
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Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp Ser Leu Lys Thr His
255 260 265
cca ttg tgg agg cta tgg aag aag ata tgg gcc gtc ccc agc cct gag 912
Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val Pro Ser Pro Glu
270 275 280
cgg ttc ttc atg ccc ctg tac aag ggc tgc agc gga gac ttc aag aaa 960
Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser Gly Asp Phe Lys Lys
285 290 295 300
15'
tgg gtg ggt gca ccc ttc act ggc tcc agc ctg gag ctg gga ccc tgg 1008
Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu Glu Leu Gly Pro Trp
305 310 315
agc cca gag gtg ccc tcc acc ctg gag gtg tac agc tgc cac cca cca 1056
Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr Ser Cys His Pro Pro
320 325 330
2 0 cgg agc ccg gcc aag agg ctg cag ctc acg gag cta caa gaa cca gca 1104
Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu Pro Ala
335 340 345
gag ctg gtg gag tct gac ggt gtg ccc aag ccc agc ttc tgg ccg aca 1152
25 Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe Trp Pro Thr
350 355 360
gcc cag aac tcg ggg ggc tca get tac agt gag gag agg gat cgg cca 1200
Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu Glu Arg Asp Arg Pro
3 0 365 370 375 380
tac ggc ctg gtg tcc att gac aca gtg act gtg cta gat gca gag ggg 1248
Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val Leu Asp Ala Glu Gly
385 390 395
cca tgc acc tgg ccc tgc agc tgt gag gat gac ggc tac cca gcc ctg 1296
Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro Ala Leu
400 405 410
4 0 gac ctg gat get ggc ctg gag ccc agc cca ggc cta gag gac cca ctc 1344
Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp Pro Leu
415 420 425
ttg gat gca ggg acc aca gtc ctg tcc tgt ggc tgt gtc tca get ggc 1392
4 5 Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val Ser Ala Gly
430 435 440
agc cct ggg cta gga ggg ccc ctg gga agc ctc ctg gac aga cta aag 1440
Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu Asp Arg Leu Lys
50 445 450 455 460
cca ccc ctt gca gat ggg gag gac tgg get ggg gga ctg ccc tgg ggt 1488
Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly Leu Pro Trp Gly
465 470 475
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ggc cgg tca cct gga ggg gtc tca gag agt gag gcg ggc tca ccc ctg 1536
Gly Arg Ser Pro Gly Gly Val Ser Glu Ser Glu Ala Gly Ser Pro Leu
480 485 490
gcc ggc ctg gat atg gac acg ttt gac agt ggc ttt gtg ggc tct gac 1584
Ala Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly Ser Asp
495 500 505
tgc agc agc cct gtg gag tgt gac ttc acc agc ccc ggg gac gaa gga 1632
Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly Asp Glu Gly
510 515 520
ccc ccc cgg agc tac ctc cgc cag tgg gtg gtc att cct ccg cca ctt 1680
Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile Pro Pro Pro Leu
15' 525 530 535 540
tcg agc cct gga ccc cag gcc agc taa 1707
Ser Ser Pro Gly Pro Gln Ala Ser
545
MPRGWAAPLLLLLLQGALEGMERKLCSPKPPPTKASLPTDPPGWGCPDLVCYTDYLQTVICILEMWNLHPSTLTLTW
ILSNNTGCYIKDRTLDLRQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECG
SFLLAESRQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR
AGPMPGSSYQGTWSEWSDPVIFQTQSEELKEGWNPHLLLLLLLVIVFIPAFWSLKTHPLWRLWKKIWAVPSPERFFM
PLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTA
QNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVS
AGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTS
PGDEGPPRSYLRQWWIPPPLSSPGPQAS
Nucleotide and polypeptide sequences of DNAX Cytokine Receptor Subunit like
embodiments (DCRS3.2; SEQ ID N0: 24 and 25):
atg ccg cgt ggc tgg gcc gcc ccc ttg ctc ctg ctg ctg ctc cag gga 48
Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly
3 5 -20 -15 -10 -5
ggc tgg ggc tgc ccc gac ctc gtc tgc tac acc gat tac ctc cag acg 96
Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr
-1 1 5 10
gtc atc tgc atc ctg gaa atg tgg aac ctc cac ccc agc acg ctc acc 144
Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr
15 20 25
4 5 ctt acc tgg caa gac cag tat gaa gag ctg aag gac gag gcc acc tcc 192
Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser
30 35 40
tgc agc ctc cac agg tcg gcc cac aat gcc acg cat gcc acc tac acc 240
Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr
45 50 55 60
tgc cac atg gat gta ttc cac ttc atg gcc gac gac att ttc agt gtc 288
Cys His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val
65 70 75
aac atc aca gac cag tct ggc aac tac tcc cag gan tgt ggc agc ttt 336
Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Xaa Cys Gly Ser Phe
80 85 90
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ctc ctg get gag agc atc aag ccg get ccc cct ttc aac gtg act gtg 384
Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val
95 100 105
5 acc ttc tca gga cag tat aat atn tcc tgg cgc tca gat tac gaa gac 432
Thr Phe Ser Gly Gln Tyr Asn Xaa Ser Trp Arg Ser Asp Tyr Glu Asp
110 115 120
cct gcc ttc tac atg ctg aaa ggc aag ctt caa tat gag ctg cag tac 480
10 Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr
125 130 135 140
agg aac cgg gga gac ccc tgg get gtg agt ccg agg aga aag ctg atc 528
Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile
15' 145 150 155
tca gtg gac tca aga agt gtc tcc ctc ctc ccc ctg gag ttc cgc aaa 576
Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys
160 165 170
gac tcg agc tat gag ctg can gtg cgg gca ggg ccc atg cct ggc tcc 624
Asp Ser Ser Tyr Glu Leu Xaa Val Arg Ala Gly Pro Met Pro Gly Ser
175 180 185
2 5 tcc tac cag ggg acc tgg agt gaa tgg agt gac ccg gtc atc tgt cag 672
Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Cys Gln
190 195 200
acc cag tca gag gag tta aag gaa ggc tgg aac cct cac ctg ctg ctt 720
3 0 Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu Leu
205 210 215 220
ctc ctc ctg ctt gtc ata gtc ttc att cct gcc ttc tgg agc ctg aag 768
Leu Leu Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp Ser Leu Lys
35 225 230 235
acc cat cca ttg tgg agg cta tgg aag aag ata tgg gcc gtc ccc agc 816
Thr His Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val Pro Ser
240 245 250
cct gag cgg ttc ttc atg ccc ctg tac aag ggc tgc agc gga gac ttc 864
Pro Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser Gly Asp Phe
255 260 265
4 5 aag aaa tgg gtg ggt gca ccc ttc act ggc tcc agc ctg gag ctg gga 912
Lys Lys Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu Glu Leu Gly
270 275 280
ccc tgg agc cca gag gtg ccc tcc acc ctg gag gtg tac agc tgc cac 960
Pro Trp Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr Ser Cys His
285 290 295 300
cca cca cgg agc ccg gcc aag agg ctg cag ctc acg gag cta caa gaa 1008
Pro Pro Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu
305 310 315
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cca gca gag ctg gtg gag tct gac ggt gtg ccc aag ccc agc ttc tgg 1056
Pro Ala Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe Trp
320 325 330
ccg aca gcc cag aac tcg ggg ggc tca get tac agt gag gag agg gat 1104
Pro Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu Glu Arg Asp
335 340 345
cgg cca tac ggc ctg gtg tcc att gac aca gtg act gtg cta gat gca 1152
Arg Pro Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val Leu Asp Ala
350 355 360
gag ggg cca tgc acc tgg ccc tgc agc tgt gag gat gac ggc tac cca 1200
15' Glu Gly Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro
365 370 375 380
gcc ctg gac ctg gat get ggc ctg gag ccc agc cca ggc cta gag gac 1248
Ala Leu Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp
385 390 395
cca ctc ttg gat gca ggg acc aca gtc ctg tcc tgt ggc tgt gtc tca 1296
Pro Leu Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val Ser
400 405 410
get ggc agc cct ggg cta gga ggg ccc ctg gga agc ctc ctg gac aga 1344
Ala Gly Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu Asp Arg
415 420 425
3 0 cta aag cca ccc ctt gca gat ggg gag gac tgg get ggg gga ctg ccc 1392
Leu Lys Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly Leu Pro
430 435 440
tgg ggt ggc cgg tca cct gga ggg gtc tca gag agt gag gcg ggc tca 1440
3 5 Trp Gly Gly Arg Ser Pro Gly Gly Val Ser Glu Ser Glu Ala Gly Ser
445 450 455 460
ccc ctg gcc ggc ctg gat atg gac acg ttt gac agt ggc ttt gtg ggc 1488
Pro Leu Ala Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly
40 465 470 475
tct gac tgc agc agc cct gtg gag tgt gac ttc acc agc ccc ggg gac 1536
Ser Asp Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly Asp
480 485 490
gaa gga ccc ccc cgg agc tac ctc cgc cag tgg gtg gtc att cct ccg 1584
Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile Pro Pro
495 500 505
cca ctt tcg agc cct gga ccc cag gcc agc taa 1617
Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser
510 515
MPRGWAAPLLLLLLQGGWGCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHA
TYTCHMDVFHFMADDIFSVNITDQSGNYSQXCGSFLLAESIKPAPPFNVTVTFSGQYNXSWRSDYEDPAFYMLKGKL
QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELXVRAGPMPGSSYQGTWSEWSDPVICQTQSEELK
EGWNPHLLLLLLLVIVFIPAFWSLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSP
EVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAE
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GPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGG
LPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWWIPPPLSSPGPQAS
Polypeptide sequence comparison of DCRS3.2 and DCRS3.1:
DCRS3.2 1 MPRGWAAPLLLLLLQG--------------------------GWGCPDLV 24
DCRS3.1 1 MPRGWAAPLLLLLLQGALEGMERKLCSPKPPPTKASLPTDPPGWGCPDLV 50
**************** ********
DCRS3.2 25 CYTDYLQTVICILEMWNLHPSTLTLTW------------------QDQYE 56
DCRS3.1 51 CYTDYLQTVICILEMWNLHPSTLTLTWILSNNTGCYIKDRTLDLRQDQYE 100
*************************** *****
DCRS3.2 57 ELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYS 106
15' DCRS3.1 101 ELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYS 150
**************************************************
DCRS3.2 107 QXCGSFLLAESIKPAPPFNVTVTFSGQYNXSWRSDYEDPAFYMLKGKLQY 156
DCRS3.1 151 QECGSFLLAE--------------SRQYNISWRSDYEDPAFYMLKGKLQY 186
2 0 * ******** * *** ********************
DCRS3.2 157 ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELXVRAGPMP 206
DCRS3.1 187 ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMP 236
****************************************** *******
DCRS3.2 207 GSSYQGTWSEWSDPVICQTQSEELKEGWNPHLLLLLLLVIVFIPAFWSLK 256
DCRS3.1 237 GSSYQGTWSEWSDPVIFQTQSEELKEGWNPHLLLLLLLVIVFIPAFWSLK 286
**************** *********************************
3 O DCRS3.2 257 THPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPW 306
DCRS3.1 287 THPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPW 336
**************************************************
DCRS3.2 307 SPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQ 356
3 5 DCRS3.1 337 SPEVPSTLEWSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQ 386
**************************************************
DCRS3.2 357 NSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDA 406
DCRS3.1 387 NSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDA 436
4 0 **************************************************
DCRS3.2 407 GLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADG 456
DCRS3.1 437 GLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADG 486
**************************************************
DCRS3.2 457 EDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECD 506
DCRS3.1 487 EDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECD 536
**************************************************
5 O DCRS3.2 507 FTSPGDEGPPRSYLRQWWIPPPLSSPGPQAS 538
DCRS3.1 537 FTSPGDEGPPRSYLRQWWIPPPLSSPGPQAS 568
********************************
Table 2: Reverse Translation of primate, e.g., human, DCRS3.1 (SEQ ID NO:
3). N may be A, C, G, or T.
ATGCCNMGNGGNTGGGCNGCNCCNYTNYTNYTNYTNYTNYTNCARGGNGCNYTNGARGGNATGGARMGNAARYTNTG
YWSNCCNAARCCNCCNCCNACNAARGCNWSNYTNCCNACNGAYCCNCCNGGNTGGGGNTGYCCNGAYYTNGTNTGYT
AYACNGAYTAYYTNCARACNGTNATHTGYATHYTNGARATGTGGAAYYTNCAYCCNWSNACNYTNACNYTNACNTGG
ATHYTNWSNAAYAAYACNGGNTGYTAYATHAARGAYMGNACNYTNGAYYTNMGNCARGAYCARTAYGARGARYTNAA
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RGAYGARGCNACNWSNTGYWSNYTNCAYMGNWSNGCNCAYAAYGCNACNCAYGCNACNTAYACNTGYCAYATGGAYG
TNTTYCAYTTYATGGCNGAYGAYATHTTYWSNGTNAAYATHACNGAYCARWSNGGNAAYTAYWSNCARGARTGYGGN
WSNTTYYTNYTNGCNGARWSNMGNCARTAYAAYATHWSNTGGMGNWSNGAYTAYGARGAYCCNGCNTTYTAYATGYT
NAARGGNAARYTNCARTAYGARYTNCARTAYMGNAAYMGNGGNGAYCCNTGGGCNGTNWSNCCNMGNMGNAARYTNA
THWSNGTNGAYWSNMGNWSNGTNWSNYTNYTNCCNYTNGARTTYMGNAARGAYWSNWSNTAYGARYTNCARGTNMGN
GCNGGNCCNATGCCNGGNWSNWSNTAYCARGGNACNTGGWSNGARTGGWSNGAYCCNGTNATHTTYCARACNCARWS
NGARGARYTNAARGARGGNTGGAAYCCNCAYYTNYTNYTNYTNYTNYTNYTNGTNATHGTNTTYATHCCNGCNTTYT
GGWSNYTNAARACNCAYCCNYTNTGGMGNYTNTGGAARAARATHTGGGCNGTNCCNWSNCCNGARMGNTTYTTYATG
CCNYTNTAYAARGGNTGYWSNGGNGAYTTYAARAARTGGGTNGGNGCNCCNTTYACNGGNWSNWSNYTNGARYTNGG
NCCNTGGWSNCCNGARGTNCCNWSNACNYTNGARGTNTAYWSNTGYCAYCCNCCNMGNWSNCCNGCNAARMGNYTNC
ARYTNACNGARYTNCARGARCCNGCNGARYTNGTNGARWSNGAYGGNGTNCCNAARCCNWSNTTYTGGCCNACNGCN
CARAAYWSNGGNGGNWSNGCNTAYWSNGARGARMGNGAYMGNCCNTAYGGNYTNGTNWSNATHGAYACNGTNACNGT
NYTNGAYGCNGARGGNCCNTGYACNTGGCCNTGYWSNTGYGARGAYGAYGGNTAYCCNGCNYTNGAYYTNGAYGCNG
GNYTNGARCCNWSNCCNGGNYTNGARGAYCCNYTNYTNGAYGCNGGNACNACNGTNYTNWSNTGYGGNTGYGTNWSN
15'
GCNGGNWSNCCNGGNYTNGGNGGNCCNYTNGGNWSNYTNYTNGAYMGNYTNAARCCNCCNYTNGCNGAYGGNGARGA
YTGGGCNGGNGGNYTNCCNTGGGGNGGNMGNWSNCCNGGNGGNGTNWSNGARWSNGARGCNGGNWSNCCNYTNGCNG
GNYTNGAYATGGAYACNTTYGAYWSNGGNTTYGTNGGNWSNGAYTGYWSNWSNCCNGTNGARTGYGAYTTYACNWSN
CCNGGNGAYGARGGNCCNCCNMGNWSNTAYYTNMGNCARTGGGTNGTNATHCCNCCNCCNYTNWSNWSNCCNGGNCC
NCARGCNWSN
Reverse Translation of primate, e.g., human, DCRS3.2 (SEQ ID NO: 26). N may
be A, C, G, or T.
2 5
ATGCCNMGNGGNTGGGCNGCNCCNYTNYTNYTNYTNYTNYTNCARGGNGGNTGGGGNTGYCCNGAYYTNGTNTGYTA
YACNGAYTAYYTNCARACNGTNATHTGYATHYTNGARATGTGGAAYYTNCAYCCNWSNACNYTNACNYTNACNTGGC
ARGAYCARTAYGARGARYTNAARGAYGARGCNACNWSNTGYWSNYTNCAYMGNWSNGCNCAYAAYGCNACNCAYGCN
ACNTAYACNTGYCAYATGGAYGTNTTYCAYTTYATGGCNGAYGAYATHTTYWSNGTNAAYATHACNGAYCARWSNGG
NAAYTAYWSNCARNNNTGYGGNWSNTTYYTNYTNGCNGARWSNATHAARCCNGCNCCNCCNTTYAAYGTNACNGTNA
3 O
CNTTYWSNGGNCARTAYAAYNNNWSNTGGMGNWSNGAYTAYGARGAYCCNGCNTTYTAYATGYTNAARGGNAARYTN
CARTAYGARYTNCARTAYMGNAAYMGNGGNGAYCCNTGGGCNGTNWSNCCNMGNMGNAARYTNATHWSNGTNGAYWS
NMGNWSNGTNWSNYTNYTNCCNYTNGARTTYMGNAARGAYWSNWSNTAYGARYTNNNNGTNMGNGCNGGNCCNATGC
CNGGNWSNWSNTAYCARGGNACNTGGWSNGARTGGWSNGAYCCNGTNATHTGYCARACNCARWSNGARGARYTNAAR
GARGGNTGGAAYCCNCAYYTNYTNYTNYTNYTNYTNYTNGTNATHGTNTTYATHCCNGCNTTYTGGWSNYTNAARAC
35
NCAYCCNYTNTGGMGNYTNTGGAAR.AARATHTGGGCNGTNCCNWSNCCNGARMGNTTYTTYATGCCNYTNTAYAARG
GNTGYWSNGGNGAYTTYAARAARTGGGTNGGNGCNCCNTTYACNGGNWSNWSNYTNGARYTNGGNCCNTGGWSNCCN
GARGTNCCNWSNACNYTNGARGTNTAYWSNTGYCAYCCNCCNMGNWSNCCNGCNAARMGNYTNCARYTNACNGARYT
NCARGARCCNGCNGARYTNGTNGARWSNGAYGGNGTNCCNAARCCNWSNTTYTGGCCNACNGCNCARAAYWSNGGNG
GNWSNGCNTAYWSNGARGARMGNGAYMGNCCNTAYGGNYTNGTNWSNATHGAYACNGTNACNGTNYTNGAYGCNGAR
4 O
GGNCCNTGYACNTGGCCNTGYWSNTGYGARGAYGAYGGNTAYCCNGCNYTNGAYYTNGAYGCNGGNYTNGARCCNWS
NCCNGGNYTNGARGAYCCNYTNYTNGAYGCNGGNACNACNGTNYTNWSNTGYGGNTGYGTNWSNGCNGGNWSNCCNG
GNYTNGGNGGNCCNYTNGGNWSNYTNYTNGAYMGNYTNAARCCNCCNYTNGCNGAYGGNGARGAYTGGGCNGGNGGN
YTNCCNTGGGGNGGNMGNWSNCCNGGNGGNGTNWSNGARWSNGARGCNGGNWSNCCNYTNGCNGGNYTNGAYATGGA
YACNTTYGAYWSNGGNTTYGTNGGNWSNGAYTGYWSNWSNCCNGTNGARTGYGAYTTYACNWSNCCNGGNGAYGARG
4 5 GNCCNCCNMGNWSNTAYYTNMGNCARTGGGTNGTNATHCCNCCNCCNYTNWSNWSNCCNGGNCCNCARGCNWSN
Nucleic acid sequence comparison of two DCRS3 embodiments:
DCRS3.2 1 ATGCCGCGTGGCTGGGCCGCCCCCTTGCTCCTGCTGCTGCTCCAGGGAGC 50
50 DCRS3.1 1 ATGCCGCGTGGCTGGGCCGCCCCCTTGCTCCTGCTGCTGCTCCAGGGA-- 49
************************************************
DCRS3.2 51 CCTCGAGGGGATGGAGAGGAAGCTCTGCAGTCCCAAGCCACCCCCCACCA 100
DCRS3.1 49 -_________________________________________________ 49
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DCRS3.2 101 AGGCCTCTCTCCCCACTGACCCTCCAGGCTGGGGCTGCCCCGACCTCGTC 150
DCRS3.1 50 ---------------------------GCTGGGGCTGCCCCGACCTCGTC 72
***********************
DCRS3.2 151 TGCTACACCGATTACCTCCAGACGGTCATCTGCATCCTGGAAATGTGGAA 200
DCRS3.1 73 TGCTACACCGATTACCTCCAGACGGTCATCTGCATCCTGGAAATGTGGAA 122
**************************************************
DCRS3.2 201 CCTCCACCCCAGCACGCTCACCCTTACCTGGATACTTTCTAATAATACTG 250
DCRS3.1 123 CCTCCACCCCAGCACGCTCACCCTTACCTGG------------------- 153
*******************************
DCRS3.2 251 GGTGCTATATCAAGGACAGAACACTGGACCTCAGGCAAGACCAGTATGAA 300
15' DCRS3.1 154 -----------------------------------C~GACCAGTATGAA 168
***************
DCRS3.2 301 GAGCTGAAGGACGAGGCCACCTCCTGCAGCCTCCACAGGTCGGCCCACAA 350
DCRS3.1 169 GAGCTGAAGGACGAGGCCACCTCCTGCAGCCTCCACAGGTCGGCCCACAA 218
2 0 **************************************************
DCRS3.2 351 TGCCACGCATGCCACCTACACCTGCCACATGGATGTATTCCACTTCATGG 400
DCRS3.1 219 TGCCACGCATGCCACCTACACCTGCCACATGGATGTATTCCACTTCATGG 268
**************************************************
DCRS3.2 401 CCGACGACATTTTCAGTGTCAACATCACAGACCAGTCTGGCAACTACTCC 450
DCRS3.1 269 CCGACGACATTTTCAGTGTCAACATCACAGACCAGTCTGGCAACTACTCC 318
**************************************************
3 O DCRS3.2 451 CAGGAGTGTGGCAGCTTTCTCCTGGCTGAGAGCA---------------- 484
DCRS3.1 319 CAGGANTGTGGCAGCTTTCTCCTGGCTGAGAGCATCAAGCCGGCTCCCCC 368
***** ****************************
DCRS3.2 485 --------------------------GACAGTATAATATCTCCTGGCGCT 508
DCRS3.1 369 TTTCAACGTGACTGTGACCTTCTCAGGACAGTATAATATNTCCTGGCGCT 418
************* **********
DCRS3.2 509 CAGATTACGAAGACCCTGCCTTCTACATGCTGAAGGGCAAGCTTCAGTAT 558
DCRS3.1 419 CAGATTACGAAGACCCTGCCTTCTACATGCTGAAAGGCAAGCTTCAATAT 468
4 0 ********************************** *********** ***
DCRS3.2 559 GAGCTGCAGTACAGGAACCGGGGAGACCCCTGGGCTGTGAGTCCGAGGAG 608
DCRS3.1 469 GAGCTGCAGTACAGGAACCGGGGAGACCCCTGGGCTGTGAGTCCGAGGAG 518
**************************************************
DCRS3.2 609 AAAGCTGATCTCAGTGGACTCAAGAAGTGTCTCCCTCCTCCCCCTGGAGT 658
DCRS3.1 519 AAAGCTGATCTCAGTGGACTCAAGAAGTGTCTCCCTCCTCCCCCTGGAGT 568
**************************************************
DCRS3.2 659 TCCGCAAAGACTCGAGCTATGAGCTGCAGGTGCGGGCAGGGCCCATGCCT 708
DCRS3.1 569 TCCGCAAAGACTCGAGCTATGAGCTGCANGTGCGGGCAGGGCCCATGCCT 618
**************************** *********************
DCRS3.2 709 GGCTCCTCCTACCAGGGGACCTGGAGTGAATGGAGTGACCCGGTCATCTT 758
DCRS3.1 619 GGCTCCTCCTACCAGGGGACCTGGAGTGAATGGAGTGACCCGGTCATCTG 668
*************************************************
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DCRS3.2 759 TCAGACCCAGTCAGAGGAGTTAAAGGAAGGCTGGAACCCTCACCTGCTGC 808
DCRS3.1 669 TCAGACCCAGTCAGAGGAGTTAAAGGAAGGCTGGAACCCTCACCTGCTGC 718
**************************************************
DCRS3.2 809 TTCTCCTCCTGCTTGTCATAGTCTTCATTCCTGCCTTCTGGAGCCTGAAG 858
DCRS3.1 719 TTCTCCTCCTGCTTGTCATAGTCTTCATTCCTGCCTTCTGGAGCCTGAAG 768
**************************************************
10 DCRS3.2 859 ACCCATCCATTGTGGAGGCTATGGAAGAAGATATGGGCCGTCCCCAGCCC 908
DCRS3.1 769 ACCCATCCATTGTGGAGGCTATGGAAGAAGATATGGGCCGTCCCCAGCCC 818
**************************************************
DCRS3.2 909 TGAGCGGTTCTTCATGCCCCTGTACAAGGGCTGCAGCGGAGACTTCAAGA 958
15' DCRS3.1 819 TGAGCGGTTCTTCATGCCCCTGTACAAGGGCTGCAGCGGAGACTTCAAGA 868
**************************************************
DCRS3.2 959 AATGGGTGGGTGCACCCTTCACTGGCTCCAGCCTGGAGCTGGGACCCTGG 1008
DCRS3.1 869 AATGGGTGGGTGCACCCTTCACTGGCTCCAGCCTGGAGCTGGGACCCTGG 918
2 0 **************************************************
DCRS3.2 1009 AGCCCAGAGGTGCCCTCCACCCTGGAGGTGTACAGCTGCCACCCACCACG 1058
DCRS3.1 919 AGCCCAGAGGTGCCCTCCACCCTGGAGGTGTACAGCTGCCACCCACCACG 968
**************************************************
DCRS3.2 1059 GAGCCCGGCCAAGAGGCTGCAGCTCACGGAGCTACAAGAACCAGCAGAGC 1108
DCRS3.1 969 GAGCCCGGCCAAGAGGCTGCAGCTCACGGAGCTACAAGAACCAGCAGAGC 1018
**************************************************
3 0 DCRS3.2 1109 TGGTGGAGTCTGACGGTGTGCCCAAGCCCAGCTTCTGGCCGACAGCCCAG 1158
DCRS3.1 1019 TGGTGGAGTCTGACGGTGTGCCCAAGCCCAGCTTCTGGCCGACAGCCCAG 1068
**************************************************
DCRS3.2 1159 AACTCGGGGGGCTCAGCTTACAGTGAGGAGAGGGATCGGCCATACGGCCT 1208
3 5 DCRS3.1 1069 AACTCGGGGGGCTCAGCTTACAGTGAGGAGAGGGATCGGCCATACGGCCT 1118
**************************************************
DCRS3.2 1209 GGTGTCCATTGACACAGTGACTGTGCTAGATGCAGAGGGGCCATGCACCT 1258
DCRS3.1 1119 GGTGTCCATTGACACAGTGACTGTGCTAGATGCAGAGGGGCCATGCACCT 1168
4 0 **************************************************
DCRS3.2 1259 GGCCCTGCAGCTGTGAGGATGACGGCTACCCAGCCCTGGACCTGGATGCT 1308
DCRS3.1 1169 GGCCCTGCAGCTGTGAGGATGACGGCTACCCAGCCCTGGACCTGGATGCT 1218
**************************************************
DCRS3.2 1309 GGCCTGGAGCCCAGCCCAGGCCTAGAGGACCCACTCTTGGATGCAGGGAC 1358
DCRS3.1 1219 GGCCTGGAGCCCAGCCCAGGCCTAGAGGACCCACTCTTGGATGCAGGGAC 1268
**************************************************
5 O DCRS3.2 1359 CACAGTCCTGTCCTGTGGCTGTGTCTCAGCTGGCAGCCCTGGGCTAGGAG 1408
DCRS3.1 1269 CACAGTCCTGTCCTGTGGCTGTGTCTCAGCTGGCAGCCCTGGGCTAGGAG 1318
**************************************************
DCRS3.2 1409 GGCCCCTGGGAAGCCTCCTGGACAGACTAAAGCCACCCCTTGCAGATGGG 1458
5 5 DCRS3.1 1319 GGCCCCTGGGAAGCCTCCTGGACAGACTAAAGCCACCCCTTGCAGATGGG 1368
**************************************************
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DCRS3.2 1459 GAGGACTGGGCTGGGGGACTGCCCTGGGGTGGCCGGTCACCTGGAGGGGT 1508
DCRS3.1 1369 GAGGACTGGGCTGGGGGACTGCCCTGGGGTGGCCGGTCACCTGGAGGGGT 1418
**************************************************
DCRS3.2 1509 CTCAGAGAGTGAGGCGGGCTCACCCCTGGCCGGCCTGGATATGGACACGT 1558
DCRS3.1 1419 CTCAGAGAGTGAGGCGGGCTCACCCCTGGCCGGCCTGGATATGGACACGT 1468
**************************************************
DCRS3.2 1559 TTGACAGTGGCTTTGTGGGCTCTGACTGCAGCAGCCCTGTGGAGTGTGAC 1608
DCRS3.1 1469 TTGACAGTGGCTTTGTGGGCTCTGACTGCAGCAGCCCTGTGGAGTGTGAC 1518
**************************************************
DCRS3.2 1609 TTCACCAGCCCCGGGGACGAAGGACCCCCCCGGAGCTACCTCCGCCAGTG 1658
15' DCRS3.1 1519 TTCACCAGCCCCGGGGACGAAGGACCCCCCCGGAGCTACCTCCGCCAGTG 1568
**************************************************
DCRS3.2 1659 GGTGGTCATTCCTCCGCCACTTTCGAGCCCTGGACCCCAGGCCAGCTAA 1707
DCRS3.1 1569 GGTGGTCATTCCTCCGCCACTTTCGAGCCCTGGACCCCAGGCCAGCTAA 1617
2 0 *************************************************
Table 3: Nucleotide and polypeptide sequences of DNAX Cytokine Receptor
Subunit like embodiment 4 (DCRS4.1; cytor). Primate, e.g., human embodiment
(see SEQ ID N0: 4 and 5). Predicted signal sequence indicated, but may vary
2 5 by a few positions and depending upon cell type.
atg atg cct aaa cat tgc ttt cta ggc ttc ctc atc agt ttc ttc ctt 48
Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu
-20 -15 -10
act ggt gta gca gga act cag tca acg cat gag tct ctg aag cct cag 96
Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln
-5 -1 1 5 10
3 5 agg gta caa ttt cag tcc cga aat ttt cac aac att ttg caa tgg cag 144
Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln
15 20 25
cct ggg agg gca ctt act ggc aac agc agt gtc tat ttt gtg cag tac 192
4 0 Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr
30 35 40
aaa ata tat gga cag aga caa tgg aaa aat aaa gaa gac tgt tgg ggt 240
Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly
45 45 50 55
act caa gaa ctc tct tgt gac ctt acc agt gaa acc tca gac ata cag 288
Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln
65 70 75
gaa cct tat tac ggg agg agg ggc aaa aat aaa aat aaa ggg aat cct 336
Glu Pro Tyr Tyr Gly Arg Arg Gly Lys Asn Lys Asn Lys Gly Asn Pro
80 85 90
55 tgg ggg cca aaa caa agt aaa cgg aaa tca aag ggg aac cag aag acc 384
Trp Gly Pro Lys Gln Ser Lys Arg Lys Ser Lys Gly Asn Gln Lys Thr
95 100 105
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aac aca gtg act gcc cca get gcc ctg aag gca ttt get gga tgt gca 432
Asn Thr Val Thr Ala Pro Ala Ala Leu Lys Ala Phe Ala Gly Cys Ala
110 115 120
aaa ata gat cct cca gtc atg aat ata acc caa gtc aat ggc tct ttg 480
Lys Ile Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu
125 130 135
ttg gta att ctc cat get cca aat tta cca tat aga tac caa aag gaa 528
Leu Val Ile Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu
140 145 150 155
aaa aat gta tct ata gaa gat tac tat gaa cta cta tac cga gtt ttt 576
15' Lys Asn Val Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe
160 165 170
ata att aac aat tca cta gaa aag gag caa aag gtt tat gaa ggg get 624
Ile Ile Asn Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala
175 180 185
cac aga gcg gtt gaa att gaa get cta aca cca cac tcc agc tac tgt 672
His Arg Ala Val Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys
190 195 200
gta gtg get gaa ata tat cag ccc atg tta gac aga aga agt cag aga 720
Val Val Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg
205 210 215
3 0 agt gaa gag aga tgt gtg gaa att cca tga 750
Ser Glu Glu Arg Cys Val Glu Ile Pro
220 225
MMPKHCFLGFLISFFLTGVAGTQSTHESLKPQRVQFQSRNFHNILQWQPGRALTGNSSVYFVQYKIYGQRQWKNKED
CWGTQELSCDLTSETSDIQEPYYGRRGKNKNKGNPWGPKQSKRKSKGNQKTNTVTAPAALKAFAGCAKIDPPVMNIT
QVNGSLLVILHAPNLPYRYQKEKNVSIEDYYELLYRVFIINNSLEKEQKVYEGAHRAVEIEALTPHSSYCWAEIYQ
PMLDRRSQRSEERCVEIP.
Nucleotide and polypeptide sequences of DNAX Cytokine Receptor Subunit like
4 0 embodiments (DCRS4.2, cytorX700; SEQ ID NO: 27 and 28):
ATGATGCCTAAACATTGCTTTCTAGGCTTCCTCATCAGTTTCTTCCTTACTGGTGTAGCAGGAACTCAGTCAACGCA
TGAGTCTCTGAAGCCTCAGAGGGTACAATTTCAGTCCCGAAATTTTCACAACATTTTGCAATGGCAGCCCGGGAGGG
CACTTACTGGCAACAGCAGTGTCTATTTTGTGCAGTACAAAATATATGGACAGAGACAATGGAAAAATAAAGAAGAC
4 5
TGTTGGGGTACTCAAGAACTCTCTTGTGACCTTACCAGTGAAACCTCAGACATACAGGAACCTTATTACGGGAGGGT
GAGGGCGGCCTCGGCTGGGAGCTACTCAGAATGGAGCATGACGCCGCGGTTCACTCCCTGGTGGGAAACAAAAATAG
ATCCTCCAGTCATGAATATAACCCAAGTCAATGGCTCTTTGTTGGTAATTCTCCATGCTCCAAATTTACCATATAGA
TACCAAAAGGAAAAA.AATGTATCTATAGAAGATTACTATGAACTACTATACCGAGTTTTTATAATTAACAATTCACT
AGAAAAGGAGCAAAAGGTTTATGAAGGGGCTCACAGAGCGGTTGAAATTGAAGCTCTAACACCACACTCCAGCTACT
50
GTGTAGTGGCTGAAATATATCAGCCCATGTTAGACAGAAGAAGTCAGAGAAGTGAAGAGAGATGTGTGGAAATTCCA
TGA
atg atg cct aaa cat tgc ttt cta ggc ttc ctc atc agt ttc ttc ctt 48
Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu
55 -20 -15 -to
act ggt gta gca gga act cag tca acg cat gag tct ctg aag cct cag 96
Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln
-5 -1 1 5 10
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agg gta caa ttt cag tcc cga aat ttt cac aac att ttg caa tgg cag 144
Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln
15 20 25
ccc ggg agg gca ctt act ggc aac agc agt gtc tat ttt gtg cag tac 192
Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr
30 35 40
aaa ata tat gga cag aga caa tgg aaa aat aaa gaa gac tgt tgg ggt 240
Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly
45 50 55
act caa gaa ctc tct tgt gac ctt acc agt gaa acc tca gac ata cag 288
Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln
15' 60 65 70 75
gaa cct tat tac ggg agg gtg agg gcg gcc tcg get ggg agc tac tca 336
Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser
80 85 90
gaa tgg agc atg acg ccg cgg ttc act ccc tgg tgg gaa aca aaa ata 384
Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu Thr Lys Ile
95 100 105
2 5 gat cct cca gtc atg aat ata acc caa gtc aat ggc tct ttg ttg gta 432
Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu Leu Val
110 115 120
att ctc cat get cca aat tta cca tat aga tac caa aag gaa aaa aat 480
3 0 Ile Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn
125 130 135
gta tct ata gaa gat tac tat gaa cta cta tac cga gtt ttt ata att 528
Val Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile
3 5 140 145 150 155
aac aat tca cta gaa aag gag caa aag gtt tat gaa ggg get cac aga 576
Asn Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala His Arg
4 0 160 165 170
gcg gtt gaa att gaa get cta aca cca cac tcc agc tac tgt gta gtg 624
Ala Val Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val Val
175 180 185
get gaa ata tat cag ccc atg tta gac aga aga agt cag aga agt gaa 672
Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg Ser Glu
190 195 200
gag aga tgt gtg gaa att cca tga 696
Glu Arg Cys Val Glu Ile Pro
205 210
>cytorX700
MMPKHCFLGFLISFFLTGVAGTQSTHESLKPQRVQFQSRNFHNILQWQPGRALTGNSSVYFVQYKIYGQRQWKNKED
CWGTQELSCDLTSETSDIQEPYYGRVRAASAGSYSEWSMTPRFTPWWETKIDPPVMNITQVNGSLLVILHAPNLPYR
YQKEKNVSIEDYYELLYRVFIINNSLEKEQKVYEGAHRAVEIEALTPHSSYCWAEIYQPMLDRRSQRSEERCVEIP
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Nucleotide and polypeptide sequences of DNAX Cytokine Receptor Subunit like
embodiments (DCRS4.3 cytorX600; SEQ ID NO: 30 and 31):
ATGATGCCTAAACATTGCTTTCTAGGCTTCCTCATCAGTTTTTTCCTTACTGGTGTAGCAGGAACTCAGTCAACGCA
TGAGTCTCTGAAGCCTCAGAGGGTACAATTTCAGTCCCGAAATTTTCACAACATTTTGCAATGGCAGCCTGGGAGGG
CACTTACTGGCAACAGCAGTGTCTATTTTGTGCAGTACAAAATATATGGACAGAGACAATGGAAAAATAAAGAAGAC
TGTTGGGGTACTCAAGAACTCTCTTGTGACCTTACCAGTGAAACCTCAGACATACAGGAATCTTATTACGGGAGGGT
GAGGGCGGCCTCGGCTGGGAGCTACTCAGAATGGAGCATGACGCCGCGGTTCACTCCCTGGTGGGAAAGAGCAAAAG
GTTTATGAAGGGGCTCACAGAGCGGTTGAAATTGAAGCTCTAACACCACACTCCAGCTACTGTGTAGTGGCTGAAAT
ATATCAGCCCACGTTAGACAGAAGAAGTCAGAGAAGTGAAGAGAGATGTGTGGAAATTCCATGA
atg atg cct aaa cat tgc ttt cta ggc ttc ctc atc agt ttt ttc ctt 48
Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu
-20 -15 -10
15'
act ggt gta gca gga act cag tca acg cat gag tct ctg aag cct cag 96
Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln
-5 -1 1 5 10
2 0 agg gta caa ttt cag tcc cga aat ttt cac aac att ttg caa tgg cag 144
Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln
20 25
cct ggg agg gca ctt act ggc aac agc agt gtc tat ttt gtg cag tac 192
2 5 Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr
30 35 40
aaa ata tat gga cag aga caa tgg aaa aat aaa gaa gac tgt tgg ggt 240
Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly
30 45 50 55
act caa gaa ctc tct tgt gac ctt acc agt gaa acc tca gac ata cag 288
Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln
60 65 70 75
gaa tct tat tac ggg agg gtg agg gcg gcc tcg get ggg agc tac tca 336
Glu Ser Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser
80 85 90
4 0 gaa tgg agc atg acg ccg cgg ttc act ccc tgg tgg gaa aga gca aaa 384
Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu Arg Ala Lys
95 100 105
ggt tta tgaaggggct cacagagcgg ttgaaattga agctctaaca ccacactcca 440
4 5 Gly Leu
gctactgtgt agtggctgaa atatatcagc ccacgttaga cagaagaagt cagagaagtg 500
aagagagatg tgtggaaatt ccatga 526
>cytorX600
MMPKHCFLGFLISFFLTGVAGTQSTHESLKPQRVQFQSRNFHNILQWQPGRALTGNSSVYFVQYKIYGQRQWKNKED
CWGTQELSCDLTSETSDIQESYYGRVRAASAGSYSEWSMTPRFTPWWERAKGL.
5 5 Polypeptide sequence comparison of DCRS4.1, DCRS4.2 and DCRS4.3:
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DCRS4.1 1 MMPKHCFLGFLISFFLTGVAGTQSTHESLKPQRVQFQSRNFHNILQWQPG 50
DCRS4.2 1 MMPKHCFLGFLISFFLTGVAGTQSTHESLKPQRVQFQSRNFHNILQWQPG 50
DCRS4.3 1 MMPKIICFLGFLISFFLTGVAGTQSTHESLKPQRVQFQSRNFHNILQWQPG 50
5 **************************************************
DCRS4.1 51 RALTGNSSWFVQYKIYGQRQWKNKEDCWGTQELSCDLTSETSDIQEPYY 100
DCRS4.2 51 RALTGNSSWFVQYKIYGQRQWKNKEDCWGTQELSCDLTSETSDIQEPYY 100
DCRS4.3 51 RALTGNSSWFVQYKIYGQRQWKNKEDCWGTQELSCDLTSETSDIQESYY 100
10 *********************************************** **
DCRS4.1 101 GR-RGKNKNKGNPWGPKQSKRKSKGNQKTNTVTAPAALKAFAGCAKIDPP 149
DCRS4.2 101 GRVRAASAGSYSEWS--MTPRFTP-----------------WWETKIDPP 131
DCRS4.3 101 GRVRAASAGSYSEWS--MTPRFTP-----------------WWE------ 119
15' ** * _ * . * .
DCRS4.1 150 VMNITQVNGSLLVILHAPNLPYRYQKEKNVSIEDYYELLYRVFIINNSLE 199
DCRS4.2 132 VMNITQVNGSLLVILHAPNLPYRYQKEKNVSIEDYYELLYRVFIINNSLE 181
DCRS4.3 123 -------------------------RAKGL 130
20 .. *..
DCRS4.1 200 KEQKWEGAHRAVEIEALTPHSSYCWAEIYQPMLDRRSQRSEERCVEIP 249
DCRS4.2 182 KEQKVYEGAHRAVEIEALTPHSSYCWAEIYQPMLDRRSQRSEERCVEIP 231
DCRS4.3 131 130
Table 4: Reverse Translation of primate, e.g., human, DCRS4.1 (SEQ ID N0: 6).
N may be A, C, G, or T.
3 O
ATGATGCCNAARCAYTGYTTYYTNGGNTTYYTNATHWSNTTYTTYYTNACNGGNGTNGCNGGNACNCARWSNACNCA
YGARWSNYTNAARCCNCARMGNGTNCARTTYCARWSNMGNAAYTTYCAYAAYATHYTNCARTGGCARCCNGGNMGNG
CNYTNACNGGNAAYWSNWSNGTNTAYTTYGTNCARTAYAARATHTAYGGNCARMGNCARTGGAARAAYAARGARGAY
TGYTGGGGNACNCARGARYTNWSNTGYGAYYTNACNWSNGARACNWSNGAYATHCARGARCCNTAYTAYGGNMGNMG
NGGNAARAAYAARAAYAARGGNAAYCCNTGGGGNCCNAARCARWSNAARMGNAARWSNAARGGNAAYCARAAR.ACNA
3 5
AYACNGTNACNGCNCCNGCNGCNYTNAARGCNTTYGCNGGNTGYGCNAARATHGAYCCNCCNGTNATGAAYATHACN
CARGTNAAYGGNWSNYTNYTNGTNATHYTNCAYGCNCCNAAYYTNCCNTAYMGNTAYCARAARGARAARAAYGTNWS
NATHGARGAYTAYTAYGARYTNYTNTAYMGNGTNTTYATHATHAAYAAYWSNYTNGARAARGARCARAARGTNTAYG
ARGGNGCNCAYMGNGCNGTNGARATHGARGCNYTNACNCCNCAYWSNWSNTAYTGYGTNGTNGCNGARATHTAYCAR
CCNATGYTNGAYMGNMGNWSNCARMGNWSNGARGARMGNTGYGTNGARATHCCN
Reverse Translation of primate, e.g., human, DCRS4.2 (SEQ ID NO: 29).
N may be A, C, G, or T.
4 5
ATGATGCCNAARCAYTGYTTYYTNGGNTTYYTNATHWSNTTYTTYYTNACNGGNGTNGCNGGNACNCARWSNACNCA
YGARWSNYTNAARCCNCARMGNGTNCARTTYCARWSNMGNAAYTTYCAYAAYATHYTNCARTGGCARCCNGGNMGNG
CNYTNACNGGNAAYWSNWSNGTNTAYTTYGTNCARTAYAARATHTAYGGNCARMGNCARTGGAARAAYAARGARGAY
TGYTGGGGNACNCARGARYTNWSNTGYGAYYTNACNWSNGARACNWSNGAYATHCARGARCCNTAYTAYGGNMGNGT
NMGNGCNGCNWSNGCNGGNWSNTAYWSNGARTGGWSNATGACNCCNMGNTTYACNCCNTGGTGGGARACNAARATHG
5 O
AYCCNCCNGTNATGAAYATHACNCARGTNAAYGGNWSNYTNYTNGTNATHYTNCAYGCNCCNAAYYTNCCNTAYMGN
TAYCARAARGA~2AARAAYGTNWSNATHGARGAYTAYTAYGARYTNYTNTAYMGNGTNTTYATHATHAAYAAYWSNYT
NGARAARGARCARAARGTNTAYGARGGNGCNCAYMGNGCNGTNGARATHGARGCNYTNACNCCNCAYWSNWSNTAYT
GYGTNGTNGCNGARATHTAYCARCCNATGYTNGAYMGNMGNWSNCARMGNWSNGARGARMGNTGYGTNGARATHCCN
55 Reverse Translation of primate, e.g., human, DCRS4.3 (SEQ ID N0: 32). N may
be A, C, G, or T.
ATGATGCCNAARCAYTGYTTYYTNGGNTTYYTNATHWSNTTYTTYYTNACNGGNGTNGCNGGNACNCARWSNACNCA
YGARWSNYTNAARCCNCARMGNGTNCARTTYCARWSNMGNAAYTTYCAYAAYATHYTNCARTGGCARCCNGGNMGNG
60
CNYTNACNGGNAAYWSNWSNGTNTAYTTYGTNCARTAYAARATHTAYGGNCARMGNCARTGGAARAAYAARGARGAY
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TGYTGGGGNACNCARGARYTNWSNTGYGAYYTNACNWSNGARACNWSNGAYATHCARGARWSNTAYTAYGGNMGNGT
NMGNGCNGCNWSNGCNGGNWSNTAYWSNGARTGGWSNATGACNCCNMGNTTYACNCCNTGGTGGGARMGNGCNAARG
GNYTN
Nucleic acid sequence comparison of three DCRS4 embodiments:
DCRS4.1 1 ATGATGCCTAAACATTGCTTTCTAGGCTTCCTCATCAGTTTCTTCCTTAC 50
DCRS4.2 1 ATGATGCCTAAACATTGCTTTCTAGGCTTCCTCATCAGTTTCTTCCTTAC 50
DCRS4.3 1 ATGATGCCTAAACATTGCTTTCTAGGCTTCCTCATCAGTTTTTTCCTTAC 50
***************************************** ********
DCRS4.1 51 TGGTGTAGCAGGAACTCAGTCAACGCATGAGTCTCTGAAGCCTCAGAGGG 100
DCRS4.2 51 TGGTGTAGCAGGAACTCAGTCAACGCATGAGTCTCTGAAGCCTCAGAGGG 100
DCRS4.3 51 TGGTGTAGCAGGAACTCAGTCAACGCATGAGTCTCTGAAGCCTCAGAGGG 100
15' **************************************************
DCRS4.1 101 TACAATTTCAGTCCCGAAATTTTCACAACATTTTGCAATGGCAGCCTGGG 150
DCRS4.2 101 TACAATTTCAGTCCCGAAATTTTCACAACATTTTGCAATGGCAGCCCGGG 150
DCRS4.3 101 TACAATTTCAGTCCCGAAATTTTCACAACATTTTGCAATGGCAGCCTGGG 150
2 0 ********************************************** ***
DCRS4.1 151 AGGGCACTTACTGGCAACAGCAGTGTCTATTTTGTGCAGTACAAAATATA 200
DCRS4.2 151 AGGGCACTTACTGGCAACAGCAGTGTCTATTTTGTGCAGTACAAAATATA 200
DCRS4.3 151 AGGGCACTTACTGGCAACAGCAGTGTCTATTTTGTGCAGTACAAAATATA 200
2 5 **************************************************
DCRS4.1 201 TGGACAGAGACAATGGAAAAATAAAGAAGACTGTTGGGGTACTCAAGAAC 250
DCRS4.2 201 TGGACAGAGACAATGGAAAAATAAAGAAGACTGTTGGGGTACTCAAGAAC 250
DCRS4.3 201 TGGACAGAGACAATGGAAAAATAAAGAAGACTGTTGGGGTACTCAAGAAC 250
3 0 **************************************************
DCRS4.1 251 TCTCTTGTGACCTTACCAGTGAAACCTCAGACATACAGGAACCTTATTAC 300
DCRS4.2 251 TCTCTTGTGACCTTACCAGTGAAACCTCAGACATACAGGAACCTTATTAC 300
DCRS4.3 251 TCTCTTGTGACCTTACCAGTGAAACCTCAGACATACAGGAATCTTATTAC 300
3 5 ***************************************** ********
DCRS4.1 301 GGGAGGAGGGGCAAAAATAAAAATAAAGGGAATCCTTGGGGGCCAAAACA 350
DCRS4.2 301 GGGAGGGTG-----------------AGGGCGGCCTCGGC---------- 323
DCRS4.3 301 GGGAGGGTG-----------------AGGGCGGCCTCGGC---------- 323
4 0 ****** * **** *** **
DCRS4.1 351 AAGTAAACGGAAATCAAAGGGGAACCAGAAGACCAACACAGTGACTGCCC 400
DCRS4.2 324 ---TGGGAGCTACTCAGAATGGAGCATGA-------CGCCGCGGTTCACT 363
DCRS4.3 324 ---TGGGAGCTACTCAGAATGGAGCATGA-------CGCCGCGGTTCACT 363
45 * * * *** * *** * **
DCRS4.1 401 CAGCTGCCCTGAAGGCATTTGCTGGATGTGCAAAAATAGATCCTCCAGTC 450
DCRS4.2 364 C------CCTGGTGGGAA-----------ACAAAAATAGATCCTCCAGTC 396
DCRS4.3 364 C------CCTGGTGGGAAAGAGCAAAAGGTTTATGAAGGGGCTCACAGA- 406
50 * **** ** * * * * * ***
DCRS4.1 451 ATGAATATAACCCAAGTC--AATGGCTCTTTGTTGGTAATTCTCCATGCT 498
DCRS4.2 397 ATGAATATAACCCAAGTC--AATGGCTCTTTGTTGGTAATTCTCCATGCT 444
DCRS4.3 407 GCGGTTGAAATTGAAGCTCTAACACCACACTCCAGCTACTGTGTAGTGGC 456
55 * * ** *** ** * * * * ** * **
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DCRS4.1 499 CCAAATTTACCATATAGATACCAAAAGGAAAAAAATGTATCTATAGAAGA 548
DCRS4.2 445 CCAAATTTACCATATAGATACCAAAAGGAAAAAAATGTATCTATAGAAGA 494
DCRS4.3 457 TGAAATATATCA-GCCCACGTTAGACAGAAGAAGTCAGAGAAGT-GAAGA 504
**** ** ** * * * *** ** * * *****
DCRS4.1 549 TTACTATGAACTACTATACCGAGTTTTTATAATTAACAATTCACTAGAAA 598
DCRS4.2 495 TTACTATGAACTACTATACCGAGTTTTTATAATTAACAATTCACTAGAAA 544
DCRS4.3 505 GAGATGTGTGGAAATTCCATGA 526
* ** * * **
DCRS4.1 599 AGGAGCAAAAGGTTTATGAAGGGGCTCACAGAGCGGTTGAAATTGAAGCT 648
DCRS4.2 545 AGGAGCAAAAGGTTTATGAAGGGGCTCACAGAGCGGTTGAAATTGAAGCT 594
DCRS4.3 527 526
15~
DCRS4.1 649 CTAACACCACACTCCAGCTACTGTGTAGTGGCTGAAATATATCAGCCCAT 698
DCRS4.2 595 CTAACACCACACTCCAGCTACTGTGTAGTGGCTGAAATATATCAGCCCAT 644
DCRS4.3 527 526
DCRS4.1 699 GTTAGACAGAAGAAGTCAGAGAAGTGAAGAGAGATGTGTGGAAATTCCAT 748
DCRS4.2 645 GTTAGACAGAAGAAGTCAGAGAAGTGAAGAGAGATGTGTGGAAATTCCAT 694
DCRS4.3 527 526
DCRS4.1 749 GA 750
DCRS4.2 695 GA 696
DCRS4.3 527 526
Table 5: Alignment of various cytokine receptor subunits with DCRS3.1.
IL-2R is SEQ ID NO: 7; IL-9R is SEQ ID NO: 8; GM/IL-3/5 receptor b
subunit common (ILRbc) is SEQ ID N0: 9; TPOR is SEQ ID NO: 10; and IL-7R
3 5 is SEQ ID NO: 11 (see GenBank).
IL-2R HU VNG--TSQFTC---FYNSRANISCVWSQ-DGALQDTSCQVHAWPDRRRWN-------QTC
DCRS3_HU LCS--PKPPPT----KASLPTDPPGWGC-PDLVCYTDYLQTVICILEMWN--LHP--STL
IL-9R HU ICI----C-TC-----VCLGVSVTGEGQGPRSRTFTCLTNNILRIDCHWS---APELGQG
4 O ILRbc HU ILTPNGNEDTTADFFLTTMPTDSLSVST-LPLPEVQCFVFNVEYMNCTWNSSSEPQPTNL
TPOR_HU LLASDSEPLKC---FSRTFEDLTCFWDE-EEAAPSGTYQLLYAYPREKPR--ACP--LSS
IL-7R HU VSGESGYAQNG---DLEDAELDDYSFSC-YSQLEVNGSQHSLTCAFEDPD--------VN
4 5 IL-2R ELLPVSQASWACN----------LILG---------APDS--QKLTTVD---------IV
HU
HU TLTWILSNNTGCYIKDR-----TLDLRQ-DQYE--ELKDEA-TSCSLHR-----SAHNAT
DCRS3
_ SSPWLLFTSNQAPG----G-THKCILR--GSECTWLPPE--AVLVPSD--------NFT
IL-9R HU
HU TLHYWYKNSDNDK-------VQKCSHY--------LFSEEITSGCQLQK-K---EIHLYQ
ILRbc
_ QSMPHFGTRYVCQFPDQ--EEVRLFFPLHLWVKNVFLNQTRTQRVLFVDSVGLPAPPSII
TPOR
HU
_ TTNLEFEICGALV------EVKCLNFR--------KLQEIYFIETKKFL---------LI
50 IL-7R
HU
IL-2R HU TLRVLCREGVRWRV---MAIQDFKPFENLRLMAPISLQV----VHVETHRCNIS---WEI
HU HATYTCHMDVFHF----MADDIFS--VNITDQSGNYSQECGSFLLAESRQYNIS---WRS
DCRS3
_ ITFHHCMSGREQVS---LVDPEYLPRRHVKLDPPSDLQS-----NISSGHCILT---WSI
55 IL-9R
HU
HU TFWQLQDPREPRR---QATQMLKLQNLVIPWAPENLTL----HKLSESQLELN---WNN
ILRbc
_ KAMGGSQPGELQISWEEPAPEISDFLRYELRYGPRDPKNS---TGPTVIQLIATETCCPA
HU
TPOR
_ GKSNICVK-VGEKS---LTCKKIDLTTIVKPEAPFDLSVI---YREGANDFWT---FNT
IL-7R HU
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IL-2R HU SQASHYFERHLE---FEARTLSPGHTWEEAPLLTLK-------QKQEWICLETLT-PDTQ
HU DYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLIS-----VDSRSVSLLPLEFRKDSS
DCRS3
_ SPALEPMTTLLS---YELAFKKQEEAWEQAQHRDHIV-----G-VTWLILEAFELDPGFI
IL-9R HU
HU RFLNHCLEHLV-------QYRTD---WDHSWTEQSV------DYRHKFSLPSVDGQKRYT
ILRbc
_ LQRPHSASALD-----QSPCAQPTMPWQDGPKQTSPSREASALTAEGGSCLISGLQPGNS
HU
TPOR
_ SHLQKKWKVLMHD-VAYRQEKDENKWTHVNLSST---------KLTLLQRK--LQPAAM
IL-7R HU
1O IL-2R HU YEFQVRVKPLQGEFT---------TWSPWSQPLAFRTKPAALG
DCRS3_HU YELQVRAGPMPGSSYQG-------TWSEWSDPVIFQTQSEELK
IL-9R HU HEARLRVQMATLEDDWEEERYTGQWSEWSQPVCFQAPQRQGP
ILRbc_HU FRVRSRFNPLCGSAQ---------HWSEWSHPIHWGSNTSKEN
TPOR_HU YWLQLRSEPDGISLGG--------SWGSWSLPVTVDLPGDAVA
15' IL-7R HU YEIKVRSIPDHYFKG---------FWSEWSPSYYFRTPEINNS
- . . * *, ** ,
Alignment of various cytokine receptor subunits with DCRS4.1. IL-lORb is the
beta subunit of IL-lOR, human is SEQ ID N0: 12, mouse is SEQ ID NO: 13; INaRl
2 0 is the beta subunit of IFNa with human SEQ ID N0: 14 and mouse SEQ ID N0:
15;
INgR is interferon gamma receptor subunit alpha with human SEQ ID NO: 16 and
mouse SEQ ID NO: 17; IL-lORa is the alpha receptor subunit with mouse SEQ ID
N0: 18 and human SEQ ID NO: 19; INgS (SEQ ID NO: 20) is the beta receptor
subunit for INFg; Zcytor7 (SEQ ID N0: 21) and CYTOR11 (SEQ ID NO: 22) are
2 5 from patent filings from Zymogenetics, and INaR2 (SEQ ID NO: 23) is the
beta
subunit of the receptor for IFNa.
IL-lORb Hu PENVRMNSVNFKNILQWES-PAFAKGNL--TFTAQYLSY---------RIFQDKCMNTTL
Mu PEKVRMNSVNFKNILQWEV-PAFPKTNL--TFTAQYESY---------RSFQDHCKRTAS
3 O IL-lORb
_ PQKVEVDIIDDNFILRWNR-SDESVGNV--TFSFDYQKTGMD-----NWIKLSGCQNITS
HU
INaRl
_ PENIDWIIDDNYTLKWSS-HGESMGSV--TFSAEYRTKDEA-----KWLKVPECQHTTT
INaRl
MU
_ PTNVTIESYNMNPIVYWEY-QIMPQVP---VFTVEVKNYGVK-----NSEWIDACINISH
INgR HU
INgR MU PTNVLIKSYNLNPWCWEY-QNMSQTP---IFTVQVKVY--------SGSWTDSCTNISD
Mu PSYVWFEARFFQHILHWKP-IPNQSEST--YYEVALKQYGNS-----TWNDIHICRKAQA
3 5 IL-lORa
_ PPSVWFEAEFFHHILHWTP-IPNQSEST--CYEVALLRYGIE-----SWNSISNC--SQT
IL-lORa Hu
INgS HU PLNPRLHLYNDEQILTWEP-SPSSNDPRPWYQVEYSFIDGSW----HRLLEPNCTDITE
Zcytor7 Hu PANITFLSINMKNVLQWTPPEGLQGVKV--TYTVQYFIYGQK-----KWLNKSECRNINR
HU LQHVKFQSSNFENILTWDS-GPEGTPDT--VYSIEYKTYGER-----DWVAKKGCQRITR
CYTOR11
_ SCTFKISLRNFRSILSWEL-KNHSIVPTHYTLLYTIMSKPE------DLKWKNCANTTR
HU
4 0 INaR2
_ PQRVQFQSRNFHNILQWQPGRALTGNSS--VYFVQYKIYGQR-----QWKNKEDCWGTQE
DCRS4 1 HU
Hu TECDFSSLSK------YGDHTLRVRAEFADEHSDWVNIT-FCPVDDTIIGPPG--MQVEV
iL-lORb
_ TQCDFSHLSK------YGDYTVRVRAELADEHSEWVNVT-FCPVEDTIIGPPE--MQIES
Mu
4 5 IL-lORb
_ TKCNFSSLKLN----VYEEIKLRIRAEKEN-TSSWYEVDSFTPFRKAQIGPPE--VHLEA
HU
INaRl
_ TKCEFSLLDTN----VYIKTQFRVRAEEGNSTSSWNEVDPFIPFYTAHMSPPE--VRLEA
INaRl
MU
_ HYCNISDHVGDP----SNSLWVRVKARVGQKESAYAKSEEFAVCRDGKIGPPKLDIR-KE
INgR HU
INgR MU HCCNIYGQIMYP----DVSAWARVKAKVGQKESDYARSKEFLMCLKGKVGPPGLEIRRKK
Mu LSCDLTTFTLDLYHR-SYGYRARVRAVDNSQYSNWTTTETRFTVDEVILTVDS--VTLKA
5 O IL-lORa
_ LSYDLTAVTLDLYH--SNGYRARVRAVDGSRHSNWTVTNTRFSVDEVTLTVGS--VNLEI
Hu
IL-lORa
_ TKCDLTGGGRLKLFPHPFTVFLRVRAKRGNLTSKWVGLEPFQHYENVTVGPPKN-ISVTP
INgS HU
Zcytor7 Hu TYCDLSAETSDY----EHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPE--VALTT
HU KSCNLTVETGN----LTELYYARVTAVSAGGRSATKMTDRFSSLQHTTLKPPDV-TCISK
CYTOR11
_ SFCDLTDEWRS-----THEAYVTVLEGFSGNTTLFSCSHNFWLAIDMSFEPPE--FEIVG
HU
55 INaR2
_ LSCDLTSETSD-----IQEPYYGRRGKNKNKGNPWGPKQSKRKSKGNQKTNTVT-APAAL
DCRS4 1 HU
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Hu LADSLHMRFLAPKIENEYE---TWTMKNVYNSWTYNVQYWKNGTDEKFQ-ITPQYDFEVL
IL-lORb
_ LAESLHLRFSAPQIENEPE---TWTLKNIYDSWAYRVQYWKNGTNEKFQ-WSPYDSEVL
Mu
IL-lORb
_ EDKAIVIHISPGTKDSV-----MWALD--GLSFTYSLLIWKNSSGVEER-IENIYSRHKI
HU
INaRl
_ EDKAILVHISPPGQDGN-----MWALE--KPSFSYTIRIWQKSSSDKKT-INSTYYVEKI
MU
INaRl
_ EKQIMIDIFHPSVFVNGDEQEVDYDPETTCYIRWNVYVRMNGS-EIQY-KILTQKEDDC
INgR HU
INgR MU EEQLSVLVFHPEVWNGESQGTMFGDGSTCYTFDYTVWEHNRSGEILH-TKHTVEKEEC
Mu MDGIIYGTIHPPRPTITPA--GDEYEQVFKDLRWKISIRKFS--ELKN-ATKRVKQETF
IL-lORa
_ HNGFILGKIQLPRPKMAPA--NDTYESIFSHFREYEIAIRKVPG-NFTF-THKKVKHENF
IL-lORa Hu
INgS HU GKGSLVIHFSPPFDVFHG------------ATFQYLVHYWEKSETQQEQ-VEGPFKSNSI
Zcytor7 Hu DEKSISWLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWS-QCVTNHTLVL
HU VRSIQMIVHPTPTPIRAGDG-HRLTLEDIFHDLFYHLELQVNRTYQMHL-GGKQREYEFF
CYTOR11
_ FTNHINVMVKFPSIVEEEL-------Q---FDLSLVIEEQSEGIVKKHKPEIKGNMSGNF
HU
INaR2
_ KAFAGCAKIDPPVMNITQ------------VNGSLLVILHAPNLPYRYQ-KEKNVSIEDY
DCRS4 1 HU
15'
IL-lORb_Hu RN-----------------LEPWTTYCVQVRGFLPDRN----------KAGEWSEPVCEQ
IL-lORb_Mu RN-----------------LEPWTTYCIQVQGFLLDQN----------RTGEWSEPICER
INaRl HU YK-----------------LSPETTYCLKVKAALLTSW----------KIGWSPVHCIK
MU PE-----------------LLPETTYCLEVKAIHPSLK----------KHSNYSTVQCIS
2 0 INaRl
_ DEIQCQLAI--------PVSSLNSQYCVSAEGVLHVWG----------VTTEKSKEVCIT
INgR HU
INgR MU NETLCELNI--------SVSTLDSRYCISVDGISSFWQ----------VRTEKSKDVCIP
Mu TLT---------------VPIGVRKFCVKVLPRLESRI----------NKAEWSEEQCLL
IL-lORa
_ SLL---------------TSGEVGEFCVQVKPSVASRS----------NKGMWSKEECIS
Hu
IL-lORa
_ VLG---------------NLKPYRWCLQTEAQLILKNKK------IRPHGLLSNVSCHE
2 5 INgS HU
Hu TW-----------------LEPNTLYCVHVESFVPGPP----------RRAQPSEKQCAR
Zcytor7
_ GLTPDTEFLGTIMICVPTWAKESAPYMCRVKTLPDRTWTYSFSGAFLFSMGFLVAVLCYL
HU
CYTOR11
_ TYIID-------------KLIPNTNYCVSVYLEHSDEQ-----------AVIKSPLKCTL
INaR2
HU
_ YE-_--_-__-_____-_____--_LLYRVFIINNSLE-_--_-_-_-~Q~yEGAHRA
DCRS4 1 HU
30
IL-lORb Hu TTHDETVP-
IL-lORb_Mu TGNDEITP-
INaRl_HU TTVENELPP
3 5 INaRl_MU TTVANKMPV
INgR HU IFNSSIKG-
INgR MU PFHDDRKD-
IL-lORa_Mu ITTEQYFT-
IL-lORa_Hu LT-RQYFT-
4 0 INgS HU TTANASAR
Zcytor7_Hu TLKDQSSE
CYTOR11_HU SYRYVTKPP
INaR2_HU LPPGQESES
DCRS4 1 HU VEIEALTP
Table 5 shows comparison of sequences of cytokine receptor
subunits with the primate, e.g., human, DCRS3.1 (50R), and
DCRS4.1 (cytor). Both of the new genes are likely alpha type
receptor subunits, and thus should bind to ligand without the
need for a beta subunit. Based upon structural features, the
ligand for the DCRS3 subunits are likely to be a member of the
family of cytokines which includes IL-2, IL-4, IL-7, IL-9, and
the additional cytokines which signal through IL-2y common
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receptor-like subunits IL-13, IL-15, and the TSLP ligand.
Similarly, the ligand for the DCRS4 receptor subunits are
probably a ligand in the IL-10 or IFN families, which may be a
multi-subunit cytokine, analogous to IL-6 and IL-12.
5 As used herein, the term DCRS3 shall be used to describe a
protein comprising an amino acid sequence shown in Table 1;
likewise with DCRS4 and Table 3. In many cases, a substantial
fragment thereof will be functionally or structurally
equivalent, including, e.g., an extracellular or intracellular
10~ domain. The invention also includes a protein variation of a
DCRS3 allele whose sequence is provided, e.g., a mutein or
soluble extracellular construct. Typically, such agonists or
antagonists will exhibit less than about 10°s sequence
differences, and thus will often have between 1- and 11-fold
15 substitutions, e.g., 2-, 3-, 5-, 7-fold, and others. It also
encompasses allelic and other variants, e.g., natural
polymorphic, of the protein described. Typically, it will bind
to its corresponding biological ligand, perhaps in a dimerized
state with an alpha receptor subunit, with high affinity, e.g.,
20 at least about 100 nM, usually better than about 30 nM,
preferably better than about 10 nM, and more preferably at
better than about 3 nM. The term shall also be used herein to
refer to related naturally occurring forms, e.g., alleles,
polymorphic variants, and metabolic variants of the mammalian
25 protein. Preferred forms of the receptor complexes will bind
the appropriate ligand with an affinity and selectivity
appropriate for a ligand-receptor interaction.
This invention also encompasses combinations of proteins or
peptides having substantial amino acid sequence identity with
30 the amino acid sequences in Tables 1 or 3. It will include
sequence variants with relatively few substitutions, e.g.,
preferably less than about 3-5.
A substantial polypeptide "fragment", or "segment", is a
stretch of amino acid residues of at least about 8 amino acids,
generally at least 10 amino acids, more generally at least 12
amino acids, often at least 14 amino acids, more often at least
16 amino acids, typically at least 18 amino acids, more
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31
typically at least 20 amino acids, usually at least 22 amino
acids, more usually at least 24 amino acids, preferably at least
26 amino acids, more preferably at least 28 amino acids, and, in
particularly preferred embodiments, at least about 30 or more
amino acids. Sequences of segments of different proteins can be
compared to one another over appropriate length stretches. In
many situations, fragments may exhibit functional properties of
the intact subunits, e.g., the extracellular domain of the
transmembrane receptor may retain the ligand binding features,
10~ and may be used to prepare a soluble receptor-like complex.
Amino acid sequence homology, or sequence identity, is
determined by optimizing residue matches. In some comparisons,
gaps may be introduces, as required. See, e.g., Needleham, et
al., (1970) J. Mol. Biol. 48:443-453; Sankoff, et al., (1983)
chapter one in Time Warps String Edits and Macromolecules: The
Theory and Practice of Seauence Comparison, Addison-Wesley,
Reading, MA; and software packages from IntelliGenetics,
Mountain View, CA; and the University of Wisconsin Genetics
Computer Group (GCG), Madison, WI; each of which is incorporated
herein by reference. This changes when considering conservative
substitutions as matches. Conservative substitutions typically
include substitutions within the following groups: glycine,
alanine; valine, isoleucine, leucine; aspartic acid, glutamic
acid; asparagine, glutamine; serine, threonine; lysine,
arginine; and phenylalanine, tyrosine. Homologous amino acid
sequences are intended to include natural allelic and
interspecies variations in the cytokine sequence. Typical
homologous proteins or peptides will have from 50-100% homology
(if gaps can be introduced), to 60-100% homology (if
conservative substitutions are included) with an amino acid
sequence segment of Table 1 or 3. Homology measures will be at
least about 70%, generally at least 76%, more generally at least
81%, often at least 85%, more often at least 88%, typically at
least 90%, more typically at least 92%, usually at least 94%,
more usually at least 95%, preferably at least 96%, and more
preferably at least 97%, and in particularly preferred
embodiments, at least 98% or more. The degree of homology will
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32
vary with the length of the compared segments. Homologous
proteins or peptides, such as the allelic variants, will share
most biological activities with the embodiments described in
Table 1 or 3.
As used herein, the term "biological activity" is used to
describe, without limitation, effects on inflammatory responses,
innate immunity, and/or morphogenic development by cytokine-like
ligands. For example, these receptors should mediate
phosphatase or phosphorylase activities, which activities are
10~ easily measured by standard procedures. See, e.g., Hardie, et
al. (eds. 1995) The Protein Kinase FactBook vols. I and II,
Academic Press, San Diego, CA; Hanks, et al. (1991) Meth.
Enzymol. 200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin
(1990) Cell 61:743-752; Pines, et al. (1991) Cold Spring Harbor
Sump. Ouant. Biol. 56:449-463; and Parker, et al. (1993) Nature
363:736-738. The receptors, or portions thereof, may be useful
as phosphate labeling enzymes to label general or specific
substrates. The subunits may also be functional immunogens to
elicit recognizing antibodies, or antigens capable of binding
antibodies.
The terms ligand, agonist, antagonist, and analog of, e.g.,
a DCRS3, include molecules that modulate the characteristic
cellular responses to cytokine ligand proteins, as well as
molecules possessing the more standard structural binding
competition features of ligand-receptor interactions, e.g.,
where the receptor is a natural receptor or an antibody. The
cellular responses likely are typically mediated through
receptor tyrosine kinase pathways.
Also, a ligand is a molecule which serves either as a
natural ligand to which said receptor, or an analog thereof,
binds, or a molecule which is a functional analog of the natural
ligand. The functional analog may be a ligand with structural
modifications, or may be a wholly unrelated molecule which has a
molecular shape which interacts with the appropriate ligand
binding determinants. The ligands may serve as agonists or
antagonists, see, e.g., Goodman, et al. (eds. 1990) Goodman &
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33
Gilman's~ The Pharmacological Bases of Therapeutics, Pergamon
Press, New York.
Rational drug design may also be based upon structural
studies of the molecular shapes of a receptor or antibody and
other effectors or ligands. See, e.g., Herz, et al. (1997) J.
ReceQt Sianal Transduct. Res. 17:671-776; and Chaiken, et al.
(1996) Trends Biotechnol. 14:369-375. Effectors may be other
proteins which mediate other functions in response to ligand
binding, or other proteins which normally interact with the
10~ receptor. One means for determining which sites interact with
specific other proteins is a physical structure determination,
e.g., x-ray crystallography or 2 dimensional NMR techniques.
These will provide guidance as to which amino acid residues form
molecular contact regions. For a detailed description of
protein structural determination, see, e.g., Blundell and
Johnson (1976) Protein Crystallography, Academic Press, New
York, which is hereby incorporated herein by reference.
II. Activities
The cytokine receptor-like proteins will have a number of
different biological activities, e.g., modulating cell
proliferation, or in phosphate metabolism, being added to or
removed from specific substrates, typically proteins. Such will
generally result in modulation of an inflammatory function,
other innate immunity response, or a morphological effect, as
typical of cytokine or interleukin signaling. The subunit may
have a specific low affinity binding to the ligand.
The receptors may signal through the JAK pathway. See,
e.g., Ihle, et al. (1997) Stem Cells 15(suppl. 1):105-111;
Silvennoinen, et al. (1997) APMIS 105:497-509; Levy (1997)
Cytokine Growth Factor Review 8:81-90; Winston and Hunter (1996)
Current Biol. 6:668-671; Barrett (1996) Baillieres Clin.
Gastroenterol. 10:1-15; and Briscoe, et al. (1996) Philos.
Trans. R. Soc. Lond. B. Biol. Sci. 351:167-171.
The biological activities of the cytokine receptor subunits
will be related to addition or removal of phosphate moieties to
substrates, typically in a specific manner, but occasionally in
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a non specific manner. Substrates may be identified, or
conditions for enzymatic activity may be assayed by standard
methods, e.g., as described in Hardie, et al. (eds. 1995) The
Protein Kinase FactBook vols. I and II, Academic Press, San
Diego, CA; Hanks, et al. (1991) Meth. Enzymol. 200:38-62;
Hunter, et al. (1992) Cell 70:375-388; Lewin (1990) Cell 61:743-
752; Pines, et al. (1991) Cold Spring Harbor Symp. Ouant. Biol.
56:449-463; and Parker, et al. (1993) Nature 363:736-738.
The receptor subunits may combine with other subunits,
10~ e.g., beta subunits, to form functional complexes, e.g., which
may be useful for binding ligand or preparing antibodies. These
will have substantial diagnostic uses, including detection or
quantitation.
ILI. Nucleic Acids
This invention contemplates use of isolated nucleic acid or
fragments, e.g., which encode these or closely related proteins,
or fragments thereof, e.9., to encode a corresponding
polypeptide_, preferably one which is biologically active. In
addition, this invention covers isolated or recombinant DNAs
which encode combinations of such proteins or polypeptides
having characteristic sequences, e.g., of DCRS3s or DCRS4s.
Typically, the nucleic acid is capable of hybridizing, under
appropriate conditions, with a nucleic acid sequence segment
shown in Tables 1 or 3, but preferably not with a corresponding
segment of other receptors, e.g., described in Table 5. Said
biologically active protein or polypeptide can be a full length
protein, or fragment, and will typically have a segment of amino
acid sequence highly homologous, e.g., exhibiting significant
stretches of identity, to ones shown in Tables 1 or 3. Further,
this invention covers the use of isolated or recombinant nucleic
acid, or fragments thereof, which encode proteins having
fragments which are equivalent to DCRS3 or DCRS4 proteins. The
isolated nucleic acids can have the respective regulatory
sequences in the 5' and 3' flanks, e.g., promoters, enhancers,
poly-A addition signals, and others from the natural gene.
Combinations, as described, are also provided.
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An "isolated" nucleic acid is a nucleic acid, e.g., an RNA,
DNA, or a mixed polymer, which is substantially pure, e.g.,
separated from other components which naturally accompany a
native sequence, such as ribosomes, polymerases, and flanking
5 genomic sequences from the originating species. The term
embraces a nucleic acid sequence which has been removed from its
naturally occurring environment, and includes recombinant or
cloned DNA isolates, which are thereby distinguishable from
naturally occurring compositions, and chemically synthesized
10~ analogs or analogs biologically synthesized by heterologous
systems. A substantially pure molecule includes isolated forms
of the molecule, either completely or substantially pure.
An isolated nucleic acid will generally be a homogeneous
composition of molecules, but will, in some embodiments, contain
15 heterogeneity, preferably minor. This heterogeneity is
typically found at the polymer ends or portions not critical to
a desired biological function or activity.
A "recombinant" nucleic acid is typically defined either by
its method of production or its structure. In reference to its
20 method of production, e.g., a product made by a process, the
process is use of recombinant nucleic acid techniques, e.g.,
involving human intervention in the nucleotide sequence.
Typically this intervention involves in vitro manipulation,
although under certain circumstances it may involve more
25 classical animal breeding techniques. Alternatively, it can be
a nucleic acid made by generating a sequence comprising fusion
of two fragments which are not naturally contiguous to each
other, but is meant to exclude products of nature, e.g.,
naturally occurring mutants as found in their natural state.
30 Thus, for example, products made by transforming cells with an
unnaturally occurring vector is encompassed, as are nucleic
acids comprising sequence derived using any synthetic
oligonucleotide process. Such a process is often done to
replace a codon with a redundant codon encoding the same or a
35 conservative amino acid, while typically introducing or removing
a restriction enzyme sequence recognition site. Alternatively,
the process is performed to join together nucleic acid segments
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36
of desired functions to generate a single genetic entity
comprising a desired combination of functions not found in the
commonly available natural forms, e.g., encoding a fusion
protein. Restriction enzyme recognition sites are often the
target of such artificial manipulations, but other site specific
targets, e.g., promoters, DNA replication sites, regulation
sequences, control sequences, or other useful features may be
incorporated by design. A similar concept is intended for a
recombinant, e.g., fusion, polypeptide. This will include a
10~ dimeric repeat. Specifically included are synthetic nucleic
acids which, by genetic code redundancy, encode equivalent
polypeptides to fragments of DCRS3 or DCRS4 and fusions of
sequences from various different related molecules, e.g., other
cytokine receptor family members.
A "fragment" in a nucleic acid context is a contiguous
segment of at least about 17 nucleotides, generally at least 21
nucleotides, more generally at least 25 nucleotides, ordinarily
at least 30 nucleotides, more ordinarily at least 35
nucleotides, often at least 39 nucleotides, more often at least
45 nucleotides, typically at least 50 nucleotides, more
typically at least 55 nucleotides, usually at least 60
nucleotides, more usually at least 66 nucleotides, preferably at
least 72 nucleotides, more preferably at least 79 nucleotides,
and in particularly preferred embodiments will be at least 85 or
more nucleotides. Typically, fragments of different genetic
sequences can be compared to one another over appropriate length
stretches, particularly defined segments such as the domains
described below.
A nucleic acid which codes for a DCRS3 or DCRS4 will be
particularly useful to identify genes, mRNA, and cDNA species
which code for itself or closely related proteins, as well as
DNAs which code for polymorphic, allelic, or other genetic
variants, e.g., from different individuals or related species.
Preferred probes for such screens are those regions of the
interleukin which are conserved between different polymorphic
variants or which contain nucleotides which lack specificity,
and will preferably be full length or nearly so. In other
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37
situations, polymorphic variant specific sequences will be more
useful .
This invention further covers recombinant nucleic acid
molecules and fragments having a nucleic acid sequence identical
to or highly homologous to the isolated DNA set forth herein.
In particular, the sequences will often be operably linked to
DNA segments which control transcription, translation, and DNA
replication. These additional segments typically assist in
expression of the desired nucleic acid segment.
10~ Homologous, or highly identical, nucleic acid sequences,
when compared to one another, e.g., DCRS3 sequences, exhibit
significant similarity. The standards for homology in nucleic
acids are either measures for homology generally used in the art
by sequence comparison or based upon hybridization conditions.
Comparative hybridization conditions are described in greater
detail below.
Substantial identity in the nucleic acid sequence
comparison context means either that the segments, or their
complementary strands, when compared, are identical when
optimally aligned, with appropriate nucleotide insertions or
deletions, in at least about 60% of the nucleotides, generally
at least 66%, ordinarily at least 71%, often at least 76%, more
often at least 80%, usually at least 84%, more usually at least
88%, typically at least 91%, more typically at least about 93%,
preferably at least about 95%, more preferably at least about 96
to 98% or more, and in particular embodiments, as high at about
99% or more of the nucleotides, including, e.g., segments
encoding structural domains such as the segments described
below. Alternatively, substantial identity will exist when the
segments will hybridize under selective hybridization
conditions, to a strand or its complement, typically using a
sequence derived from Tables 1 or 3. Typically, selective
hybridization will occur when there is at least about 55%
homology over a stretch of at least about 14 nucleotides, more
typically at least about 65%, preferably at least about 75%, and
more preferably at least about 90%. See, Kanehisa (1984) Nucl.
Acids Res. 12:203-213, which is incorporated herein by
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reference. The length of homology comparison, as described, may
be over longer stretches, and in certain embodiments will be
over a stretch of at least about 17 nucleotides, generally at
least about 20 nucleotides, ordinarily at least about 24
nucleotides, usually at least about 28 nucleotides, typically at
least about 32 nucleotides, more typically at least about 40
nucleotides, preferably at least about 50 nucleotides, and more
preferably at least about 75 to 100 or more nucleotides. This
includes, e.g., 125, 150, 175, 200, 225, 250, 275, 300, 325,
10~ 350, 375, 400, 425, 450, 475, 500, 525, 544, and other lengths.
Stringent conditions, in referring to homology in the
hybridization context, will be stringent combined conditions of
salt, temperature, organic solvents, and other parameters
typically controlled in hybridization reactions. Stringent
temperature conditions will usually include temperatures in
excess of about 30 C, more usually in excess of about 37 C,
typically in excess of about 45 C, more typically in excess of
about 55 C, preferably in excess of about 65 C, and more
preferably in excess of about 70 C. Stringent salt conditions
will ordinarily be less than about 500 mM, usually less than
about 400 mM, more usually less than about 300 mM, typically
less than about 200 mM, preferably less than about 100 mM, and
more preferably less than about 80 mM, even down to less than
about 20 mM. However, the combination of parameters is much
more important than the measure of any single parameter. See,
e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370, which
is hereby incorporated herein by reference.
The isolated DNA can be readily modified by nucleotide
substitutions, nucleotide deletions, nucleotide insertions, and
inversions of nucleotide stretches. These modifications result
in novel DNA sequences which encode this protein or its
derivatives. These modified sequences can be used to produce
mutant proteins (muteins) or to enhance the expression of
variant species. Enhanced expression may involve gene
amplification, increased transcription, increased translation,
and other mechanisms. Such mutant DCRS-like derivatives include
predetermined or site-specific mutations of the protein or its
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fragments, including silent mutations using genetic code
degeneracy. "Mutant DCRS3" as used herein encompasses a
polypeptide otherwise falling within the homology definition of
the DCRS3 as set forth above, but having an amino acid sequence
which differs from that of other cytokine receptor-like proteins
as found in nature, whether by way of deletion, substitution, or
insertion. In particular, "site specific mutant DCRS3"
encompasses a protein having substantial sequence identity with
a protein of Table 1, and typically shares most of the
10~ biological activities or effects of the forms disclosed herein.
Likewise in reference to DCRS4.
Although site specific mutation sites are predetermined,
mutants need not be site specific. Mammalian DCRS3 mutagenesis
can be achieved by making amino acid insertions or deletions in
the gene, coupled with expression. Substitutions, deletions,
insertions, or many combinations may be generated to arrive at a
final construct. Insertions include amino- or carboxy- terminal
fusions. Random mutagenesis can be conducted at a target codon
and the expressed mammalian DCRS3 mutants can then be screened
for the desired activity, providing some aspect of a structure-
activity relationship. Methods for making substitution
mutations at predetermined sites in DNA having a known sequence
are well known in the art, e.g., by M13 primer mutagenesis. See
also Sambrook, et al. (1989) and Ausubel, et al. (1987 and
periodic Supplements).
The mutations in the DNA normally should not place coding
sequences out of reading frames and preferably will not create
complementary regions that could hybridize to produce secondary
mRNA structure such as loops or hairpins.
The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce
suitable synthetic DNA fragments. A double stranded fragment
will often be obtained either by synthesizing the complementary
strand and annealing the strand together under appropriate
conditions or by adding the complementary strand using DNA
polymerase with an appropriate primer sequence.
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Polymerase chain reaction (PCR) techniques can often be
applied in mutagenesis. Alternatively, mutagenesis primers are
commonly used methods for generating defined mutations at
predetermined sites. See, e.g., Innis, et al. (eds. 1990) PCR
5 Protocols: A Guide to Methods and Applications Academic Press,
San Diego, CA; and Dieffenbach and Dveksler (1995; eds.) PCR
Primer: A Laboratory Manual Cold Spring Harbor Press, CSH, NY.
Certain embodiments of the invention are directed to
combination compositions comprising the receptor or ligand
10~ sequences described. In other embodiments, functional portions
of the sequences may be joined to encode fusion proteins. In
other forms, variants of the described sequences may be
substituted.
15 IV. Proteins, Peptides
As described above, the present invention encompasses
primate DCRS3, e.g., whose sequences are disclosed in Table l,
and described above. Allelic and other variants are also
contemplated, including, e.g., fusion proteins combining
20 portions of such sequences with others, including, e.g., epitope
tags and functional domains.
The present invention also provides recombinant proteins,
e.g., heterologous fusion proteins using segments from these
primate or rodent proteins. A heterologous fusion protein is a
25 fusion of proteins or segments which are naturally not normally
fused in the same manner. Thus, the fusion product of a DCRS3
with another cytokine receptor is a continuous protein molecule
having sequences fused in a typical peptide linkage, typically
made as a single translation product and exhibiting properties,
30 e.g., sequence or antigenicity, derived from each source
peptide. A similar concept applies to heterologous nucleic acid
sequences. Combinations of various designated proteins into
complexes are also provided.
In addition, new constructs may be made from combining
35 similar functional or structural domains from other related
proteins, e.g., cytokine receptors or Toll-like receptors,
including species variants. For example, ligand-binding or
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other segments may be "swapped" between different new fusion
polypeptides or fragments. See, e.g., Cunningham, et al. (1989)
Science 243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem.
263:15985-15992, each of which is incorporated herein by
reference. Thus, new chimeric polypeptides exhibiting new
combinations of specificities will result from the functional
linkage of receptor-binding specificities. For example, the
ligand binding domains from other related receptor molecules may
be added or substituted for other domains of this or related
10~ proteins. The resulting protein will often have hybrid function
and properties. For example, a fusion protein may include a
targeting domain which may serve to provide sequestering of the
fusion protein to a particular subcellular organelle.
Candidate fusion partners and sequences can be selected
from various sequence data bases, e.g., GenBank, c/o
IntelliGenetics, Mountain View, CA; and BCG, University of
Vdisconsin Biotechnology Computing Group, Madison, WI, which are
each incorporated herein by reference. In particular,
combinations of polypeptide sequences provided in Tables 1 and 3
are particularly preferred. Variant forms of the proteins may
be substituted in the described combinations.
The present invention particularly provides muteins which
bind cytokine-like ligands, and/or which are affected in signal
transduction. Structural alignment of human DCRS3 or DCRS4 with
other members of the cytokine receptor family show conserved
features/residues. See Table 5. Alignment of human DCRS3 or
DCRS4 sequence with other members of the cytokine receptor
family indicates various structural and functionally shared
features. See also, Bazan, et al. (1996) Nature 379:591; Lodi,
et al. (1994) Science 263:1762-1766; Sayle and Milner-White
(1995) TIBS 20:374-376; and Gronenberg, et al. (1991) Protein
Enaineerina 4:263-269.
Substitutions with either mouse sequences or human
sequences are particularly preferred. Conversely, conservative
substitutions away from the ligand binding interaction regions
will probably preserve most signaling activities; and
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conservative substitutions away from the intracellular domains
will probably preserve most ligand binding properties.
"Derivatives" of primate DCRS3 include amino acid sequence
mutants, glycosylation variants, metabolic derivatives and
covalent or aggregative conjugates with other chemical moieties.
Covalent derivatives can be prepared by linkage of
functionalities to groups which are found in DCRS3 amino acid
side chains or at the N- or C- termini, e.g., by means which are
well known in the art. These derivatives can include, without
10~ limitation, aliphatic esters or amides of the carboxyl terminus,
or of residues containing carboxyl side chains, O-acyl
derivatives of hydroxyl group-containing residues, and N-acyl
derivatives of the amino terminal amino acid or amino-group
containing residues, e.g., lysine or arginine. Acyl groups are
selected from the group of alkyl-moieties, including C3 to C18
normal alkyl, thereby forming alkanoyl aroyl species.
In particular, glycosylation alterations are included,
e.g., made by modifying the glycosylation patterns of a
polypeptide during its synthesis and processing, or in further
processing steps. Particularly preferred means for
accomplishing this are by exposing the polypeptide to
glycosylating enzymes derived from cells which normally provide
such processing, e.g., mammalian glycosylation enzymes.
Deglycosylation enzymes are also contemplated. Also embraced are
versions of the same primary amino acid sequence which have
other minor modifications, including phosphorylated amino acid
residues, e.g., phosphotyrosine, phosphoserine, or
phosphothreonine.
A major group of derivatives are covalent conjugates of the
receptors or fragments thereof with other proteins of
polypeptides. These derivatives can be synthesized in
recombinant culture such as N- or C-terminal fusions or by the
use of agents known in the art for their usefulness in
cross-linking proteins through reactive side groups. Preferred
derivatization sites with cross-linking agents are at free amino
groups, carbohydrate moieties, and cysteine residues.
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Fusion polypeptides between the receptors and other
homologous or heterologous proteins are also provided.
Homologous polypeptides may be fusions between different
receptors, resulting in, for instance, a hybrid protein
exhibiting binding specificity for multiple different cytokine
ligands, or a receptor which may have broadened or weakened
specificity of substrate effect. Likewise, heterologous fusions
may be constructed which would exhibit a combination of
properties or activities of the derivative proteins. Typical
10~ examples are fusions of a reporter polypeptide, e.g.,
luciferase, with a segment or domain of a receptor, e.g., a
ligand-binding segment, so that the presence or location of a
desired ligand may be easily determined. See, e.g., Dull, et
al., U.S. Patent No. 4,859,609, which is hereby incorporated
herein by reference. Other gene fusion partners include
glutathione-S-transferase (GST), bacterial f~-galactosidase,
trpE, Protein A, f3-lactamase, alpha amylase, alcohol
dehydrogenase, and yeast alpha mating factor. See, e.g.,
Godowski, et al. (1988) Science 241:812-816. Labeled proteins
will often be substituted in the described combinations of
proteins.
The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce
suitable synthetic DNA fragments. A double stranded fragment
will often be obtained either by synthesizing the complementary
strand and annealing the strand together under appropriate
conditions or by adding the complementary strand using DNA
polymerase with an appropriate primer sequence.
Such polypeptides may also have amino acid residues which
have been chemically modified by phosphorylation, sulfonation,
biotinylation, or the addition or removal of other moieties,
particularly those which have molecular shapes similar to
phosphate groups. In some embodiments, the modifications will
be useful labeling reagents, or serve as purification targets,
e.9., affinity ligands.
Fusion proteins will typically be made by either
recombinant nucleic acid methods or by synthetic polypeptide
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methods. Techniques for nucleic acid manipulation and
expression are described generally, for example, in Sambrook, et
al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.),
Vols. 1-3, Cold Spring Harbor Laboratory, and Ausubel, et al.
(eds. 1987 and periodic supplements) Current Protocols in
Molecular Bioloay, Greene/Wiley, New York, which are each
incorporated herein by reference. Techniques for synthesis of
polypeptides -are described, for example, in Merrifield (1963) J.
Amer. Chem. Soc. 85:2149-2156; Merrifield (1986) Science 232:
10~ 341-347; and Atherton, et al. (1989) Solid Phase Peptide
Synthesis: A Practical Approach, IRL Press, Oxford; each of
which is incorporated herein by reference. See also Dawson, et
al. (1994) Science 266:776-779 for methods to make larger
polypeptides.
This invention also contemplates the use of derivatives of
a DCRS3 or DCRS4 other than variations in amino acid sequence or
glycosylation. Such derivatives may involve covalent or
aggregative association with chemical moieties. These
derivatives generally fall into three classes: (1) salts, (2)
side chain and terminal residue covalent modifications, and (3)
adsorption complexes, for example with cell membranes. Such
covalent or aggregative derivatives are useful as immunogens, as
reagents in immunoassays, or in purification methods such as for
affinity purification of a receptor or other binding molecule,
e.g., an antibody. For example, a cytokine ligand can be
immobilized by covalent bonding to a solid support such as
cyanogen bromide-activated Sepharose, by methods which are well
known in the art, or adsorbed onto polyolefin surfaces, with or
without glutaraldehyde cross-linking, for use in the assay or
purification of a cytokine receptor, antibodies, or other
similar molecules. The ligand can also be labeled with a
detectable group, for example radioiodinated by the chloramine T
procedure, covalently bound to rare earth chelates, or
conjugated to another fluorescent moiety for use in diagnostic
assays.
A combination, e.g., including a DCRS3 or DCRS4, of this
invention can be used as an immunogen for the production of
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antisera or antibodies specific, e.g., capable of distinguishing
between other cytokine receptor family members, for the
combinations described. The complexes can be used to screen
monoclonal antibodies or antigen-binding fragments prepared by
5 immunization with various forms of impure preparations
containing the protein. In particular, the term "antibodies"
also encompasses antigen binding fragments of natural
antibodies, e.g., Fab, Fab2, Fv, etc. A purified DCRS3 can also
be used as a reagent to detect antibodies generated in response
10~ to the presence of elevated levels of expression, or
immunological disorders which lead to antibody production to the
endogenous receptor. Additionally, DCRS3 fragments may also
serve as immunogens to produce the antibodies of the present
invention, as described immediately below. For example, this
15 invention contemplates antibodies having binding affinity to or
being raised against the amino acid sequences shown in Table 1,
fragments thereof, or various homologous peptides. In
particular, this invention contemplates antibodies having
binding affinity to, or having been raised against, specific
20 fragments which are predicted to be, or actually are, exposed at
the exterior protein surface of a native DCRS3. Complexes of
combinations of proteins will also be useful, and antibody
preparations thereto can be made.
The blocking of physiological response to the receptor
25 ligands may result from the inhibition of binding of the ligand
to the receptor, likely through competitive inhibition. Thus,
in vitro assays of the present invention will often use
antibodies or antigen binding segments of these antibodies, or
fragments attached to solid phase substrates. These assays will
30 also allow for the diagnostic determination of the effects of
either ligand binding region mutations and modifications, or
other mutations and modifications, e.g., which affect signaling
or enzymatic function.
This invention also contemplates the use of competitive
35 drug screening assays, e.g., where neutralizing antibodies to
the receptor complexes or fragments compete with a test compound
for binding to a ligand or other antibody. In this manner, the
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neutralizing antibodies or fragments can be used to detect the
presence of a polypeptide which shares one or more binding sites
to a receptor and can also be used to occupy binding sites on a
receptor that might otherwise bind a ligand.
V. Making Nucleic Acids and Protein
DNA which encodes the protein or fragments thereof can be
obtained by chemical synthesis, screening cDNA libraries, or by
screening genomic libraries prepared from a wide variety of cell
10~ lines or tissue samples. Natural sequences can be isolated
using standard methods and the sequences provided herein, e.g.,
in Tables 1 or 3. Other species counterparts can be identified
by hybridization techniques, or by various PCR techniques,
combined with or by searching in sequence databases, e.g.,
GenBank.
This DNA can be expressed in a wide variety of host cells
for the synthesis of a full-length receptor or fragments which
can in turn, for example, be used to generate polyclonal or
monoclonal antibodies; for binding studies; for construction and
expression of modified ligand binding or kinase/phosphatase
domains; and for structure/function studies. Variants or
fragments can be expressed in host cells that are transformed or
transfected with appropriate expression vectors. These
molecules can be substantially free of protein or cellular
contaminants, other than those derived from the recombinant
host, and therefore are particularly useful in pharmaceutical
compositions when combined with a pharmaceutically acceptable
carrier and/or diluent. The protein, or portions thereof, may
be expressed as fusions with other proteins. Combinations of
the described proteins, or nucleic acids encoding them, are
particularly interesting.
Expression vectors are typically self-replicating DNA or
RNA constructs containing the desired receptor gene or its
fragments, usually operably linked to suitable genetic control
elements that are recognized in a suitable host cell. These
control elements are capable of effecting expression within a
suitable host. The multiple genes may be coordinately
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expressed, and may be on a polycistronic message. The specific
type of control elements necessary to effect expression will
depend upon the eventual host cell used. Generally, the genetic
control elements can include a prokaryotic promoter system or a
eukaryotic promoter expression control system, and typically
include a transcriptional promoter, an optional operator to
control the onset of transcription, transcription enhancers to
elevate the level of mRNA expression, a sequence that encodes a
suitable ribosome binding site, and sequences that terminate
10~ transcription and translation. Expression vectors also usually
contain an origin of replication that allows the vector to
replicate independently of the host cell.
The vectors of this invention include those which contain
DNA which encodes a combination of proteins, as described, or a
biologically active equivalent polypeptide. The DNA can be
under the control of a viral promoter and can encode a selection
marker. This invention further contemplates use of such
expression vectors which are capable of expressing eukaryotic
cDNAs coding for such proteins in a prokaryotic or eukaryotic
host, where the vector is compatible with the host and where the
eukaryotic cDNAs are inserted into the vector such that growth
of the host containing the vector expresses the cDNAs in
question. Usually, expression vectors are designed for stable
replication in their host cells or for amplification to greatly
increase the total number of copies of the desirable gene per
cell. It is not always necessary to require that an expression
vector replicate in a host cell, e.g., it is possible to effect
transient expression of the protein or its fragments in various
hosts using vectors that do not contain a replication origin
that is recognized by the host cell. It is also possible to use
vectors that cause integration of the protein encoding portions
into the host DNA by recombination.
Vectors, as used herein, comprise plasmids, viruses,
bacteriophage, integratable DNA fragments, and other vehicles
which enable the integration of DNA fragments into the genome of
the host. Expression vectors are specialized vectors which
contain genetic control elements that effect expression of
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operably linked genes. Plasmids are the most commonly used form
of vector but all other forms of vectors which serve an
equivalent function and which are, or become, known in the art
are suitable for use herein. See, e.g., Pouwels, et al. (1985
and Supplements) Cloning Vectors: A Laboratory Manual, Elsevier,
N.Y., and Rodriguez, et al. (eds. 1988) Vectors: A Survey of
Molecular Cloninct Vectors and Their Uses, Buttersworth, Boston,
which are incorporated herein by reference.
Transformed cells are cells, preferably mammalian, that
10~ have been transformed or transfected with vectors constructed
using recombinant DNA techniques. Transformed host cells
usually express the desired proteins, but for purposes of
cloning, amplifying, and manipulating its DNA, do not need to
express.the subject proteins. This invention further
contemplates culturing transformed cells in a nutrient medium,
thus permitting the proteins to accumulate. The proteins can be
recovered, either from the culture or, in certain instances,
from the culture medium.
For purposes of this invention, nucleic sequences are
operably linked when they are functionally related to each
other. For example, DNA for a presequence or secretory leader
is operably linked to a polypeptide if it is expressed as a
preprotein or participates in directing the polypeptide to the
cell membrane or in secretion of the polypeptide. A promoter is
operably linked to a coding sequence if it controls the
transcription of the polypeptide; a ribosome binding site is
operably linked to a coding sequence if it is positioned to
permit translation. Usually, operably linked means contiguous
and in reading frame, however, certain genetic elements such as
repressor genes are not contiguously linked but still bind to
operator sequences that in turn control expression.
Suitable host cells include prokaryotes, lower eukaryotes,
and higher eukaryotes. Prokaryotes include both gram negative
and gram positive organisms, e.g., E. coli and B. subtilis.
Lower eukaryotes include yeasts, e.g., S. cerevisiae and Pichia,
and species of the genus Dictyostelium. Higher eukaryotes
include established tissue culture cell lines from animal cells,
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both of non-mammalian origin, e.g., insect cells, and birds, and
of mammalian origin, e.g., human, primates, and rodents.
Prokaryotic host-vector systems include a wide variety of
vectors for many different species. As used herein, E. coli and
its vectors will be used generically to include equivalent
vectors used in other prokaryotes. A representative vector for
amplifying DNA is pBR322 or many of its derivatives. Vectors
that can be used to express the receptor or its fragments
include, but are not limited to, such vectors as those
10~ containing the lac promoter (pUC-series); trp promoter
(pBR322-trp); Ipp promoter (the pIN-series); lambda-pP or pR
promoters (pOTS); or hybrid promoters such as ptac (pDR540).
See Brosius, et al. (1988) "Expression Vectors Employing
Lambda-, trp-, lac-, and Ipp-derived Promoters", in Vectors: A
Survey of Molecular Clonincl Vectors and Their Uses, (eds.
Rodriguez and Denhardt), Buttersworth, Boston, Chapter 10, pp.
205-236, which is incorporated herein by reference.
Lower eukaryotes, e.g., yeasts and Dictyostelium, may be
transformed with DCRS3 or DCRS4 sequence containing vectors.
For purposes of this invention, the most common lower eukaryotic
host is the baker's yeast, Saccharomvces cerevisiae. It will be
used to generically represent lower eukaryotes although a number
of other strains and species are also available. Yeast vectors
typically consist of a replication origin (unless of the
integrating type), a selection gene, a promoter, DNA encoding
the receptor or its fragments, and sequences for translation
termination, polyadenylation, and transcription termination.
Suitable expression vectors for yeast include such constitutive
promoters as 3-phosphoglycerate kinase and various other
glycolytic enzyme gene promoters or such inducible promoters as
the alcohol dehydrogenase 2 promoter or metallothionine
promoter. Suitable vectors include derivatives of the following
types: self-replicating low copy number (such as the
YRp-series), self-replicating high copy number (such as the
YEp-series); integrating types (such as the YIp-series), or
mini-chromosomes (such as the YCp-series).
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Higher eukaryotic tissue culture cells are normally the
preferred host cells for expression of the functionally active
interleukin or receptor proteins. In principle, many higher
eukaryotic tissue culture cell lines are workable, e.g., insect
5 baculovirus expression systems, whether from an invertebrate or
vertebrate source. However, mammalian cells are preferred.
Transformation or transfection and propagation of such cells has
become a routine procedure. Examples of useful cell lines
include HeLa cells, Chinese hamster ovary (CHO) cell lines, baby
10~ rat kidney (BRK) cell lines, insect cell lines, bird cell lines,
and monkey (COS) cell lines. Expression vectors for such cell
lines usually include an origin of replication, a promoter, a
translation initiation site, RNA splice sites (if genomic DNA is
used), a polyadenylation site, and a transcription termination
15 site. These vectors also usually contain a selection gene or
amplification gene. Suitable expression vectors may be
plasmids, viruses, or retroviruses carrying promoters derived,
e.g., from such sources as from adenovirus, SV40, parvoviruses,
vaccinia virus, or cytomegalovirus. Representative examples of
20 suitable expression vectors include pCDNAl; pCD, see Okayama, et
al. (1985) Mol. Cell Biol. 5:1136-1142; pMClneo PolyA, see
Thomas, et al. (1987) Cell 51:503-512; and a baculovirus vector
such as pAC 373 or pAC 610.
For secreted proteins and some membrane proteins, an open
25 reading frame usually encodes a polypeptide that consists of a
mature or secreted product covalently linked at its N-terminus
to a signal peptide. The signal peptide is cleaved prior to
secretion of the mature, or active, polypeptide. The cleavage
site can be predicted with a high degree of accuracy from
30 empirical rules, e.g., von-Heijne (1986) Nucleic Acids Research
14:4683-4690 and Nielsen, et al. (1997) Protein Ena. 10:1-12,
and the precise amino acid composition of the signal peptide
often does not appear to be critical to its function, e.g.,
Randall, et al. (1989) Science 243:1156-1159; Kaiser et al.
35 (1987) Science 235:312-317. The mature proteins of the
invention can be readily determined using standard methods.
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It will often be desired to express these polypeptides in a
system which provides a specific or defined glycosylation
pattern. In this case, the usual pattern will be that provided
naturally by the expression system. However, the pattern will
be modifiable by exposing the polypeptide, e.g., an
unglycosylated form, to appropriate glycosylating proteins
introduced into a heterologous expression system. For example,
the receptor gene may be co-transformed with one or more genes
encoding mammalian or other glycosylating enzymes. Using this
10~ approach, certain mammalian glycosylation patterns will be
achievable in prokaryote or other cells. Expression in
prokaryote cells will typically lead to unglycosylated forms of
protein.
The source of DCRS3 or DCRS4 can be a eukaryotic or
prokaryotic host expressing recombinant DCRS, such,as is
described above. The source can also be a cell line, but other
mammalian cell lines are also contemplated by this invention,
with the preferred cell line being from the human species.
Now that the sequences are known, a primate DCRS3 or DCRS4,
fragments, or derivatives thereof can be prepared by
conventional processes for synthesizing peptides. These include
processes such as are described in Stewart and Young (1984)
Solid Phase Pe,~tide Synthesis, Pierce Chemical Co., Rockford,
IL; Bodanszky and Bodanszky (1984) The Practice of Peptide
Synthesis, Springer-Verlag, New York; and Bodanszky (1984) The
Principles of Peptide Synthesis, Springer-Verlag, New York; all
of each which are incorporated herein by reference. For
example, an azide process, an acid chloride process, an acid
anhydride process, a mixed anhydride process, an active ester
process (for example, p-nitrophenyl ester, N-hydroxysuccinimide
ester, or cyanomethyl ester), a carbodiimidazole process, an
oxidative-reductive process, or a dicyclohexylcarbodiimide
(DCCD)/additive process can be used. Solid phase and solution
phase syntheses are both applicable to the foregoing processes.
Similar techniques can be used with partial DCRS3 or DCRS4
sequences.
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DCRS3 or DCRS4 proteins, fragments, or derivatives are
suitably prepared in accordance with the above processes as
typically employed in peptide synthesis, generally either by a
so-called stepwise process which comprises condensing an amino
acid to the terminal amino acid, one by one in sequence, or by
coupling peptide fragments to the terminal amino acid. Amino
groups that are not being used in the coupling reaction
typically must be protected to prevent coupling at an incorrect
location.
10~ If a solid phase synthesis is adopted, the C-terminal amino
acid is bound to an insoluble carrier or support through its
carboxyl group. The insoluble carrier is not particularly
limited as long as it has a binding capability to a reactive
carboxyl group. Examples of such insoluble carriers include
halomethyl resins, such as chloromethyl resin or bromomethyl
resin, hydroxymethyl resins, phenol resins,
tert-alkyloxycarbonylhydrazidated resins, and the like.
An amino group-protected amino acid is bound in sequence
through condensation of its activated carboxyl group and the
reactive amino group of the previously formed peptide or chain,
to synthesize the peptide step by step. After synthesizing the
complete sequence, the peptide is split off from the insoluble
carrier to produce the peptide. This solid-phase approach is
generally described by Merrifield, et al. (1963) in J. Am. Chem.
Soc. 85:2149-2156, which is incorporated herein by reference.
The prepared protein and fragments thereof can be isolated
and purified from the reaction mixture by means of peptide
separation, for example, by extraction, precipitation,
electrophoresis, various forms of chromatography, and the like.
The receptors of this invention can be obtained in varying
degrees of purity depending upon desired uses. Purification can
be accomplished by use of the protein purification techniques
disclosed herein, see below, or by the use of the antibodies
herein described in methods of immunoabsorbant affinity
chromatography. This immunoabsorbant affinity chromatography is
carried out by first linking the antibodies to a solid support
and then contacting the linked antibodies with solubilized
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lysates of appropriate cells, lysates of other cells expressing
the receptor, or lysates or supernatants of cells producing the
protein as a result of DNA techniques, see below.
Generally, the purified protein will be at least about 40%
pure, ordinarily at least about 50% pure, usually at least about
60% pure, typically at least about 70% pure, more typically at
least about 80% pure, preferable at least about 90% pure and
more preferably at least about 95% pure, and in particular
embodiments, 97%-99% or more. Purity will usually be on a
10~ weight basis, but can also be on a molar basis. Different
assays will be applied as appropriate. Individual proteins may
be purified and thereafter combined.
VI. Antibodies
Antibodies can be raised to various mammalian, e.g.,
primate DCRS3 or DCRS4 proteins and fragments thereof, both in
naturally occurring native forms and in their recombinant forms,
the difference being that antibodies to the active receptor are
more likely to recognize epitopes which are only present in the
native conformations. Denatured antigen detection can also be
useful in, e.g., Western analysis. Anti-idiotypic antibodies
are also contemplated, which would be useful as agonists or
antagonists of a natural receptor or an antibody.
Antibodies, including binding fragments and single chain
versions, against predetermined fragments of the protein can be
raised by immunization of animals with conjugates of the
fragments with immunogenic proteins. Monoclonal antibodies are
prepared from cells secreting the desired antibody. These
antibodies can be screened for binding to normal or defective
protein, or screened for agonistic or antagonistic activity.
These monoclonal antibodies will usually bind with at least a KD
of about 1 mM, more usually at least about 300 ~M, typically at
least about 100~M, more typically at least about 30 ~M,
preferably at least about 10 ~M, and more preferably at least
about 3 ~M or better.
The antibodies, including antigen binding fragments, of
this invention can have significant diagnostic or therapeutic
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value. They can be potent antagonists that bind to the receptor
and inhibit binding to ligand or inhibit the ability of the
receptor to elicit a biological response, e.g., act on its
substrate. They also can be useful as non-neutralizing
antibodies and can be coupled to toxins or radionuclides to bind
producing cells, or cells localized to the source of the
interleukin. Further, these antibodies can be conjugated to
drugs or other therapeutic agents, either directly or indirectly
by means of a linker.
10~ The antibodies of this invention can also be useful in
diagnostic applications. As capture or non-neutralizing
antibodies, they might bind to the receptor without inhibiting
ligand or substrate binding. As neutralizing antibodies, they
can be useful in competitive binding assays. They will also be
useful in detecting or quantifying ligand. They may be used as
reagents for Western blot analysis, or for immunoprecipitation
or immunopurification of the respective protein. Likewise,
nucleic acids and proteins may be immobilized to solid
substrates for affinity purification or detection methods. The
substrates may be, e.g., solid resin beads or sheets of plastic.
Protein fragments may be joined to other materials,
particularly polypeptides, as fused or covalently joined
polypeptides to be used as immunogens. Mammalian cytokine
receptors and fragments may be fused or covalently linked to a
variety of immunogens, such as keyhole limpet hemocyanin, bovine
serum albumin, tetanus toxoid, etc. See Microbiolocrv, Hoeber
Medical Division, Harper and Row, 1969; Landsteiner (1962)
Specificity of Serological Reactions, Dover Publications, New
York; and Williams, et al. (1967) Methods in Immunology and
Immunochemistry, Vol. 1, Academic Press, New York; each of which
are incorporated herein by reference, for descriptions of
methods of preparing polyclonal antisera. A typical method
involves hyperimmunization of an animal with an antigen. The
blood of the animal is then collected shortly after the repeated
immunizations and the gamma globulin is isolated.
In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
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primates, humans, etc. Description of techniques for preparing
such monoclonal antibodies may be found in, e.g., Stites, et al.
(eds.) Basic and Clinical Immunoloav (4th ed.), Lange Medical
Publications, Los Altos, CA, and references cited therein;
5 Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH
Press; Goding (1986) Monoclonal Antibodies: Principles and
Practice (2d ed.) Academic Press, New York; and particularly in
Kohler and Milstein (1975) in Nature 256: 495-497, which
discusses one method of generating monoclonal antibodies. Each
10~ of these references is incorporated herein by reference.
Summarized briefly, this method involves injecting an animal
with an immunogen. The animal is then sacrificed and cells
taken from its spleen, which are then fused with myeloma cells.
The result is a hybrid cell or "hybridoma" that is capable of
15 reproducing in vitro. The population of hybridomas is then
screened to isolate individual clones, each of which secrete a
single antibody species to the immunogen. In this manner, the
individual antibody species obtained are the products of
immortalized and cloned single B cells from the immune animal
20 generated in response to a specific site recognized on the
immunogenic substance.
Other suitable techniques involve in vitro exposure of
lymphocytes to the antigenic polypeptides or alternatively to
selection of libraries of antibodies in phage or similar
25 vectors. See, Huse, et al. (1989) "Generation of a Large
Combinatorial Library of the Immunoglobulin Repertoire in Phage
Lambda," Science 246:1275-1281; and Ward, et al. (1989) Nature
341:544-546, each of which is hereby incorporated herein by
reference. The polypeptides and antibodies of the present
30 invention may be used with or without modification, including
chimeric or humanized antibodies. Frequently, the polypeptides
and antibodies will be labeled by joining, either covalently or
non-covalently, a substance which provides for a detectable
signal. A wide variety of labels and conjugation techniques are
35 known and are reported extensively in both the scientific and
patent literature. Suitable labels include radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent
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moieties, chemiluminescent moieties, magnetic particles, and the
like. Patents, teaching the use of such labels include U.S.
Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant or
chimeric immunoglobulins may be produced, see Cabilly, U.S.
Patent No. 4,816,567; or made in transgenic mice, see Mendez, et
al. (1997) Nature Genetics 15:146-156. These references are
incorporated herein by reference.
The antibodies of this invention can also be used for
10~ affinity chromatography in isolating DCRS3 proteins or peptides.
Columns can be prepared where the antibodies are linked to a
solid support, e.g., particles, such as agarose, Sephadex, or
the like, where a cell lysate may be passed through the column,
the column washed, followed by increasing concentrations of a
mild denaturant, whereby the purified protein will be released.
Alternatively, the protein may be used to purify antibody.
Appropriate cross absorptions or depletions may be applied.
The antibodies may also be used to screen expression
libraries for particular expression products. Usually the
antibodies used in such a procedure will be labeled with a
moiety allowing easy detection of presence of antigen by
antibody binding.
Antibodies raised against a cytokine receptor will also be
used to raise anti-idiotypic antibodies. These will be useful
in detecting or diagnosing various immunological conditions
related to expression of the protein or cells which express the
protein. They also will be useful as agonists or antagonists of
the ligand, which may be competitive inhibitors or substitutes
for naturally occurring ligands.
A cytokine receptor protein that specifically binds to or
that is specifically immunoreactive with an antibody generated
against a defined immunogen, such as an immunogen consisting of
the amino acid sequence of SEQ ID NO: 2, 25, 5, 28, or 31, is
typically determined in an immunoassay. The immunoassay
typically uses a polyclonal antiserum which was raised, e.g., to
a protein of SEQ ID NO: 2, 25, 5, 28, or 31. This antiserum is
selected to have low crossreactivity against other cytokine
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receptor family members, e.g., IL-11 receptor subunit alpha, IL-
6 receptor subunit alpha, or p40, preferably from the same
species, and any such crossreactivity is removed by
immunoabsorption prior to use in the immunoassay.
In order to produce antisera for use in an immunoassay, the
protein, e.g., of SEQ ID NO: 2, 25, 5, 28, or 31, is isolated as
described herein. For example, recombinant protein may be
produced in a mammalian cell line. An appropriate host, e.g.,
an inbred strain of mice such as Balb/c, is immunized with the
10~ selected protein, typically using a standard adjuvant, such as
Freund's adjuvant, and a standard mouse immunization protocol
(see Harlow and Lane, supra). Alternatively, a synthetic
peptide derived from the sequences disclosed herein and
conjugated to a carrier protein can be used an immunogen.
Polyclonal sera are collected and titered against the immunogen
protein in an immunoassay, e.g., a solid phase immunoassay with
the immunogen immobilized on a solid support. Polyclonal
antisera with a titer of 104 or greater are selected and tested
for their cross reactivity against other cytokine receptor
family members, e.g., IL-2, IL-7, IL-9, or EPO receptor subunit,
using a competitive binding immunoassay such as the one
described in Harlow and Lane, supra, at pages 570-573.
Preferably at least two cytokine receptor family members are
used in this determination. These cytokine receptor family
members can be produced as recombinant proteins and isolated
using standard molecular biology and protein chemistry
techniques as described herein.
Immunoassays in the competitive binding format can be used
for the crossreactivity determinations. For example, the
protein of SEQ ID N0: 2, 25, 5, 28, or 31 can be immobilized to
a solid support. Proteins added to the assay compete with the
binding of the antisera to the immobilized antigen. The ability
of the above proteins to compete with the binding of the
antisera to the immobilized protein is compared to the proteins,
e.g., of IL-2, IL-7, IL-9, or EPO receptor subunit. The percent
crossreactivity for the above proteins is calculated, using
standard calculations. Those antisera with less than 10°s
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58
crossreactivity with each of the proteins listed above are
selected and pooled. The cross-reacting antibodies are then
removed from the pooled antisera by immunoabsorption with the
above-listed proteins.
The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second protein to the immunogen protein (e. g., DCRS3 like
protein of SEQ ID NO: 2). In order to make this comparison, the
two proteins are each assayed at a wide range of concentrations
10~ and the amount of each protein required to inhibit 50°s of the
binding of the antisera to the immobilized protein is
determined. If the amount of the second protein required is
less than twice the amount of the protein of the selected
protein or proteins that is required, then the second protein is
said to specifically bind to an antibody generated to the
immunogen.
It is understood that these cytokine receptor proteins are
members of a family of homologous proteins that comprise at
least 6 so far identified genes. For a particular gene product,
such as a DCRS3 or DCRS4, the term refers not only to the amino
acid sequences disclosed herein, but also to other proteins that
are allelic, non-allelic, or species variants. It is also
understood that the terms include nonnatural mutations
introduced by deliberate mutation using conventional recombinant
technology such as single site mutation, or by excising short
sections of DNA encoding the respective proteins, or by
substituting new amino acids, or adding new amino acids. Such
minor alterations typically will substantially maintain the
immunoidentity of the original molecule and/or its biological
activity. Thus, these alterations include proteins that are
specifically immunoreactive with a designated naturally
occurring DCRS3 or DCRS4 protein. The biological properties of
the altered proteins can be determined by expressing the protein
in an appropriate cell line and measuring the appropriate
effect, e.g., upon transfected lymphocytes. Particular protein
modifications considered minor would include conservative
substitution of amino acids with similar chemical properties, as
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described above for the cytokine receptor family as a whole. By
aligning a protein optimally with the protein of the cytokine
receptors and by using the conventional immunoassays described
herein to determine immunoidentity, one can determine the
protein compositions of the invention.
VII. Kits and quantitation
Both naturally occurring and recombinant forms of~the
cytokine receptor like molecules of this invention are
10~ particularly useful in kits and assay methods. For example,
these methods would also be applied to screening for binding
activity, e.g., ligands for these proteins. Several methods of
automating assays have been developed in recent years so as to
permit screening of tens of thousands of compounds per year.
See, e.g., a BIOMEK automated workstation, Beckman Instruments,
Palo Alto, California, and Fodor, et al. (1991) Science 251:767-
773, which is incorporated herein by reference. The latter
describes means for testing binding by a plurality of defined
polymers synthesized on a solid substrate. The development of
suitable assays to screen for a ligand or agonist/antagonist
homologous proteins can be greatly facilitated by the
availability of large amounts of purified, soluble cytokine
receptors in an active state such as is provided by this
invention.
Purified DCRS3 or DCRS4 can be coated directly onto plates
for use in the aforementioned ligand screening techniques.
However, non-neutralizing antibodies to these proteins can be
used as capture antibodies to immobilize the respective receptor
on the solid phase, useful, e.9., in diagnostic uses.
This invention also contemplates use of DCRS3 or DCRS4,
fragments thereof, peptides, and their fusion products in a
variety of diagnostic kits and methods for detecting the
presence of the protein or its ligand. Alternatively, or
additionally, antibodies against the molecules may be
incorporated into the kits and methods. Typically the kit will
have a compartment containing either a DCRS3 or DCRS4 peptide or
gene segment or a reagent which recognizes one or the other.
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Typically, recognition reagents, in the case of peptide, would
be a receptor or antibody, or in the case of a gene segment,
would usually be a hybridization probe.
A preferred kit for determining the concentration of, e.g.,
5 DCRS3 in a sample would typically comprise a labeled compound,
e.g., ligand or antibody, having known binding affinity for
DCRS3, a source of DCRS3 (naturally occurring or recombinant) as
a positive control, and a means for separating the bound from
free labeled compound, for example a solid phase for
10~ immobilizing DCRS3 in the test sample. Compartments containing
reagents, and instructions, will normally be provided.
Appropriate nucleic acid or protein containing kits are also
provided.
Antibodies, including antigen binding fragments, specific
15 for mammalian DCRS3 or a peptide fragment, or receptor fragments
are useful in diagnostic applications to detect the presence of
elevated levels of ligand and/or its fragments. Diagnostic
assays may be homogeneous (without a separation step between
free reagent and antibody-antigen complex) or heterogeneous
20 (with a separation step). Various commercial assays exist, such
as radioimmunoassay (RIA), enzyme-linked immunosorbent assay
(ELISA), enzyme immunoassay (EIA), enzyme-multiplied immunoassay
technique (EMIT), substrate-labeled fluorescent immunoassay
(SLFIA) and the like. For example, unlabeled antibodies can be
25 employed by using a second antibody which is labeled and which
recognizes the antibody to a cytokine receptor or to a
particular fragment thereof. These assays have also been
extensively discussed in the literature. See, e.g., Harlow and
Lane (1988) Antibodies: A Laboratory Manual, CSH., and Coligan
30 (ed. 1991 and periodic supplements) Current Protocols In
Immunoloctv Greene/Wiley, New York.
Anti-idiotypic antibodies may have similar use to serve as
agonists or antagonists of cytokine receptors. These should be
useful as therapeutic reagents under appropriate circumstances.
35 Frequently, the reagents for diagnostic assays are supplied
in kits, so as to optimize the sensitivity of the assay. For
the subject invention, depending upon the nature of the assay,
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the protocol, and the label, either labeled or unlabeled
antibody, or labeled ligand is provided. This is usually in
conjunction with other additives, such as buffers, stabilizers,
materials necessary for signal production such as substrates for
enzymes, and the like. Preferably, the kit will also contain
instructions for proper use and disposal of the contents after
use. Typically the kit has compartments for each useful
reagent, and will contain instructions for proper use and
disposal of reagents. Desirably, the reagents are provided as a
10~ dry lyophilized powder, where the reagents may be reconstituted
in an aqueous medium having appropriate concentrations for
performing the assay.
The aforementioned constituents of the diagnostic assays
may be used without modification or may be modified in a variety
of ways. For example, labeling may be achieved by covalently or
non-covalently joining a moiety which directly or indirectly
provides a detectable signal. In many of these assays, a test
compound, cytokine receptor, or antibodies thereto can be
labeled either directly or indirectly. Possibilities for direct
labeling include label groups: radiolabels such as 125I, enzymes
(U. S. Pat. No. 3,645,090) such as peroxidase and alkaline
phosphatase, and fluorescent labels (U. S. Pat. No. 3,940,475)
capable of monitoring the change in fluorescence intensity,
wavelength shift, or fluorescence polarization. Both of the
patents are incorporated herein by reference. Possibilities for
indirect labeling include biotinylation of one constituent
followed by binding to avidin coupled to one of the above label
groups.
There are also numerous methods of separating the bound
from the free ligand, or alternatively the bound from the free
test compound. The cytokine receptor can be immobilized on
various matrixes followed by washing. Suitable matrices include
plastic such as an ELISA plate, filters, and beads. Methods of
immobilizing the receptor to a matrix include, without
limitation, direct adhesion to plastic, use of a capture
antibody, chemical coupling, and biotin-avidin. The last step
in this approach involves the precipitation of antibody/antigen
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complex by any of several methods including those utilizing,
e.g., an organic solvent such as polyethylene glycol or a salt
such as ammonium sulfate. Other suitable separation techniques
include, without limitation, the fluorescein antibody
magnetizable particle method described in Rattle, et al. (1984)
Clin. Chem. 30:1457-1461, and the double antibody magnetic
particle separation as described in U.S. Pat. No. 4,659,678,
each of which is incorporated herein by reference.
The methods for linking protein or fragments to various
10~ labels have been extensively reported in the literature and do
not require detailed discussion here. Many of the techniques
involve the use of activated carboxyl groups either through the
use of carbodiimide or active esters to form peptide bonds, the
formation of thioethers by reaction of a mercapto group with an
activated halogen such as chloroacetyl, or an activated olefin
such as maleimide, for linkage, or the like. Fusion proteins
will also find use in these applications.
Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the
sequence of an cytokine receptor. These sequences can be used
as probes for detecting levels of the respective cytokine
receptor in patients suspected of having an immunological
disorder. The preparation of both RNA and DNA nucleotide
sequences, the labeling of the sequences, and the preferred size
of the sequences has received ample description and discussion
in the literature. Normally an oligonucleotide probe should
have at least about 14 nucleotides, usually at least about 18
nucleotides, and the polynucleotide probes may be up to several
kilobases. Various labels may be employed, most commonly
radionuclides, particularly 32P. However, other techniques may
also be employed, such as using biotin modified nucleotides for
introduction into a polynucleotide. The biotin then serves as
the site for binding to avidin or antibodies, which may be
labeled with a wide variety of labels, such as radionuclides,
fluorescers, enzymes, or the like. Alternatively, antibodies
may be employed which can recognize specific duplexes, including
DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, or
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DNA-protein duplexes. The antibodies in turn may be labeled and
the assay carried out where the duplex is bound to a surface, so
that upon the formation of duplex on the surface, the presence
of antibody bound to the duplex can be detected. The use of
probes to the novel anti-sense RNA may be carried out in
conventional techniques such as nucleic acid hybridization, plus
and minus screening, recombinational probing, hybrid released
translation (HRT), and hybrid arrested translation (HART). This
also includes amplification techniques such as polymerase chain
10~ reaction (PCR).
Diagnostic kits which also test for the qualitative or
quantitative presence of other markers are also contemplated.
Diagnosis or prognosis may depend on the combination of multiple
indications used as markers. Thus, kits may test for
combinations of markers. See, e.9., Viallet, et al. (1989)
Progress in Growth Factor Res. 1:89-97.
VIII. Therapeutic Utility
This invention provides reagents with significant
therapeutic value. See, e.g., Levitzk_i (1996) Curr. Opin. Cell
Biol. 8:239-244. The cytokine receptors (naturally occurring or
recombinant), fragments thereof, mutein receptors, and
antibodies, along with compounds identified as having binding
affinity to the receptors or antibodies, should be useful in the
treatment of conditions exhibiting abnormal expression of the
receptors of their ligands. Such abnormality will typically be
manifested by immunological disorders. Additionally, this
invention should provide therapeutic value in various diseases
or disorders associated with abnormal expression or abnormal
triggering of response to the ligand. For example, the IL-1
ligands have been suggested to be involved in morphologic
development, e.g., dorso-ventral polarity determination, and
immune responses, particularly the primitive innate responses.
See, e.g., Sun, et al. (1991) Eur. J. Biochem. 196:247-254; and
Hultmark (1994) Nature 367:116-117.
Recombinant cytokine receptors, muteins, agonist or
antagonist antibodies thereto, or antibodies can be purified and
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then administered to a patient. These reagents can be combined
for therapeutic use with additional active ingredients, e.g., in
conventional pharmaceutically acceptable carriers or diluents,
along with physiologically innocuous stabilizers and excipients.
These combinations can be sterile, e.g., filtered, and placed
into dosage forms as by lyophilization in dosage vials or
storage in stabilized aqueous preparations. This invention also
contemplates use of antibodies or. binding fragments thereof
which are not complement binding.
10~ Ligand screening using cytokine receptor or fragments
thereof can be performed to identify molecules having binding
affinity to the receptors. Subsequent biological assays can
then be utilized to determine if a putative ligand can provide
competitive binding, which can block intrinsic stimulating
activity. Receptor fragments can be used as a blocker or
antagonist in that it blocks the activity of ligand. Likewise,
a compound having intrinsic stimulating activity can activate
the receptor and is thus an agonist in that it simulates the
activity of ligand, e.g., inducing signaling. This invention
further contemplates the therapeutic use of antibodies to
cytokine receptors as antagonists.
The quantities of reagents necessary for effective therapy
will depend upon many different factors, including means of
administration, target site, reagent physiological life,
pharmacological life, physiological state of the patient, and
other medicants administered. Thus, treatment dosages should be
titrated to optimize safety and efficacy. Typically, dosages
used in vitro may provide useful guidance in the amounts useful
for in situ administration of these reagents. Animal testing of
effective doses for treatment of particular disorders will
provide further predictive indication of human dosage. Various
considerations are described, e.g., in Gilman, et al. (eds.
1990) Goodman and Gilman's~ The Pharmacological Bases of
Therapeutics, 8th Ed., Pergamon Press; and Reminaton's
Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co.,
Easton, Penn.; each of which is hereby incorporated herein by
reference. Methods for administration are discussed therein and
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below, e.g., for oral, intravenous, intraperitoneal, or
intramuscular administration, transdermal diffusion, and others.
Pharmaceutically acceptable carriers will include water, saline,
buffers, and other compounds described, e.g., in the Merck
5 Index, Merck & Co., Rahway, New Jersey. Because of the likely
high affinity binding, or turnover numbers, between a putative
ligand and its receptors, low dosages of these reagents would be
initially expected to be effective. And the signaling pathway
suggests extremely low amounts of ligand may have effect. Thus,
10~ dosage ranges would ordinarily be expected to be in amounts
lower than 1 mM concentrations, typically less than about 10 ~M
concentrations, usually less than about 100 nM, preferably less
than about 10 pM (picomolar), and most preferably less than
about 1 fM (femtomolar), with an appropriate carrier. Slow
15 release formulations, or slow release apparatus will often be
utilized for continuous administration.
Cytokine receptors, fragments thereof, and antibodies or
its fragments, antagonists, and agonists, may be administered
directly to the host to be treated or, depending on the size of
20 the compounds, it may be desirable to conjugate them to carrier
proteins such as ovalbumin or serum albumin prior to their
administration. Therapeutic formulations may be administered in
many conventional dosage formulations. While it is possible for
the active ingredient to be administered alone, it is preferable
25 to present it as a pharmaceutical formulation. Formulations
comprise at least one active ingredient, as defined above,
together with one or more acceptable carriers thereof. Each
carrier must be both pharmaceutically and physiologically
acceptable in the sense of being compatible with the other
30 ingredients and not injurious to the patient. Formulations
include those suitable for oral, rectal, nasal, or parenteral
(including subcutaneous, intramuscular, intravenous and
intradermal) administration. The formulations may conveniently
be presented in unit dosage form and may be prepared by methods
35 well known in the art of pharmacy. See, e.g., Gilman, et al.
(eds. 1990) Goodman and Gilman's: The Pharmacological Bases of
Therapeutics, 8th Ed., Pergamon Press; and Reminaton's
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Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co.,
Easton, Penn.; Avis, et al. (eds. 1993) Pharmaceutical Dosage
Forms: Parenteral Medications Dekker, NY; Lieberman, et al.
(eds. 1990) Pharmaceutical Dosage Forms: Tablets Dekker, NY; and
Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms:
Disperse Systems Dekker, NY. The therapy of this invention may
be combined with or used in association with other therapeutic
agents, particularly agonists or antagonists of other cytokine
receptor family members.
10~
IX. Screening
Drug screening using DCRS3 or DCRS4 or fragments thereof
can be performed to identify compounds having binding affinity
to the receptor subunit, including isolation of associated
components. Subsequent biological assays can then be utilized
to determine if the compound has intrinsic stimulating activity
and is therefore a blocker or antagonist in that it blocks the
activity of the ligand. Likewise, a compound having intrinsic
stimulating activity can activate the receptor and is thus an
agonist in that it simulates the activity of a cytokine ligand.
This invention further contemplates the therapeutic use of
antibodies to the receptor as cytokine agonists or antagonists.
Similarly, complexes comprising multiple proteins may be
used to screen for ligands or reagents capable of recognizing
the complex. Most cytokine receptors comprise at least two
subunits, which may be the same, or distinct. Alternatively,
the transmembrane receptor may bind to a complex comprising a
cytokine-like ligand associated with another soluble protein
serving, e.g., as a second receptor subunit.
One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing, e.g., a DCRS3 in
combination with another cytokine receptor subunit. Cells may
be isolated which express a receptor in isolation from other
functional receptors. Such cells, either in viable or fixed
form, can be used for standard antibody/antigen or
ligand/receptor binding assays. See also, Parce, et al. (1989)
Science 246:243-247; and Owicki, et al. (1990) Proc. Nat'1 Acad.
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Sci. USA 87:4007-4011, which describe sensitive methods to
detect cellular responses. Competitive assays are particularly
useful, where the cells (source of putative ligand) are
contacted and incubated with a labeled receptor or antibody
having known binding affinity to the ligand, such as 125I_
antibody, and a test sample whose binding affinity to the
binding composition is being measured. The bound and free
labeled binding compositions are then separated to assess the
degree of ligand binding. The amount of test compound bound is
10~ inversely proportional to the amount of labeled receptor binding
to the known source. Many techniques can be used to separate
bound from free ligand to assess the degree of ligand binding.
This separation step could typically involve a procedure such as
adhesion to filters followed by washing, adhesion to plastic
followed by washing, or centrifugation of the cell membranes.
Viable cells could also be used to screen for the effects of
drugs on cytokine mediated functions, e.g., second messenger
levels, i.e., Ca++; cell proliferation; inositol phosphate pool
changes; and others. Some detection methods allow for
elimination of a separation step, e.g., a proximity sensitive
detection system. Calcium sensitive dyes will be useful for
detecting Ca++ levels, with a fluorimeter or a fluorescence cell
sorting apparatus.
X. Ligands
The descriptions of DCRS3 or DCRS4 herein provide means to
identify ligands, as described above. Such ligand should bind
specifically to the respective receptor with reasonably high
affinity. Various constructs are made available which allow
either labeling of the receptor to detect its ligand. For
example, directly labeling cytokine receptor, fusing onto it
markers for secondary labeling, e.g., FLAG or other epitope
tags, etc., will allow detection of receptor. This can be
histological, as an affinity method for biochemical
purification, or labeling or selection in an expression cloning
approach. A two-hybrid selection system may also be applied
making appropriate constructs with the available cytokine
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receptor sequences. See, e.g., Fields and Song (1989) Nature
340:245-246.
The broad scope of this invention is best understood with
reference to the following examples, which are not intended to
limit the inventions to the specific embodiments.
EXAMPLES
I. General Methods
10' Some of the standard methods are described or referenced,
e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press;
Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual,
(2d ed.), vols. 1-3, CSH Press, NY; or Ausubel, et al. (1987 and
Supplements) Current Protocols in Molecular Biology,
Greene/Wiley, New York. Methods for protein purification
include such methods as ammonium sulfate precipitation, column
chromatography, electrophoresis, centrifugation,
crystallization, and others. See, e.g., Ausubel, et al. (1987
and periodic supplements); Coligan, et al. (ed. 1996) and
periodic supplements, Current Protocols In Protein Science
Greene/Wiley, New York; Deutscher (1990) "Guide to Protein
Purification" in Methods in Enzymoloay, vol. 182, and other
volumes in this series; and manufacturer's literature on use of
protein purification products, e.g., Pharmacia, Piscataway,
N.J., or Bio-Rad, Richmond, CA. Combination with recombinant
techniques allow fusion to appropriate segments, e.g., to a FLAG
sequence or an equivalent which can be fused via a protease-
removable sequence. See, e.g., Hochuli (1990) "Purification of
Recombinant Proteins with Metal Chelate Absorbent" in Setlow
(ed.) Genetic Engineering, Principle and Methods 12:87-98,
Plenum Press, N.Y.; and Crowe, et al. (1992) QIAexpress: The
High Level Expression & Protein Purification System QUIAGEN,
Inc., Chatsworth, CA.
Computer sequence analysis is performed, e.g., using
available software programs, including those from the GCG (U.
Wisconsin) and GenBank sources. Public sequence databases were
also used, e.g., from GenBank and others.
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Many techniques applicable to IL-10 receptors may be
applied to DCRS3 or DCRS4, as described, e.g., in USSN
08/110,683 (IL-10 receptor), which is incorporated herein by
reference.
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II. Computational Analysis
Human sequences related to cytokine receptors were
identified from genomic sequence database using, e.g., the BLAST
5 server (Altschul, et al. (1994) Nature Genet. 6:119-129).
Standard analysis programs may be used to evaluate structure,
e.g., PHD (Rost and Sander (1994) Proteins 19:55-72) and DSC
(King and Sternberg (1996) Protein Sci. 5:2298-2310). Standard
comparison software includes, e.9., Altschul, et al. (1990) J.
10~ Mol. Biol. 215:403-10; Waterman (1995) Introduction to
Computational Bioloq_y~ Maps Seauences and Genomes Chapman &
Hall; Lander and Waterman (eds. 1995) Calculatin4 the Secrets of
Life Applications of the Mathematical Sciences in Molecular
Biolocry National Academy Press; and Speed and Waterman (eds.
15 1996) Genetic Ma~pina and DNA Seauencing (IMA Volumes in
Mathematics and Its Applications, Vol 81) Springer Verlag.
III. Cloning of full-length DCRS3 or DCRS4 cDNAs; Chromosomal
localization
20 PCR primers derived from DCRS3 or DCRS4 sequence are used
to probe a human cDNA library. Sequences may be derived, e.g.,
from Table 1 or 3, preferably those adjacent the ends of
sequences. Full length cDNAs for primate, rodent, or other
species are cloned, e.g., by DNA hybridization screening of
25 7~gt10 phage. PCR reactions are conducted using T. aquaticus
Taqplus DNA polymerase (Stratagene) under appropriate
conditions.
For experimental confirmation of localization, chromosome
spreads are prepared. In situ hybridization is performed on
30 chromosome preparations obtained from phytohemagglutinin-
stimulated human lymphocytes cultured for 72 h. 5-
bromodeoxyuridine was added for the final seven hours of culture
(60 ~g/ml of medium), to ensure a posthybridization chromosomal
banding of good quality.
35 A PCR fragment, amplified with the help of primers, is
cloned into an appropriate vector. The vector is labeled by
nick-translation with 3H. The radiolabeled probe is hybridized
to metaphase spreads at final concentration of 200 ng/ml of
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hybridization solution as described in Mattei, et al. (1985)
Hum. Genet. 69:327-331.
After coating with nuclear track emulsion (KODAK NTB2),
slides are exposed. To avoid any slipping of silver grains
during the banding procedure, chromosome spreads are first
stained with buffered Giemsa solution and metaphase
photographed. R-banding is then performed by the fluorochrome-
photolysis-Giemsa (FPG) method and metaphases rephotographed
before analysis.
10~ Similar appropriate methods are used for other species.
IV. Localization of DCRS mRNA
Human multiple tissue (Cat# 1, 2) and cancer cell line
blots (Cat# 7757-1), containing approximately 2 ~g of poly(A)+
RNA per lane, are purchased from Clontech (Palo Alto, CA).
Probes are radiolabeled with [a-32P] dATP, e.g., using the
Amersham Rediprime random primer labeling kit (RPN1633).
Prehybridization and hybridizations are performed, e.g., at 65°
C in 0.5 M Na2HP04, 7% SDS, 0.5 M EDTA (pH 8.0). High
stringency washes are conducted, e.g., at 65° C with two initial
washes in 2 x SSC, 0.1% SDS for 40 min followed by a subsequent
wash in 0.1 x SSC, O.lo SDS for 20 min. Membranes are then
exposed at -70° C to X-Ray film (Kodak) in the presence of
intensifying screens. More detailed studies by cDNA library
Southerns are performed with selected appropriate human DCRS3
clones to examine their expression in hemopoietic or other cell
subsets.
Alternatively, two appropriate primers are selected from
Tables 1 or 3. RT-PCR is used on an appropriate mRNA sample
selected for the presence of message to produce a cDNA, e.g., a
sample which expresses the gene.
Full length clones may be isolated by hybridization of cDNA
libraries from appropriate tissues pre-selected by PCR signal.
Northern blots can be performed.
Message for genes encoding DCRS3 or DCRS4 will be assayed
by appropriate technology, e.g., PCR, immunoassay,
hybridization, or otherwise. Tissue and organ cDNA preparations
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are available, e.g., from Clontech, Mountain View, CA.
Identification of sources of natural expression are useful, as
described. And the identification of functional receptor
subunit pairings will allow for prediction of what cells express
the combination of receptor subunits which will result in a
physiological responsiveness to each of the cytokine ligands.
For mouse distribution, e.g., Southern Analysis can be
performed: DNA (5 fig) from a primary amplified cDNA library was
digested with appropriate restriction enzymes to release the
10~ inserts, run on a to agarose gel and transferred to a nylon
membrane (Schleicher and Schuell, Keene, NH).
Samples for mouse mRNA isolation may include: resting mouse
fibroblastic L cell line (C200); Braf:ER (Braf fusion to
estrogen receptor) transfected cells, control (C201); T cells,
TH1 polarized (Me114 bright, CD4+ cells from spleen, polarized
for 7 days with IFN-y and anti IL-4; T200); T cells, TH2
polarized (Me114 bright, CD4+ cells from spleen, polarized for 7
days with IL-4 and anti-IFN-y; T201); T cells, highly TH1
polarized (see Openshaw, et al. (1995) J. Exp. Med. 182:1357-
1367; activated with anti-CD3 for 2, 6, 16 h pooled; T202); T
cells, highly TH2 polarized (see Openshaw, et al. (1995) J. Exp.
Med. 182:1357-1367; activated with anti-CD3 for 2, 6, 16 h
pooled; T203); CD44- CD25+ pre T cells, sorted from thymus
(T204); TH1 T cell clone D1.1, resting for 3 weeks after last
stimulation with antigen (T205); TH1 T cell clone D1.1, 10 ~g/ml
ConA stimulated 15 h (T206); TH2 T cell clone CDC35, resting for
3 weeks after last stimulation with antigen (T207); TH2 T cell
clone CDC35, 10 ~g/ml ConA stimulated 15 h (T208); Me114+ naive
T cells from spleen, resting (T209); Me114+ T cells, polarized
to Thl with IFN-y/IL-12/anti-IL-4 for 6, 12, 24 h pooled (T210);
Me114+ T cells, polarized to Th2 with IL-4/anti-IFN-y for 6, 13,
24 h pooled (T211); unstimulated mature B cell leukemia cell
line A20 (B200); unstimulated B cell line CH12 (B201);
unstimulated large B cells from spleen (B202); B cells from
total spleen, LPS activated (B203); metrizamide enriched
dendritic cells from spleen, resting (D200); dendritic cells
from bone marrow, resting (D201); monocyte cell line RAW 264.7
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activated with LPS 4 h (M200); bone-marrow macrophages derived
with GM and M-CSF (M201); macrophage cell line J774, resting
(M202); macrophage cell line J774 + LPS + anti-IL-10 at 0.5, 1,
3, 6, 12 h pooled (M203); macrophage cell line J774 + LPS + IL-
10 at 0.5, 1, 3, 5, 12 h pooled(M204); aerosol challenged mouse
lung tissue, Th2 primers, aerosol OVA challenge 7, 14, 23 h
pooled (see Garlisi, et al. (1995) Clinical Immunology and
Immunopathologv 75:75-83; X206); Nippostrongulus-infected lung
tissue (see Coffman, et al. (1989) Science 245:308-310; X200);
10~ total adult lung, normal (0200); total lung, rag-1 (see Schwarz,
et al. (1993) Immunodeficiency 4:249-252; 0205); IL-10 K.O.
spleen (see Kuhn, et al. (1991) Cell 75:263-274; X201); total
adult spleen, normal (0201); total spleen, rag-1 (0207); IL-10
K.O. Peyer's patches (0202); total Peyer's patches, normal
(0210); IL-10 K.O. mesenteric lymph nodes (X203); total
mesenteric lymph nodes, normal (0211); IL-10 K.O. colon (X203);
total colon, normal (0212); NOD mouse pancreas (see Makino, et
al. (1980) Jikken Dobutsu 29:1-13; X205); total thymus, rag-1
(0208); total kidney, rag-1 (0209); total heart, rag-1 (0202);
total brain, rag-1 (0203); total testes, rag-1 (0204); total
liver, rag-1 (0206); rat normal joint tissue (0300); and rat
arthritic joint tissue (X300).
Samples for human mRNA isolation may include: peripheral
blood mononuclear cells (monocytes, T cells, NK cells,
granulocytes, B cells), resting (T100); peripheral blood
mononuclear cells, activated with anti-CD3 for 2, 6, 12 h pooled
(T101); T cell, THO clone Mot 72, resting (T102); T cell, THO
clone Mot 72, activated with anti-CD28 and anti-CD3 for 3, 6, 12
h pooled (T103); T cell, THO clone Mot 72, anergic treated with
specific peptide for 2, 7, 12 h pooled (T104); T cell, TH1 clone
HY06, resting (T107); T cell, TH1 clone HY06, activated with
anti-CD28 and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1
clone HY06, anergic treated with specific peptide for 2, 6, 12 h
pooled (T109); T cell, TH2 clone HY935, resting (T110); T cell,
TH2 clone HY935, activated with anti-CD28 and anti-CD3 for 2, 7,
12 h pooled (T111); T cells CD4+CD45R0- T cells polarized 27
days in anti-CD28, IL-4, and anti IFN-y, TH2 polarized, activated
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with anti-CD3 and anti-CD28 4 h (T116); T cell tumor lines
Jurkat and Hut78, resting (T117); T cell clones, pooled AD130.2,
Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting (T118); T cell
random ys T cell clones, resting (T119); Splenocytes, resting
(B100); Splenocytes, activated with anti-CD40 and IL-4 (B101); B
cell EBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY,
resting (B102); B cell line JY, activated with PMA and ionomycin
for 1, 6 h pooled (B103); NK 20 clones pooled, resting (K100);
NK 20 clones pooled, activated with PMA and ionomycin for 6 h
10~ (K101); NKL clone, derived from peripheral blood of LGL leukemia
patient, IL-2 treated (K106); NK cytotoxic clone 640-A30-1,
resting (K107); hematopoietic precursor line TF1, activated with
PMA and ionomycin for 1, 6 h pooled (C100); U937 premonocytic
line, resting (M100); U937 premonocytic line, activated with PMA
and ionomycin for 1, 6 h pooled (M101); elutriated monocytes,
activated with LPS, IFNy, anti-IL-10 for 1, 2, 6, 12, 24 h pooled
(M102); elutriated monocytes, activated with LPS, IFNy, IL-10 for
l, 2, 6, 12, 24 h. pooled (M103); elutriated monocytes, activated
with LPS, IFNy, anti-IL-10 for 4, 16 h pooled (M106); elutriated
monocytes, activated with LPS, IFNy, IL-10 for 4, 16 h pooled
(M107); elutriated monocytes, activated LPS for 1 h (M108);
elutriated monocytes, activated LPS for 6 h (M109); DC 70%
CDla+, from CD34+ GM-CSF, TNFa 12 days, resting (D101); DC 70%
CDla+, from CD34+ GM-CSF, TNFa 12 days, activated with PMA and
ionomycin for 1 hr (D102); DC 70% CDla+, from CD34+ GM-CSF, TNFa
12 days, activated with PMA and ionomycin for 6 hr (D103); DC
95% CDla+, from CD34+ GM-CSF, TNFa 12 days FACS sorted,
activated with PMA and ionomycin for 1, 6 h pooled (D104); DC
95% CD14+, ex CD34+ GM-CSF, TNFa 12 days FACS sorted, activated
with PMA and ionomycin 1, 6 hr pooled (D105); DC CDla+ CD86+,
from CD34+ GM-CSF, TNFa 12 days FACS sorted, activated with PMA
and ionomycin for l, 6 h pooled (D106); DC from monocytes GM-
CSF, IL-4 5 days, resting (D107); DC from monocytes GM-CSF, IL-4
5 days, resting (D108); DC from monocytes GM-CSF, IL-4 5 days,
activated LPS 4, 16 h pooled (D109); DC from monocytes GM-CSF,
IL-4 5 days, activated TNFa, monocyte supe for 4, 16 h pooled
(D110); leiomyoma L11 benign tumor (X101); normal myometrium M5
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(0115); malignant leiomyosarcoma GS1 (X103); lung fibroblast
sarcoma line MRC5, activated with PMA and ionomycin for 1, 6 h
pooled (C101); kidney epithelial carcinoma cell line CHA,
activated with PMA and ionomycin for 1, 6 h pooled (C102);
5 kidney fetal 28 wk male (0100); lung fetal 28 wk male (0101);
liver fetal 28 wk male (0102); heart fetal 28 wk male (0103);
brain fetal 28 wk male (0104); gallbladder fetal 28 wk male
(0106); small intestine fetal 28 wk male (0107); adipose tissue
fetal 28 wk male (0108); ovary fetal 25 wk female (0109); uterus
10~ fetal 25 wk female (0110); testes fetal 28 wk male (0111);
spleen fetal 28 wk male (0112); adult placenta 28 wk (0113); and
tonsil inflamed, from 12 year old (X100).
Similar samples may isolated in other species for
evaluation.
V. Cloning of species counterparts of DCRS3 or DCRS4
Various strategies are used to obtain species counterparts
of DCRS3 or DCRS4, preferably from other primates or rodents.
One method is by cross hybridization using closely related
species DNA probes. It may be useful to go into evolutionarily
similar species as intermediate steps. Another method is by
using specific PCR primers based on the identification of blocks
of similarity or difference between genes, e.g., areas of highly
conserved or nonconserved polypeptide or nucleotide sequence.
Antibody based screening methods are also available, e.g., in
expression cloning.
VI. Production of mammalian DCRS3 or DCRS4 protein
An appropriate, e.g., GST, fusion construct is engineered
for expression, e.g., in E. coli. For example, a mouse IGIF
pGex plasmid is constructed and transformed into E. coli.
Freshly transformed cells are grown, e.g., in LB medium
containing 50 ~g/ml ampicillin and induced with IPTG (Sigma, St.
Louis, MO). After overnight induction, the bacteria are
harvested and the pellets containing, e.g., DCRS3, protein are
isolated. The pellets are homogenized, e.9., in TE buffer (50
mM Tris-base pH 8.0, 10 mM EDTA and 2 mM pefabloc) in 2 liters.
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This material is passed through a microfluidizer (Microfluidics,
Newton, MA) three times. The fluidized supernatant is spun down
on a Sorvall GS-3 rotor for 1 h at 13,000 rpm. The resulting
supernatant containing the cytokine receptor protein is filtered
and passed over a glutathione-SEPHAROSE column equilibrated in
50 mM Tris-base pH 8Ø The fractions containing the DCRS3-GST
fusion protein are pooled and cleaved, e.g., with thrombin
(Enzyme Research Laboratories, Inc., South Bend, IN). The
cleaved pool is then passed over a Q-SEPHAROSE column
10~ equilibrated in 50 mM Tris-base. Fractions containing DCRS3 are
pooled and diluted in cold distilled H20, to lower the
conductivity, and passed back over a fresh Q-Sepharose column,
alone or in succession with an immunoaffinity antibody column.
Fractions containing DCRS3 protein are pooled, aliquoted, and
stored in the -70° C freezer.
Comparison of the CD spectrum with cytokine receptor
protein may suggest that the protein is correctly folded. See
Hazuda, et al. (1969) J. Biol. Chem. 264:1689-1693.
VII. Preparation of antibodies specific for DCRS3 or DCRS4
Inbred Balb/c mice are immunized intraperitoneally with
recombinant forms of the protein, e.g., purified DCRS3 or stable
transfected NIH-3T3 cells. Animals are boosted at appropriate
time points with protein, with or without additional adjuvant,
to further stimulate antibody production. Serum is collected,
or hybridomas produced with harvested spleens.
Alternatively, Balb/c mice are immunized with cells
transformed with the gene or fragments thereof, either
endogenous or exogenous cells, or with isolated membranes
enriched for expression of the antigen. Serum is collected at
the appropriate time, typically after numerous further
administrations. Various gene therapy techniques may be useful,
e.g., in producing protein in situ, for generating an immune
response. Serum or antibody preparations may be cross-absorbed
or immunoselected to prepare substantially purified antibodies
of defined specificity and high affinity.
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Monoclonal antibodies may be made. For example,
splenocytes are fused with an appropriate fusion partner and
hybridomas are selected in growth medium by standard procedures.
Hybridoma supernatants are screened for the presence of
antibodies which bind to DCRS3, e.g., by ELISA or other assay.
Antibodies which specifically recognize specific DCRS3
embodiments may also be selected or prepared.
In another method, synthetic peptides or purified protein
are presented to an immune system to generate monoclonal or
10~ polyclonal antibodies. See, e.g., Coligan (ed. 1991) Current
Protocols in Immunoloay Wiley/Greene; and Harlow and Lane (1989)
Antibodies: A Laboratory Manual Cold Spring Harbor Press. In
appropriate situations, the binding reagent is either labeled as
described above, e.g., fluorescence or otherwise, or immobilized
to a substrate for panning methods. Nucleic acids may also be
introduced into cells in an animal to produce the antigen, which
serves to elicit an immune response. See, e.g., Wang, et al.
(1993) Proc. Nat'1. Acad. Sci. 90:4156-4160; Barry, et al.
(1994) BioTechnigues 16:616-619; and Xiang, et al. (1995)
Immunity 2: 129-135.
VIII. Production of fusion proteins with DCRS
Various fusion constructs are made with DCRS. A portion of
the appropriate gene is fused to an epitope tag, e.g., a FLAG
tag, or to a two hybrid system construct. See, e.g., Fields and
Song (1989) Nature 340:245-246.
The epitope tag may be used in an expression cloning
procedure with detection with anti-FLAG antibodies to detect a
binding partner, e.g., ligand for the respective cytokine
receptor. The two hybrid system may also be used to isolate
proteins which specifically bind to DCRS.
IX. Structure activity relationship
Information on the criticality of particular residues is
determined using standard procedures and analysis. Standard
mutagenesis analysis is performed, e.g., by generating many
different variants at determined positions, e.g., at the
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positions identified above, and evaluating biological activities
of the variants. This may be performed to the extent of
determining positions which modify activity, or to focus on
specific positions to determine the residues which can be
substituted to either retain, block, or modulate biological
activity.
Alternatively, analysis of natural variants can indicate
what positions tolerate natural mutations. This may result from
populational analysis of variation among individuals, or across
10~ strains or species. Samples from selected individuals are
analyzed, e.g., by PCR analysis and sequencing. This allows
evaluation of population polymorphisms.
X. Isolation of a ligand for DCRS
A cytokine receptor can be used as a specific binding
reagent to 'identify its binding partner, by taking advantage of
its specificity of binding, much like an antibody would be used.
The binding receptor may be a heterodimer of receptor subunits;
or may involve, e.g., a complex of the DCRS with another
subunit. A binding reagent is either labeled as described
above, e.g., fluorescence or otherwise, or immobilized to a
substrate for panning methods.
The binding composition is used to screen an expression
library made from a cell line which expresses a binding partner,
i.e., ligand, preferably membrane associated. Standard staining
techniques are used to detect or sort surface expressed ligand,
or surface expressing transformed cells are screened by panning.
Screening of intracellular expression is performed by various
staining or immunofluorescence procedures. See also McMahan, et
al. (1991) EMBO J. 10:2821-2832.
For example, on day 0, precoat 2-chamber permanox slides
with 1 ml per chamber of fibronectin, 10 ng/ml in PBS, for 30
min at room temperature. Rinse once with PBS. Then plate COS
cells at 2-3 x 105 cells per chamber in 1.5 ml of growth media.
Incubate overnight at 37°C.
On day 1 for each sample, prepare 0.5 ml of a solution of
66 ~g/ml DEAE-dextran, 66 ~M chloroquine, and 4 ~g DNA in serum
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free DME. For each set, a positive control is prepared, e.g.,
of DCRS-FLAG cDNA at 1 and 1/200 dilution, and a negative mock.
Rinse cells with serum free DME. Add the DNA solution and
incubate 5 hr at 37°C. Remove the medium and add 0.5 ml 10%
DMSO in DME for 2.5 min. Remove and wash once with DME. Add
1.5 ml growth medium and incubate overnight.
On day 2, change the medium. On days 3 or 4, the cells are
fixed and stained. Rinse the cells twice with Hank's Buffered
Saline Solution (HBSS) and fix in 4% paraformaldehyde
10~ (PFA)/glucose for 5 min. Wash 3X with HBSS. The slides may be
stored at -80°C after all liquid is removed. For each chamber,
0.5 ml incubations are performed as follows. Add HBSS/saponin
(0.1%) with 32 ~1/ml of 1 M NaN3 for 20 min. Cells are then
washed with HBSS/saponin 1X. Add appropriate DCRS or
DCRS/antibody complex to cells and incubate for 30 min. wash
cells twice with HBSS/saponin. If appropriate, add first
antibody for 30 min. Add second antibody, e.g., Vector anti-
mouse antibody, at 1/200 dilution, and incubate for 30 min.
Prepare ELISA solution, e.9., Vector Elite ABC horseradish
peroxidase solution, and preincubate for 30 min. Use, e.g., 1
drop of solution A (avidin) and 1 drop solution B (biotin) per
2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin. Add
ABC HRP solution and incubate for 30 min. Wash cells twice with
HBSS, second wash for 2 min, which closes cells. Then add
Vector diaminobenzoic acid (DAB) for 5 to 10 min. Use 2 drops
of buffer plus 4 drops DAB plus 2 drops of H202 per 5 ml of
glass distilled water. Carefully remove chamber and rinse slide
in water. Air dry for a few minutes, then add 1 drop of Crystal
Mount and a cover slip. Bake for 5 min at 85-90°C.
Evaluate positive staining of pools and progressively
subclone to isolation of single genes responsible for the
binding.
Alternatively, receptor reagents are used to affinity
purify or sort out cells expressing a putative ligand. See,
e.g., Sambrook, et al. or Ausubel, et al.
Another strategy is to screen for a membrane bound receptor
by panning. The receptor cDNA is constructed as described
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above. Immobilization may be achieved by use of appropriate
antibodies which recognize, e.g., a FLAG sequence of a DCRS
fusion construct, or by use of antibodies raised against the
first antibodies. Recursive cycles of selection and
5 amplification lead to enrichment of appropriate clones and
eventual isolation of receptor expressing clones.
Phage expression libraries can be screened by mammalian
DCRS. Appropriate label techniques, e.g., anti-FLAG antibodies,
will allow specific labeling of appropriate clones.
10~
All citations herein are incorporated herein by reference to
the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference.
15 Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited by the terms of the appended claims,
20 along with the full scope of equivalents to which such claims
are entitled; and the invention is not to be limited by the
specific embodiments that have been presented herein by way of
example.
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1
SEQUENCE LISTING
<110> Schering Corporation
<120> Mammalian Receptor Proteins; Related Reagents and
Methods
<130> DX01086K PCT
<140>
<141>
<150> US 09/443,060
<151> 1999-11-18
<160> 32
<170> PatentIn Ver. 2.0
<210> 1
<211> 1707
<212> DNA
<213> primate; surmised Homo Sapiens
<220>
<221> CDS
<222> (1)..(1704)
<220>
<221> mat peptide
<222> (61)..(1704)
<400> 1
atg ccg cgt ggc tgg gcc gcc ccc ttg ctc ctg ctg ctg ctc cag gga 48
Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly
-20 -15 -10 -5
gcc ctc gag ggg atg gag agg aag ctc tgc agt ccc aag cca ccc ccc 96
Ala Leu Glu Gly Met Glu Arg Lys Leu Cys Ser Pro Lys Pro Pro Pro
-1 1 5 10
acc aag gcc tct ctc ccc act gac cct cca ggc tgg ggc tgc ccc gac 144
Thr Lys Ala Ser Leu Pro Thr Asp Pro Pro Gly Trp Gly Cys Pro Asp
15 20 25
ctc gtc tgc tac acc gat tac ctc cag acg gtc atc tgc atc ctg gaa 192
Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys Ile Leu Glu
30 35 40
atg tgg aac ctc cac ccc agc acg ctc acc ctt acc tgg ata ctt tct 240
Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp Ile Leu Ser
45 50 55 60
aat aat act ggg tgc tat atc aag gac aga aca ctg gac ctc agg caa 288
Asn Asn Thr Gly Cys Tyr Ile Lys Asp Arg Thr Leu Asp Leu Arg Gln
65 70 75
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gac cag tat gaa gag ctg aag gac gag gcc acc tcc tgc agc ctc cac 336
Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu His
80 85 90
agg tcg gcc cac aat gcc acg cat gcc acc tac acc tgc cac atg gat 384
Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met Asp
95 100 105
gta ttc cac ttc atg gcc gac gac att ttc agt gtc aac atc aca gac 432
Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr Asp
110 115 120
cag tct ggc aac tac tcc cag gag tgt ggc agc ttt ctc ctg get gag 480
Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala Glu
125 130 135 140
agc aga cag tat aat atc tcc tgg cgc tca gat tac gaa gac cct gcc 528
Ser Arg Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp Pro Ala
145 150 155
ttc tac atg ctg aag ggc aag ctt cag tat gag ctg cag tac agg aac 576
Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn
160 165 170
cgg gga gac ccc tgg get gtg agt ccg agg aga aag ctg atc tca gtg 624
Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile Ser Val
175 180 185
gac tca aga agt gtc tcc ctc ctc ccc ctg gag ttc cgc aaa gac tcg 672
Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser
190 195 200
agc tat gag ctg cag gtg cgg gca ggg ccc atg cct ggc tcc tcc tac 720
Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr
205 210 215 220
cag ggg acc tgg agt gaa tgg agt gac ccg gtc atc ttt cag acc cag 768
Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln
225 230 235
tca gag gag tta aag gaa ggc tgg aac cct cac ctg ctg ctt ctc ctc 816
Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu Leu Leu Leu
240 245 250
ctg ctt gtc ata gtc ttc att cct gcc ttc tgg agc ctg aag acc cat 864
Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp Ser Leu Lys Thr His
255 260 265
cca ttg tgg agg cta tgg aag aag ata tgg gcc gtc ccc agc cct gag 912
Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val Pro Ser Pro Glu
270 275 280
cgg ttc ttc atg ccc ctg tac aag ggc tgc agc gga gac ttc aag aaa 960
Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser Gly Asp Phe Lys Lys
285 290 295 300
tgg gtg ggt gca ccc ttc act ggc tcc agc ctg gag ctg gga ccc tgg 1008
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Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu Glu Leu Gly Pro Trp
305 310 315
agc cca gag gtg ccc tcc acc ctg gag gtg tac agc tgc cac cca cca 1056
Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr Ser Cys His Pro Pro
320 325 330
cgg agc ccg gcc aag agg ctg cag ctc acg gag cta caa gaa cca gca 1104
Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu Pro Ala
335 340 345
gag ctg gtg gag tct gac ggt gtg ccc aag ccc agc ttc tgg ccg aca 1152
Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe Trp Pro Thr
350 355 360
gcc cag aac tcg ggg ggc tca get tac agt gag gag agg gat cgg cca 1200
Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu Glu Arg Asp Arg Pro
365 370 375 380
tac ggc ctg gtg tcc att gac aca gtg act gtg cta gat gca gag ggg 1248
Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val Leu Asp Ala Glu Gly
385 390 395
cca tgc acc tgg ccc tgc agc tgt gag gat gac ggc tac cca gcc ctg 1296
Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro Ala Leu
400 405 410
gac ctg gat get ggc ctg gag ccc agc cca ggc cta gag gac cca ctc 1344
Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp Pro Leu
415 420 425
ttg gat gca ggg acc aca gtc ctg tcc tgt ggc tgt gtc tca get ggc 1392
Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val Ser Ala Gly
430 435 440
agc cct ggg cta gga ggg ccc ctg gga agc ctc ctg gac aga cta aag 1440
Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu Asp Arg Leu Lys
445 450 455 460
cca ccc ctt gca gat ggg gag gac tgg get ggg gga ctg ccc tgg ggt 1488
Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly Leu Pro Trp Gly
465 470 475
ggc cgg tca cct gga ggg gtc tca gag agt gag gcg ggc tca ccc ctg 1536
Gly Arg Ser Pro Gly Gly Val Ser Glu Ser Glu Ala Gly Ser Pro Leu
480 485 490
gcc ggc ctg gat atg gac acg ttt gac agt ggc ttt gtg ggc tct gac 1584
Ala Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly Ser Asp
495 500 505
tgc agc agc cct gtg gag tgt gac ttc acc agc ccc ggg gac gaa gga 1632
Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly Asp Glu Gly
510 515 520
ccc ccc cgg agc tac ctc cgc cag tgg gtg gtc att cct ccg cca ctt 1680
Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile Pro Pro Pro Leu
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525 530 535 540
tcg agc cct gga ccc cag gcc agc taa 1707
Ser Ser Pro Gly Pro Gln Ala Ser
545
<210> 2
<211> 568
<212> PRT
<213> primate; surmised Homo sapiens
<400> 2
Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly
-20 -15 -10 -5
Ala Leu Glu Gly Met Glu Arg Lys Leu Cys Ser Pro Lys Pro Pro Pro
-1 1 5 10
Thr Lys Ala Ser Leu Pro Thr Asp Pro Pro Gly Trp Gly Cys Pro Asp
15 20 25
Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys Ile Leu Glu
30 35 40
Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp Ile Leu Ser
45 50 55 60
Asn Asn Thr Gly Cys Tyr Ile Lys Asp Arg Thr Leu Asp Leu Arg Gln
65 70 75
Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu His
80 85 90
Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met Asp
95 100 105
Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr Asp
110 115 120
Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala Glu
125 130 135 140
Ser Arg Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp Pro Ala
145 150 155
Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn
160 165 170
Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile Ser Val
175 180 185
Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser
190 195 200
Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr
205 210 215 220
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Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln
225 230 235
Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu Leu Leu Leu
240 245 250
Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp Ser Leu Lys Thr His
255 260 265
Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val Pro Ser Pro Glu
270 275 280
Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser Gly Asp Phe Lys Lys
285 290 295 300
Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu Glu Leu Gly Pro Trp
305 310 315
Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr Ser Cys His Pro Pro
320 325 330
Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu Pro Ala
335 340 345
Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe Trp Pro Thr
350 355 360
Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu Glu Arg Asp Arg Pro
365 370 375 380
Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val Leu Asp Ala Glu Gly
385 390 395
Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro Ala Leu
400 405 410
Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp Pro Leu
415 420 425
Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val Ser Ala Gly
430 435 440
Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu Asp Arg Leu Lys
445 450 455 460
Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly Leu Pro Trp Gly
465 470 475
Gly Arg Ser Pro Gly Gly Val Ser Glu Ser Glu Ala Gly Ser Pro Leu
480 485 490
Ala Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly Ser Asp
495 500 505
Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly Asp Glu Gly
510 515 520
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Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile Pro Pro Pro Leu
525 530 535 540
Ser Ser Pro Gly Pro Gln Ala Ser
545
<210> 3
<211> 1704
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: reverse
translation
<220>
<221> misc_feature
<222> (6). (1704)
<223> n may be a, c, g, or t
<400> 3
atgccnmgng gntgggcngc nccnytnytn ytnytnytny tncarggngc nytngarggn 60
atggarmgna arytntgyws nccnaarccn ccnccnacna argcnwsnyt nccnacngay 120
ccnccnggnt ggggntgycc ngayytngtn tgytayacng aytayytnca racngtnath 180
tgyathytng aratgtggaa yytncayccn wsnacnytna cnytnacntg gathytnwsn 240
aayaayacng gntgytayat haargaymgn acnytngayy tnmgncarga ycartaygar 300
garytnaarg aygargcnac nwsntgywsn ytncaymgnw sngcncayaa ygcnacncay 360
gcnacntaya cntgycayat ggaygtntty cayttyatgg cngaygayat httywsngtn 420
aayathacng aycarwsngg naaytaywsn cargartgyg gnwsnttyyt nytngcngar 480
wsnmgncart ayaayathws ntggmgnwsn gaytaygarg ayccngcntt ytayatgytn 540
aarggnaary tncartayga rytncartay mgnaaymgng gngayccntg ggcngtnwsn 600
ccnmgnmgna arytnathws ngtngaywsn mgnwsngtnw snytnytncc nytngartty 660
mgnaargayw snwsntayga rytncargtn mgngcnggnc cnatgccngg nwsnwsntay 720
carggnacnt ggwsngartg gwsngayccn gtnathttyc aracncarws ngargarytn 780
aargarggnt ggaayccnca yytnytnytn ytnytnytny tngtnathgt nttyathccn 840
gcnttytggw snytnaarac ncayccnytn tggmgnytnt ggaaraarat htgggcngtn 900
ccnwsnccng armgnttytt yatgccnytn tayaarggnt gywsnggnga yttyaaraar 960
tgggtnggng cnccnttyac nggnwsnwsn ytngarytng gnccntggws nccngargtn 1020
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ccnwsnacny tngargtnta ywsntgycay ccnccnmgnw snccngcnaa rmgnytncar 1080
ytnacngary tncargarcc ngcngarytn gtngarwsng ayggngtncc naarccnwsn 1140
ttytggccna cngcncaraa ywsnggnggn wsngcntayw sngargarmg ngaymgnccn 1200
tayggnytng tnwsnathga yacngtnacn gtnytngayg cngarggncc ntgyacntgg 1260
ccntgywsnt gygargayga yggntayccn gcnytngayy tngaygcngg nytngarccn 1320
wsnccnggny tngargaycc nytnytngay gcnggnacna cngtnytnws ntgyggntgy 1380
gtnwsngcng gnwsnccngg nytnggnggn ccnytnggnw snytnytnga ymgnytnaar 1440
ccnccnytng cngayggnga rgaytgggcn ggnggnytnc cntggggngg nmgnwsnccn 1500
ggnggngtnw sngarwsnga rgcnggnwsn ccnytngcng gnytngayat ggayacntty 1560
gaywsnggnt tygtnggnws ngaytgywsn wsnccngtng artgygaytt yacnwsnccn 1620
ggngaygarg gnccnccnmg nwsntayytn mgncartggg tngtnathcc nccnccnytn 1680
wsnwsnccng gnccncargc nwsn 1704
<210> 4
<211> 750
<212> DNA
<213> primate; surmised Homo Sapiens
<220>
<221> CDS
<222> (1)..(747)
<220>
<221> mat peptide
<222> (64)..(747)
<400> 4
atg atg cct aaa cat tgc ttt cta ggc ttc ctc atc agt ttc ttc ctt 48
Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu
-20 -15 -10
act ggt gta gca gga act cag tca acg cat gag tct ctg aag cct cag 96
Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln
-5 -1 1 5 10
agg gta caa ttt cag tcc cga aat ttt cac aac att ttg caa tgg cag 144
Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln
15 20 25
cct ggg agg gca ctt act ggc aac agc agt gtc tat ttt gtg cag tac 192
Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr
30 35 40
aaa ata tat gga cag aga caa tgg aaa aat aaa gaa gac tgt tgg ggt 240
Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly
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45 50 55
act caa gaa ctc tct tgt gac ctt acc agt gaa acc tca gac ata cag 288
Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln
60 65 70 75
gaa cct tat tac ggg agg agg ggc aaa aat aaa aat aaa ggg aat cct 336
Glu Pro Tyr Tyr Gly Arg Arg Gly Lys Asn Lys Asn Lys Gly Asn Pro
80 85 90
tgg ggg cca aaa caa agt aaa cgg aaa tca aag ggg aac cag aag acc 384
Trp Gly Pro Lys Gln Ser Lys Arg Lys Ser Lys Gly Asn Gln Lys Thr
95 100 105
aac aca gtg act gcc cca get gcc ctg aag gca ttt get gga tgt gca 432
Asn Thr Val Thr Ala Pro Ala Ala Leu Lys Ala Phe Ala Gly Cys Ala
110 115 120
aaa ata gat cct cca gtc atg aat ata acc caa gtc aat ggc tct ttg 480
Lys Ile Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu
125 130 135
ttg gta att ctc cat get cca aat tta cca tat aga tac caa aag gaa 528
Leu Val Ile Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu
140 145 150 155
aaa aat gta tct ata gaa gat tac tat gaa cta cta tac cga gtt ttt 576
Lys Asn Val Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe
160 165 170
ata att aac aat tca cta gaa aag gag caa aag gtt tat gaa ggg get 624
Ile Ile Asn Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala
175 180 185
cac aga gcg gtt gaa att gaa get cta aca cca cac tcc agc tac tgt 672
His Arg Ala Val Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys
190 195 200
gta gtg get gaa ata tat cag ccc atg tta gac aga aga agt cag aga 720
Val Val Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg
205 210 215
agt gaa gag aga tgt gtg gaa att cca tga 750
Ser Glu Glu Arg Cys Val Glu Ile Pro
220 225
<210> 5
<211> 249
<212> PRT
<213> primate; surmised Homo sapiens
<400> 5
Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu
-20 -15 -10
Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln
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-5 -1 1 5 10
Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln
15 20 25
Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr
30 35 40
Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly
45 50 55
Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln
60 65 70 75
Glu Pro Tyr Tyr Gly Arg Arg Gly Lys Asn Lys Asn Lys Gly Asn Pro
80 85 90
Trp Gly Pro Lys Gln Ser Lys Arg Lys Ser Lys.Gly Asn Gln Lys Thr
95 100 105
Asn Thr Val Thr Ala Pro Ala Ala Leu Lys Ala Phe Ala Gly Cys Ala
110 115 120
Lys Ile Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu
125 130 135
Leu Val Ile Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu
140 145 150 155
Lys Asn Val Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe
160 165 170
Ile Ile Asn Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala
175 180 185
His Arg Ala Val Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys
190 195 200
Val Val Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg
205 210 215
Ser Glu Glu Arg Cys Val Glu Ile Pro
220 225
<210> 6
<211> 747
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: reverse
translation
<220>
<221> misc_feature
<222> (1). (747)
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<223> n may be a, c, g, or t
<400> 6
atgatgccna arcaytgytt yytnggntty ytnathwsnt tyttyytnac nggngtngcn 60
ggnacncarw snacncayga rwsnytnaar ccncarmgng tncarttyca rwsnmgnaay 120
ttycayaaya thytncartg gcarccnggn mgngcnytna cnggnaayws nwsngtntay 180
ttygtncart ayaarathta yggncarmgn cartggaara ayaargarga ytgytggggn 240
acncargary tnwsntgyga yytnacnwsn garacnwsng ayathcarga rccntaytay 300
ggnmgnmgng gnaaraayaa raayaarggn aayccntggg gnccnaarca rwsnaarmgn 360
aarwsnaarg gnaaycaraa racnaayacn gtnacngcnc cngcngcnyt naargcntty 420
gcnggntgyg cnaarathga yccnccngtn atgaayatha cncargtnaa yggnwsnytn 480
ytngtnathy tncaygcncc naayytnccn taymgntayc araargaraa raaygtnwsn 540
athgargayt aytaygaryt nytntaymgn gtnttyatha thaayaayws nytngaraar 600
garcaraarg tntaygargg ngcncaymgn gcngtngara thgargcnyt nacnccncay 660
wsnwsntayt gygtngtngc ngarathtay carccnatgy tngaymgnmg nwsncarmgn 720
wsngargarm gntgygtnga rathccn 747
<210> 7
<211> 210
<212> PRT
<213> primate
<400> 7
Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala Asn
1 5 10 15
Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser Cys
25 30
Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys Glu
35 40 45
Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu Gly
50 55 60
Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu Arg
65 70 75 80
Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln Asp
85 90 95
Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu Gln
100 105 110
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Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile Ser
115 120 125
Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg Thr
130 135 140
Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu Lys
145 150 155 160
Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr Gln
165 170 175
Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr Thr
180 185 190
Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala Ala
195 200 205
Leu Gly
210
<210> 8
<211> 231
<212> PRT
<213> primate
<400> 8
Ile Cys Ile Cys Thr Cys Val Cys Leu Gly Val Ser Val Thr Gly Glu
1 5 10 15
Gly Gln Gly Pro Arg Ser Arg Thr Phe Thr Cys Leu Thr Asn Asn Ile
20 25 30
Leu Arg Ile Asp Cys His Trp Ser Ala Pro Glu Leu Gly Gln Gly Ser
35 40 45
Ser Pro Trp Leu Leu Phe Thr Ser Asn Gln Ala Pro Gly Gly Thr His
50 55 60
Lys Cys Ile Leu Arg Gly Ser Glu Cys Thr Val Val Leu Pro Pro Glu
65 70 75 80
Ala Val Leu Val Pro Ser Asp Asn Phe Thr Ile Thr Phe His His Cys
85 90 95
Met Ser Gly Arg Glu Gln Val Ser Leu Val Asp Pro Glu Tyr Leu Pro
100 105 110
Arg Arg His Val Lys Leu Asp Pro Pro Ser Asp Leu Gln Ser Asn Ile
115 120 125
Ser Ser Gly His Cys Ile Leu Thr Trp Ser Ile Ser Pro Ala Leu Glu
130 135 140
Pro Met Thr Thr Leu Leu Ser Tyr Glu Leu Ala Phe Lys Lys Gln Glu
145 150 155 160
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Glu Ala Trp Glu Gln Ala Gln His Arg Asp His Ile Val Gly Val Thr
165 170 175
Trp Leu Ile Leu Glu Ala Phe Glu Leu Asp Pro Gly Phe Ile His Glu
180 185 190
Ala Arg Leu Arg Val Gln Met Ala Thr Leu Glu Asp Asp Val Val Glu
195 200 205
Glu Glu Arg Tyr Thr Gly Gln Trp Ser Glu Trp Ser Gln Pro Val Cys
210 215 220
Phe Gln Ala Pro Gln Arg Gln
225 230
<210> 9
<211> 216
<212> PRT
<213> primate
<400> 9
Ile Cys Ile Cys Thr Cys Val Cys Leu Gly Val Ser Val Thr Gly Glu
1 5 10 15
Gly Gln Gly Pro Arg Ser Arg Thr Phe Thr Cys Leu Thr Asn Asn Ile
20 25 30
Leu Arg Ile Asp Cys His Trp Ser Ala Pro Glu Leu Gly Gln Gly Thr
35 40 45
Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln Lys Cys
50 55 60
Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln Leu Gln
65 70 75 80
Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu Gln Asp
85 90 95
Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu Gln Asn
100 105 110
Leu Val Ile Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys Leu Ser
115 120 125
Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn His Cys
130 135 140
Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser Trp Thr
145 150 155 160
Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser Val Asp
165 170 175
Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn Pro Leu
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180 185 190
Cys Gly Ser Ala Gln His Trp Ser Glu Trp Ser His Pro Ile His Trp
195 200 205
Gly Ser Asn Thr Ser Lys Glu Asn
210 215
<210> 10
<211> 257
<212> PRT
<213> primate
<400> 10
Leu Leu Ala Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe
1 5 10 15
Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly
20 25 30
Thr Tyr Gln Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg Ala Cys
35 40 45
Pro Leu Ser Ser Gln Ser Met Pro His Phe Gly Thr Arg Tyr Val Cys
50 55 60
Gln Phe Pro Asp Gln Glu Glu Val Arg Leu Phe Phe Pro Leu His Leu
65 70 75 80
Trp Val Lys Asn Val Phe Leu Asn Gln Thr Arg Thr Gln Arg Val Leu
85 90 95
Phe Val Asp Ser Val Gly Leu Pro Ala Pro Pro Ser Ile Ile Lys Ala
100 105 110
Met Gly Gly Ser Gln Pro Gly Glu Leu Gln Ile Ser Trp Glu Glu Pro
115 120 125
Ala Pro Glu Ile Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro
130 135 140
Arg Asp Pro Lys Asn Ser Thr Gly Pro Thr Val Ile Gln Leu Ile Ala
145 150 155 160
Thr Glu Thr Cys Cys Pro Ala Leu Gln Arg Pro His Ser Ala Ser Ala
165 170 175
Leu Asp Gln Ser Pro Cys Ala Gln Pro Thr Met Pro Trp Gln Asp Gly
180 185 190
Pro Lys Gln Thr Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu
195 200 205
Gly Gly Ser Cys Leu Ile Ser Gly Leu Gln Pro Gly Asn Ser Tyr Trp
210 215 220
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Leu Gln Leu Arg Ser Glu Pro Asp Gly Ile Ser Leu Gly Gly Ser Trp
225 230 235 240
Gly Ser Trp Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val
245 250 255
Ala
<210> 11
<211> 217
<212> PRT
<213> primate
<400> 11
Val Ser Gly Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp Ala
1 5 10 15
Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val Asn
20 25 30
Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val Asn
35 40 45
Thr Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val Lys
50 55 60
Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr Lys
65 70 75 80
Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly Glu
85 90 95
Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys Pro
100 105 110
Glu Ala Pro Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn Asp
115 120 125
Phe Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val Lys
130 135 140
Val Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn Lys
145 150 155 160
Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln Arg
165 170 175
Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile Pro
180. 185 190
Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr Tyr
195 200 205
Phe Arg Thr Pro Glu Ile Asn Asn Ser
210 215
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<210> 12
<211> 196
<212> PRT
<213> primate
<400> 12
Pro Glu Asn Val Arg Met Asn Ser Val Asn Phe Lys Asn Ile Leu Gln
1 5 10 15
Trp Glu Ser Pro Ala Phe Ala Lys Gly Asn Leu Thr Phe Thr Ala Gln
25 30
Tyr Leu Ser Tyr Arg Ile Phe Gln Asp Lys Cys Met Asn Thr Thr Leu
35 40 45
Thr Glu Cys Asp Phe Ser Ser Leu Ser Lys Tyr Gly Asp His Thr Leu
50 55 60
Arg Val Arg Ala Glu Phe Ala Asp Glu His Ser Asp Trp Val Asn Ile
65 70 75 80
Thr Phe Cys Pro Val Asp Asp Thr Ile Ile Gly Pro Pro Gly Met Gln
85 90 95
Val Glu Val Leu Ala Asp Ser Leu His Met Arg Phe Leu Ala Pro Lys
100 105 110
Ile Glu Asn Glu Tyr Glu Thr Trp Thr Met Lys Asn Val Tyr Asn Ser
115 120 125
Trp Thr Tyr Asn Val Gln Tyr Trp Lys Asn Gly Thr Asp Glu Lys Phe
130 135 140
Gln Ile Thr Pro Gln Tyr Asp Phe Glu Val Leu Arg Asn Leu Glu Pro
145 150 155 160
Trp Thr Thr Tyr Cys Val Gln Val Arg Gly Phe Leu Pro Asp Arg Asn
165 170 175
Lys Ala Gly Glu Trp Ser Glu Pro Val Cys Glu Gln Thr Thr His Asp
180 185 190
Glu Thr Val Pro
195
<210> 13
<211> 196
<212> PRT
<213> rodent
<400> 13
Pro Glu Lys Val Arg Met Asn Ser Val Asn Phe Lys Asn Ile Leu Gln
1 5 10 15
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Trp Glu Val Pro Ala Phe Pro Lys Thr Asn Leu Thr Phe Thr Ala Gln
20 25 30
Tyr Glu Ser Tyr Arg Ser Phe Gln Asp His Cys Lys Arg Thr Ala Ser
35 40 45
Thr Gln Cys Asp Phe Ser His Leu Ser Lys Tyr Gly Asp Tyr Thr Val
50 55 60
Arg Val Arg Ala Glu Leu Ala Asp Glu His Ser Glu Trp Val Asn Val
65 70 75 80
Thr Phe Cys Pro Val Glu Asp Thr Ile Ile Gly Pro Pro Glu Met Gln
85 90 95
Ile Glu Ser Leu Ala Glu Ser Leu His Leu Arg Phe Ser Ala Pro Gln
100 105 110
Ile Glu Asn Glu Pro Glu Thr Trp Thr Leu Lys Asn Ile Tyr Asp Ser
115 120 125
Trp Ala Tyr Arg Val Gln Tyr Trp Lys Asn Gly Thr Asn Glu_Lys Phe
130 135 140
Gln Val Val Ser Pro Tyr Asp Ser Glu Val Leu Arg Asn Leu Glu Pro
145 150 155 160
Trp Thr Thr Tyr Cys Ile Gln Val Gln Gly Phe Leu Leu Asp Gln Asn
165 170 175
Arg Thr Gly Glu Trp Ser Glu Pro Ile Cys Glu Arg Thr Gly Asn Asp
180 185 190
Glu Ile Thr Pro
195
<210> 14
<211> 199
<212> PRT
<213> primate
<400> 14
Pro Gln Lys Val Glu Val Asp Ile Ile Asp Asp Asn Phe Ile Leu Arg
1 5 10 15
Trp Asn Arg Ser Asp Glu Ser Val Gly Asn Val Thr Phe Ser Phe Asp
20 25 30
Tyr Gln Lys Thr Gly Met Asp Asn Trp Ile Lys Leu Ser Gly Cys Gln
35 40 45
Asn Ile Thr Ser Thr Lys Cys Asn Phe Ser Ser Leu Lys Leu Asn Val
50 55 60
Tyr Glu Glu Ile Lys Leu Arg Ile Arg Ala Glu Lys Glu Asn Thr Ser
65 70 75 80
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Ser Trp Tyr Glu Val Asp Ser Phe Thr Pro Phe Arg Lys Ala Gln Ile
85 90 95
Gly Pro Pro Glu Val His Leu Glu Ala Glu Asp Lys Ala Ile Val Ile
100 105 110
His Ile Ser Pro Gly Thr Lys Asp Ser Val Met Trp Ala Leu Asp Gly
115 120 125
Leu Ser Phe Thr Tyr Ser Leu Leu Ile Trp Lys Asn Ser Ser Gly Val
130 135 140
Glu Glu Arg Ile Glu Asn Ile Tyr Ser Arg His Lys Ile Tyr Lys Leu
145 150 155 160
Ser Pro Glu Thr Thr Tyr Cys Leu Lys Val Lys Ala Ala Leu Leu Thr
165 170 175
Ser Trp Lys Ile Gly Val Tyr Ser Pro Val His Cys Ile Lys Thr Thr
180 185 190
Val Glu Asn Glu Leu Pro Pro
195
<210> 15
<211> 200
<212> PRT
<213> rodent
<400> 15
Pro Glu Asn Ile Asp Val Tyr Ile Ile Asp Asp Asn Tyr Thr Leu,Lys
1 5 10 15
Trp Ser Ser His Gly Glu Ser Met Gly Ser Val Thr Phe Ser Ala Glu
20 25 30
Tyr Arg Thr Lys Asp Glu Ala Lys Trp Leu Lys Val Pro Glu Cys Gln
35 40 45
His Thr Thr Thr Thr Lys Cys Glu Phe Ser Leu Leu Asp Thr Asn Val
50 55 60
Tyr Ile Lys Thr Gln Phe Arg Val Arg Ala Glu Glu Gly Asn Ser Thr
65 70 75 80
Ser Ser Trp Asn Glu Val Asp Pro Phe Ile Pro Phe Tyr Thr Ala His
85 90 95
Met Ser Pro Pro Glu Val Arg Leu Glu Ala Glu Asp Lys Ala Ile Leu
100 105 110
Val His Ile Ser Pro Pro Gly Gln Asp Gly Asn Met Trp Ala Leu Glu
115 120 125
Lys Pro Ser Phe Ser Tyr Thr Ile Arg Ile Trp Gln Lys Ser Ser Ser
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130 135 140
Asp Lys Lys Thr Ile Asn Ser Thr Tyr Tyr Val Glu Lys Ile Pro Glu
145 150 155 160
Leu Leu Pro Glu Thr Thr Tyr Cys Leu Glu Val Lys Ala Ile His Pro
165 170 175
Ser Leu Lys Lys His Ser Asn Tyr Ser Thr Val Gln Cys Ile Ser Thr
180 185 190
Thr Val Ala Asn Lys Met Pro Val
195 200
<210> 16
<211> 214
<212> PRT
<213> primate
<400> 16
Pro Thr Asn Val Thr Ile Glu Ser Tyr Asn Met Asn Pro Ile Val Tyr
1 5 10 15
Trp Glu Tyr Gln Ile Met Pro Gln Val Pro Val Phe Thr Val Glu Val
20 25 30
Lys Asn Tyr Gly Val Lys Asn Ser Glu Trp Ile Asp Ala Cys Ile Asn
35 40 45
Ile Ser His His Tyr Cys Asn Ile Ser Asp His Val Gly Asp Pro Ser
50 55 60
Asn Ser Leu Trp Val Arg Val,Lys Ala Arg Val Gly Gln Lys Glu Ser
65 70 75 80
Ala Tyr Ala Lys Ser Glu Glu Phe Ala Val Cys Arg Asp Gly Lys Ile
85 90 95
Gly Pro Pro Lys Leu Asp Ile Arg Lys Glu Glu Lys Gln Ile Met Ile
100 105 110
Asp Ile Phe His Pro Ser Val Phe Val Asn Gly Asp Glu Gln Glu Val
115 120 125
Asp Tyr Asp Pro Glu Thr Thr Cys Tyr Ile Arg Val Tyr Asn Val Tyr
130 135 140
Val Arg Met Asn Gly Ser Glu Ile Gln Tyr Lys Ile Leu Thr Gln Lys
145 150 155 160
Glu Asp Asp Cys Asp Glu Ile Gln Cys Gln Leu Ala Ile Pro Val Ser
165 170 175
Ser Leu Asn Ser Gln Tyr Cys Val Ser Ala Glu Gly Val Leu His Val
180 185 190
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Trp Gly Val Thr Thr Glu Lys Ser Lys Glu Val Cys Ile Thr Ile Phe
195 200 205
Asn Ser Ser Ile Lys Gly
210
<210> 17
<211> 213
<212> PRT
<213> rodent
<400> 17
Pro Thr Asn Val Leu Ile Lys Ser Tyr Asn Leu Asn Pro Val Val Cys
1 5 10 15
Trp Glu Tyr Gln Asn Met Ser Gln Thr Pro Ile Phe Thr Val Gln Val
20 25 30
Lys Val Tyr Ser Gly Ser Trp Thr Asp Ser Cys Thr Asn Ile Ser Asp
35 40 45
His Cys Cys Asn Ile Tyr Gly Gln Ile Met Tyr Pro Asp Val Ser Ala
50 55 60
Trp Ala Arg Val Lys Ala Lys Val Gly Gln Lys Glu Ser Asp Tyr Ala
65 70 75 80
Arg Ser Lys Glu Phe Leu Met Cys Leu Lys Gly Lys Val Gly Pro Pro
85 90 95
Gly Leu Glu Ile Arg Arg Lys Lys Glu Glu Gln Leu Ser Val Leu Val
100 105 110
Phe His Pro Glu Val Val Val Asn Gly Glu Ser Gln Gly Thr Met Phe
115 120 125
Gly Asp Gly Ser Thr Cys Tyr Thr Phe Asp Tyr Thr Val Tyr Val Glu
130 135 140
His Asn Arg Ser Gly Glu Ile Leu His Thr Lys His Thr Val Glu Lys
145 150 155 160
Glu Glu Cys Asn Glu Thr Leu Cys Glu Leu Asn Ile Ser Val Ser Thr
165 170 175
Leu Asp Ser Arg Tyr Cys Ile Ser Val Asp Gly Ile Ser Ser Phe Trp
180 185 190
Gln Val Arg Thr Glu Lys Ser Lys Asp Val Cys Ile Pro Pro Phe His
195 200 205
Asp Asp Arg Lys Asp
210
<210> 18
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<211> 207
<212> PRT
<213> rodent
<400> 18
Pro Ser Tyr Val Trp Phe Glu Ala Arg Phe Phe Gln His Ile Leu His
1 5 10 15
Trp Lys Pro Ile Pro Asn Gln Ser Glu Ser Thr Tyr Tyr Glu Val Ala
20 25 30
Leu Lys Gln Tyr Gly Asn Ser Thr Trp Asn Asp Ile His Ile Cys Arg
35 40 45
Lys Ala Gln Ala Leu Ser Cys Asp Leu Thr Thr Phe Thr Leu Asp Leu
50 55 60
Tyr His Arg Ser Tyr Gly Tyr Arg Ala Arg Val Arg Ala Val Asp Asn
65 70 75 80
Ser Gln Tyr Ser Asn Trp Thr Thr Thr Glu Thr Arg Phe Thr Val Asp
85 90 95
Glu Val Ile Leu Thr Val Asp Ser Val Thr Leu Lys Ala Met Asp Gly
100 105 110
Ile Ile Tyr Gly Thr Ile His Pro Pro Arg Pro Thr Ile Thr Pro Ala
115 120 125
Gly Asp Glu Tyr Glu Gln Val Phe Lys Asp Leu Arg Val Tyr Lys Ile
130 135 140
Ser Ile Arg Lys Phe Ser Glu Leu Lys Asn Ala Thr Lys Arg Val Lys
145 150 155 160
Gln Glu Thr Phe Thr Leu Thr Val Pro Ile Gly Val Arg Lys Phe Cys
165 170 175
Val Lys Val Leu Pro Arg Leu Glu Ser Arg Ile Asn Lys Ala Glu Trp
180 185 190
Ser Glu Glu Gln Cys Leu Leu Ile Thr Thr Glu Gln Tyr Phe Thr
195 200 205
<210> 19
<211> 204
<212> PRT
<213> primate
<400> 19
Pro Pro Ser Val Trp Phe Glu Ala Glu Phe Phe His His Ile Leu His
1 5 10 15
Trp Thr Pro Ile Pro Asn Gln Ser Glu Ser Thr Cys Tyr Glu Val Ala
20 25 30
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Leu Leu Arg Tyr Gly Ile Glu Ser Trp Asn Ser Ile Ser Asn Cys Ser
35 40 45
Gln Thr Leu Ser Tyr Asp Leu Thr Ala Val Thr Leu Asp Leu Tyr His
50 55 60
Ser Asn Gly Tyr Arg Ala Arg Val Arg Ala Val Asp Gly Ser Arg His
65 70 75 80
Ser Asn Trp Thr Val Thr Asn Thr Arg Phe Ser Val Asp Glu Val Thr
85 90 95
Leu Thr Val Gly Ser Val Asn Leu Glu Ile His Asn Gly Phe Ile Leu
100 105 110
Gly Lys Ile Gln Leu Pro Arg Pro Lys Met Ala Pro Ala Asn Asp Thr
115 120 125
Tyr Glu Ser Ile Phe Ser His Phe Arg Glu Tyr Glu Ile Ala Ile Arg
130 135 140
Lys Val Pro Gly Asn Phe Thr Phe Thr His Lys Lys Val Lys His Glu
145 150 155 160
Asn Phe Ser Leu Leu Thr Ser Gly Glu Val Gly Glu Phe Cys Val Gln
165 170 175
Val Lys Pro Ser Val Ala Ser Arg Ser Asn Lys Gly Met Trp Ser Lys
180 185 190
Glu Glu Cys Ile Ser Leu Thr Arg Gln Tyr Phe Thr
195 200
<210> 20
<211> 208
<212> PRT
<213> primate
<400> 20
Pro Leu Asn Pro Arg Leu His Leu Tyr Asn Asp Glu Gln Ile Leu Thr
1 5 10 15
Trp Glu Pro Ser Pro Ser Ser Asn Asp Pro Arg Pro Val Val Tyr Gln
20 25 30
Val Glu Tyr Ser Phe Ile Asp Gly Ser Trp His Arg Leu Leu Glu Pro
35 40 45
Asn Cys Thr Asp Ile Thr Glu Thr Lys Cys Asp Leu Thr Gly Gly Gly
50 55 60
Arg Leu Lys Leu Phe Pro His Pro Phe Thr Val Phe Leu Arg Val Arg
65 70 75 80
Ala Lys Arg Gly Asn Leu Thr Ser Lys Trp Val Gly Leu Glu Pro Phe
85 90 95
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Gln His Tyr Glu Asn Val Thr Val Gly Pro Pro Lys Asn Ile Ser Val
100 105 110
Thr Pro Gly Lys Gly Ser Leu Val Ile His Phe Ser Pro Pro Phe Asp
115 120 125
Val Phe His Gly Ala Thr Phe Gln Tyr Leu Val His Tyr Trp Glu Lys
130 135 140
Ser Glu Thr Gln Gln Glu Gln Val Glu Gly Pro Phe Lys Ser Asn Ser
145 150 155 160
Ile Val Leu Gly Asn Leu Lys Pro Tyr Arg Val Tyr Cys Leu Gln Thr
165 170 175
Glu Ala Gln Leu Ile Leu Lys Asn Lys Lys Ile Arg Pro His Gly Leu
180 185 190
Leu Ser Asn Val Ser Cys His Glu Thr Thr Ala Asn Ala Ser Ala Arg
195 200 205
<210> 21
<211> 207
<212> PRT
<213> primate
<400> 21
Pro Ala Asn Ile Thr Phe Leu Ser Ile Asn Met Lys Asn Val Leu Gln
1 5 10 15
Trp Thr Pro Pro Glu Gly Leu Gln Gly Val Lys Val Thr Tyr Thr Val
20 25 30
Gln Tyr Phe Ile Tyr Gly Gln Lys Lys Trp Leu Asn Lys Ser Glu Cys
35 40 45
Arg Asn Ile Asn Arg Thr Tyr Cys Asp Leu Ser Ala Glu Thr Ser Asp
50 55 60
Tyr Glu His Gln Tyr Tyr Ala Lys Val Lys Ala Ile Trp Gly Thr Lys
65 70 75 80
Cys Ser Lys Trp Ala Glu Ser Gly Arg Phe Tyr Pro Phe Leu Glu Thr
85 90 95
Gln Ile Gly Pro Pro Glu Val Ala Leu Thr Thr Asp Glu Lys Ser Ile
100 105 110
Ser Val Val Leu Thr Ala Pro Glu Lys Trp Lys Arg Asn Pro Glu Asp
115 120 125
Leu Pro Val Ser Met Gln Gln Ile Tyr Ser Asn Leu Lys Tyr Asn Val
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130 135 140
Ser Val Leu Asn Thr Lys Ser Asn Arg Thr Trp Ser Gln Cys Val Thr
145 150 155 160
Asn His Thr Leu Val Leu Thr Trp Leu Glu Pro Asn Thr Leu Tyr Cys
165 170 175
Val His Val Glu Ser Phe Val Pro Gly Pro Pro Arg Arg Ala Gln Pro
180 185 190
Ser Glu Lys Gln Cys Ala Arg Thr Leu Lys Asp Gln Ser Ser Glu
195 200 205
<210> 22
<211> 234
<212> PRT
<213> primate
<400> 22
Leu Gln His Val Lys Phe Gln Ser Ser Asn Phe Glu Asn Ile Leu Thr
1 5 10 15
Trp Asp Ser Gly Pro Glu Gly Thr Pro Asp Thr Val Tyr Ser Ile Glu
20 25 30
Tyr Lys Thr Tyr Gly Glu Arg Asp Trp Val Ala Lys Lys Gly Cys Gln
35 40 45
Arg Ile Thr Arg Lys Ser Cys Asn Leu Thr Val Glu Thr Gly Asn Leu
50 55 60
Thr Glu Leu Tyr Tyr Ala Arg Val Thr Ala Val Ser Ala Gly Gly Arg
65 70 75 80
Ser Ala Thr Lys Met Thr Asp Arg Phe. Ser Ser Leu Gln His Thr Thr
85 90 95
Leu Lys Pro Pro Asp Val Thr Cys Ile Ser Lys Val Arg Ser Ile Gln
100 105 110
Met Ile Val His Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly His
115 120 125
Arg Leu Thr Leu Glu Asp Ile Phe His Asp Leu Phe Tyr His Leu Glu
130 135 140
Leu Gln Val Asn Arg Thr Tyr Gln Met His Leu Gly Gly Lys Gln Arg
145 150 155 160
Glu Tyr Glu Phe Phe Gly Leu Thr Pro Asp Thr Glu Phe Leu Gly Thr
165 170 175
Ile Met Ile Cys Val Pro Thr Trp Ala Lys Glu Ser Ala Pro Tyr Met
180 185 190
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Cys Arg Val Lys Thr Leu Pro Asp Arg Thr Trp Thr Tyr Ser Phe Ser
195 200 205
Gly Ala Phe Leu Phe Ser Met Gly Phe Leu Val Ala Val Leu Cys Tyr
210 215 220
Leu Ser Tyr Arg Tyr Val Thr Lys Pro Pro
225 230
<210> 23
<211> 201
<212> PRT
<213> primate
<400> 23
Ser Cys Thr Phe Lys Ile Ser Leu Arg Asn Phe Arg Ser Ile Leu Ser
1 5 10 15
Trp Glu Leu Lys Asn His Ser Ile Val Pro Thr His Tyr Thr Leu Leu
20 25 30
Tyr Thr Ile Met Ser Lys Pro Glu Asp Leu Lys Val Val Lys Asn Cys
35 40 45
Ala Asn Thr Thr Arg Ser Phe Cys Asp Leu Thr Asp Glu Trp Arg Ser
50 55 60
Thr His Glu Ala Tyr Val Thr Val Leu Glu Gly Phe Ser Gly Asn Thr
65 70 75 80
Thr Leu Phe Ser Cys Ser His Asn Phe Trp Leu Ala Ile Asp Met Ser
85 90 95
Phe Glu Pro Pro Glu Phe Glu Ile Val Gly Phe Thr Asn His Ile Asn
100 105 110
Val Met Val Lys Phe Pro Ser Ile Val Glu Glu Glu Leu Gln Phe Asp
115 120 125
Leu Ser Leu Val Ile Glu Glu Gln Ser Glu Gly Ile Val Lys Lys His
130 135 140
Lys Pro Glu Ile Lys Gly Asn Met Ser Gly Asn Phe Thr Tyr Ile Ile
145 150 155 160
Asp Lys Leu Ile Pro Asn Thr Asn Tyr Cys Val Ser Val Tyr Leu Glu
165 170 175
His Ser Asp Glu Gln Ala Val Ile Lys Ser Pro Leu Lys Cys Thr Leu
180 185 190
Leu Pro Pro Gly Gln Glu Ser Glu Ser
195 200
<210> 24
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<211> 1617
<212> DNA
<213> primate; surmised Homo sapiens
<220>
<221> CDS
<222> (1)..(1614)
<220>
<221> mat~eptide
<222> (61)..(1614)
<220>
<221> misc_feature
<222> (1). (1617)
<223> n may be a, c, g, or t; translated amino acid
depends on genetic code
<400> 24
atg ccg cgt ggc tgg gcc gcc ccc ttg ctc ctg ctg ctg ctc cag gga 48
Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly
-20 -15 -10 -5
ggc tgg ggc tgc ccc gac ctc gtc tgc tac acc gat tac ctc cag acg 96
Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr
-1 1 5 10
gtc atc tgc atc ctg gaa atg tgg aac ctc cac ccc agc acg ctc acc 144
Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr
15 20 25
ctt acc tgg caa gac cag tat gaa gag ctg aag gac gag gcc acc tcc 192
Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser
35 40
tgc agc ctc cac agg tcg gcc cac aat gcc acg cat gcc acc tac acc 240
Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr
45 50 55 60
tgc cac atg gat gta ttc cac ttc atg gcc gac gac att ttc agt gtc 288
Cys His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val
65 70 75
aac atc aca gac cag tct ggc aac tac tcc cag gan tgt ggc agc ttt 336
Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Xaa Cys Gly Ser Phe
80 85 90
ctc ctg get gag agc atc aag ccg get ccc cct ttc aac gtg act gtg 384
Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val
95 100 105
acc ttc tca gga cag tat aat atn tcc tgg cgc tca gat tac gaa gac 432
Thr Phe Ser Gly Gln Tyr Asn Xaa Ser Trp Arg Ser Asp Tyr Glu Asp
110 115 120
cct gcc ttc tac atg ctg aaa ggc aag ctt caa tat gag ctg cag tac 480
Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr
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125 130 135 140
agg aac cgg gga gac ccc tgg get gtg agt ccg agg aga aag ctg atc 528
Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile
145 150 155
tca gtg gac tca aga agt gtc tcc ctc ctc ccc ctg gag ttc cgc aaa 576
Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys
160 165 170
gac tcg agc tat gag ctg can gtg cgg gca ggg ccc atg cct ggc tcc 624
Asp Ser Ser Tyr Glu Leu Xaa Val Arg Ala Gly Pro Met Pro Gly Ser
175 180 185
tcc tac cag ggg acc tgg agt gaa tgg agt gac ccg gtc atc tgt cag 672
Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Cys Gln
190 195 200
acc cag tca gag gag tta aag gaa ggc tgg aac cct cac ctg ctg ctt 720
Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu Leu
205 210 215 220
ctc ctc ctg ctt gtc ata gtc ttc att cct gcc ttc tgg agc ctg aag 768
Leu Leu Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp Ser Leu Lys
225 230 235
acc cat cca ttg tgg agg cta tgg aag aag ata tgg gcc gtc ccc agc 816
Thr His Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val Pro Ser
240 245 250
cct gag cgg ttc ttc atg ccc ctg tac aag ggc tgc agc gga gac ttc 864
Pro Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser Gly Asp Phe
255 260 265
aag aaa tgg gtg ggt gca ccc ttc act ggc tcc agc ctg gag ctg gga 912
Lys Lys Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu Glu Leu Gly
270 275 280
ccc tgg agc cca gag gtg ccc tcc acc ctg gag gtg tac agc tgc cac 960
Pro Trp Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr Ser Cys His
285 290 295 300
cca cca cgg agc ccg gcc aag agg ctg cag ctc acg gag cta caa gaa 1008
Pro Pro Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu
305 310 315
cca gca gag ctg gtg gag tct gac ggt gtg ccc aag ccc agc ttc tgg 1056
Pro Ala Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe Trp
320 325 330
ccg aca gcc cag aac tcg ggg ggc tca get tac agt gag gag agg gat 1104
Pro Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu Glu Arg Asp
335 340 345
cgg cca tac ggc ctg gtg tcc att gac aca gtg act gtg cta gat gca 1152
Arg Pro Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val Leu Asp Ala
350 355 360
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gag ggg cca tgc acc tgg ccc tgc agc tgt gag gat gac ggc tac cca 1200
Glu Gly Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro
365 370 375 380
gcc ctg gac ctg gat get ggc ctg gag ccc agc cca ggc cta gag gac 1248
Ala Leu Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp
385 390 395
cca ctc ttg gat gca ggg acc aca gtc ctg tcc tgt ggc tgt gtc tca 1296
Pro Leu Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val Ser
400 405 410
get ggc agc cct ggg cta gga ggg ccc ctg gga agc ctc ctg gac aga 1344
Ala Gly Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu Asp Arg
415 420 425
cta aag cca ccc ctt gca gat ggg gag gac tgg get ggg gga ctg ccc 1392
Leu Lys Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly Leu Pro
430 435 440
tgg ggt ggc cgg tca cct gga ggg gtc tca gag agt gag gcg ggc tca 1440
Trp Gly Gly Arg Ser Pro Gly Gly Val Ser Glu Ser Glu Ala Gly Ser
445 450 455 460
ccc ctg gcc ggc ctg gat atg gac acg ttt gac agt ggc ttt gtg ggc 1488
Pro Leu Ala Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly
465 470 475
tct gac tgc agc agc cct gtg gag tgt gac ttc acc agc ccc ggg gac 1536
Ser Asp Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly Asp
480 485 490
gaa gga ccc ccc cgg agc tac ctc cgc cag tgg gtg gtc att cct ccg 1584
Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile Pro Pro
495 500 505
cca ctt tcg agc cct gga ccc cag gcc agc taa 1617
Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser
510 515
<210> 25
<211> 538
<212> PRT
<213> primate; surmised Homo Sapiens
<400> 25
Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly
-20 -15 -10 -5
Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr
-1 1 5 10
Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr
15 20 25
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Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser
30 35 40
Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr
45 50 55 60
Cys His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val
65 70 75
Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Xaa Cys Gly Ser Phe
80 85 90
Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val
95 100 105
Thr Phe Ser Gly Gln Tyr Asn Xaa Ser Trp Arg Ser Asp Tyr Glu Asp
110 115 120
Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr
125 130 135 140
Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile
145 150 155
Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys
160 165 170
Asp Ser Ser Tyr Glu Leu Xaa Val Arg Ala Gly Pro Met Pro Gly Ser
175 180 185
Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Cys Gln
190 195 200
Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu Leu
205 210 215 220
Leu Leu Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp Ser Leu Lys
225 230 235
Thr His Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val Pro Ser
240 245 250
Pro Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser Gly Asp Phe
255 260 265
Lys Lys Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu Glu Leu Gly
270 275 280
Pro Trp Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr Ser Cys His
285 290 295 300
Pro Pro Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu
305 310 315
Pro Ala Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe Trp
320 325 330
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Pro Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu Glu Arg Asp
335 340 345
Arg Pro Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val Leu Asp Ala
350 355 360
Glu Gly Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro
365 370 375 380
Ala Leu Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp
385 390 395
Pro Leu Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val Ser
400 405 410
Ala Gly Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu Asp Arg
415 420 425
Leu Lys Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly Leu Pro
430 435 440
Trp Gly Gly Arg Ser Pro Gly Gly Val Ser Glu Ser Glu Ala Gly Ser
445 450 455 460
Pro Leu Ala Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly
465 470 475
Ser Asp Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly Asp
480 485 490
Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile Pro Pro
495 500 505
Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser
510 515
<210> 26
<211> 1614
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1) . (1614)
<223> n may be a, c, g, or t
<220>
<223> Description of Artificial Sequence: reverse
translation
<400> 26
atgccnmgng gntgggcngc nccnytnytn ytnytnytny tncarggngg ntggggntgy 60
ccngayytng tntgytayac ngaytayytn caracngtna thtgyathyt ngaratgtgg 120
aayytncayc cnwsnacnyt nacnytnacn tggcargayc artaygarga rytnaargay 180
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gargcnacnw sntgywsnyt ncaymgnwsn gcncayaayg cnacncaygc nacntayacn 240
tgycayatgg aygtnttyca yttyatggcn gaygayatht tywsngtnaa yathacngay 300
carwsnggna aytaywsnca rnnntgyggn wsnttyytny tngcngarws nathaarccn 360
gcnccnccnt tyaaygtnac ngtnacntty wsnggncart ayaaynnnws ntggmgnwsn 420
gaytaygarg ayccngcntt ytayatgytn aarggnaary tncartayga rytncartay 480
mgnaaymgng gngayccntg ggcngtnwsn ccnmgnmgna arytnathws ngtngaywsn 540
mgnwsngtnw snytnytncc nytngartty mgnaargayw snwsntayga rytnnnngtn 600
mgngcnggnc cnatgccngg nwsnwsntay carggnacnt ggwsngartg gwsngayccn 660
gtnathtgyc aracncarws ngargarytn aargarggnt ggaayccnca yytnytnytn 720
ytnytnytny tngtnathgt nttyathccn gcnttytggw snytnaarac ncayccnytn 780
tggmgnytnt ggaaraarat htgggcngtn ccnwsnccng armgnttytt yatgccnytn 840
tayaarggnt gywsnggnga yttyaaraar tgggtnggng cnccnttyac nggnwsnwsn 900
ytngarytng gnccntggws nccngargtn ccnwsnacny tngargtnta ywsntgycay 960
ccnccnmgnw snccngcnaa rmgnytncar ytnacngary tncargarcc ngcngarytn 1020
gtngarwsng ayggngtncc naarccnwsn ttytggccna cngcncaraa ywsnggnggn 1080
wsngcntayw sngargarmg ngaymgnccn tayggnytng tnwsnathga yacngtnacn 1140
gtnytngayg cngarggncc ntgyacntgg ccntgywsnt gygargayga yggntayccn 1200
gcnytngayy tngaygcngg nytngarccn wsnccnggny tngargaycc nytnytngay 1260
gcnggnacna cngtnytnws ntgyggntgy gtnwsngcng gnwsnccngg nytnggnggn 1320
ccnytnggnw snytnytnga ymgnytnaar ccnccnytng cngayggnga rgaytgggcn 1380
ggnggnytnc cntggggngg nmgnwsnccn ggnggngtnw sngarwsnga rgcnggnwsn 1440
ccnytngcng gnytngayat ggayacntty gaywsnggnt tygtnggnws ngaytgywsn 1500
wsnccngtng artgygaytt yacnwsnccn ggngaygarg gnccnccnmg nwsntayytn 1560
mgncartggg tngtnathcc nccnccnytn wsnwsnccng gnccncargc nwsn 1614
<210> 27
<211> 696
<212> DNA
<213> primate; surmised Homo sapiens
<220>
<221> CDS
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<222> (1)..(693)
<220>
<221> mat~eptide
<222> (64)..(693)
<400> 27
atg atg cct aaa cat tgc ttt cta ggc ttc ctc atc agt ttc ttc ctt 48
Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu
-20 -15 -10
act ggt gta gca gga act cag tca acg cat gag tct ctg aag cct cag 96
Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln
-5 -1 1 5 10
agg gta caa ttt cag tcc cga aat ttt cac aac att ttg caa tgg cag 144
Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln
15 20 25
ccc ggg agg gca ctt act ggc aac agc agt gtc tat ttt gtg cag tac 192
Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr
30 35 40
aaa ata tat gga cag aga caa tgg aaa aat aaa gaa gac tgt tgg ggt 240
Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly
45 50 55
act caa gaa ctc tct tgt gac ctt acc agt gaa acc tca gac ata cag 288
Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln
60 65 70 75
gaa cct tat tac ggg agg gtg agg gcg gcc tcg get ggg agc tac tca 336
Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser
80 85 90
gaa tgg agc atg acg ccg cgg ttc act ccc tgg tgg gaa aca aaa ata 384
Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu Thr Lys Ile
95 100 105
gat cct cca gtc atg aat ata acc caa gtc aat ggc tct ttg ttg gta 432
Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu Leu Val
110 115 120
att ctc cat get cca aat tta cca tat aga tac caa aag gaa aaa aat 480
Ile Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn
125 130 135
gta tct ata gaa gat tac tat gaa cta cta tac cga gtt ttt ata att 528
Val Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile
140 145 150 155
aac aat tca cta gaa aag gag caa aag gtt tat gaa ggg get cac aga 576
Asn Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala His Arg
160 165 170
gcg gtt gaa att gaa get cta aca cca cac tcc agc tac tgt gta gtg 624
Ala Val Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val Val
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175 180 185
get gaa ata tat cag ccc atg tta gac aga aga agt cag aga agt gaa 672
Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg Ser Glu
190 195 200
gag aga tgt gtg gaa att cca tga 696
Glu Arg Cys Val Glu Ile Pro
205 210
<210> 28
<211> 231
<212> PRT
<213> primate; surmised Homo sapiens
<400> 28
Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu
-20 -15 -10
Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln
-5 -1 1 5 10
Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln
15 20 25
Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr
30 35 40
Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly
45 50 55
Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln
60 65 70 75
Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser
80 85 90
Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu Thr Lys Ile
95 100 105
Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu Leu Val
110 115 120
Ile Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn
125 130 135
Val Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile
140 145 150 155
Asn Asn Ser Leu Glu.Lys Glu Gln Lys Val Tyr Glu Gly Ala His Arg
160 165 170
Ala Val Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val Val
175 180 185
Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg Ser Glu
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190 195 200
Glu Arg Cys Val Glu Ile Pro
205 210
<210> 29
<211> 693
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: reverse
translation
<220>
<221> misc_feature
<222> (1) . (693)
<223> n may be a, c, g, or t
<400> 29
atgatgccna arcaytgytt yytnggntty ytnathwsnt tyttyytnac nggngtngcn 60
ggnacncarw snacncayga rwsnytnaar ccncarmgng tncarttyca rwsnmgnaay 120
ttycayaaya thytncartg gcarccnggn mgngcnytna cnggnaayws nwsngtntay 180
ttygtncart ayaarathta yggncarmgn cartggaara ayaargarga ytgytggggn 240
acncargary tnwsntgyga yytnacnwsn garacnwsng ayathcarga rccntaytay 300
ggnmgngtnm gngcngcnws ngcnggnwsn taywsngart ggwsnatgac nccnmgntty 360
acnccntggt gggaracnaa rathgayccn ccngtnatga ayathacnca rgtnaayggn 420
wsnytnytng tnathytnca ygcnccnaay ytnccntaym gntaycaraa rgaraaraay 480
gtnwsnathg argaytayta ygarytnytn taymgngtnt tyathathaa yaaywsnytn 540
garaargarc araargtnta ygarggngcn caymgngcng tngarathga rgcnytnacn 600
ccncaywsnw sntaytgygt ngtngcngar athtaycarc cnatgytnga ymgnmgnwsn 660
carmgnwsng argarmgntg ygtngarath ccn 693
<210> 30
<211> 526
<212> DNA
<213> primate; surmised Homo Sapiens
<220>
<221> CDS
<222> (1)..(390)
<220>
<221> mat~eptide
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<222> (64)..(390)
<400> 30
atg atg cct aaa cat tgc ttt cta ggc ttc ctc atc agt ttt ttc ctt 48
Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu
-20 -15 -10
act ggt gta gca gga act cag tca acg cat gag tct ctg aag cct cag 96
Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln
-5 -1 1 5 10
agg gta caa ttt cag tcc cga aat ttt cac aac att ttg caa tgg cag 144
Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln
15 20 25
cct ggg agg gca ctt act ggc aac agc agt gtc tat ttt gtg cag tac 192
Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr
30 35 40
aaa ata tat gga cag aga caa tgg aaa aat aaa gaa gac tgt tgg ggt 240
Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly
45 50 55
act caa gaa ctc tct tgt gac ctt acc agt gaa acc tca gac ata cag 288
Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln
60 65 70 75
gaa tct tat tac ggg agg gtg agg gcg gcc tcg get ggg agc tac tca 336
Glu Ser Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser
80 85 90
gaa tgg agc atg acg ccg cgg ttc act ccc tgg tgg gaa aga gca aaa 384
Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu Arg Ala Lys
95 100 105
ggt tta tgaaggggct cacagagcgg ttgaaattga agctctaaca ccacactcca 440
Gly Leu
gctactgtgt agtggctgaa atatatcagc ccacgttaga cagaagaagt cagagaagtg 500
aagagagatg tgtggaaatt ccatga 526
<210> 31
<211> 130
<212> PRT
<213> primate; surmised Homo Sapiens
<400> 31
Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu
-20 -15 -10
Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln
-5 -1 1 5 10
Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln
15 20 25
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Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr
30 35 40
Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly
50 55
Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln
60 65 70 75
Glu Ser Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser
80 85 90
Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu Arg Ala Lys
95 100 105
Gly Leu
<210> 32
<211> 390
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: reverse
translation
<220>
<221> misc_feature
<222> (1). (390)
<223> n may be a, c, g,or t
<400> 32
atgatgccna arcaytgytt yytnggntty ytnathwsnt tyttyytnac nggngtngcn 60
ggnacncarw snacncayga rwsnytnaar ccncarmgng tncarttyca rwsnmgnaay 120
ttycayaaya thytncartg gcarccnggn mgngcnytna cnggnaayws nwsngtntay 180
ttygtncart ayaarathta yggncarmgn cartggaara ayaargarga ytgytggggn 240
acncargary tnwsntgyga yytnacnwsn garacnwsng ayathcarga rwsntaytay 300
ggnmgngtnm gngcngcnws ngcnggnwsn taywsngart ggwsnatgac nccnmgntty 360
acnccntggt gggarmgngc naarggnytn 390
