Note: Descriptions are shown in the official language in which they were submitted.
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OB PROTEIN RECEPTOR AND RELATED COMPOSITIONS AND METHODS
FIELD OF THE INVENTION
The present invention relates to OB protein
receptors, related compositions and methods of making
and using such receptors and related compositions.
BACKGROUND
Although the molecular basis for obesity is
largely unknown, the identification of the "OB gene",and
protein encoded ("OB protein") has shed some light on
mechanisms the body uses to regulate body fat deposi-
tion. Zhang et al., Nature 72: 42t5-432 (1994); see
also, the Correction at Nature 374: 479 (1995). The OB
protein is active i. vivo in both ob/ob mutant mice
(mice obese due to a defect in the production of the 0B
gene product) as well as in normal, wild type mice. The
biological activity manifests itself in, among other
things, weight loss. 5= generallv, Barinaga, "Obese"
Protein Slims Mice, Science 2&2: 475-476 (1995) .5= PCT
International Publication Number WO 96/05309,
"Modulators of Body Weight, Corresponding Nucleic Acids
and Proteins, and Diagnostic and Therapeutic Uses
Thereof,";
The other biological effects of OB protein are
not well characterized. It is known, for instance, that
in ob/ob mutant mice, administration of OB protein
results in a decrease in serum insulin levels, and serum
glucose levels. It is also known that administration of
OB protein results in a decrease in body fat. This was
observed in both ob/ob mutant mice, as well as non-obese
normal mice. Pelleymounter et al., Science 2-U: 540-543
(1995); Halaas et al., Science 2U: 543-546 (1995).
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also, Campfield et al., Science 269: 546-549 (1995)
(Peripheral and central administration of microgram
doses of OB protein reduced food intake and body weight
of ob/ob and diet-induced obese mice but not in db/db
obese mice.) In none of these reports have toxicities been observed, even at
the highest doses.
Despite the promise of clinical application
of the OB protein, the mode of action of the OB protein
In, vivo is not clearly elucidated, in part due to the
absence of information on the OB receptor. High affinity
binding of the OB protein has been detected in the rat
hypothalamus, reportedly indicating OB receptor loca-
tion. Stephens et al., Nature 377: 530-532 (1995). The
db/db mouse displays the identical phenotype as the
ob/ob mouse, extreme obesity and Type II diabetes;
this phenotype is thought to be due to a defective OB
receptor, particularly since db/db mice fail to respond
to OB protein administration. See Stephens et al.,
su ra.
Identification of the OB protein receptor is
key in determining the pathway of signal transduction.
Moreover, identification of the OB protein receptor
would provide powerful application in diagnostic uses,
for example, to determine if individuals would benefit
from OB protein therapy. Furthermore, the OB receptor
could be a key component in an assay for determining
additional molecules which bind to the receptor and
result in desired biological activity. Further, such
soluble receptor could enhance or alter the effective-
ness of OB protein (or analog or derivative thereof).
SLTMMARY OF THE IjJVENTION
The present invention relates to a novel class of protein receptors, herein
denominated "OB protein
receptors" or "OB receptors", which are thought to
selectively bind OB protein. As such, the novel OB
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receptor family is provided, as well as novel members of
such family. Also provided are nucleic acids, vectors
and host cells containing such nucleic acids, related
antisense nucleic acids, molecules which selectively
bind to the OB protein receptor, and related composi-
tions of matter, such as OB receptor protein/OB protein
complexes. In other aspects, the present invention
relates to methods of using the above compositions, such
as therapeutic and/or diagnostic methods, and methods
for preparing OB receptor ligands.
DETAILED DESCRIPTION
A novel family of OB receptors is provided.
This novel family resulted from identification of a PCR
fragment isolated from a human liver cell cDNA library.
The original PCR fragment, from which primers were
isolated, contained a "WSXWS" motif, common to cytokine
receptors. As illustrated by the working examples
below, using this fragment four members of this OB
protein receptor family have been identified. These
members, herein designated as "A", "B", and "C", and "D"
are identical at amino acid position 1-891 (using the
numbering of Seq. ID No. 1), but diverge at position 892
through the C-terminus. They vary in length at the
C-terminus beyond amino acid 891, and the different
forms appear to have different tissue distribution.
Using hydrophobicity analysis, the leader
sequence is likely to comprise amino acids (Seq. ID.
No. 1) 1-21, 1-22, or 1-28. The first amino acid of the
mature protein is likely to be 22 (F), 23 (N) or 29 (T).
Most likely, based on analysis of eucaryotic cell
expression (CHO cell expression see Example 8, infra),
the first amino acid of the mature protein is 22(F).
The beginning of the transmembrane domain appears to be
located at position 840 (A) or 842 (L). The end of the
transmembrane domain appears to be located at position
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862 (I) , 863 (S) or 864 (H). Thus, based on predictions
from hydrophobicity analysis, for OB protein binding, at
a minimum what is needed is the extracellular domain of
the mature protein, amino acids 22, 23 or 29 through
amino acids 839 (D) or 841 (G). Therefore, the present
class of OB receptor proteins includes those having
amino acids (according to Seq. ID No. 1):
(a) 1-896;
(b) 22-896;
(c) 23-896;
(d) 29-896;
(e) 1-839;
(f) 22-839;
(h) 1-841;
(i) 22-841;
(j) 23-841;
(k) 29-841;
(1) 1-891;
(m) 22-891;
(n) 23-891;
(o) 29-891;
(p) the amino acids of subparts (1)
through (o) having the C-terminal amino acids selected
from among:
(i) OB receptor B (Seq. ID No. 3)
positions 892-904;
(ii) OB receptor C (Seq. ID No. 5)
positions 892- 958; and,
(iii) OB receptor D (Seq. ID No. 7)
positions 892-1165;
(q) amino acids of subparts b, c,
d, f, g, i, j, k, m, n, o, and any of (p) lacking a
leader sequence, which have an N-terminal methionyl
residue.
Also provided herein is what is thought to be
a human splice variant of a soluble OB receptor. This
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splice variant includes the extracellular domain at
least up to amino acid 798 (of Seq. ID No. 1, for
example) and has a unique 6 amino acid C-terminus at
positions 799-804: G K F T I L.
The functional domains of the 0B receptor may
be predicted using the information contained in Bazan et
al., PNAS-USA $7: 6934-6938 (1990)
For the present OB receptor, there are
two hematopoietin domains, a random coil region, the
transmembrane domain, and the intracellular domain. The
overall geography may be illustrated as follows:
.- 4.:v~v_=4i-.v '4 ~ \:i
.'.yr. a:: ..;~= ' =i~i:~
="i~;<,~ ~~'.; ~~y~:
RC /
H1 H2 TM Box 1 Box2 Box3
IC
Using the information provided by Bazan,
Zupra, the domains may be predicted, with essentially an
error of approximately plus or minus three base pairs
(as applied to all amino acid location specified for
purposes of identifying the Bazan predicted domains).
The precise locations may be determined empirically by
methods known in the art, such as preparing and
expressing modified recombinant DNAs. The structural
characteristics are though to be important for
maintaining the structural integrity of the molecule,
and therefore, to the extent that such structure is
important for function, for functional characteristics
as well.
The hematopoietin domains (Hi and H2) are
thought to have two fibronectin type 3 repeats each, one
set of paired cysteine residues each (thought to form a
disulfide bridge), and one "WSXWS box" (referring to the
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single letter amino acid abbreviation, with "X" being
any amino acid). The fibrinectin type 3 domains may be
identified by location of a double proline ("PP"), which marks the beginning
of the second fibronectin type 3
repeat; the actual beginning of such second fibronectin
type 3 repeat is likely to begin about 3 amino acids
upstream of that double proline.
The first hematopoietin domain is likely to
begin at amino acid 123 (using the numbering according
to Seq. ID No. 1, for example), which is an isoleucine
residue M. The last amino acid of the hematopoietin
domain is likely to be amino acid 339, which is a lysine
(K) residue. The two fibronectin type 3 repeats are
likely to be located at (about) amino acids 123 through
235 and 236 through 339. There is a single pair of
cysteine residues which likely form a disulfide bridge,
located at position 131 and position 142. The "WSXWS
box" is located at position 319 through 323.
The second hematopoietin domain is likely to
begin at position 428, which is an isoleucine (I) and
end at position 642 which is a glycine (G). The paired
fibronectin type 3 repeats are located at about position
428 through position 535 and about position 536 through
about position 642. One pair of cysteines is located at
position 436 and position 447, and the second pair is
located at position 473 and 488. The "WSXWS box" is
located at position 622-626.
Between the first and the second hematopoietin
domain (amino acids 339-428, approximately) is a region
of unknown functional significance.
The random coil domain ("RC" between the H2
and the transmembrane domain, "TM") is likely to begin
at the amino acid following the end of the second
hematopoietin domain, and is likely to end at the
beginning of the transmembrane domain. This is likely
to be from about amino acid 642 through amino acid 839
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or 841 (with the transmembrane domain beginning at
position 840 (A) or 842 (L)). The intracellular domain
("IC") is likely to begin at position 861 (L), 862 (I),
863 (S) or 864 (H).
The intracellular domain ("IC") contains three
regions, or "boxes," thought to participate in signal
transduction (two "JAK" boxes and a single "STAT" box,
"Box 1", "Box 2", and "Box 3"). With respect to the
numbering of the amino acid positions of the "D" form of
the OB receptor (Seq. ID No.7, below), box 1 is located
at amino acid 871 (F) through 878 (P). Box 2.is located
at approximately amino acid number 921 (I) through
931 (K). Box 3 on the "D" form is located at approxi-
mately position 1141 through 1144t(amino acids YMPQ, as
the "STAT" box is typically a conserved region of "YXXQ"
wherein "X" designates any amino acid). The intracel-
lular domain is thought to be responsible for signal
transduction. One possible mode of action is via
phosphorylation of various residues. ~g Ihle et al.,
Cell 4 : 331-334 (1996).
One possible mode of action is that upon
ligand binding (here, OB protein binding), the OB
receptor dimerizes with another receptor. A kinase
("JAK") binds to box 1, and becomes phosphorylated.
(The JAK may already be bound prior to dimerization.)
Also, "STATS" bind to box 3 and become phosphorylated on
a specific tyrosine. It is thought that this
phosphorylation results, probably indirectly, in DNA
binding protein production, which results in altered DNA
transcription, and therefore altered expression. As seen
below in Example 6, one measurement of the capability of
an OB receptor to transduce signal is the degree of
phosphorylation of JAK/STAT molecules.
The C-terminus region is intracellular (of
cell-bound 0B receptor). The differences in the C-
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terminus among members of the present OB receptor family
may result in differences in signal transduction among
the species. Thus, the present OB receptors include at
least the extracellular domain which is important for OB
protein ligand binding. Nucleic acids encoding the
present OB receptors, vectors, and host cells are also
provided for herein.
The extracellular domain may be modified and
still retain the function of ligand binding,
particularly by one or more of the following
modifications: (a) the random coil domain (as indicated
above, occuring downstream of the second hematopoietic
domain through the beginning of the transmembrane
domain) may be deleted (this may be approximately
positions 642 through 839 or 841); (b) the "WSXWS" box
may be modified by (i) substitution of the first serine
with another amino acid, particularly conserved in terms
of hydrophobicity and/or charge, such as a glycine; (ii)
the last serine may be substituted with another amino
acid, such as a threonine; (iii) the first tryptophan
may be substituted with another amino acid, for example,
a tyrosine.
Human genomic DNA encoding OB receptor protein
is also provided herein. The genomic DNA has been
localized to human chromosome 1P31, which is believed to
correspond to mouse chromosome 4, the location of the
mouse db locus.
Tissue distribution analysis demonstrates the
presence of OB receptor nucleic acids is fairly
ubiquitous, and particularly noted in the liver. It is
also observed in the ovary, and heart; and, to a lesser
extent, in small intestine, lung, skeletal muscle,
kidney, and, to an even lesser extent, spleen, thymus,
prostate, testes, placenta and pancreas (Example 2,
below). There may also be one or more forms of the OB
receptor present in serum, such as soluble OB receptor,
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which may be complexed to one or more forms of the OB
protein.
Amino Acid Secruences and Compositions
According to the present invention, novel OB
protein receptors and DNA sequences coding for all or
part of such OB receptors are provided. The present
invention provides purified and isolated polypeptide
products having part or all of the primary structural
conformation (i.e., continuous sequence of amino acid
residues) and one or more of the biological properties
(e.g., immunological properties and in vitro biological
activity) and physical properties (e.g., molecular
weight) of naturally-occurring mammalian OB receptor
including allelic variants thereof. The term "purified
and isolated" herein means substantially free of
unwanted substances so that the present polypeptides are
useful for an intended purpose. For example, one may
have a recombinant human OB receptor substantially free
of human proteins or pathological agents. These
polypeptides are also characterized by being a product
of mammalian cells, or the product of chemical synthetic
procedures or of procaryotic or eucaryotic host
expression (e.g., by bacterial, yeast, higher plant,
insect and mammalian cells in culture) of exogenous DNA
sequences obtained by genomic or cDNA cloning or by gene
synthesis. The products of expression in typical yeast
(e.g., Saccharomyces cerevisiae), insect, or procaryote
(e.g., L. coli) host cells are free of association with
any mammalian proteins. The products of expression in
vertebrate (e.g., non-human mammalian (e.g. COS or CHO)
and avian) cells are free of association with any human
proteins. Depending upon the host employed, and other
factors, polypeptides of the invention may be
glycosylated with mammalian or other eucaryotic
carbohydrates or may be non-glycosylated. One may modify
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the nucleic acid so that glycosylation sites are
included in the resultant polypeptide. One may choose to
partially or fully deglycosylate a glycosylated
polypeptide. Polypeptides of the invention may also
include an initial methionine amino acid residue (at position -1 with respect
to the first amino acid residue
of the mature polypeptide).
In addition to naturally-occurring allelic
forms of OB receptor, the present invention also
embraces other OB receptor products such as polypeptide
analogs of OB receptor and fragments of OB receptor.
Following the procedures of the above noted published
application by Alton et al. (WO 83/04053), one can
readily design and manufacture genes coding for
microbial expression of polypeptides having primary
conformations which differ from that herein specified
for in terms of the identity or location of one or more
residues (e.g., substitutions, terminal and intermediate
additions and deletions). Alternately, modifications of
cDNA and genomic genes may be readily accomplished by
well-known site-directed mutagenesis techniques and
employed to generate analogs and derivatives of OB
receptor. Such products would share at least one of the
biological properties of mammalian OB receptor but may
differ in others. As examples, projected products of
the invention include those which are foreshortened by
e.g., deletions; or those which are more stable to
hydrolysis (and, therefore, may have more pronounced or
longer lasting effects than naturally-occurring); or
which have been altered to delete one or more potential
sites for glycosylation (which may result in higher
activities for yeast-produced products); or which have
one or more cysteine residues deleted or replaced by,
e.g., alanine or serine residues and are potentially
more easily isolated in active form from microbial
systems; or which have one or more tyrosine residues
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replaced by phenylalanine; or have an altered lysine
composition (such as those prepared for purposes of
derivatization). Included are those polypeptides with
amino acid substitutions which are "conservative"
according to acidity, charge, hydrophobicity, polarity,
size or any other characteristic known to those skilled
in the art. 0= g ne Praliv_, Creighton, Proteins, W.H.
Freeman and Company, N.Y., (1984) 498 pp. plus index,
2assim. One may make changes in selected amino acids so
long as such changes preserve the overall folding or
activity of the protein, (see Table 1, below). Small
amino terminal extensions, such as an amino-terminal
methionine residue, a small linker peptide of up to
about 20-25 residues, or a small extension that facili-
tates purification, such as a poly-histidine tract, an
antigenic epitope or a binding domain, may also be
present. 5.aa, in general Ford et al., Protein Exnression
and Purification 2: 95-107, 1991.
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Table 1
Conservative Amino Acid Substitutions
Basic: arginine
lysine
histidine
Acidic: glutamic acid
aspartic acid
Polar: glutamine
as ara ine
Hydrophobic: leucine
isoleucine
valine
Aromatic: phenylalanine
tryptophan
tyrosine
Small: glycine
alanine
serine
threonine
methionine
Also comprehended are polypeptide fragments
duplicating only a part of the continuous amino acid
sequence or secondary conformations within OB receptor,
which fragments may possess one activity of mammalian
(particularly human) OB receptor (e.g., immunological
activity) and not others (e.g., OB protein binding
activity).
Of applicability to OB receptor fragments and
polypeptide analogs of the invention are reports of the
immunological activity of synthetic peptides which
substantially duplicate the amino acid sequence extant
in naturally-occurring proteins, glycoproteins and
nucleoproteins. More specifically, relatively low
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molecular weight polypeptides have been shown to
participate in immune reactions which are similar in
duration and extent to the immune reactions of
physiologically significant proteins such as viral
antigens, polypeptide hormones, and the like. Included
among the immune reactions of such polypeptides is the
provocation of the formation of specific antibodies in
immunologically active animals. See, e.a., Lerner et
al., Cell 2a: 309-310 (1891) ; Ross et al., Nature 294 :
654-656 (1891); Walter et al., PNAS-USA Z: 5197-5200
(1980); Lerner et al., PNAS-USA, 2$: 3403-3407 (1891);
Walter et al., PNAS-USA Z$: 4882-4886 (1891); Wong et
al., PNAS-USA 79: 5322-5326 (1982); Baron et al., Cell
Z$: 395-404 (1982); Dressman et al., Nature 295: 185-160
(1982); and Lerner, Scientific American 248: 66-74
(1983). See, also, Kaiser et al. Science ,2,22: 249-255
(1984) relating to biological and immunological
activities of synthetic peptides which approximately
share secondary structures of peptide hormones but may
not share their primary structural conformation. The
present invention also includes that class of polypep-
tides coded for by portions of the DNA complementary to
the protein-coding strand of the human cDNA or genomic
DNA sequences of OB receptor i.e., "complementary
inverted proteins" as described by Tramontano et al.
Nucleic Acid Res. IZ: 5049-5059 (1984). Polypeptides or
analogs thereof may also contain one or more amino acid
analogs, such as peptidomimetics.
Thus, the present class of OB receptor
proteins includes those having amino acids (according to
Seq. ID No. 1):
(a) 1-896;
(b) 22-8 96;
(c) 23-896;
(d) 29-896
(e) 1-839;
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(f) 22-839;
(g) 29-839;
(h) 1-841;
(i) 22-841;
(j) 23-841;
(k) 29-841;
(1) 1-891;
(m) 22-891;
(n) 23-891;
(o) 29-891;
(p) the amino acids of subparts (1)
through (o) having the C-terminal amino acid sequence
beginning at position 892 of OB receptor B (Seq. ID
No. 3) or C (Seq. ID. No. 5);
(q) amino acids of subparts b, c, d, f,
g, i, j, k, m, n, o, and any of (p) lacking a leader
sequence, which have an N-terminal methionyl residue.
Also provided is a longer form of an OB
receptor protein, herein denominated the "D" form, which
has an amino acid sequence selected from among
(according to Seq. ID No. 7):
(a) amino acids 1-1165;
(b) amino acids 22-1165;
(c) amino acids 23-1165;
(d) amino acids 29-1165;
(e) amino acids of subparts (b), (c) or
(d) having an N-terminal methionyl residue.
As set forth above, one may prepare soluble
receptor by elimination of the transmembrane and intra-
cellular regions. Examples of soluble receptors include
those set forth in Seq. ID Nos. 10 and 13. What is
thought to be a native, secreted form of a soluble human
OB receptor is also provided herein. This form of OB
receptor protein has an amino acid sequence selected
from among (according to Seq. ID No. 13):
(a) amino acids 1-804;
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(b) amino acids 22-804;
(c) amino acids 23-804;
(d) amino acids 29-804; and,
(e) amino acids of subparts (b), (c) or
(d) having an N-terminal methionyl residue.
In addition, since the C-terminus region of
the above polyeptides diverges at position 892 (with
respect to Seq. ID Nos. 1, 3, 5, 7 and 13) one may
desire to prepare only the polypeptides which are
divergent:
(a) those having only amino acids 892-896
of Seq. ID No. 1;
(b) those having only amino acids 892-904
of Seq. ID No. 3;
(c) those having only amino acids 892-958
of Seq. ID No. 5;
(d) those having only amino acids 892-
1165 of Seq. ID No. 7; and,
(e) those having only amino acids 799-804
of Seq. ID No. 13.
The above polypeptides which have an
extracellular domain may be modified, as indicated
above, and still retain the function of ligand binding.
Such modification may include one or more of the
following:
(a) the random coil domain (as indicated
above, occuring downstream of the second hematopoietic
domain through the beginning of the transmembrane
domain) may be deleted (this may be approximately
positions 642 through 839 or 841);
(b) the "WSXWS" box may be modified by
(i) substitution of the first serine with another amino
acid, particularly conserved in terms of hydrophobicity
and/or charge, such as a glycine; (ii) the last serine
may be substituted with another amino acid, such as a
threonine; (iii) the first tryptophan may be
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substituted with another amino acid, for example, a
tyrosine.
Thus, the present polypeptides include
(according to the numbering of Seq. ID No. 7):
(a) 1-896;
(b) 22-896;
(c) 23-896;
(d) 29-896
(e) 1-839;
(f) 22-839;
(g) 29-839;
(h) 1-841;
(i) 22-841;
( j ) 23-841;
(k) 29-841;
(1) 1-891;
(m) 22-891;
(n) 23-891;
(o) 29-891;
(p) the amino acids of subparts (1)
through (o) having the C-terminal amino acids selected
from the C-terminal amino acids of OB receptor B (Seq.
ID No. 3), C (Seq. ID. No. 5) and D (Seq ID No. 7);
(q) the amino acids (according to Seq. ID
No. 13) selected from the group consisting of 22-804;
23-804 and 29-804;
(r) amino acids of subparts b, c, d, f,
g, i, j, k, m, n, o, any of (p) lacking a leader
sequence, and (q) which have an N-terminal methionyl
residue; and
(s) amino acids of subparts (a) through
(r) which above having at least one of the following
modifications:
(i) for amino acids of subparts (a)
through (p) and those of subpart (r) which are not amino
acids according to subpart (q), deletion of (or
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substitution of amino acid(s) or other modifications of)
a random coil domain sequence selected from
(a) 640 through 839 (using
the numbering according to Seq. ID No. 1);
(b) 641 through 839;
(c) 642 through 839;
(d) 640 through 841;
(e) 641 through 841; and
(f) 642 through 841;
(ii) for amino acids of subpart (q)
and those of subpart (r) which contain the sequence of
subpart (q), deletion of of (or substitution of amino
acid(s) or other modifications of) a random coil domain
sequence selected from among:
(a) 640 through 804;
(b) 641 through 804; and,
(c) 642 through 804;
and,
(iii) modification of a "WSXWS"
sequence which is
(a) substitution of the first
serine with another amino acid, particularly conserved
in terms of hydrophobicity and/or charge, such as a
glycine;
(b) substition of the last
serine with another amino acid, such as a threonine;
and
(c) substitution of the first
tryptophan with another amino acid, for example, a
tyrosine.
One may modify the OB receptor to create a
fusion molecule with other peptide sequence. For
example, if one desired to "tag" the OB receptor with an
immunogenic peptide, one could construct a DNA which
35' would result in such fusion protein. The tag may be at
the N-terminus. Also, since it is apparent that the
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C-terminus is not necessary for ligand binding activity,
one may chemically modify the C-terminus of, for
example, a soluble OB receptor. One may desire, for
example, a preparation whereby one or more polymer
molecules such as polyethylene glycol molecules are
attached. Thus, another aspect of the present invention
is chemically modified OB receptor protein (also further
described infra).
An example of such "tag" is provided herein
using the C-terminus of a recombinant soluble OB
receptor. Seq. ID No. 12 provides a"FLAG-tag" version
of such soluble OB receptor (the nucleic acid sequence
is provided, which may be transcribed to prepare the
polypeptide). Such "FLAG-tag" may also be attached to
the N-terminus or other region of an OB receptor
protein. This type of "tagging" is useful to bind the
protein using reagents, such as antibodies, which are
selective for such tag. Such binding may be for
detection of the location or amount of protein, or for
protein capturing processes where, for example, an
affinity column is used to bind the tag, and thus the
desired protein. Other types of detectable labels, such
as radioisotopes, light-emitting (e.g., fluorescent or
phosporescent compounds), enzymatically cleavable,
detectable antibody (or modification thereof), or other
substances may be used for such labelling of the present
proteins. Detecting protein via use of the labels may
be useful for identifying the presence or amount of OB
receptor protein or a compound containing such protein
(e.g., OB protein complexed to OB receptor). Moreover,
such labelled protein may be useful for distinguishing
exogenous OB receptor protein from the endogenous form.
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Nucleic Acids
Novel nucleic acid sequences of the invention
include sequences useful in securing expression in
procaryotic or eucaryotic host cells of polypeptide
products having at least a part of the primary struc-
tural conformation and one or more of the biological
properties of recombinant human 0B receptor. The
nucleic acids may be purified and isolated, so that the
desired coding region is useful to produce the present
polypeptides, for example, or for diagnostic purposes,
as described more fully below. DNA sequences of the
invention specifically comprise: (a) any of the DNA
sequences set forth in Seq. ID No.2, 4, 8, 9, 11,
12, and 14 (and complementary strands); (b) a DNA
sequence which hybridizes (under hybridization
conditions disclosed in the cDNA library screening
section below, using the 300 bp PCR fragment as
described to selectively hybridize to a cDNA encoding an
0B receptor protein in a human liver cDNA library, or
equivalent conditions or more stringent conditions) to
the DNA sequence in subpart (a) or to fragments thereof;
and (c) a DNA sequence which, but for the degeneracy of
the genetic code, would hybridize to the DNA sequence in
subpart (a). Specifically comprehended in parts (b) and
(c) are genomic DNA sequences encoding allelic variant
forms of human 0B receptor and/or encoding 0B receptor
from other mammalian species, and manufactured DNA
sequences encoding 0B receptor, fragments of OB
receptor, and analogs of 0B receptor which DNA sequences
may incorporate codons facilitating transcription and
translation of messenger RNA in microbial hosts. Such
manufactured sequences may readily be constructed
according to the methods of Alton et al., PCT published
application WO 83/04053.
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Genomic DNA, such as that of Seq. ID No. 9,
encoding the present OB receptors may contain additional
non-coding bases, or introns, and such genomic DNAs are
obtainable by hybridizing all or part of the cDNA,
illustrated in Seq. ID Nos. 2,. 4, 6, 8, 11, and 14 to a
genomic DNA source, such as a human genomic DNA library.
Such genomic DNA will encode functional OB receptor
polypeptide; however, use of the cDNAs may be more
practicable in that, since only the coding region is
involved, recombinant manipulation is facilitated. The
intron/exon location of genomic DNA is set forth in Seq.
ID No. 9, infra.
Nucleic acid sequences include the
incorporation of codons which enhance expression by
selected nonmammalian hosts; the provision of sites for
cleavage by restriction endonuclease enzymes; and the
provision of additional initial, terminal or
intermediate DNA sequences which facilitate construction
of cloning and/or expression vectors.
The present invention also provides DNA
sequences coding for polypeptide analogs or derivatives
of OB receptor which differ from naturally-occurring
forms in terms as described above. The leader sequence
DNA may be substituted with another leader sequence for
ease in expression or for other purposes.
Also, one may prepare antisense nucleic acids
against the present DNAs. Such antisense nucleic acids
may be useful in modulating the effects of OB receptor
protein ,jn vivo. For example, one may prepare an
antisense nucleic acid which effectively disables the
ability of a cell to produce OB receptor by binding to
the nucleic acid which encodes such OB receptor.
DNA sequences of the invention are also
suitable materials for use as labeled probes in
isolating human genomic DNA encoding OB receptor, as mentioned above, and
related proteins as well as cDNA
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and genomic DNA sequences of other mammalian species.
DNA sequences may also be useful in various alternative
methods of protein synthesis (e.g., in insect cells) or,
as described infra, in genetic therapy in humans and
other mammals. DNA sequences of the invention are
expected to be useful in developing transgenic mammalian
species which may serve as eucaryotic "hosts" for
production of OB receptor and OB receptor products in
quantity. See, generally, Palmiter et al., Science 22,Z:
809-814 (1983).
Vectors and Host Cells
According to another aspect of the present
invention, the DNA sequences described herein which
encode OB receptor polypeptides are valuable for the
information which they provide concerning the amino acid
sequence of the mammalian protein which have heretofore
been unavailable. Put another way, DNA sequences
provided by the invention are useful in generating new
and useful viral and circular plasmid DNA vectors, new
and useful transformed and transfected procaryotic and
eucaryotic host cells (including bacterial cells, yeast
cells, insect cells, and mammalian cells grown in
culture), and new and useful methods for cultured growth
of such host cells capable of expression of OB receptor
and its related products.
The DNA provided herein (or corresponding
RNAs) may also be used for gene therapy for, example,
treatment of conditions characterized by the
overexpression of OB protein, such as anorexia or
cachexia. Alternatively, gene therapy may be used in
cases where increased sensitivity to OB protein is
desired, such as in cases where an individual has a
condition characterized by OB protein receptors
defective in ability to bind or retain the binding of OB
protein. Currently, vectors suitable for gene therapy
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(such as retroviral or adenoviral vectors modified for
gene therapy purposes and of purity and pharmaceutical
acceptability) may be administered for delivery into the
lung, for example. Such vectors may incorporate nucleic
acid encoding the present polypeptides for expression in
a desired location. Gene therapy may involve more than
one gene for a desired protein or different desired
proteins.
Alternatively, one may use no vector so as to
facilitate relatively stable presence in the host. For
example, homologous recombination of a DNA as provided
herein or of a suitable transcription or translation
control region may facilitate integration into or
expression from a host genome. (T,his may be performed
for production purposes as well, e.g., U.S. Patent
No. 5,272,071 and WO 91/09955.) The nucleic acid may be
placed within a pharmaceutically acceptable carrier to
facilitate cellular uptake, such as a lipid solution
carrier (e.g., a charged lipid), a liposome, or
polypeptide carrier (e.g., polylysine). A review
article on gene therapy is Verma, Scientific American,
November 1990, pages 68-84.
Thus, the present invention provides for a
population of cells expressing an OB receptor of the
present OB receptor family. Such cells are suitable for
transplantation or implantation into an individual for
therapeutic purposes. For example, one may prepare a
population of cells to overexpress OB receptor (such as
one identified in the Sequence ID's or otherwise denoted
herein), or to express a desired form of OB receptor,
such as one which is particularly sensitive to OB
protein (i.e., a form which has a desired capacity for
signal transduction). One may then implant such cells
into an individual to increase that individual's
sensitivity to OB protein. Such cells may, for example,
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be liver cells, bone marrow cells, or cells derived from
umbillical cord. Alternatively, one may wish to use
overexpressing circulating cells such as blood
progenitor cells, T cells or other blood cells. For
humans, human cells may be used. Cells may be in the
form of tissue. Such cells may be cultured prior to
transplantation or implantation. Such OB receptor
overexpression, or expression of particularly sensitive
forms of OB receptor may be accomplished by, for
example, altering the regulatory mechanism for
expression of OB receptor, such as using homologous
recombination techniques as described oupra. Thus,
provided is a population of host cells modified so that
expression of endogenous OB receptor DNA is enhanced.
The cells to be transferred to the recipient
may be cultured using one or more factors affecting the
growth or proliferation of such cells if appropriate.
Hematopoietic factors may be us'ed in culturing
hematopoietic cells. Such factors include G-CSF, EPO,
MGDF, SCF, Flt-3 ligand, interleukins (e.g., ILl-IL13),
GM-CSF, LIF, and analogs and derivatives thereof as
available to one skilled in the art.
Nerve cells, such as neurons or glia, may also
be used, and these may be cultured with neurotrophic
factors such as BDNF, CNTF, GDNF, NT3, or others.
There may be a co-gene therapy involving the
transplantation of cells expressing more than one
desired protein. For example, cells expressing OB
receptor protein may be used in conjunction,
simultaneously or !a serriatim with cells expressing OB
protein.
For gene therapy dosages, one will generally
use between one copy and several thousand copies of the
present nucleic acid per cell, depending on the vector,
the expression system, the age, weight and condition of
the recipient and other factors which will be apparent
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to those skilled in the art. The cellular delivery of
such protein may be designed to last for a selected
period of time, such as a period of days, weeks, months
or years. At the end of the effective time period, the
recipient of such transformed cells may receive another
"dose" (e.g., transplantation of cells). Cells may be
selected for their lifespan, their time period of
expression of the desired protein, or their ability to
be reisolated from an individual (i.e., for blood cells,
leukaphoresis may be used to retrieve transformed cells
using markers present on the cell surface). Vectors may
be similiarly designed using, for example, viruses which
have a known period of expression of DNAs contained
therein.
The desired cells or vectors may be stored
using techniques, such as freezing, available to those
in the art.
Thus, the present invention also contemplates
a method for administering OB receptor protein to an
individual, wherein the source of said OB receptor
protein is selected from (i) a population of cells
expressing OB receptor protein and (ii) a population of
vectors expressing OB receptor protein. Said OB
receptor protein may be selected from among those
described herein. Said vectors may be virus vectors
capable of infecting human cells. Said cells may be
selected from among tissue or individual cells. Said
individual cells may be selected from among adipocytes,
fibroblasts, bone marrow cells, peripheral blood
progenitor cells, red blood cells, and white blood
cells, including T cells and nerve cells. Said
population of cells or vectors may be co-administered
with a population of cells or vectors which express OB
protein or another desired protein. Said cells or
vectors may be stored for use in an individual. Storage
may be by freezing
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Complexes
In addition to the OB receptor protein as
described herein, one may prepare complexes of OB
receptor protein and OB protein, analog or derivative.
The OB protein may be selected from those
described in PCT publication WO 96/05309, above.
Figure 3 of that publication (Seq. ID No. 4, as cited
therein) depicts the full deduced amino acid sequence
derived for the human OB gene. The amino acids arel
numbered from 1 to 167. A signal sequence cleavage site
is located after amino acid 21 (Ala) so that the mature
protein extends from amino acid Z2 (Val) to amino acid
167 (Cys). For the present disclosure, a different
numbering is used herein, where the amino acid position
1 is the Valine residue which is at the beginning of the
mature protein.
Generally, the OB protein for use will be
capable of complexing to the OB protein receptor
selected. Thus, one may empirically test the binding
capability (to all or part of the extracellular domain
of the 0B receptor as indicated above) to determine
which 0B protein forms may be used. Generally,
modifications generally applicable as indicated above
for OB receptor protein may also be applied here.
As set forth in WO 96 05309, OB protein in its native form,
or fragments (such as enzyme cleavage products) or other
truncated forms, analogs, and derivatives all retain
biological activity. Such forms may be used so long as
the form binds to at least a portion of the
extracellular domain of the present OB receptor
proteins.
An effective amount of an 0B protein, analog
or derivative thereof may be selected from among
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according to the amino acid sequence as presented in PCT
WO 96/05309, Figure 3 numbered so that the first amino
acid of the mature protein is number 1:
(a) the amino acid sequence 1-146,
optionally lacking a glutaminyl residue at position 28,
and further optionally having a methionyl residue at the
N-terminus;
(b) an amino acid sequence of subpart
(a) having a different amino acid substituted in one or
more of the following positions: 4, 8, 32, 33, 35, 48,
50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97,
100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138,
142, and 145;
(c) a truncated OB protein analog
selected from among: (using the numbering of subpart (a)
above ) :
(i) amino acids 98-146
(ii) amino acids 1-32
(iii) amino acids 1-35
(iv) amino acids 40-116
(v) amino acids 1-99 and 112-146
(vi) amino acids 1-99 and 112-146
having one or more of amino acids 100-111
sequentially placed between amino acids 99 and 112;
and,
(vii) the truncated OB analog of
subpart (i) having one or more of amino acids 100,
102, 105, 106, 107, 108, 111, 112, 118, 136, 138,
142, and 145 substituted with another amino acid;
(viii) the truncated analog of subpart
(ii) having one or more of amino acids 4, 8 and 32
substituted with another amino acid;
(ix) the truncated analog of subpart
(iv) having one or more of amino acids 50, 53, 60,
64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102,
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105, 106, 107, 108, 111 and 112 replaced with
another amino acid;
(x) the truncated analog of subpart
(v) having one or more of amino acids 4, 8, 32, 33,
35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77,
78, 89, 97, 112, 118, 136, 138, 142, and 145
replaced with another amino acid;
(xi) the truncated analog of subpart
(vi) having one or more of amino acids 4, 8,32, 33,
35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77,
78, 89, 97, 100, 102, 105, 106, 107, 108, 111,
112, 118, 136, 138, 142, and 145 replaced with
another amino acid;
(xii) the truncated analog of any of
subparts (i)-(xi) having an N-terminal methionyl
residue; and
(d) the OB protein or analog derivative
of any of subparts (a) through (c) comprised of a
chemical moiety connected to the protein moiety;
(e) a derivative of subpart (d) wherein
said chemical moiety is a water soluble polymer moiety;
(f) a derivative of subpart (e) wherein
said water soluble polymer moiety is polyethylene
glycol;
(g) A derivative of subpart (f) wherein
said water soluble polymer moiety is a polyamino acid
moiety;
(h) a derivative of subpart (g) wherein
said water soluble polymer moiety is attached at solely
the N-terminus of said protein moiety;
(i) an OB protein, analog or derivative
of any of subparts (a) through (h) in a pharmaceutically
acceptable carrier.
OB proteins, analogs and related molecules are
also reported in the following publications; however, no
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representation is made with regard to the activity of
any composition reported:
U.S.Patent Nos. 5,521,283; 5,532,336;
5,552,522; 5,552,523; 5,552,524; 5,554,727;
5, 559, 208; 5, 563, 243; 5, 563, 244; 5, 563, 245;
5, 567, 678; 5, 567, 803; 5, 569, 744; 5,569,743
(all assigned to Eli Lilly and.Company);
PCT W096/23517; W096/23515; W096/23514;
W096/24670; W096/23513; W096/23516;
W096/23518; W096/23519; W096/23520;
W096/23815; W096/24670; W096/27385 (all
assigned to Eli Lilly and Company);
PCT W096/22308 (assigned to Zymogenetics);
PCT W096/29405 (assigned to Ligand
Pharmaceuticals, Inc.);
PCT W096/31526 (assigned to Amyin
Pharmaceuticals, Inc.);
PCT W096/34885 (assigned to Smithkline Beecham
PLC) ;
PCT W096/35787 (assigned to Chiron);
EP 0 725 079 (assigned to Eli Lilly and
Company);
EP 0 725 078 (assigned to Eli Lilly and
Company);
EP 0 736 599 (assigned to Takeda);
EP 0 741 187 (assigned to F. Hoffman LaRoche).
To the extent these references provide for
useful OB proteins or analogs or derivatives thereof, or
associated compositions or methods, such compositions
and/or methods may be used in conjunction with the
present OB receptor proteins, such as for co-
admi.nistration (together or separately, in a selected
dosage schedule) or by complexing compositions to the
present OB protein receptors.
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Derivatives and Formulations
The present OB protein receptor and/or OB
protein (herein the term "protein" is used to include
"peptide" and OB protein or receptor analogs, such as
those recited infra, unless otherwise indicated) may
also be derivatized by the attachment of one or more
chemical moieties to the protein moiety. If the present
pharmaceutical compositions contain as the active
ingredient a complex of OB protein receptor and OB
protein, one or both of such proteins may be
derivatized. The chemically modified derivatives may be
further formulated for intraarterial, intraperitoneal,
intramuscular, subcutaneous, intravenous, oral, nasal,
pulmonary, topical or other routes of administration.
Chemical modification of biologically active proteins
has been found to provide additional advantages under
certain circumstances, such as increasing the stability
and circulation time of the therapeutic protein and
decreasing immunogenicity. See U.S. Patent
No. 4,179,337, Davis et al., issued December 18, 1979.
For a review, see Abuchowski et al., la Enzymes as
Drugs. (J.S. Holcerberg and J. Roberts, eds.
pp. 367-383 (1891)). A review article describing
protein modification and fusion proteins is Francis,
Focus on Growth Factors 3-: 4-10 (May 1992) (published by
Mediscript, Mountview Court, Friern Barnet Lane, London
N20, OLD, UK ) .
Preferably, for therapeutic use of the
end-product preparation, the chemical moiety for
derivatization will be pharmaceutically acceptable. A
polymer may be used. One skilled in the art will be
able to select the desired polymer based on such
considerations as whether the polymer/protein conjugate
will be used therapeutically, and if so, the desired
dosage, circulation time, resistance to proteolysis, and
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other considerations. For the present proteins and
peptides, the effectiveness of the derivatization may be
ascertained by administering the derivative, in the
desired form (i.e., by osmotic pump, or by injection or
infusion, or, further formulated for oral, pulmonary or
nasal delivery, for example), and observing biological
effects as described herein.
The chemical moieties suitable for
derivatization may be selected from among various water
soluble polymers. The polymer selected should be water
soluble so that the protein to which it is attached so
that it is miscible in an aqueous environment, such as a
physiological environment. The water soluble polymer
may be selected from the group consisting of, for
example, polyethylene glycol, copolymers of ethylene
glycol/propylene glycol, carboxymethylcellulose,
dextran, polyvinyl alcohol, polyvinyl pyrolidone,
poly-1, 3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids
(either homopolymers or random or non-random copolymers
(see supra regarding fusion molecules), and dextran or
poly(n-vinyl pyrolidone)polyethylene glycol, propylene
glycol homopolymers, polypropylene oxide/ethylene oxide
co-polymers, polyoxyethylated polyols,
polystyrenemaleate and polyvinyl alcohol. Polyethylene
glycol propionaldenhyde may have advantages in
manufacturing due to its stability in water.
Fusion proteins may be prepared by attaching
polyaminoacids to the OB protein receptor or OB protein
(or analog or complex) moiety. For example, the
polyamino acid may be a carrier protein which serves to
increase the circulation half life of the protein. For
the present therapeutic or cosmetic purposes, such
polyamino acid should be those which do not create
neutralizing antigenic response, or other adverse
response. Such polyamino acid may be selected from the
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group consisting of serum album (such as human serum
albumin), an antibody or portion thereof (such as an
antibody constant region, sometimes called "Fc") or
other polyamino acids. As indicated below, the location
of attachment of the polyamino. acid may be at the
N-terminus of the OB protein moiety, or other place, and
also may be connected by a chemical "linker" moiety to
the OB protein.
The polymer may be of any molecular weight,
and may be branched or unbranched. For polyethylene
glycol, the preferred molecular weight is between about
2 kDa and about 100 kDa (the term "about" indicating
that in preparations of polyethylene glycol, some
molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and
manufacturing. Other sizes may be used, depending on
the desired therapeutic profile (e.g., the duration of
sustained release desired, the effects, if any on
biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the
polyethylene glycol to a therapeutic protein or analog).
The number of polymer molecules so attached
may vary, and one skilled in the art will be able to
ascertain the effect on function. One may mono-
derivatize, or may provide for a di-, tri-, tetra- or
some combination of derivatization, with the same or
different chemical moieties (e.g., polymers, such as
different weights of polyethylene glycols). The
proportion of polymer molecules to protein (or peptide)
molecules will vary, as will their concentrations in the
reaction mixture. In general, the optimum ratio (in
terms of efficiency of reaction in that there is no
excess unreacted protein or polymer) will be determined
by factors such as the desired degree of derivatization
(e.g., mono, di-, tri-, etc.), the molecular weight of
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the polymer selected, whether the polymer is branched or
unbranched, and the reaction conditions.
The chemical moieties should be attached to
the protein with consideration of effects on functional
or antigenic domains of the protein. There are a number
of attachment methods available to those skilled in the
,art. E.a., EP 0 401 384
(coupling PEG to G-CSF), see Also Malik et al., Exp.
Hematol. 2,Q: 1028-1035 (1992) (reporting pegylation of
GM-CSF using tresyl chloride). For example,
polyethylene glycol may be covalently bound through
amino acid residues via a reactive group, such as, a
free amino or carboxyl group. Reactive groups are those
to which an activated polyethylene glycol molecule (or
other chemical moiety) may be bound. The amino acid
residues having a free amino group may include lysine
residues and the N-terminal amino acid residue. Those
having a free carboxyl group may include aspartic acid
residues, glutamic acid residues, and the C-terminal
amino acid residue. Sulfhydrl groups may also be used
as a reactive group for attaching the polyethylene
glycol molecule(s) (or other chemical moiety).
Preferred for therapeutic manufacturing purposes is
attachment at an amino group, such as attachment at the
N-terminus or lysine group. Attachment at residues
important for receptor binding should be avoided if
receptor binding is desired.
One may specifically desire N-terminally
chemically modified protein. Using polyethylene glycol
as an illustration of the present compositions, one may
select from a variety of polyethylene glycol molecules
(by molecular weight, branching, etc.), the proportion
of polyethylene glycol molecules to protein molecules in
the reaction mix, the type of pegylation reaction to be
performed, and the method of obtaining the selected
N-terminally pegylated protein. The method of obtaining
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the N-terminally pegylated preparation (i.e.,
separating this moiety from other monopegylated moieties
if necessary) may be by purification of the N-terminally
pegylated material from a population of pegylated
protein molecules. Selective N-terminal chemical
modification may be accomplished by reductive alkylation
which exploits differential reactivity of different
types of primary amino groups (lysine versus the
N-terminal) available for derivatization in a particular
protein. 5= PCT WO 96/11953.
Under the appropriate reaction conditions,
substantially selective derivatization of the protein at
the N-terminus with a carbonyl group containing polymer
is achieved. For example, one may selectively
N-terminally pegylate the protein by performing the
reaction at a pH which allows one to take advantage of
the pKa differences between the e-amino group of the
lysine residues and that of the a-amino group of the
N-terminal residue of the protein. By such selective
derivatization, attachment of a polymer to a protein is
controlled: the conjugation with the polymer takes
place predominantly at the N-terminus of the protein and
no significant modification of other reactiv~e groups,
such as the lysine side chain amino groups, occurs.
Using reductive alkylation, the polymer may be of the
type described above, and should have a single reactive
aldehyde for coupling to the protein. Polyethylene
glycol propionaldehyde, containing a single reactive
aldehyde, may be used.
An N-terminally chemically modified derivative
is preferred (over other forms of chemical modification)
for ease in production of a therapeutic. N-terminal
chemical modification ensures a homogenous product as
characterization of the product is simplified relative
to di-, tri- or other multi-derivatized products. The
use of the above reductive alkylation process for
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preparation of an N-terminally chemically modified
product is preferred for ease in commercial
manufacturing.
In yet another aspect of the present
invention, provided are methods of using pharmaceutical
compositions of the proteins, and derivatives. Such
,pharmaceutical compositions may be for administration by
injection, or for oral, pulmonary, nasal, transdermal or
other forms of administration. In general, comprehended
by the invention are pharmaceutical compositions
comprising effective amounts of protein or derivative
products of the invention together with pharmaceutically
acceptable diluents, preservatives, solubilizers,
emulsifiers, adjuvants and/or carriers. Such
compositions include diluents of various buffer content
(e.g., Tris-HC1, acetate, phosphate), pH and ionic
strength; additives such as detergents and solubilizing
agents (e.g., Tween*80, Polysorbate 80), anti-oxidants
(e.g., ascorbic acid, sodium metabisulfite),
preservatives (e.g., Thimersol, benzyl alcohol) and
bulking substances (e.g., lactose, mannitol);
incorporation of the material into particulate
preparations of polymeric compounds such as polylactic
acid, polyglycolic acid, etc. or into liposomes. Lg,
e.a., PCT W096/29989, Collins et al., "Stable protein:
phospholipid compositions and methods," published
October 3, 1996,
Hylauronic acid may also be used, and this may have the
effect of promoting sustained duration in the
circulation. Such compositions may influence the
physical state, stability, rate of in vivo release, and
rate of in vivo clearance of the present proteins and
derivatives. 5ag, e.g., Remington's Pharmaceutical
Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,
PA 18042) pages 1435-1712.
The compositions may be prepared in
* Trademark
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liquid form, or may be in dried powder, such as
lyophilized form. Implantable sustained release
formulations are also contemplated, as are transdermal
formulations.
Specifically contemplated are oral dosage
forms of the above derivatized proteins. Protein may be
chemically modified so that oral delivery of the
derivative is efficacious. Generally, the chemical
modification contemplated is the attachment of at least
one moiety to the protein (or peptide) molecule itself,
where said moiety permits (a) inhibition of proteolysis;
and (b) uptake into the blood stream from the stomach or
intestine. Also desired is the increase in overall
stability of the protein and increase in circulation
time in the body. ,~L= PCT W095/21629, Habberfield,
"Oral Delivery of Chemically Modified Proteins"
(published August 17, 1995)
and U.S. Patent No. 5,574,018, Habberfield et
al., "Conjugates of Vitamin B12 and Proteins," issued
November 12, 1996.
Also contemplated herein is pulmonary delivery
of the present protein, or derivative thereof. The
protein (derivative) is delivered to the lungs of a
mammal while inhaling and traverses across the lung
epithelial lining to the blood stream. See, PCT
W094/20069, Niven et al., "Pulmonary administration of
granulocyte colony stimulating factor," published
September 15, 1994.
Nasal delivery of the protein (or analog or
derivative) is also contemplated. Nasal delivery allows
the passage of the protein to the blood stream directly
- after administering the therapeutic product to the nose,
without the necessity for deposition of the product in
the lung. Formulations for nasal delivery include those
with absorption enhancing agents, such as dextran or
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cyclodextran. Delivery via transport across other
mucous membranes is also contemplated.
Dosaqes
One skilled in the art will be able to
ascertain effective dosages by administration and
observing the desired therapeutic effect. Preferably,
the formulation of the molecule or complex in a
pharmaceutical composition will be such that between
about .10 g/kg/day and 10 mg/kg/day will yield the
desired therapeutic effect. The effective dosages may
be determined using diagnostic tools over time. For
example, a diagnostic for measuring the amount of OB
protein or OB receptor protein in the blood (or plasma
or serum) may first be used to determine endogenous
levels of OB protein (or receptor). Such diagnostic
tool may be in the form of an antibody assay, such as an
antibody sandwich assay. The amount of endogenous OB
receptor protein (such as soluble receptor) is
quantified initially, and a baseline is determined. The
therapeutic dosages are determined as the quantification
of endogenous and exogenous OB receptor protein (that
is, protein, analog or derivative found within the body,
either self-produced or administered) is continued over
the course of therapy. The dosages may therefore vary
over the course of therapy, with a relatively high
dosage being used initially, until therapeutic benefit
is seen, and lower dosages used to maintain the
therapeutic benefits.
During an ini.tial course of therapy of an
obese person, dosages may be administered whereby weight
loss and concomitant fat tissue decrease increase is
achieved. Once sufficient weight loss is achieved, a
dosage sufficient to prevent re-gaining weight, yet
sufficient to maintain desired weight or fat mass may be
administered. These dosages can be determined
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empirically, as the effects of OB protein are
reversible. E.cr., Campfield et al., Science 269: 546-549
(1995) at 547. Thus, if a dosage resulting in weight
loss is observed when weight loss is not desired, one
would administer a lower dose, yet maintain the desired
weight.
Therapeutic Compositions and Methods
The present OB receptor proteins, alone, or in
combination with an OB protein, and nucleic acids may be
used for methods of treatment, or for methods of
manufacturing medicaments for treatment. Such treatment
includes conditions characterized by excessive
production of OB protein, wherein the present OB
receptors, particularly in soluble form, may be used to
complex to and therefore inactivate such excessive OB
protein. Or, such OB receptor protein, particularly in
soluble form, may act to protect the activity of OB
protein. While not wishing to be bound by theory, one
may postulate that OB protein receptor agonist activity
may be accomplished by a protective effect achieved when
OB protein receptor (particularly soluble receptor) is
complexed to OB protein. Such effect may prolong the
serum half life of OB protein in vivo. Such treatments
may be accomplished by preparing soluble receptor (e.g.,
use of an extracellular domain as described supra) and
administering such composition to an individual in need
thereof or by preparation of a population of cells
containing or expressing such OB receptor, and
transplanting such cells into the individual in need
thereof.
The present OB receptors may also be used for
treatment of those having defective OB receptors. For
example, one may treat an individual having defective OB
receptors by preparation of a population of cells
containing such non-defective OB receptor, and
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transplanting such cells into an individual. Or, an
individual may have an inadequate number of OB
receptors, and cells containing such receptors may be
transplanted in order to increase the number of OB
receptors available to an individual.
The present OB receptor proteins and related
compositions such as OB receptor protein/OB protein
complex, provide for weight loss, fat loss, increase in
lean mass, increase in insulin sensitivity, increase in
overall strength, increase in red blood cells (and
oxygenation in the blood), decrease in bone resportion
or osteoporosis, decreased or maintained serum
cholesterol level, decreased or maintained triglyceride
(LDL or VLDL) levels, prevention or reduction in
arterial plaque formation, treatment of hypertension,
and prevention or reduction of gall stone formation. As
body fat composition may be correlated with certain
types of cancers, the present compositions may be useful
for the prevention or amelioration of certain types of
cancers. The present invention also includes methods
for manufacture of a medicament for use in conjunction
with the cosmetic/therapeutic conditions described
herein, containing at least one of the present
compositions.
The present compositions and methods may be
used in conjunction with other medicaments, such as
those useful for the treatment of diabetes (e.g.,
insulin or analogs thereof, thiazolidinediones or other
antihyperglycemic agents, and possibly amylin or
antagonists there of), cholesterol and blood pressure
lowering medicaments (such as those which reduce blood
lipid levels or other cardiovascular medicaments), and
activity increasing medicaments (e.g., amphetamines).
Appetite suppressants may also be used (such as
serotonin modulators and neuropeptide Y antagonists).
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Such administration may be simultaneous or may be in
seriatim.
In addition, the present methods may be used
in conjunction with surgical procedures, such as
cosmetic surgeries designed to alter the overall
appearance of a body (e.g., liposuction or laser
surgeries designed to reduce body mass, or implant
surgeries designed to increase the appearance of body
mass). The health benefits of cardiac surgeries, such
as bypass surgeries or other surgeries designed to
relieve a deleterious condition caused by blockage of
blood vessels by fatty deposits, such as arterial
plaque, may be increased with concomitant use of the
present compositions and methods. Methods to eliminate
gall stones, such as ultrasonic or laser methods, may
also be used either prior to, during or after a course
of the present therapeutic methods. Furthermore, the
present methods may be used as an adjunct to surgeries
or therapies for broken bones, damaged muscle, or other
therapies which would be improved by an increase in lean
tissue mass.
In yet another aspect, the present invention
provides for methods of manufacture of a medicament for
the treatment of obesity, type II diabetes, excess blood
lipid, or cholesterol levels, increasing sensitivity to
insulin, increasing lean mass, and other conditions as
set forth above. Also provided are solely cosmetic
treatments for individuals wishing to improve appearance
by weight loss, and more specifically, loss of fat
deposits, even in the absence of any therapeutic
benefit.
DiaanoGtic Compositions and Methods
As indicated supra, polypeptide products of
the invention may be "labeled" by association with a
detectable marker substance (e.g., radiolabeled with
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125I, fluorescent, chemiluminescent, enzyme) to provide
reagents useful in detection and quantification of OB
receptor (or complexes) in solid tissue and fluid
samples such as blood or urine. Nucleic acid products
of the invention may also be labeled with detectable
markers (such as radiolabels and non-isotopic labels
such as biotin) and employed in hybridization processes
to locate the human OB receptor gene position and/or the
position of any related gene family in a chromosomal
map. Nucleic acid sequences which selectively bind the
human OB receptor gene are useful for this purpose.
They may also be used for identifying human OB receptor
gene disorders at the DNA level and used as gene markers
for identifying neighboring genes and their disorders.
Such nucleic acid sequences may be sued for detection or
measurement of OB receptor mRNA level from a biological
sample. Contemplated herein are kits containing such
labelled materials.
The protein and/or nucleic acids provided
herein may also be embodied as part of a kit or article
of manufacture. Contemplated is an article of
manufacture comprising a packaging material and one or
more preparations of the presently provided
compositions. Such packaging material will comprise a
label indicating that the protein or nucleic acid
preparation is useful for detecting and/or quantifying
the amount of OB receptor in a biological sample, or OB
receptor defects in a biological sample. As such, the
kit may optionally include materials to carry out such
testing, such as reagents useful for performing DNA or
RNA hybridization analysis, or PCR analysis on blood,
urine, or tissue samples.
A further embodiment of the invention is
selective binding molecules, such as monoclonal
antibodies selectively binding OB receptor. The
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hybridoma technique described originally by Kohler and
Milstein Eur. J. Immunol. ,~, 511-519 (1976) has been
widely applied to produce hybrid cell lines that secrete
high levels of monoclonal antibodies against many
specific antigens. Recombinant antibodies, (see Huse et
al., Science 246: 1275 (1989)) may also be prepared.
Such recombinant antibodies may be further modified,
such as by modification of complementarity determining
regions to increase or alter affinity, or "humanizing"
such antibodies. Such antibodies may be incorporated
into a kit for diagnostic purposes, for example. A
diagnostic kit may be employed to determine the location
and/or amount or OB receptor of an individual.
Diagnostic kits may also be used to determine if an
individual has receptors which bind OB protein, or those
which, to varying degrees, have reduced binding capacity
or ability. As stated infra, such antibodies may be
prepared using immunogenic portions of an OB receptor
protein. Such selective binding molecules may
themselves be alternatives to OB protein, and may be
formulated for pharmaceutical composition.
Such proteins and/or nucleic acids may be used
for tissue distribution assays (for example, as provided
in the working example below) or for other assays to
determine the location of OB receptor.
The present OB receptor protein family may be
used in methods to obtain OB protein analogs, mimetics
or small molecules. One would simply prepare a desired
OB receptor protein, particularly one with capability of
binding to native OB protein, and assay the test
molecule, which may be labelled with a detectable label
substance, for ability to bind to such receptor. Other
parameters, such as affinity, and location of binding,
may also be ascertained by methods available to those
skilled in the art. For example, one could use portions
of the present OB receptors, particularly portions in
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the extracellular domain which are necessary for ligand
binding, to determine the location of such binding. One
could prepare OB receptors which have various truncations or deletions of
regions of the extracellular
domain which could be used to determine the location of
test molecule binding. One could use an OB receptor
known to be defective in native OB binding, such as
potentially one from an individual having such defective
receptors, and use this as the basis for ascertaining OB
protein which would be effective to result in desired
biological activity (i.e., weight loss, reduction in
blood dyslipidemias or lowering of cholesterol levels,
reduction in incidence or severity of diabetes). Other
uses include solely cosmetic uses for alteration of body
appearance, particularly the removal of fat.
The present OB receptor protein or nucleic
acids may also be useful to identify substances which
"up-regulate" OB protein or receptor. For instance, the
temporal expression of OB receptor In vivo may be useful
to determine if an administered substance causes an
increase or decrease in OB receptor. One may conclude
that an increase in OB receptor expression results in
modultion of weight or lipid metabolism.
The divergence in the C-terminus may represent
OB receptors with different signal transduction
abilities. Therefore the different receptor family
members may be used for different assays, depending on
the type of signal transduction observed. It is thought
that at least a portion of the intracellular domain is
necessary for signal transduction (see supra).
The following examples are offered to more
fully illustrate the invention, but are not to be
construed as limiting the scope thereof.
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FXAMmr,E 1: IDENTIFICATION OF HUMAN OB RECEPTOR PROTEIN
Human OB receptor protein DNA was identified
in a human liver cDNA library in two steps. The first
step used two primers in polymerase chain reaction (PCR)
to amplify a selected 300 base pair region from the
human liver cDNA library. The second step used the PCR
fragment as a probe to screen the human liver cDNA
library. Thirteen clones were obtained, but these were
incomplete at the 5' end. A procedure was performed to
complete the 5' end to make complete clones. Twelve
clones were sequenced. These twelve clones were
identified as either "A", "B" or "C" as denoted by the
C-terminus of the predicted amino acid sequence.
Polymerase Chain Reaction.
The original PCR primer was based on the 5'
end and the 3' end of a 416 base pair sequence having
GenBank Database Accession No. T73849. This sequence
was selected on the basis of a known motif present in
cytokine receptors, "WSXWS".
The 5' primer had the sequence 73-96 of the
416 bp sequence. The 3' primer had the sequence 337-360
of the 416 bp sequence.
These primers were used to probe a human cDNA
liver library (Stratagene). Standard methods were used.
This resulted in a PCR fragment having the
sequence 73-360 of the 416 bp fragment.
Hybridization.
The 300 bp PCR fragment was used to probe a
human liver cDNA library (Stratagene) using standard
methods. This second hybridization resulted in 13
positive clones. These were partial clones, incomplete
at the 5' end.
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Completion of the 5' end.
Rapid .Amplification of cDNA End ("RACE", kit,
GIBCO/BRL) was used to obtain the full length clones.
,5acruenci ncr results.
Sequencing revealed the three types of OB
receptor DNAs. Of the thirteen clones, 4 clones were
the "A" type (Seq. ID Nos. 1 and 2); 1 clone was the "B"
type (Seq. ID Nos. 3 and 4) and 4 clones were of the "C"
type (Seq. ID Nos. 5 and 6).
As can be seen from the Sequence
Identifications (below), OB receptor A is 896 amino
acids long, "B" is 904 amino acids long, and "C" is 958
amino acids long. These different OB receptors are
identical at amino acid positions 1-891, and diverge
almost completely beginning at position 892. The leader
sequence is postulated to be, by hydrophobicity
analysis, amino acids 1-21(M-A), 1-22(M-F) or 1-28(M-I),
with the mature protein beginning at positions 22(F),
23(N) or 29(T). Based on hydrophobicity analysis, the
leader sequence is most likely to be at positions 1-21(M
through A). Chinese Hamster Ovary Cell ("CHO") cell
production of the secreted form of OB receptor protein
also produced a protein having amino acid number 22 as
the first amino acid of the mature protein. The
transmembrane region is likely to begin at either
position 840 (A) or 842(L) through position 862(I),
863(S) or 864(H). For OB receptor type "A", the last
amino acid is located at position 896 and is a lysine
(L). For OB receptor type "B", the last amino acid is
located at position 904 and is a glutamine (Q). For OB
receptor type "C", the last amino acid is located at
position 958 and is glutamic acid (E).
For OB receptor protein type "C", the C-
terminal region possesses high homology to a known human
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transposable element. From nucleotide 2737 through 2947
of the present human OB receptor protein type "C", there
is a 98.1% homology with a 211 base section of a human
retrotransposable element described in Ono et al., Nucl.
Acids Res. 15: 8725-8737 (1987) (bases 520 through 731,
SINE-R11, GENBANK accession no. x07417).
FXAEPT,E 2: TISSUE DISTRIBUTION
Tissue distribution was ascertained using two
methods. The first method involved using the entire
type "A" OB receptor. The second method involved using
probes which are specific to the C-terminal region of
the protein. Since these C terminal regions are diver-
gent, the second method detected the tissue distribution
of the different members of the OB receptor family.
The first method used a Northern Blot kit
(Clontech), using the entire type A OB receptor DNA as a
probe. The second method used PCR with primers specific
to the nucleic acids encoding the divergent C terminus
of the three types. Standard methods were used.
Table 2 shows the results for the Northern
Blot and the PCR methods. The "+ indicates the
investigator's subjective determination of the strength
of signal. For the Northern Blot analysis, a triple
11 +++" indicates that a result (a dark "band" on the X-
ray film) was seen upon overnight exposure of the film.
A double "++" indicates that bands were seen at two
weeks of exposure. A single "+" indicates that the
bands were seen after three weeks of exposure. In
addition, using this method, two molecular weights were
observed, one at 4 Kb and one at 6.2 Kb. Although
distribution was ubiquitous, the strongest signals were
seen for ovary, heart and liver. For the PCR analysis,
OB receptor "A" was seen in all tissue types tested
(prostate, ovary, small intestine, heart, lung, liver
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and skeletal muscle), type "B" was seen only in lung and
liver, and type "C" was seen in ovary, heart, lung and
liver.
Table 2
Tisstae Distribution of thc~ Novel OB R Cep _nr
Northern Blot PCR
4 Kb 6.2 Kb A B C
Spleen - +
Thymus - +
Prostate - + + - -
Testis - +
Ovary - +++ + - +
Small - ++ + - -
Intestine
Colon - -
Peripheral - -
blood
Leukoc te
Heart - +++ + - +
Brain - -
Placenta - +
Lung + ++ + + +
Liver +++ +++ + + +
Skeletal - ++ + - -
Muscle
Kidney - ++
Pancreas - +
EXAMPLE 3: IDENTIFICATION OF HUMAN OB RECEPTOR GENOMIC
DNA AND CHROMOSOME LOCALIZATION; IDENTIFICATION OF HUMAN
OB RECEPTOR "D"
The full length human OB receptor genomic DNA
was also prepared. OB receptor "A" cDNA, in its
entirety, was used as a probe against a human genomic
DNA library, using materials and methods from a
commercially available kit (Genome Systems, using a
human genomic library in a Pl vector). A single
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positive clone was detected. There are introns located
at (with respect to OB receptor "A" DNA) base pair
number: 559, 1059, 1350, 1667, 1817, 1937, 2060, 2277,
2460, 2662, and 2738.
The human OB receptor gene was localized to
human chromosome 1P31 by FISH analysis (Genome Systems).
Human chromosome 1 is thought to correspond to mouse
chromosome 4C7, which is presumed to be the location of
the db locus.
A further chromosomal sequence was isolated.
This chromosomal DNA sequence was isolated from a human
genomic library as described above. This chromosomal
sequence encodes what is here denominated human OB
receptor "D", and the encoded amino acid sequence is set
forth in SEQ. ID No. 7. A cDNA encoding this amino acid
sequence is set forth in SEQ. ID No. 8. The chromosomal
DNA intron/exon junction map is set forth as SEQ. ID
No. 9.
As with forms "A", "B", and "C", for the
present form "D" OB receptor protein, the first amino
acid of the mature protein is likely (using
hydrophobicity analysis) to begin at position 22 (F), 23
(N) or 29 (T). The last amino acid of the protein is at
position 1165 and is a valine residue. As with the
other forms, the extracellular domain extends from
position 22 (F), 23 (N) or 29 (T) to position 839 (D) or
841 (G). The transmembrane domain appears to begin at
position 840 (A) or 842 (L). The end of the
transmembrane domain appears to be located at position
862 (I), 863 (S) or 864 (H) . The C-terminal region,
beyond the transmembrane region, is likely to be
involved in signal transduction, and is located at
position 863 (S), 864 (H) or 865 (Q) through position
1165 (V).
The present OB receptor form "D" is identical
to that published by Tartaglia et al, Cell $a: 1263-1271
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(December 29, 1995) with the exception of a single amino
acid change at amino acid position 976 (nucleotide codon
begining at position 3022). The present type "D" amino acid at position 976 is
aspartic acid, and the published
amino acid corresponding to the same position is
alanine. This is a non-conservative substitution, see
infra, and since the location of the substitution is
within a region thought important for signal trans-
duction, this change could affect the function of the
molecule.
EXAMPLE 4: PREPARATION OF SOLUBLE OB RECEPTOR
Three forms of soluble human OB receptor have
been prepared:
1. Leader + Extracellular Domain (Seq.
ID Nos. 10 and 11): A recombinant form of the soluble
human OB receptor was prepared. This form encompasses,
in the immature protein, the leader sequence and the
extracellular domain (amino acids 1-839). The mature
protein would have the leader sequence deleted, and the
first amino acid of the mature recombinant soluble human
OB receptor would be 22 (F), 23 (N) or 29 (T). This
protein was expressed as described below.
2. Leader + Extracellular Domain + C-
terminal FLAG (Seq. ID No. 12): A second form of the
recombinant soluble human OB receptor was also prepared.
This form had a"FLAG tag located at the "C" terminus
of the protein. The "FLAG" peptide is a useful research
tool as it allows one to follow the protein using an
antibody which recognizes the "FLAG" peptide. Such
reagents are commercially available (IBI, New Haven,
CT). This protein was expressed as described below.
3. Native Splice Variant (Seq. ID Nos.
13 and 14): This form is believed to the the
recombinant form of a naturally occurring secreted,
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soluble human OB receptor. This form has most of the
amino acids found in the extracellular domain (amino
acids 22-798), and a unique 6 amino acid sequence at the
carboxyl terminus. Beginning at amino acid position 799
of Seq. ID No. 13, the amino acid sequence of this
native splice variant human OB receptor protein is "G K
F T I L."
EXA.NlPLE 5: PREPARATION OF EXPRESSION VECTORS
Recombinant human OB receptor expression
vectors have been prepared for expression in mammalian
cells. As indicated above, expression may also be in
non-mammalian cells, such as bacterial cells. The type
"A" cDNA (Seq. ID No. 2) was placed into a commercially
available mammalian vector (pCEP4, Invitrogen) for
expression in mammalian cells, including the commercial-
ly available human embryonic kidney cell line, "293".
Recombinant human OB receptor expression
vectors have been prepared for expression of recombinant
soluble OB receptor, consisting of the leader sequence
and the extracellular domain (Seq. ID Nos. 10 and 11),
using the same system as above (the commercialy
available mammalian vector pCEP4, and "293" cells).
This recombinant soluble human OB receptor was also
expressed in CHO cells in a similar way.
The "FLAG-tagged" form (Seq. ID No. 12) of the
recombinant soluble human OB receptor, and the "D" form
(Seq. ID No. 7) were also expressed in "293" cells in a
similar fashion as above.
Detection of desired protein was accomplished
using BIACORE (Pharmacia) analysis. This analysis is
analogous to that described in Bartley et al., Nature
368: 558-560 (1994).
Essentially, the BIACORE machine measures
affinity interactions between two proteins. In this
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case, the OB protein was immobilized on the machine, and
conditioned media from cell lines expressing the OB
receptor was added to the machine. Any receptor protein present in the
conditioned media bound to the OB protein
surface. The BIACORE machine gave a read-out indicating
that receptor protein was being expressed. For
recombinant soluble receptor (Seq. ID No. 10) expression
in "293" cells, the read-out was 191.0 relative to a
baseline readout of 0. For recombinant soluble receptor
(SEq. ID No. 10) expression in CHO cells, the read-out
was 150.9 relative to a baseline readout of 0. For
recombinant soluble receptor with a C-terminal FLAG-tag
(Seq. ID. No. 12), the read-out was 172.0 relative to a
baseline of 0.
For expression in bacterial cells, one would
typically eliminate that portion encoding the leader
sequence (e.g., potentially amino acids 1-21, 1-22 or 1-
28). One may add an additional methionyl at the
N-terminus for bacterial expression. Additionally, one
may substitute the native leader sequence with a
different leader sequence, or other sequence for
cleavage for ease of expression.
EXAMPLE 6: DEMONSTRATION OF SIGNAL TRANSDUCTION
This example demonstrates that the "D" form is
active to produce a signal within a cell, whereas in the
same cell type, the "A" form does not. The signal
transduction assay was performed by the use of "293"
cells transiently expressing either the "A" or the "D"
form (see above for preparation of the "293" expression
clones). Phosphorylation of molecules predicted to be
involved in signal transduction within the cell was
examined upon OB protein binding to the OB receptor
protein tested. The results demonstrate that upon
binding of OB protein to the extracellular domain, the
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"D" form of the present OB protein receptor transduces a
signal sufficient to initiate phosphorylation of
signalling molecules.
Methods
1. OB receptor molecules. As indicated
above, the "A" form (Seq. ID No. 1) and the "D" form
(Seq. ID. No. 7) were studied.
2. Expression system. The pCEP 4 system (as
described above) having inserted DNA encoding the "A"
form (Seq. ID No. 2) or the "D" form (Seq. ID No. 8) was
used to transfect "293" cells. These cells did not
allow for the pCEP4 vector to integrate into the genome,
so such expression was transient. Non-recombinant
(mock-transfected) cells were also prepared as controls.
3. Detection of phosphorylation. Mock
transfected cells and cells expressing the "A" form or
the "D" form were analyzed. Prior to treatment the
cells were serum-starved by incubation in media with
0.5% serum for 16 hours prior to the treatments. The
cells were treated with the OB protein (10 mg/ml) for 15
minutes at 370C, after which the cells were lysed in
modified NP40 buffer (50 mM Tris, pH 8.0, 150 mM sodium
chloride, 1% NP40, 10 mg/ml aprotinin, 5mM EDTA, 200 mM
sodium orthovanadate). Phosphotyrosine containing
proteins were immunoprecipitated (Anti-phosphotyrosine
antibody 4G10, UBI, Lake Placid, NY), and separated by
SDS polyacrylamide gel electrophoresis. After
electrophoresis and electroblotting to membranes the
immunoprecipitates were probed with antibodies to
various signal transduction molecules. Antibodies to
STATs, JAKs and ERKs were purchased from Santa Cruz
Biotechnology Inc. Immune complexes were detected by
horseradish peroxidase conjugated secondary reagents
using chemiluminescence as described by the manufacturer
(ECL, Amersham). As a positive control, 32D cells were
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treated with IL-3, which is known to activate by
tyrosine phosphorylation most of the molecules being
analyzed.
4. Results. Results are presented in Table
3, below. As can be seen, only the "D" form was able to
respond to either mouse or human OB protein as detected
by phosphorylation of JAK and STAT molecules. A +"
designation indicates signal was detected, a""
designation means that no signal was observed.
TABLE 3
Signal 293 293/D 293/D 293/A 293/A 32D
/AB* Alone hrOB* mrOB** hrOB# mrOB## IL-3
STAT1 - +
STAT3 - + + - - +
STAT5 - + + +
JAK1 - + + - - +
JAK2 - + + - - +
JAK3 - - - -
TYK2 - + + -
ERKs - - - - - +
1 2
$ Antibody detection target
* 293 cells expressing receptor form "D", treated with
recombinant human OB
** 293 cells expressing receptor form "D" treated with
recombinant murine OB
# 293 cells expressing receptor form "A" treated with
recombinant human OB
## 293 cells expressing receptor form "A" treated with
recombinant murine OB
The "D" form is capable of initiating
signalling through the JAK/STAT pathways in 293 cells,
whereas the "A" form cannot.
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EXAtvrPLE 7: USE OF SOLUBLE OB RECEPTOR AS A THERAPEUTIC
This example demonstrates that soluble OB
receptor protein acts to protect the activity of OB
protein. Below, soluble OB receptor and/or OB protein
was delivered to a mammal via "gene transplant" -- that
is, via bone marrow cells engineered to express the
desired DNAs. When soluble OB receptor combined with OB
protein was delivered, the animals lost more weight than
delivery of OB protein alone. This demonstrates the
protective activity of OB receptor protein.
While not wishing to be bound by theory, one
explanation of the mode of action is that soluble OB
receptor protein acts to protect the OB protein in serum
from agents or conditions which could diminish its
activity. The protective action appears to increase
circulating half-life of the protein. As such, the
present example demonstrates that OB receptor either
alone, or administered as a complex with OB protein (or
analog or derivative thereof) could act as a therapeutic
agent.
Materials and metrods:
1. Preparation of recombinant ob retroviral
vector Packagina Cells.
Use of murine ob cDNA. Full length wild-type
murine ob cDNA was amplified by the PCR using synthetic
oligonucleotides designed from the published sequence
Zhang et al., Nature 372: 425-432 (1994).Linkers (An Eco
RI linker and a Bal II linker) were used to facilitate
subcloning.
rTsQ of soluble recombinant human OB receptor
cDNA. Methods similar to those above were used. A
construct containing the recombinant human soluble
receptor of Seq. ID No. 10 was used, and modified with
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linkers to facilitate cloning (i.e., the addition of a
Bal II restriction endonuclease recognition site).
Placement of desired cDNA into vector. PCR
products were digested with EcoRI and BglII and cloned
into similarly-digested parental vector (pMSCV2.1) under
the transcriptional control of the viral LTR promoter.
The parental MSCV vector (supplied by R. Hawley,
University of Toronto, Canada) was derived from MESV
(murine embryonic stem cell virus) and contains a
neomycin phosphotransferase resistance (neor) gene
driven by an internal mouse phosphoglycerate kinase
(PGK) promoter, as described= Hawley, et al, J. Exp.
Med. 176: 1149 -1163 (1992). The parental plasmid
pMSCV2.1 and pMSCV-OB were independently electroporated
into the GP+E-86 packaging cell line (supplied by Dr. A.
Bank, Columbia University, NY) Markowitz et al., J.
Virol. fa:1120-1124 (1988). Transient supernatants were
harvested from electroporated populations and used to
infect tunicamycin treated parental GP+E-86 cells.
Tunicamycin treatment relieves the block to
superinfection of the parental packaging cells. G418
(0.78 mg/mL, 67% active, GIBCO Laboratories, Life
Technologies, Inc., Grand Island, NY) resistant clones
were selected from each infected population and titered
by infection of NIH3T3 cells. Clones with the highest
G418 resistant titer were expanded and frozen as
aliquots. Each bone marrow infection and
transplantation experiment used aliquots from the same
passage of frozen viral packaging cells. Both the
parental and ob packaging cell lines were tested for the
presence of, and found to be free from, replication
competent virus using a sensitive marker rescue assay.
Moore, et al., (1993) im: Gene Targeting: A Practical
Approach, Joyner, Ed. (Oxford University Press, New
York, NY).
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2. prnrinc+- i on of Retroviral Supernatants.
Recombinant virus-producing packaging cell lines were
grown in 175cm2 tissue culture flasks in Iscove's
Modified Dulbecco's Medium (IMDM) (GIBCO), 10% (v/v)
FBS, at 37 C. Sub-confluent (approximately 60%)
monolayers of cells were fed with fresh medium 24h prior
to harvest of virus-containing supernatants. Viral
supernatants were removed from packaging cell lines by
aspiration, sterile filtered (0.45mM) and added directly
to bone marrow cultures. Fresh aliquots of frozen
packaging cell lines were thawed for use in each
experiment.
3. Bone Marrow Infection and Transplantation.
Eight to 12-week old female C57BL/6J (+/+) or (ob/ob)
mice were used as bone marrow donors and recipients.
All mice were purchased from The Jackson Laboratory (Bar
Harbor, ME) and housed under specific pathogen-free
conditions in a vivarium in accordance with governmental
regulations and institutional guidelines.
Bone marrow cells were harvested from femurs
and tibias of donor mice 4 days post 5-fluorouracil (5-
FU, Sigma Chemical Co., St. Louis, MO) treatment (150
mg/kg i.v.). Bone marrow cells (6 X 105/mL) were
incubated in 150mm tissue culture dishes (30mL/dish)
containing fresh viral supernatant (as described above),
15% FBS, 6 mg/mL polybrene (Sigma), 0.1% bovine serum
albumin (BSA, Fraction V, Sigma), 2.5 ng/mL recombinant
mouse IL-3 (rmIL-3), 100 ng/mL each of recombinant human
IL-6 (rhIL-6), recombinant human IL-11 (rhIL-11), and
recombinant rat SCF (rrSCF). All growth factors were
produced by Amgen, Inc. (Thousand Oaks, CA). Culture
media were replaced daily for 3 days with fresh virus-
containing supernatant and growth factors.
At the end of the infection period, total non-
adherent and adherent cells were washed and resuspended
in 1% BSA-saline and transplanted into g-irradiated (12
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Gy, Cs137) mice. Each animal was transplanted with 2.5
X 106 syngeneic cells. There were approximately 10
animals per cohort. ,
4. Analysis of QB protein expression in
trans d..11s an ansplanted animals. For
transfected bone marrow cells, Western analysis was
performed. Vector packaging cell supernatant was
resolved by SDS-PAGE (16% acrylamide), then transferred
to Hybond-ECL (Amersham, Arlington Heights, IL). The
filter was incubated with affinity-purified rabbit a-
mouse OB protein polyclonal antibody (1mg/mL) in T-TBS
buffer (20mM Tris-chloride, pH7.6, 137mM NaCl, 0.1%
Tween20) at room temperature for 45 min. Horseradish
peroxidase (HRP)-conjugated donkey a-rabbit IgG
(Amersham) was diluted in T-TBS (1:2500) and incubated
with the filter at room temperature for 45 min.
Enhanced chemiluminescence (ECL, Amersham) detection was
performed as recommended by the manufacturer.
For transplanted animals, serum was analyzed.
Animals were bled retroorbitally, under isofluorane
anesthesia. Serum from transplanted ob/ob animals was
resolved by SDS-PAGE (4-20% acrylamide) under non-
reducing and reducing conditions, then transferred to
Trans-Blot (Bio-Rad Laboratories, Hercules, CA)
membranes. The membranes were incubated for 2 hours at
room temperature with HRP-conjugated rabbit a-mouse OB
protein antibody (0.125mg/mL) in T-TBS buffer containing
5% fetal bovine serum and 1% bovine serum albumin.
Bound OB protein was detected by ECL (Amersham),
performed as recommended by the manufacturer.
For quantitation of soluble OB protein levels,
serum from transplanted animals was subjected to ELISA
analysis. Briefly, affinity-purified rabbit a-OB
protein polyclonal antibody was coated onto 96-well
plates. Standards (purified recombinant OB protein
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monomer, Pelleymounter et al., Science 269: 540-543
(1995) and experimental samples were added, and the
plates were incubated at room temperature. The plates
were washed twice and affinity-purified rabbit a-OB
protein antibody conjugated to horseradish peroxidase
was added. Following incubation at room temperature,
the plates were washed four times with TNE-Tween20.
TMB/peroxide substrate was added and the color reaction
was read at 450nm in a Molecular Devices plate reader.
OB protein concentrations in sera were estimated by
comparison to a standard curve prepared from internal
standards. OB protein levels were reliably measured in
samples containing >160 pg/mL.
5. Body Weight and Food Intake. Mice were
offered pelletized rodent chow (PMI Feeds, Inc., St.
Louis, MO) ad libitum. The body weight of individual
animals was measured daily for the first two months of
analysis, and weekly thereafter. Food consumption was
measured daily on selected groups of individually-housed
animals.
Results
Results are presented in Tables 4 and 5 below.
Administration of OB protein receptor increased the
effectiveness of OB protein. This may have been
accomplished via an increased circulation time of OB
protein in the presence of OB protein receptor.
As can be seen in the Table, animals
administered a combination of OB protein and OB protein
receptor (via genetic therapy) had a greater_weight loss
after 28 days than either composition alone. The Table
presents the results of two experiments (" / "). As
can be seen, use of the OB protein alone at day 40
resulted in animals with 87.5% and 72.2% of the starting
weight. Using OB receptor in combination with OB
protein, however, resulted in animals with 68% and
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53.6% of the starting weight. Use of the receptor alone
appeared to have little effect, if any.
TABLE 4
Treatment Weight(g) % starting % starting
decrease at weight weight
day 28 (ave) (ave) (ave)
day 28 day 40
OB alone* 6.3/12.7 87.9/75.3 87.5/72.2
Receptor** [1.4]/[0.3] 103/100.6 104.2/101.7
alone
OB + 12.6/16.8 76.3/67.5 68/53.6
Rece tor***
* 50% bone marrow cells transfected with OB protein
cDNA as described above, and 50% bone marrow cells
without genetic alteration
** 50% bone marrow cells transfected with OB receptor
protein cDNA as described above, and 50% bone marrow
cells without genetic alteration
*** 50% bone marrow cells transfected with OB protein
cDNA as described above, and 50% bone marrow cells
transfected with OB receptor protein cDNA as described
above.
Table 5, below, contains results of the OB
levels found in the serum from animals administered OB
protein alone, or administered OB protein in combination
with OB protein receptor (via the "gene therapy" method
of this example). The data reflect nanograms of OB
protein per milliliter of serum, plus or minus the
standard error of the mean.
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TABLE 5
Treatment Experiment #1* Experiment #2tt
OB alone* 2.93 +J- 0.77 9.74 +/- 1.02
Receptor** 0.08 +/- 0.05 0.12 +/- 0.07
alone
OB + 12.11 +/- 1.90 15.18 +/- 2.52
Rece tor***
* 50% bone marrow cells transfected with OB protein
cDNA as described above, and 50% bone marrow cells
without genetic alteration
** 50% bone marrow cells transfected with OB receptor
protein cDNA as described above, and 50% bone marrow
cells without genetic alteration
*** 50% bone marrow cells transfected with OB protein
cDNA as described above, and 50% bone marrow cells
transfected with OB receptor protein cDNA as described
above.
# Experiment #1 was conducted as described above,
with OB protein serum levels measured after 38 days.
$t- Experiment #2 was also conducted as described
above, with OB protein serum levels measured after 24
days.
The data demonstrate the protective effects of
OB receptor. As can be seen, in the presence of OB
receptor, OB protein has a higher accumulation in the
serum. The degree of accumulation is observed to
increase inversely with the levels of OB protein in the
serum. In Experiment #1 (with a base OB protein level
of about 2.93 ng/ml), the OB protein serum level
increased about 400% with the addition of receptor,
where in Experiment #2 (with a base of about 9.74), the
OB protein serum level increased by about 25%.
OB receptor administered either alone or in
association with OB protein (or analogs or derivatives
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thereof) may serve to increase the circulation time of
OB protein, and therefore enhance the therapeutic
efficacy of either exogenous or endogenous OB protein.
EXAMPLE 8: PREPARATION OF SELECTIVE BINDING MOLECULES Animals were immunized
for the preparation of
polyclonal antibodies using the following peptides (with
respect to the numbering of the amino acids for OB
receptor A, Seq. ID No. 1): 54-64; 91-100; 310-325;
397-406; 482-496; 874-885; and, with respect to amino
acids of OB receptor "C" (Seq. ID No. 5), 910-929. Some
of the polyclonal antibodies prepared (in rabbits) were
tested for ability to bind to recombinant human OB
receptor protein. The polyclonal antibody prepared
against amino acids 54-64 was found to have the highest
affinity for recombinant human OB receptor protein. The
polyclonal antibody prepared against amino acids 397-406
was also found to bind to recombinant human OB receptor
protein. The polyclonal antibody prepared against amino
acids 91-100 was found to slightly bind to recombinant
human OB receptor protein. The polyclonal antibody
prepared against amino acids 874-885 was found not to
bind to recombinant human OB receptor protein.
An additional study was performed which
demonstrates the expression and purification of the
extracellular domain of the OB receptor protein in CHO
cells, and antibodies which recognize this OB protein
receptor extracellular domain.
The extracellular domain of the human OB
receptor protein was expressed as a secreted, soluble
protein in CHO cells as previously described s,lvra.
Individual cell lines were isolated and grown in
increasing amounts of methotrexate to increase
selection/expression of the recombinant receptor protein
(100, 200 or 500 micrograms methotrexate per ml of
media). Conditioned media from the CHO cell lines was
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collected, and the proteins in the conditioned media
were fractionated by SDS-PAGE. The OB receptor
extracellular domain migrated as a broad band with an
apparent size range of about 140 kDa to about 200 kDa.
The OB receptor protein extracellular domain was
detected by Western Blot analysis using polyclonal
antibodies prepared against a portion of the extracel-
lular domain of the OB receptor protein. The unfolded,
bacterially expressed protein was used as an antigen to
generate antisera in rabbits. The identified OB receptor
extracellular domain was purified by affinity
chromatography. The purified protein was sequenced at
the amino terminus to confirm that it was the OB
receptor and also to determine the start of the mature
protein (after signal peptide cleavage) as expresed in
CHO cells. It was found that amino acid no. 22
(according to the amino acid sequence numbering of Seq.
ID No. 1, infra), was the first amino acid of the mature
protein as expressed in CHO cells.
Other immunogenic peptides may be used.
Polyclonal, monospecific polyclonal, monoclonal,
antibody fragments, and recombinant antibodies may be
prepared using methods available to those skilled in the
art.
One may further use recombinant techniques or
peptide synthesis methods to alter the character of such
selective binding molecules. This may be accomplished
by preparing recombinant antibodies having altered
complementarity determining regions (sometimes referred
to in the art as "CDR's") to, for example "humanize" the
antibodies by using human Fc (constant) regions. Other
types of recombinant antibodies, for example, those
having CDR's altered to enhance affinity or selectivity
to one or more members of the OB receptor family, may be
prepared and used using methods available to those
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skilled in the art. See Winter et al., Nature 349: 293-
299 (1991).
The present OB receptor protein may be used as
an assay to screen for desired selective binding
molecules. Such assay may be based on binding
capability, or biological activity, or, other means of
detecting signal transduction. For example, if one were
to prepare a series of modified antibodies, one could
test them for affinity (i.e, binding strength) against
the target OB receptor.
The selective binding molecules may be useful
for diagnostic purposes, such as tissue distribution
analysis, or to diagnose the relative affinity of an
individual's OB receptors for such selective binding
molecule to determine the functionality of an
individual's OB receptor during a course of therapy.
Selective binding molecules may be alternative
therapeutic or cosmetic products to OB protein.
EXAMPLE 9: GENE THERAPY
One may deliver the present OB receptor
protein via gene therapy, as described infra.
One may envision, using materials and methods
available to those skilled in the art and provided
herein, using T-cells as an agent carrying DNA
expressing OB receptor for gene therapy. An individual
would have T-cells selected using CD34+ selection and a
magnetic microparticles selection device. Such cells
would be transfected with the desired DNA, or the
regulation of the desired coding region may be altered
using homologous recombination or other ,jM situ
techniques. The transduced cells could be selected
empirically, using means to detect the desired protein,
or a marker may be included which permits indirect
detection (i.e., a selectable marker as is known in the
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art). Optionally, such cells could be expanded, for
example, using one or more growth factors such as SCF or
an interleukin, and such cells could be stored for
future use. In such a way, the procedure would only
have to be accomplished once or infrequently in an
individual's lifetime, for later transfer into the
individual. The cells would be re-planted into the
individual, and the individual would be monitored for
desired therapeutic effect, such as weight
loss/maintenance of weight, diabetes recurrence, blood
lipid levels, or other conditions.
T> >ust rafi ive Nucleic Acid and Amino Acid Secjuences
The below amino acid and DNA sequences are
those to which reference has been made. An asterick("*")
indicates the position of a stop codon.
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Human OB Receptor "A" Amino Acid S quence (Seq. ID No. 1(Amino
Acid. sinqle letter abbreviation):
1 MICQKFCVVL LHWEFIYVIT AFNLSYPITP WRFKLSCMPP NSTYDYFLLP
51 AGLSKNTSNS NGHYETAVEP KFNSSGTHFS NLSKTTFHCC FRSEQDRNCS
101 LCADNIEGKT FVSTVNSLVF QQIDANWNIQ CWLKGDLKLF ICYVESLFKN
151 LFRNYNYKVH LLYVLPEVLE DSPLVPQKGS FQMVHCNCSV HECCECLVPV
201 PTAKLNDTLL MCLKITSGGV IFQSPLMSVQ PINMVKPDPP LGLHMEITDD
251 GNLKISWSSP PLVPFPLQYQ VKYSENSTTV IREADKIVSA TSLLVDSILP
301 GSSYEVQVRG KRLDGPGIWS DWSTPRVFTT QDVIYFPPKI LTSVGSNVSF
351 HCIYKKENKI VPSKEIVWWM NLAEKIPQSQ YDVVSDHVSK VTFFNLNETK
401 PRGKFTYDAV YCCNEHECHH RYAELYVIDV NINISCETDG YLTKMTCRWS
451 TSTIQSLAES TLQLRYHRSS LYCSDIPSIH PISEPKDCYL QSDGFYECIF
501 QPIFLLSGYT MWIRINHSLG SLDSPPTCVL PDSVVKPLPP SSVKAEITIN
551 IGLLKISWEK PVFPENNLQF QIRYGLSGKE VQWKMYEVYD AKSKSVSLPV
601 PDLCAVYAVQ VRCKRLDGLG YWSNWSNPAY TVVMDIKVPM RGPEFWRIIN
651 GDTMKKEKNV TLLWKPLMKN DSLCSVQRYV INHHTSCNGT WSEDVGNHTK
701 FTFLWTEQAH TVTVLAINSI GASVANFNLT FSWPMSKVNI VQSLSAYPLN
751 SSCVIVSWIL SPSDYKLMYF IIEWKNLNED GEIKWLRISS SVKKYYIHDH
801 FIPIEKYQFS LYPIFMEGVG KPKIINSFTQ DDIEKHQSDA GLYVIVPVII
851 SSSILLLGTL LISHQRMKKL FWEDVPNPKN CSWAQGLNFQ KRTDIL*SLI
901 MITTDEPNVP TSQQSIEY*K IFTF*RRGAN LKKIQLNF*E LTYGGLC*FR
951 T*NRCVNLGS KCRFESSLDV *L
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liuman OB Receptor "A" DNA Sequence (Seq ID No. 2 (DNA))-
1 CCGCCGCCAT CTCTGCCTTC GGTCGAGTTG GACCCCCGGA TCAAGGTGTA
51 CTTCTCTGAA GTAAGATGAT TTGTCAAAAA TTCTGTGTGG TTTTGTTACA
101 TTGGGAATTT ATTTATGTGA TAACTGCGTT TAACTTGTCA TATCCAATTA
151 CTCCTTGGAG ATTTAAGTTG TCTTGCATGC CACCAAATTC AACCTATGAC
201 TACTTCCTTT TGCCTGCTGG ACTCTCAAAG AATACTTCAA ATTCGAATGG
251 ACATTATGAG ACAGCTGTTG AACCTAAGTT TAATTCAAGT GGTACTCACT
301 TTTCTAACTT ATCCAAAACA ACTTTCCACT GTTGCTTTCG GAGTGAGCAA
351 GATAGAAACT GCTCCTTATG TGCAGACAAC ATTGAAGGAA AGACATTTGT
401 TTCAACAGTA AATTCTTTAG TTTTTCAACA AATAGATGCA AACTGGAACA
451 TACAGTGCTG GCTAAAAGGA GACTTAAAAT TATTCATCTG TTATGTGGAG
501 TCATTATTTA AGAATCTATT CAGGAATTAT AACTATAAGG TCCATCTTTT
551 ATATGTTCTG CCTGAAGTGT TAGAAGATTC ACCTCTGGTT CCCCAAAAAG
601 GCAGTTTTCA GATGGTTCAC TGCAATTGCA GTGTTCATGA ATGTTGTGAA
651 TGTCTTGTGC CTGTGCCAAC AGCCAAACTC AACGACACTC TCCTTATGTG
701 TTTGAAAATC ACATCTGGTG GAGTAATTTT CCAGTCACCT CTAATGTCAG
751 TTCAGCCCAT AAATATGGTG AAGCCTGATC CACCATTAGG TTTGCATATG
801 GAAATCACAG ATGATGGTAA TTTAAAGATT TCTTGGTCCA GCCCACCATT
851 GGTACCATTT CCACTTCAAT ATCAAGTGAA ATATTCAGAG AATTCTACAA
901 CAGTTATCAG AGAAGCTGAC AAGATTGTCT CAGCTACATC CCTGCTAGTA
951 GACAGTATAC TTCCTGGGTC TTCGTATGAG GTTCAGGTGA GGGGCAAGAG
1001 ACTGGATGGC CCAGGAATCT GGAGTGACTG GAGTACTCCT CGTGTCTTTA
1051 CCACACAAGA TGTCATATAC TTTCCACCTA AAATTCTGAC AAGTGTTGGG
1101 TCTAATGTTT CTTTTCACTG CATCTATAAG AAGGAAAACA AGATTGTTCC
1151 CTCAAAAGAG ATTGTTTGGT GGATGAATTT AGCTGAGAAA ATTCCTCAAA
1201 GCCAGTATGA TGTTGTGAGT GATCATGTTA GCAAAGTTAC TTTTTTCAAT
1251 CTGAATGAAA CCAAACCTCG AGGAAAGTTT ACCTATGATG CAGTGTACTG
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1301 CTGCAATGAA CATGAATGCC ATCATCGCTA TGCTGAATTA TATGTGATTG
1351 ATGTCAATAT CAATATCTCA TGTGAAACTG ATGGGTACTT AACTAAAATG
1401 ACTTGCAGAT GGTCAACCAG TACAATCCAG TCACTTGCGG AAAGCACTTT
1451 GCAATTGAGG TATCATAGGA GCAGCCTTTA CTGTTCTGAT ATTCCATCTA
1501 TTCATCCCAT ATCTGAGCCC AAAGATTGCT ATTTGCAGAG TGATGGTTTT
1551 TATGAATGCA TTTTCCAGCC AATCTTCCTA TTATCTGGCT ACACAATGTG
1601 GATTAGGATC AATCACTCTC TAGGTTCACT TGACTCTCCA CCAACATGTG
1651 TCCTTCCTGA TTCTGTGGTG AAGCCACTGC CTCCATCCAG TGTGAAAGCA
1701 GAAA.TTACTA TAAACATTGG ATTATTGAAA ATATCTTGGG AA.AAGCCAGT
1751 CTTTCCAGAG AATAACCTTC AATTCCAGAT TCGCTATGGT TTAAGTGGAA
1801 AAGAAGTACA ATGGAAGATG TATGAGGTTT ATGATGCAAA ATCAAAATCT
1851 GTCAGTCTCC CAGTTCCAGA CTTGTGTGCA GTCTATGCTG TTCAGGTGCG
1901 CTGTAAGAGG CTAGATGGAC TGGGATATTG GAGTAATTGG AGCAATCCAG
1951 CCTACACAGT TGTCATGGAT ATAAAAGTTC CTATGAGAGG ACCTGAATTT
2001 TGGAGAATAA TTAATGGAGA TACTATGAAA AAGGAGAAAA ATGTCACTTT
2051 ACTTTGGAAG CCCCTGATGA AAAATGACTC ATTGTGCAGT GTTCAGAGAT
2101 ATGTGATAAA CCATCATACT TCCTGCAATG GAACATGGTC AGAAGATGTG
2151 GGAAATCACA CGAAATTCAC TTTCCTGTGG ACAGAGCAAG CACATACTGT
2201 TACGGTTCTG GCCATCAATT CAATTGGTGC TTCTGTTGCA AATTTTAATT
2251 TAACCTTTTC ATGGCCTATG AGCAAAGTAA ATATCGTGCA GTCACTCAGT
2301 GCTTATCCTT TAAACAGCAG TTGTGTGATT GTTTCCTGGA TACTATCACC
2351 CAGTGATTAC AAGCTAATGT ATTTTATTAT TGAGTGGAAA AATCTTAATG
2401 AAGATGGTGA AATAAAATGG CTTAGAATCT CTTCATCTGT TAAGAAGTAT
2451 TATATCCATG ATCATTTTAT CCCCATTGAG AAGTACCAGT TCAGTCTTTA
2501 CCCAATATTT ATGGAAGGAG TGGGAAAACC AAAGATAATT AATAGTTTCA
2551 CTCAAGATGA TATTGAAAAA CACCAGAGTG ATGCAGGTTT ATATGTAATT
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2601 GTGCCAGTAA TTATTTCCTC TTCCATCTTA TTGCTTGGAA CATTATTAAT
2651 ATCACACCAA AGAATGAAAA AGCTATTTTG GGAAGATGTT CCGAACCCCA
2701 AGAATTGTTC CTGGGCACAA GGACTTAATT TTCAGAAGAG AACGGACATT
2751 CTTTGAAGTC TAATCATGAT CACTACACAT GAACCCAATG TGCCAACTTC
2801 CCAACAGTCT ATAGAGTATT AGAAGATTTT TACATTTTGA AGAAGGGGAG
2851 CAAATCTAAA AA.AAATTCAG TTGAACTTCT GAGAGTTAAC ATATGGTGGA
2901 TTATGTTGAT TTAGAACTTA AAATAGATGT GTAAATTTGG GTTCAAAATG
2951 TAGATTTGAG TCCAGTTTGG ATGTGTGATT AATTTTCAAA TCATCTAAAG
3001 TTTAAAAGTA GTATTCATGA TTTCTGGCTT TTGATTTGCC ATATTCCTGG
3051 TCATAAAACA TTAAGAAAAT TATGGCTGTT GCTGTCATTA CATATCTATT
3101 AAATGTCATC AAATATGTAG TAGACAATTT TGTAATTAGG TGAACTCTAA
3151 AACTGCAACA TCTGACAAAT TGCTTTAAAA ATACAATGAT TAT
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Human OB Receptor "B" Amino Acid Sequence (Seqs ID No. 3(Amino
Acid) )
1 MICQKFCVVL LHWEFIYVIT AFNLSYPITP WRFKLSCMPP NSTYDYFLLP
51 AGLSKNTSNS NGHYETAVEP KFNSSGTHFS NLSKTTFHCC FRSEQDRNCS
101 LCADNIEGKT FVSTVNSLVF QQIDANWNIQ CWLKGDLKLF ICYVESLFKN
151 LFRNYNYKVH LLYVLPEVLE DSPLVPQKGS FQMVHCNCSV HECCECLVPV
201 PTAKLNDTLL MCLKITSGGV IFQSPLMSVQ PINMVKPDPP LGLHMEITDD
251 GNLKISWSSP PLVPFPLQYQ VKYSENSTTV IREADKIVSA TSLLVDSILP
301 GSSYEVQVRG KRLDGPGIWS DWSTPRVFTT QDVIYFPPKI LTSVGSNVSF
351 HCIYKKENKI VPSKEIVWWM NLAEKIPQSQ YDVVSDHVSK VTFFNLNETK
401 PRGKFTYDAV YCCNEHECHH RYAELYVIDV NINISCETDG YLTKMTCRWS
451 TSTIQSLAES TLQLRYHRSS LYCSDIPSIH PISEPKDCYL QSDGFYECIF
501 QPIFLLSGYT MWIRINHSLG SLDSPPTCVL PDSVVKPLPP SSVKAEITIN
551 IGLLKISWEK PVFPENNLQF QIRYGLSGKE VQWKMYEVYD AKSKSVSLPV
601 PDLCAVYAVQ VRCKRLDGLG YWSNWSNPAY TVVMDIKVPM RGPEFWRIIN
651 GDTMKKEKNV TLLWKPLMKN DSLCSVQRYV INHHTSCNGT WSEDVGNHTK
701 FTFLWTEQAH TVTVLAINSI GASVANFNLT FSWPMSKVNI VQSLSAYPLN
751 SSCVIVSWIL SPSDYKLMYF IIEWKNLNED GEIKWLRISS SVKKYYIHDH
801 FIPIEKYQFS LYPIFMEGVG KPKIINSFTQ DDIEKHQSDA GLYVIVPVII
851 SSSILLLGTL LISHQRMKKL FWEDVPNPKN CSWAQGLNFQ KKRLSIFLSS
901 IQHQ*HVVLF FWSLKQFQKI SVLIHHGKIK MR*CQQLWSL YFQQQILKRV
951 LFVLVTSSTV LTSLRLRVLR *PMRTKARDN PLLNTPR*SA TLNQVKLVK
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Human OB Receptor "B" DNA Secxuence (Seq ID No. 4 (DNA)):
1 CCGCCGCCAT CTCTGCCTTC GGTCGAGTTG GACCCCCGGA TCAAGGTGTA
51 CTTCTCTGAA GTAAGATGAT TTGTCAAAAA TTCTGTGTGG TTTTGTTACA
101 TTGGGAATTT ATTTATGTGA TAACTGCGTT TAACTTGTCA TATCCAATTA
151 CTCCTTGGAG ATTTAAGTTG TCTTGCATGC CACCAAATTC AACCTATGAC
201 TACTTCCTTT TGCCTGCTGG ACTCTCAAAG AATACTTCAA ATTCGAATGG
251 ACATTATGAG ACAGCTGTTG AACCTAAGTT TAATTCAAGT GGTACTCACT
301 TTTCTAACTT ATCCAAAACA ACTTTCCACT GTTGCTTTCG GAGTGAGCAA
351 GATAGAAACT GCTCCTTATG TGCAGACAAC ATTGAAGGAA AGACATTTGT
401 TTCAACAGTA AATTCTTTAG TTTTTCAACA AATAGATGCA AACTGGAACA
451 TACAGTGCTG GCTAAAAGGA GACTTAAAAT TATTCATCTG TTATGTGGAG
501 TCATTATTTA AGAATCTATT CAGGAATTAT AACTATAAGG TCCATCTTTT
551 ATATGTTCTG CCTGAAGTGT TAGAAGATTC ACCTCTGGTT CCCCAAAAAG
601 GCAGTTTTCA GATGGTTCAC TGCAATTGCA GTGTTCATGA ATGTTGTGAA
651 TGTCTTGTGC CTGTGCCAAC AGCCAAACTC AACGACACTC TCCTTATGTG
701 TTTGAAAATC ACATCTGGTG GAGTAATTTT CCAGTCACCT CTAATGTCAG
751 TTCAGCCCAT AAATATGGTG AAGCCTGATC CACCATTAGG TTTGCATATG
801 GAAATCACAG ATGATGGTAA TTTAAAGATT TCTTGGTCCA GCCCACCATT
851 GGTACCATTT CCACTTCAAT ATCAAGTGAA ATATTCAGAG AATTCTACAA
901 CAGTTATCAG AGAAGCTGAC AAGATTGTCT CAGCTACATC CCTGCTAGTA
951 GACAGTATAC TTCCTGGGTC TTCGTATGAG GTTCAGGTGA GGGGCAAGAG
1001 ACTGGATGGC CCAGGAATCT GGAGTGACTG GAGTACTCCT CGTGTCTTTA
1051 CCACACAAGA TGTCATATAC TTTCCACCTA AAATTCTGAC AAGTGTTGGG
1101 TCTAATGTTT CTTTTCACTG CATCTATAAG AAGGAAAACA AGATTGTTCC
1151 CTCAAAAGAG ATTGTTTGGT GGATGAATTT AGCTGAGAAA ATTCCTCAAA
1201 GCCAGTATGA TGTTGTGAGT GATCATGTTA GCAAAGTTAC TTTTTTCAAT
1251 CTGAATGAAA CCAAACCTCG AGGAAAGTTT ACCTATGATG CAGTGTACTG
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1301 CTGCAATGAA CATGAATGCC ATCATCGCTA TGCTGAATTA TATGTGATTG
1351 ATGTCAATAT CAATATCTCA TGTGAAACTG ATGGGTACTT AACTAAAATG
1401 ACTTGCAGAT GGTCAACCAG TACAATCCAG TCACTTGCGG AAA.GCACTTT
1451 GCAATTGAGG TATCATAGGA GCAGCCTTTA CTGTTCTGAT ATTCCATCTA
1501 TTCATCCCAT ATCTGAGCCC AAAGATTGCT ATTTGCAGAG TGATGGTTTT
1551 TATGA.ATGCA TTTTCCAGCC AATCTTCCTA TTATCTGGCT ACACAATGTG
1601 GATTAGGATC AATCACTCTC TAGGTTCACT TGACTCTCCA CCAACATGTG
1651 TCCTTCCTGA TTCTGTGGTG AAGCCACTGC CTCCATCCAG TGTGAAAGCA
1701 GAAATTACTA TAAACATTGG ATTATTGAAA ATATCTTGGG AAAAGCCAGT
1751 CTTTCCAGAG AP.TAACCTTC AATTCCAGAT TCGCTATGGT TTAAGTGGAA
1801 AAGAAGTACA ATGGAAGATG TATGAGGTTT ATGATGCAAA ATCAAAATCT
1851 GTCAGTCTCC CAGTTCCAGA CTTGTGTGCA GTCTATGCTG TTCAGGTGCG
1901 CTGTAAGAGG CTAGATGGAC TGGGATATTG GAGTAATTGG AGCAATCCAG
1951 CCTACACAGT TGTCATGGAT ATAAAAGTTC CTATGAGAGG ACCTGAATTT
2001 TGGAGAATAA TTAATGGAGA TACTATGAAA AAGGAGAAAA ATGTCACTTT
2051 ACTTTGGAAG CCCCTGATGA AAAATGACTC ATTGTGCAGT GTTCAGAGAT
2101 ATGTGATAAA CCATCATACT TCCTGCAATG GAACATGGTC AGAAGATGTG
2151 GGAAATCACA CGAAATTCAC TTTCCTGTGG ACAGAGCAAG CACATACTGT
2201 TACGGTTCTG GCCATCAATT CAATTGGTGC TTCTGTTGCA AATTTTAATT
2251 TAACCTTTTC ATGGCCTATG AGCAAAGTAA ATATCGTGCA GTCACTCAGT
2301 GCTTATCCTT TAAACAGCAG TTGTGTGATT GTTTCCTGGA TACTATCACC
2351 CAGTGATTAC AAGCTAATGT ATTTTATTAT TGAGTGGAAA AATCTTAATG
2401 AAGATGGTGA AATAAAATGG CTTAGAATCT CTTCATCTGT TAAGAAGTAT
2451 TATATCCATG ATCATTTTAT CCCCATTGAG AAGTACCAGT TCAGTCTTTA
2501 CCCAATATTT ATGGAAGGAG TGGGAAAACC AAAGATAATT AATAGTTTCA
2551 CTCAAGATGA TATTGAAAAA CACCAGAGTG ATGCAGGTTT ATATGTAATT
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2601 GTGCCAGTAA TTATTTCCTC TTCCATCTTA TTGCTTGGAA CATTATTAAT
2651 ATCACACCAA AGAATGAAAA AGCTATTTTG GGAAGATGTT CCGAACCCCA
2701 AGAATTGTTC CTGGGCACAA GGACTTAATT TTCAGAAGAA ACGTTTGAGC
2751 ATCTTTTTAT CAAGCATACA GCATCAGTGA CATGTGGTCC TCTTCTTTTG
2801 GAGCCTGAAA CAATTTCAGA AGATATCAGT GTTGATACAT CATGGAAAAA
2851 TAAAGATGAG ATGATGCCAA CAACTGTGGT CTCTCTACTT TCAACAACAG
2901 ATCTTGAAAA GGGTTCTGTT TGTTTTAGTG ACCAGTTCAA CAGTGTTAAC
2951 TTCTCTGAGG CTGAGGGTAC TGAGGTAACC TATGAGGACG AAAGCCAGAG
3001 ACAACCCTTT GTTAAATACG CCACGCTGAT CAGCAACTCT AAACCAAGTG
3051 AAACTGGTGA AGA
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Human OB Receptor "C" Amino Acid Sequence (Seq ID No. 5 (Amino
Acid))=
1 MICQKFCVVL LHWEFIYVIT AFNLSYPITP WRFKLSCMPP NSTYDYFLLP
51 AGLSKNTSNS NGHYETAVEP KFNSSGTHFS NLSKTTFHCC FRSEQDRNCS
101 LCADNIEGKT FVSTVNSLVF QQIDANWNIQ CWLKGDLKLF ICYVESLFKN
151 LFRNYNYKVH LLYVLPEVLE DSPLVPQKGS FQMVHCNCSV HECCECLVPV
201 PTAKLNDTLL MCLKITSGGV IFQSPLMSVQ PINMVKPDPP LGLHMEITDD
251 GNLKISWSSP PLVPFPLQYQ VKYSENSTTV IREADKIVSA TSLLVDSILP
301 GSSYEVQVRG KRLDGPGIWS DWSTPRVFTT QDVIYFPPKI LTSVGSNVSF
351 HCIYKKENKI VPSKEIVWWM NLAEKIPQSQ YDVVSDHVSK VTFFNLNETK
401 PRGKFTYDAV YCCNEHECHH RYAELYVIDV NINISCETDG YLTKMTCRWS
451 TSTIQSLAES TLQLRYHRSS LYCSDIPSIH PISEPKDCYL QSDGFYECIF
501 QPIFLLSGYT MWIRINHSLG SLDSPPTCVL PDSVVKPLPP SSVKAEITIN
551 IGLLKISWEK PVFPENNLQF QIRYGLSGKE VQWKMYEVYD AKSKSVSLPV
601 PDLCAVYAVQ VRCKRLDGLG YWSNWSNPAY TVVMDIKVPM RGPEFWRIIN
651 GDTMKKEKNV TLLWKPLMKN DSLCSVQRYV INHHTSCNGT WSEDVGNHTK
701 FTFLWTEQAH TVTVLAINSI GASVANFNLT FSWPMSKVNI VQSLSAYPLN
751 SSCVIVSWIL SPSDYKLMYF IIEWKNLNED GEIKWLRISS SVKKYYIHDH
801 FIPIEKYQFS LYPIFMEGVG KPKIINSFTQ DDIEKHQSDA GLYVIVPVII
851 SSSILLLGTL LISHQRMKKL FWEDVPNPKN CSWAQGLNFQ KMLEGSMFVK
901 SHHHSLISST QGHKHCGRPQ GPLHRKTRDL CSLVYLLTLP PLLSYDPAKS
951 PSVRNTQE*S IKKKKKKLEG
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Human OB Receptor "C" DNA Sequence (Seq. ID No. 6 (DNA)):
1 CCGCCGCCAT CTCTGCCTTC GGTCGAGTTG GACCCCCGGA TCAAGGTGTA
51 CTTCTCTGAA GTAAGATGAT TTGTCAAAAA TTCTGTGTGG TTTTGTTACA
101 TTGGGAATTT ATTTATGTGA TAACTGCGTT TAACTTGTCA TATCCAATTA
151 CTCCTTGGAG ATTTAAGTTG TCTTGCATGC CACCAAATTC AACCTATGAC
201 TACTTCCTTT TGCCTGCTGG ACTCTCAAAG AATACTTCAA ATTCGAATGG
251 ACATTATGAG ACAGCTGTTG AACCTAAGTT TAATTCAAGT GGTACTCACT
301 TTTCTAACTT ATCCAAAACA ACTTTCCACT GTTGCTTTCG GAGTGAGCAA
351 GATAGAAACT GCTCCTTATG TGCAGACAAC ATTGAAGGAA AGACATTTGT
401 TTCAACAGTA AATTCTTTAG TTTTTCAACA AATAGATGCA AACTGGAACA
451 TACAGTGCTG GCTAAA.AGGA GACTTAAAAT TATTCATCTG TTATGTGGAG
501 TCATTATTTA AGAATCTATT CAGGAATTAT AACTATAAGG TCCATCTTTT
551 ATATGTTCTG CCTGAAGTGT TAGAAGATTC ACCTCTGGTT CCCCAAAAAG
601 GCAGTTTTCA GATGGTTCAC TGCAATTGCA GTGTTCATGA ATGTTGTGAA
651 TGTCTTGTGC CTGTGCCAAC AGCCAAACTC AACGACACTC TCCTTATGTG
701 TTTGAAAATC ACATCTGGTG GAGTAATTTT CCAGTCACCT CTAATGTCAG
751 TTCAGCCCAT AAATATGGTG AAGCCTGATC CACCATTAGG TTTGCATATG
801 GAAATCACAG ATGATGGTAA TTTAAAGATT TCTTGGTCCA GCCCACCATT
851 GGTACCATTT CCACTTCAAT ATCAAGTGAA ATATTCAGAG AATTCTACAA
901 CAGTTATCAG AGAAGCTGAC AAGATTGTCT CAGCTACATC CCTGCTAGTA
951 GACAGTATAC TTCCTGGGTC TTCGTATGAG GTTCAGGTGA GGGGCAAGAG
1001 ACTGGATGGC CCAGGAATCT GGAGTGACTG GAGTACTCCT CGTGTCTTTA
1051 CCACACAAGA TGTCATATAC TTTCCACCTA AAATTCTGAC AAGTGTTGGG
1101 TCTAATGTTT CTTTTCACTG CATCTATAAG AAGGAAAACA AGATTGTTCC
1151 CTCAAAAGAG ATTGTTTGGT GGATGAATTT AGCTGAGAAA ATTCCTCAAA
1201 GCCAGTATGA TGTTGTGAGT GATCATGTTA GCAAAGTTAC TTTTTTCAAT
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1251 CTGAATGAAA CCAAACCTCG AGGAAAGTTT ACCTATGATG CAGTGTACTG
1301 CTGCAATGAA CATGAATGCC ATCATCGCTA TGCTGAATTA TATGTGATTG
1351 ATGTCAATAT CAATATCTCA TGTGAAACTG ATGGGTACTT AACTAAAATG
1401 ACTTGCAGAT GGTCAACCAG TACAATCCAG TCACTTGCGG AAAGCACTTT
1451 GCAATTGAGG TATCATAGGA GCAGCCTTTA CTGTTCTGAT ATTCCATCTA
1501 TTCATCCCAT ATCTGAGCCC AAAGATTGCT ATTTGCAGAG TGATGGTTTT
1551 TATGAATGCA TTTTCCAGCC AATCTTCCTA TTATCTGGCT ACACAATGTG
1601 GATTAGGATC AATCACTCTC TAGGTTCACT TGACTCTCCA CCAACATGTG
1651 TCCTTCCTGA TTCTGTGGTG AAGCCACTGC CTCCATCCAG TGTGAAAGCA
1701 GAAATTACTA TAAACATTGG ATTATTGAAA ATATCTTGGG AAAAGCCAGT
1751 CTTTCCAGAG AATAACCTTC AATTCCAGAT TCGCTATGGT TTAAGTGGAA
1801 AAGAAGTACA ATGGAAGATG TATGAGGTTT ATGATGCAAA ATCAAAATCT
1851 GTCAGTCTCC CAGTTCCAGA CTTGTGTGCA GTCTATGCTG TTCAGGTGCG
1901 CTGTAAGAGG CTAGATGGAC TGGGATATTG GAGTAATTGG AGCAATCCAG
1951 CCTACACAGT TGTCATGGAT ATAAAAGTTC CTATGAGAGG ACCTGAATTT
2001 TGGAGAATAA TTAATGGAGA TACTATGAAA AAGGAGAAAA ATGTCACTTT
2051 ACTTTGGAAG CCCCTGATGA AAAATGACTC ATTGTGCAGT GTTCAGAGAT
2101 ATGTGATAAA CCATCATACT TCCTGCAATG GAACATGGTC AGAAGATGTG
2151 GGAAATCA.CA CGAAATTCAC TTTCCTGTGG ACAGAGCAAG CACATACTGT
2201 TACGGTTCTG GCCATCAATT CAATTGGTGC TTCTGTTGCA AATTTTAATT
2251 TAACCTTTTC ATGGCCTATG AGCA.AAGTAA ATATCGTGCA GTCACTCAGT
2301 GCTTATCCTT TAAACAGCAG TTGTGTGATT GTTTCCTGGA TACTATCACC
2351 CAGTGATTAC AAGCTAATGT ATTTTATTAT TGAGTGGAAA AATCTTAATG
2401 AAGATGGTGA AATAAAATGG CTTAGAZATCT CTTCATCTGT TAAGAAGTAT
2451 TATATCCATG ATCATTTTAT CCCCATTGAG AAGTACCAGT TCAGTCTTTA
2501 CCCAATATTT ATGGAAGGAG TGGGAAAACC AAAGATAATT AATAGTTTCA
2551 CTCAAGATGA TATTGA.AAAA CACCAGAGTG ATGCAGGTTT ATATGTAATT
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2601 GTGCCAGTAA TTATTTCCTC TTCCATCTTA TTGCTTGGAA CATTATTAAT
2651 ATCACACCAA AGAATGAAAA AGCTATTTTG GGAAGATGTT CCGAACCCCA
2701 AGAATTGTTC CTGGGCACAA GGACTTAATT TTCAGAAGAT GCTTGAAGGC
2751 AGCATGTTCG TTAAGAGTCA TCACCACTCC CTAATCTCAA GTACCCAGGG
2801 ACACAAACAC TGCGGAAGGC CACAGGGTCC TCTGCATAGG AAAACCAGAG
2851 ACCTTTGTTC ACTTGTTTAT CTGCTGACCC TCCCTCCACT ATTGTCCTAT
2901 GACCCTGCCA AATCCCCCTC TGTGAGAAAC ACCCAAGAAT GATCAATAAA
2951 AAA-kAAAAAA AAAAAACTCG AGGGGG
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jliman OB R ptor "D" Amino Acid Sequence (SequencP ID No. 7)
1 MICQKFCVVL LHWEFIYVIT AFNLSYPITP WRFKLSCMPP NSTYDYFLLP 5
51 AGLSKNTSNS NGHYETAVEP KFNSSGTHFS NLSKTTFHCC FRSEQDRNCS
101 LCADNIEGKT FVSTVNSLVF QQIDANWNIQ CWLKGDLKLF ICYVESLFKN
151 LFRNYNYKVH LLYVLPEVLE DSPLVPQKGS FQMVHCNCSV HECCECLVPV
201 PTAKLNDTLL MCLKITSGGV IFQSPLMSVQ PINMVKPDPP LGLHMEITDD
251 GNLKISWSSP PLVPFPLQYQ VKYSENSTTV IREADKIVSA TSLLVDSILP
301 GSSYEVQVRG KRLDGPGIWS DWSTPRVFTT QDVIYFPPKI LTSVGSNVSF
351 HCIYKKENKI VPSKEIVWWM NLAEKIPQSQ YDVVSDHVSK VTFFNLNETK
401 PRGKFTYDAV YCCNEHECHH RYAELYVIDV NINISCETDG YLTKMTCRWS
451 TSTIQSLAES TLQLRYHRSS LYCSDIPSIH PISEPKDCYL QSDGFYECIF
501 QPIFLLSGYT MWIRINHSLG SLDSPPTCVL PDSVVKPLPP SSVKAEITIN
551 IGLLKISWEK PVFPENNLQF QIRYGLSGKE VQWKMYEVYD AKSKSVSLPV
601 PDLCAVYAVQ VRCKRLDGLG YWSNWSNPAY TVVMDIKVPM RGPEFWRIIN
651 GDTMKKEKNV TLLWKPLMKN DSLCSVQRYV INHHTSCNGT WSEDVGNHTK
701 FTFLWTEQAH TVTVLAINSI GASVANFNLT FSWPMSKVNI VQSLSAYPLN
751 SSCVIVSWIL SPSDYKLMYF IIEWKNLNED GEIKWLRISS SVKKYYIHDH
801 FIPIEKYQFS LYPIFMEGVG KPKIINSFTQ DDIEKHQSDA GLYVIVPVII
851 SSSILLLGTL LISHQRMKKL FWEDVPNPKN CSWAQGLNFQ KPETFEHLFI
901 KHTASVTCGP LLLEPETISE DISVDTSWKN KDEMMPTTVV SLLSTTDLEK
951 GSVCISDQFN SVNFSEAEGT EVTYEDESQR QPFVKYATLI SNSKPSETGE
1001 EQGLINSSVT KCFSSKNSPL KDSFSNSSWE IEAQAFFILS DQHPNIISPH
1051 LTFSEGLDEL LKLEGNFPEE NNDKKSIYYL GVTSIKKRES GVLLTDKSRV
1101 SCPFPAPCLF TDIRVLQDSC SHFVENNINL GTSSKKTFAS YMPQFQTCST
1151 QTHKIMENKM CDLTV*FH*R NLQICVIMGN IKCNRL*LWV GERKETRVKF
1201 ENNCSK*KKK KKNSRPARPD
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Human OB Recentor "D" Nucleic Acid Secquence (Sequence ID No 8)
1 GCGGCCGCCA GTGTGATGGA TATCTGCAGA ATTCGGCTTT CTCTGCCTTC
51 GGTCGAGTTG GACCCCCGGA TCAAGGTGTA CTTCTCTGAA GTAAGATGAT
101 TTGTCAAAAA TTCTGTGTGG TTTTGTTACA TTGGGAATTT ATTTATGTGA
151 TAACTGCGTT TAACTTGTCA TATCCAATTA CTCCTTGGAG ATTTAAGTTG
201 TCTTGCATGC CACCAAATTC AACCTATGAC TACTTCCTTT TGCCTGCTGG
251 GCTCTCAAAG AATACTTCAA ATTCGAATGG ACATTATGAG ACAGCTGTTG
301 AACCTAAGTT TAATTCAAGT GGTACTCACT TTTCTAACTT ATCCAAAACA
351 ACTTTCCACT GTTGCTTTCG GAGTGAGCAA GATAGAAACT GCTCCTTATG
401 TGCAGACAAC ATTGAAGGAA AGACATTTGT TTCAACAGTA AATTCTTTAG
451 TTTTTCAACA AATAGATGCA AACTGGAACA TACAGTGCTG GCTAAAAGGA
501 GACTTAAAAT TATTCATCTG TTATGTGGAG TCATTATTTA AGAATCTATT
551 CAGGAATTAT AACTATAAGG TCCATCTTTT ATATGTTCTG CCTGAAGTGT
601 TAGAAGATTC ACCTCTGGTT CCCCAAAAAG GCAGTTTTCA GATGGTTCAC
651 TGCAATTGCA GTGTTCACGA ATGTTGTGAA TGTCTTGTGC CTGTGCCAAC
701 AGCCAAACTC AACGACACTC TCCTTATGTG TTTGAAAATC ACATCTGGTG
751 GAGTAATTTT CCAGTCACCT CTAATGTCAG TTCAGCCCAT AAATATGGTG
801 AAGCCTGATC CACCATTAGG TTTGCATATG GAAATCACAG ATGATGGTAA
851 TTTAAAGATT TCTTGGTCCA GCCCACCATT GGTACCATTT CCACTTCAAT
901 ATCAAGTGAA ATATTCAGAG AATTCTACAA CAGTTATCAG AGAAGCTGAC
951 AAGATTGTCT CAGCTACATC CCTGCTAGTA GACAGTATAC TTCCTGGGTC
1001 TTCGTATGAG GTTCAGGTGA GGGGCAAGAG ACTGGATGGC CCAGGAATCT
1051 GGAGTGACTG GAGTACTCCT CGTGTCTTTA CCACACAAGA TGTCATATAC
1101 TTTCCACCTA AAATTCTGAC AAGTGTTGGG TCTAATGTTT CTTTTCACTG
1151 CATCTATAAG AAGGA.AAACA AGATTGTTCC CTCAAAAGAG ATTGTTTGGT
1201 GGATGAATTT AGCTGAGAAA ATTCCTCAAA GCCAGTATGA TGTTGTGAGT
1251 GATCATGTTA GCAAAGTTAC TTTTTTCAAT CTGAATGAAA CCAAACCTCG
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1301 AGGAAAGTTT ACCTATGATG CAGTGTACTG CTGCAATGAA CATGAATGCC
1351 ATCATCGCTA TGCTGAATTA TATGTGATTG ATGTCAATAT CAATATCTCA
1401 TGTGAAACTG ATGGGTACTT AACTAAAATG ACTTGCAGAT GGTCAACCAG
1451 TACAATCCAG TCACTTGCGG AAAGCACTTT GCAATTGAGG TATCATAGGA
1501 GCAGCCTTTA CTGTTCTGAT ATTCCATCTA TTCATCCCAT ATCTGAGCCC
1551 AAAGATTGCT ATTTGCAGAG TGATGGTTTT TATGAATGCA TTTTCCAGCC
1601 AATCTTCCTA TTATCTGGCT ACACAATGTG GATTAGGATC AATCACTCTC
1651 TAGGTTCACT TGACTCTCCA CCAACATGTG TCCTTCCTGA TTCTGTGGTG
1701 AAGCCACTGC CTCCATCCAG TGTGAAAGCA GAAATTACTA TAAACATTGG
1751 ATTATTGAAA ATATCTTGGG AAAAGCCAGT CTTTCCAGAG AATAACCTTC
1801 AATTCCAGAT TCGCTATGGT TTAAGTGGAA AAGAAGTACA ATGGAAGATG
1851 TATGAGGTTT ATGATGCAAA ATCAAAATCT GTCAGTCTCC CAGTTCCAGA
1901 CTTGTGTGCA GTCTATGCTG TTCAGGTGCG CTGTAAGAGG CTAGATGGAC
1951 TGGGATATTG GAGTAATTGG AGCAATCCAG CCTACACAGT TGTCATGGAT
2001 ATAAAAGTTC CTATGAGAGG ACCTGAATTT TGGAGAATAA TTAATGGAGA
2051 TACTATGAAA AAGGAGAAAA ATGTCACTTT ACTTTGGAAG CCCCTGATGA
2101 AAAATGACTC ATTGTGCAGT GTTCAGAGAT ATGTGATAAA CCATCATACT
2151 TCCTGCAATG GAACATGGTC AGAAGATGTG GGAAATCACA CGAAATTCAC
2201 TTTCCTGTGG ACAGAGCAAG CACATACTGT TACGGTTCTG GCCATCAATT
2251 CAATTGGTGC TTCTGTTGCA AATTTTAATT TAACCTTTTC ATGGCCTATG
2301 AGCAAAGTAA ATATCGTGCA GTCACTCAGT GCTTATCCTT TAAACAGCAG
2351 TTGTGTGATT GTTTCCTGGA TACTATCACC CAGTGATTAC AAGCTAATGT
2401 ATTTTATTAT TGAGTGGAAA AATCTTAATG AAGATGGTGA AATAAAATGG
2451 CTTAGAATCT CTTCATCTGT TAAGAAGTAT TATATCCATG ATCATTTTAT
2501 CCCCATTGAG AAGTACCAGT TCAGTCTTTA CCCAATATTT ATGGAAGGAG
2551 TGGGAAAACC AAAGATAATT AATAGTTTCA CTCAAGATGA TATTGAAAAA
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2601 CACCAGAGTG ATGCAGGTTT ATATGTAATT GTGCCAGTAA TTATTTCCTC
2651 TTCCATCTTA TTGCTTGGAA CATTATTAAT ATCACACCAA AGAATGAAAA
2701 AGCTATTTTG GGAAGATGTT CCGAACCCCA AGAATTGTTC CTGGGCACAA
2751 GGACTTAATT TTCAGAAGCC AGAAACGTTT GAGCATCTTT TTATCAAGCA
2801 TACAGCATCA GTGACATGTG GTCCTCTTCT TTTGGAGCCT GAAACAATTT
2851 CAGAAGATAT CAGTGTTGAT ACATCATGGA AAAATAAAGA TGAGATGATG
2901 CCAACAACTG TGGTCTCTCT ACTTTCAACA ACAGATCTTG AAAAGGGTTC
2951 TGTTTGTATT AGTGACCAGT TCAACAGTGT TAACTTCTCT GAGGCTGAGG
3001 GTACTGAGGT AACCTATGAG GACGAAAGCC AGAGACAACC CTTTGTTAAA
3051 TACGCCACGC TGATCAGCAA CTCTAAACCA AGTGAAACTG GTGAAGAACA
3101 AGGGCTTATA AATAGTTCAG TCACCAAGTG CTTCTCTAGC AAAAATTCTC
3151 CGTTGAAGGA TTCTTTCTCT AATAGCTCAT GGGAGATAGA GGCCCAGGCA
3201 TTTTTTATAT TATCGGATCA GCATCCCAAC ATAATTTCAC CACACCTCAC
3251 ATTCTCAGAA GGATTGGATG AACTTTTGAA ATTGGAGGGA AATTTCCCTG
3301 AAGAAAATAA TGATAAAAAG TCTATCTATT ATTTAGGGGT CACCTCAATC
3351 AAAAAGAGAG AGAGTGGTGT GCTTTTGACT GACAAGTCAA GGGTATCGTG
3401 CCCATTCCCA GCCCCCTGTT TATTCACGGA CATCAGAGTT CTCCAGGACA
3451 GTTGCTCACA CTTTGTAGAA AATAATATCA ACTTAGGAAC TTCTAGTAAG
3501 AAGACTTTTG CATCTTACAT GCCTCAATTC CAAACTTGTT CTACTCAGAC
3551 TCATAAGATC ATGGAAAACA AGATGTGTGA CCTAACTGTG TAATCTAGA
.
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Human OB Receptor Protein "D" Chromosomal DNP,. (S a ID No. 9)
Intron 1 .....taccttttccag GTG TAC TTC
CAT TGG G gtaagttatttg..... Intron 2 .....atatcctaacag AA TTT ATT
His Trp Glu Phe Ile
12 13 14 15 16
CAA ATA G gtaagcattagc..... Intron 3 .....ttttaaattoag AT GCA AAC
Gln Ile Asp Ala Asn
122 123 124 125 126
20'
TAT GTT CT gtaagtaccaaa..... Intron 4 .....ttttcaatatag G CCT GAA
Tyr Val Leu Pro Glu
163 164 165 166 167
AAT ATG G gtaagttatgca..... Intron 5 .....tttttccttaag TG AAG CCT
Asn Met Val Lys Pro
233 234 235 236 237
ATC AGA GAA gtaagtatattt..... Intron 6 .....aatatttaacag GCT GAC AAG
Ile Arg Glu Ala Asp Lys
281 282 283 284 285 286
ACA CAA G gtaggttatgta..... Intron 7 .....ccctcattacag AT GTC ATA
Thr Gin Asp Val Ile
330 331 332 333 334
GTG ATT G gtaagaaaacag..... Intron 8 .....tgtttcaaatag AT GTC AAT
Val Ile Asp Val Asn
427 428 429 430 431
TAT CAT AG gtacgC.attatt..... Intron 9 .....tatcttttaaag G AGC AGC
Tyr His Arg Ser Ser
466 467 468 469 470
TCT GTG G gtatgtcaagct..... Intron 10 .....aaaaatttctag TG AAG CCA
Ser Val Val Lys Pro
533 534 535 536 537
CAA TGG AAG gtaccttttact..... Intron 11 .....cttattttacag ATG TAT GAG
Gln Trp Lys Met Tyr Glu
582 583 584 585 586 587
ATA AAA G gtctgcagagat..... Intron 12 .....gtcattttgcag 2T CCT ATG
Ile Lys Val Pro Met
636 637 638 639 640 ~
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CTT TGG AAG gtattcccaatt..... Intron 13 .....tatttactacag CCC CTG ATG
Leu Trp Lys Pro Leu Met
663 664 665 666 667 668
AGC AAA G gtaagaagaggt..... Intron 14 .....ttttcccctcag TA AAT ATC
Ser Lys Val Asn Ile
736 737 738 739 740
ATC CAT G gtaagtttacta..... Intron 15 .....ttttctcctcag AT CAT TTT
Ile His Asp His Phe
797 798 799 800 801
ACT CAA G gtaaaaattata..... Intron 16 .....tttctttttcag AT GAT ATT
Thr Gin Asp Asp Ile
829 830 831 832 833
CAC CAA AG gtattgtacttg..... Intron 17 .....tatcctttgtag A ATG AAA
His Gln Arg Met Lys
864 865 866 867 868
Exon A
TTT CAG AAG gttgctttttca..... Intron 18 .....ttatctaaacag AGA ACG GAC
Phe Gln Lys Arg Thr Asp
889 890 891 892 893 894
Exon A Exon D
AAA TAT GAT gtacatttgtct..... Intron 18 .....cttttcttttag CCA GAA ACG
Pro Glu Thr
892 893 894
Exon B
AAA CGT TTG
Lys Arg Leu
892 893 894
Exon D Exon C
GAA ACC AGA gtatccagtgtt..... Intron 18 .....ctttttaaacag ATG CTT GAA
Met Leu Glu
892 893 894
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Human OB Receptor Protein Recombinant S rr d R ptor amino
acid sequence lSeq. ID. No. 10):
1 MICQKFCVVL LHWEFIYVIT AFNLSYPITP WRFKLSCMPP NSTYDYFLLP
51 AGLSKNTSNS NGHYETAVEP KFNSSGTHFS NLSKTTFHCC FRSEQDRNCS
101 LCADNIEGKT FVSTVNSLVF QQIDANWNIQ CWLKGDLKLF ICYVESLFKN
151 LFRNYNYKVH LLYVLPEVLE DSPLVPQKGS FQMVHCNCSV HECCECLVPV
201 PTAKLNDTLL MCLKITSGGV IFQSPLMSVQ PINMVKPDPP LGLHMEITDD
251 GNLKISWSSP PLVPFPLQYQ VKYSENSTTV IREADKIVSA TSLLVDSILP
301 GSSYEVQVRG KRLDGPGIWS DWSTPRVFTT QDVIYFPPKI LTSVGSNVSF
351 HCIYKKENKI VPSKEIVWWM NLAEKIPQSQ YDVVSDHVSK VTFFNLNETK
401 PRGKFTYDAV YCCNEHECHH RYAELYVIDV NINISCETDG YLTKMTCRWS
451 TSTIQSLAES TLQLRYHRSS LYCSDIPSIH PISEPKDCYL QSDGFYECIF
501 QPIFLLSGYT MWIRINHSLG SLDSPPTCVL PDSVVKPLPP SSVKAEITIN
551 IGLLKISWEK PVFPENNLQF QIRYGLSGKE VQWKMYEVYD AKSKSVSLPV
601 PDLCAVYAVQ VRCKRLDGLG YWSNWSNPAY TVVMDIKVPM RGPEFWRIIN
651 GDTMKKEKNV TLLWKPLMKN DSLCSVQRYV INHHTSCNGT WSEDVGNHTK
701 FTFLWTEQAH TVTVLAINSI GASVANFNLT FSWPMSKVNI VQSLSAYPLN
751 SSCVIVSWIL SPSDYKLMYF IIEWKNLNED GEIKWLRISS SVKKYYIHDH
801 FIPIEKYQFS LYPIFMEGVG KPKIINSFTQ DDIEKHQSD
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Human OB Recep~or Protein Recombinant Secreted Receptor DNA
sequPnra ( Seq ID . No. 11) :
1 GCGGCCGCCA GTGTGATGGA TATCTGCAGA ATTCGGCTTT CTCTGCCTTC
51 GGTCGAGTTG GACCCCCGGA TCAAGGTGTA CTTCTCTGAA GTAAGATGAT
101 TTGTCAAAAA TTCTGTGTGG TTTTGTTACA TTGGGAATTT ATTTATGTGA
151 TAACTGCGTT TAACTTGTCA TATCC.A.ATTA CTCCTTGGAG ATTTAAGTTG
201 TCTTGCATGC CACCAAATTC AACCTATGAC TACTTCCTTT TGCCTGCTGG
251 GCTCTCAAAG AATACTTCAA ATTCGAATGG ACATTATGAG ACAGCTGTTG
301 AACCTAAGTT TAATTCAAGT GGTACTCACT TTTCTAACTT ATCCAAAACA
351 ACTTTCCACT GTTGCTTTCG GAGTGAGCAA GATAGAAACT GCTCCTTATG
401 TGCAGACAAC ATTGAAGGAA AGACATTTGT TTCAACAGTA AATTCTTTAG
451 TTTTTCAACA AATAGATGCA AACTGGAACA TACAGTGCTG GCTAAAAGGA
501 GACTTAAAAT TATTCATCTG TTATGTGGAG TCATTATTTA AGAATCTATT
551 CAGGAATTAT AACTATAAGG TCCATCTTTT ATATGTTCTG CCTGAAGTGT
601 TAGAAGATTC ACCTCTGGTT CCCCAAAAAG GCAGTTTTCA GATGGTTCAC
651 TGCAATTGCA GTGTTCACGA ATGTTGTGAA TGTCTTGTGC CTGTGCCAAC
701 AGCCAAACTC AACGACACTC TCCTTATGTG TTTGAAAATC ACATCTGGTG
751 GAGTAATTTT CCAGTCACCT CTAATGTCAG TTCAGCCCAT AAATATGGTG
801 AAGCCTGATC CACCATTAGG TTTGCATATG GAA.ATCACAG ATGATGGTAA
851 TTTAAAGATT TCTTGGTCCA GCCCACCATT GGTACCATTT CCACTTCAAT
901 ATCAAGTGAA ATATTCAGAG AATTCTACAA CAGTTATCAG AGAAGCTGAC
951 AAGATTGTCT CAGCTACATC CCTGCTAGTA GACAGTATAC TTCCTGGGTC
1001 TTCGTATGAG GTTCAGGTGA GGGGCAAGAG ACTGGATGGC CCAGGAATCT
1051 GGAGTGACTG GAGTACTCCT CGTGTCTTTA CCACACAAGA TGTCATATAC
} 1101 TTTCCACCTA AAATTCTGAC AAGTGTTGGG TCTAATGTTT CTTTTCACTG
1151 CATCTATAAG AAGGAAAACA AGATTGTTCC CTCAAAAGAG ATTGTTTGGT
1201 GGATGAATTT AGCTGAGAAA ATTCCTCAAA GCCAGTATGA TGTTGTGAGT
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1251 GATCATGTTA GCAAAGTTAC TTTTTTCAAT CTGAATGAAA CCAAACCTCG
1301 AGGAAAGTTT ACCTATGATG CAGTGTACTG CTGCAATGAA CATGAATGCC 5
1351 ATCATCGCTA TGCTGAATTA TATGTGATTG ATGTCAATAT CAATATCTCA
1401 TGTGAAACTG ATGGGTACTT AACTAAAATG ACTTGCAGAT GGTCAACCAG
1451 TACAATCCAG TCACTTGCGG AAAGCACTTT GCAATTGAGG TATCATAGGA
1501 GCAGCCTTTA CTGTTCTGAT ATTCCATCTA TTCATCCCAT ATCTGAGCCC
1551 AAAGATTGCT ATTTGCAGAG TGATGGTTTT TATGAATGCA TTTTCCAGCC
1601 AATCTTCCTA TTATCTGGCT ACACAATGTG GATTAGGATC AATCACTCTC
1651 TAGGTTCACT TGACTCTCCA CCAACATGTG TCCTTCCTGA TTCTGTGGTG
1701 AAGCCACTGC CTCCATCCAG TGTGAAAGCA GAAATTACTA TAAACATTGG
1751 ATTATTGAAA ATATCTTGGG AAAAGCCAGT CTTTCCAGAG AATAACCTTC
1801 AATTCCAGAT TCGCTATGGT TTAAGTGGAA AAGAAGTACA ATGGAAGATG
1851 TATGAGGTTT ATGATGCAAA ATCAAAATCT GTCAGTCTCC CAGTTCCAGA
1901 CTTGTGTGCA GTCTATGCTG TTCAGGTGCG CTGTAAGAGG CTAGATGGAC
1951 TGGGATATTG GAGTAATTGG AGCAATCCAG CCTACACAGT TGTCATGGAT
2001 ATAAAAGTTC CTATGAGAGG ACCTGAATTT TGGAGAATAA TTAATGGAGA
2051 TACTATGAAA AAGGAGAAAA ATGTCACTTT ACTTTGGAAG CCCCTGATGA
2101 AAAATGACTC ATTGTGCAGT GTTCAGAGAT ATGTGATAAA CCATCATACT
2151 TCCTGCAATG GAACATGGTC AGAAGATGTG GGAAATCACA CGAAATTCAC
2201 TTTCCTGTGG ACAGAGCAAG CACATACTGT TACGGTTCTG GCCATCAATT
2251 CAATTGGTGC TTCTGTTGCA AATTTTAATT TAACCTTTTC ATGGCCTATG
2301 AGCAAAGTAA ATATCGTGCA GTCACTCAGT GCTTATCCTT TAAACAGCAG
2351 TTGTGTGATT GTTTCCTGGA TACTATCACC CAGTGATTAC AAGCTAATGT
2401 ATTTTATTAT TGAGTGGAAA AATCTTAATG AAGATGGTGA AATAAAATGG
2451 CTTAGAATCT CTTCATCTGT TAAGAAGTAT TATATCCATG ATCATTTTAT
2501 CCCCATTGAG AAGTACCAGT TCAGTCTTTA CCCAATATTT ATGGAAGGAG
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2551 TGGGAAAACC AAAGATAATT AATAGTTTCA CTCAAGATGA TATTGAAAAA
2601 CACCAGAGTG ATTGATAAGG ATCC
4
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Human CB Receptor Protein. ReCombinant S r_._d R ptor DNA
seauence with C-termi na 1 FLAG (SPq ID. No. 12) :
1 CCATTGAAGT CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGGATTT
51 CCAAAATGTC GTAATAACCC CGCCCCGTTG ACGCAAATGG GCGGTAGGCG
101 TGTACGGTGG GAGGTCTATA TAAGCAGAGC TCGTTTAGTG AACCGTCAGA
151 TCTCTAGAAG CTGGGTACCA GCTGCTAGCA AGCTTGCTAG CGGCCGCCAG
201 TGTGATGGAT ATCTGCAGAA TTCGGCTTTC TCTGCCTTCG GTCGAGTTGG
251 ACCCCCGGAT CAAGGTGTAC TTCTCTGAAG TAAGATGATT TGTCAAAAAT
301 TCTGTGTGGT TTTGTTACAT TGGGAATTTA TTTATGTGAT AACTGCGTTT
351 AACTTGTCAT ATCCAATTAC TCCTTGGAGA TTTAAGTTGT CTTGCATGCC
401 ACCAAATTCA ACCTATGACT ACTTCCTTTT GCCTGCTGGG CTCTCAAAGA
451 ATACTTCAAA TTCGAATGGA CATTATGAGA CAGCTGTTGA ACCTAAGTTT
501 AATTCAAGTG GTACTCACTT TTCTAACTTA TCCAP,AACAA CTTTCCACTG
551 TTGCTTTCGG AGTGAGCAAG ATAGAAACTG CTCCTTATGT GCAGACAACA
601 TTGAAGGAAA GACATTTGTT TCAACAGTAA ATTCTTTAGT TTTTCAACAA
651 ATAGATGCAA ACTGGAACAT ACAGTGCTGG CTAAAAGGAG ACTTAAAATT
701 ATTCATCTGT TATGTGGAGT CATTATTTAA GAATCTATTC AGGAATTATA
751 ACTATAAGGT CCATCTTTTA TATGTTCTGC CTGAAGTGTT AGAAGATTCA
801 CCTCTGGTTC CCCAAAAAGG CAGTTTTCAG ATGGTTCACT GCAATTGCAG
851 TGTTCACGAA TGTTGTGAAT GTCTTGTGCC TGTGCCAACA GCCAAACTCA
901 ACGACACTCT CCTTATGTGT TTGAAAATCA CATCTGGTGG AGTAATTTTC
951 CAGTCACCTC TAATGTCAGT TCAGCCCATA AATATGGTGA AGCCTGATCC
1001 ACCATTAGGT TTGCATATGG AAATCACAGA TGATGGTAAT TTAAAGATTT
1051 CTTGGTCCAG CCCACCATTG GTACCATTTC CACTTCAATA TCAAGTGAP.A.
1101 TATTCAGAGA ATTCTACAAC AGTTATCAGA GAAGCTGACA AGATTGTCTC
1151 AGCTACATCC CTGCTAGTAG ACAGTATACT TCCTGGGTCT TCGTATGAGG
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1201 TTCAGGTGAG GGGCAAGAGA CTGGATGGCC CAGGAATCTG GAGTGACTGG
1251 AGTACTCCTC GTGTCTTTAC CACACAAGAT GTCATATACT TTCCACCTAA
1301 AATTCTGACA AGTGTTGGGT CTAATGTTTC TTTTCACTGC ATCTATAAGA
1351 AGGAAAACAA GATTGTTCCC TCAAAAGAGA TTGTTTGGTG GATGAATTTA
1401 GCTGAGAAAA TTCCTCAAAG CCAGTATGAT GTTGTGAGTG ATCATGTTAG
1451 CAAAGTTACT TTTTTCAATC TGAATGAAAC CAAACCTCGA GGAAAGTTTA
1501 CCTATGATGC AGTGTACTGC TGCAATGAAC ATGAATGCCA TCATCGCTAT
1551 GCTGAATTAT ATGTGATTGA TGTCAATATC AATATCTCAT GTGAAACTGA
1601 TGGGTACTTA ACTAAAATGA CTTGCAGATG GTCAACCAGT ACAATCCAGT
1651 CACTTGCGGA AAGCACTTTG CAATTGAGGT ATCATAGGAG CAGCCTTTAC
1701 TGTTCTGATA TTCCATCTAT TCATCCCATA TCTGAGCCCA AAGATTGCTA
1751 TTTGCAGAGT GATGGTTTTT ATGAATGCAT TTTCCAGCCA ATCTTCCTAT
1801 TATCTGGCTA CACAATGTGG ATTAGGATCA ATCACTCTCT AGGTTCACTT
1851 GACTCTCCAC CAACATGTGT CCTTCCTGAT TCTGTGGTGA AGCCACTGCC
1901 TCCATCCAGT GTGAAAGCAG AAATTACTAT AAACATTGGA TTATTGAAAA
1951 TATCTTGGGA AAAGCCAGTC TTTCCAGAGA ATAACCTTCA ATTCCAGATT
2001 CGCTATGGTT TAAGTGGAAA AGAAGTACAA TGGAAGATGT ATGAGGTTTA
2051 TGATGCAAAA TCAAAATCTG TCAGTCTCCC AGTTCCAGAC TTGTGTGCAG
2101 TCTATGCTGT TCAGGTGCGC TGTAAGAGGC TAGATGGACT GGGATATTGG
2151 AGTAATTGGA GCAATCCAGC CTACACAGTT GTCATGGATA TAAAAGTTCC
2201 TATGAGAGGA CCTGAATTTT GGAGAATAAT TAATGGAGAT ACTATGAAAA
2251 AGGAGAAAAA TGTCACTTTA CTTTGGAAGC CCCTGATGAA AAATGACTCA
2301 TTGTGCAGTG TTCAGAGATA TGTGATAAAC CATCATACTT CCTGCAATGG
2351 AACATGGTCA GAAGATGTGG GAAATCACAC GAAATTCACT TTCCTGTGGA
2401 CAGAGCAAGC ACATACTGTT ACGGTTCTGG CCATCAATTC AATTGGTGCT
2451 TCTGTTGCAA ATTTTAATTT AACCTTTTCA TGGCCTATGA GCAAAGTAAA
2501 TATCGTGCAG TCACTCAGTG CTTATCCTTT AAACAGCAGT TGTGTGATTG
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2551 TTTCCTGGAT ACTATCACCC AGTGATTACA AGCTAATGTA TTTTATTATT
2601 GAGTGGAAAA ATCTTAATGA AGATGGTGAA ATAAAATGGC TTAGAATCTC
2651 TTCATCTGTT AAGAAGTATT ATATCCATGA TCATTTTATC CCCATTGAGA
2701 AGTACCAGTT CAGTCTTTAC CCAATATTTA TGGAAGGAGT GGGAAAACCA
2751 AAGATAATTA ATAGTTTCAC TCAAGATGAT ATTGAAAAAC ACCAGAGTGA
2801 TGCAGGTGAC TACAAGGACG ACGATGACAA GTAGGGATCC AGACATGATA
2851 AGATACATTG ATGAGTTTGG ACAACCCACA ACTAGAATGC AGTGAAAAAA
2901 ATGCTTTATT TGTGAAATTT GTGATGCTAT TGCTTTATTT GTAACCAT
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RArnmhinaõt- Human OB Receptor Protein. Natural Splice Variant
amino acid Gernuence fSecd ID No. 13)
1 MICQKFCVVL LHWEFIYVIT AFNLSYPITP WRFKLSCMPP NSTYDYFLLP
51 AGLSKNTSNS NGHYETAVEP KFNSSGTHFS NLSKTTFHCC FRSEQDRNCS
101 LCADNIEGKT FVSTVNSLVF QQIDANWNIQ CWLKGDLKLF ICYVESLFKN
151 LFRNYNYKVH LLYVLPEVLE DSPLVPQKGS FQMVHCNCSV HECCECLVPV
201 PTAKLNDTLL MCLKITSGGV IFQSPLMSVQ PINMVKPDPP LGLHMEITDD
251 GNLKISWSSP PLVPFPLQYQ VKYSENSTTV IREADKIVSA TSLLVDSILP
301 GSSYEVQVRG KRLDGPGIWS DWSTPRVFTT QDVIYFPPKI LTSVGSNVSF
351 HCIYKKENKI VPSKEIVWWM NLAEKIPQSQ YDVVSDHVSK VTFFNLNETK
401 PRGKFTYDAV YCCNEHECHH RYAELYVIDV NINISCETDG YLTKMTCRWS
451 TSTIQSLAES TLQLRYHRSS LYCSDIPSIH PISEPKDCYL QSDGFYECIF
501 QPIFLLSGYT MWIRINHSLG SLDSPPTCVL PDSVVKPLPP SSVKAEITIN
551 IGLLKISWEK PVFPENNLQF QIRYGLSGKE VQWKMYEVYD AKSKSVSLPV
601 PDLCAVYAVQ VRCKRLDGLG YWSNWSNPAY TVVMDIKVPM RGPEFWRIIN
651 GDTMKKEKNV TLLWKPLMKN DSLCSVQRYV INHHTSCNGT WSEDVGNHTK
701 FTFLWTEQAH TVTVLAINSI GASVANFNLT FSWPMSKVNI VQSLSAYPLN
751 SSCVIVSWIL SPSDYKLMYF IIEWKNLNED GEIKWLRISS SVKKYYIHGK
801 FTIL
3
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Human OB Receptor Protein, Natural Splice Variant DNA (Seq ID
No. 14)
1 GCGGCCGCCA GTGTGATGGA TATCTGCAGA ATTCGGCTTT CTCTGCCTTC 5
51 GGTCGAGTTG GACCCCCGGA TCAAGGTGTA CTTCTCTGAA GTAAGATGAT
101 TTGTCAAAAA TTCTGTGTGG TTTTGTTACA TTGGGAATTT ATTTATGTGA
151 TAACTGCGTT TAACTTGTCA TATCCAATTA CTCCTTGGAG ATTTAAGTTG
201 TCTTGCATGC CACCAAATTC AACCTATGAC TACTTCCTTT TGCCTGCTGG
251 GCTCTCAAAG AATACTTCAA ATTCGAATGG ACATTATGAG ACAGCTGTTG
301 AACCTAAGTT TAATTCAAGT GGTACTCACT TTTCTAACTT ATCCAAAACA
351 ACTTTCCACT GTTGCTTTCG GAGTGAGCAA GATAGAAACT GCTCCTTATG
401 TGCAGACAAC ATTGAAGGAA AGACATTTGT TTCAACAGTA AATTCTTTAG
451 TTTTTCAACA AATAGATGCA AACTGGAACA TACAGTGCTG GCTAAAAGGA
501 GACTTAAAAT TATTCATCTG TTATGTGGAG TCATTATTTA AGAATCTATT
551 CAGGAATTAT AACTATAAGG TCCATCTTTT ATATGTTCTG CCTGAAGTGT
601 TAGAAGATTC ACCTCTGGTT CCCCAA.AAAG GCAGTTTTCA GATGGTTCAC
651 TGCAATTGCA GTGTTCACGA ATGTTGTGAA TGTCTTGTGC CTGTGCCAAC
701 AGCCAAACTC AACGACACTC TCCTTATGTG TTTGAAAATC ACATCTGGTG
751 GAGTAATTTT CCAGTCACCT CTAATGTCAG TTCAGCCCAT AAATATGGTG
801 AAGCCTGATC CACCATTAGG TTTGCATATG GAA.ATCACAG ATGATGGTAA
851 TTTAAAGATT TCTTGGTCCA GCCCACCATT GGTACCATTT CCACTTCAAT
901 ATCAAGTGAA ATATTCAGAG AATTCTACAA CAGTTATCAG AGAAGCTGAC
951 AAGATTGTCT CAGCTACATC CCTGCTAGTA GACAGTATAC TTCCTGGGTC
1001 TTCGTATGAG GTTCAGGTGA GGGGCAAGAG ACTGGATGGC CCAGGAATCT
1051 GGAGTGACTG GAGTACTCCT CGTGTCTTTA CCACACAAGA TGTCATATAC
1101 TTTCCACCTA AAATTCTGAC AAGTGTTGGG TCTAATGTTT CTTTTCACTG
1151 CATCTATAAG AAGGAAAACA AGATTGTTCC CTCAAAAGAG ATTGTTTGGT
1201 GGATGAATTT AGCTGAGAAA ATTCCTCAAA GCCAGTATGA TGTTGTGAGT
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1251 GATCATGTTA GCAAAGTTAC TTTTTTCAAT CTGAATGAAA CCAAACCTCG
1301 AGGAAAGTTT ACCTATGATG CAGTGTACTG CTGCAATGA.A CATGA.ATGCC
1351 ATCATCGCTA TGCTGAATTA TATGTGATTG ATGTCAATAT CAATATCTCA
1401 TGTGAAACTG ATGGGTACTT AACTAAAATG ACTTGCAGAT GGTCAACCAG
1451 TACAATCCAG TCACTTGCGG AAAGCACTTT GCAATTGAGG TATCATAGGA
1501 GCAGCCTTTA CTGTTCTGAT ATTCCATCTA TTCATCCCAT ATCTGAGCCC
1551 AAAGATTGCT ATTTGCAGAG TGATGGTTTT TATGAATGCA TTTTCCAGCC
1601 AATCTTCCTA TTATCTGGCT ACACAATGTG GATTAGGATC AATCACTCTC
1651 TAGGTTCACT TGACTCTCCA CCAACATGTG TCCTTCCTGA TTCTGTGGTG
1701 AAGCCACTGC CTCCATCCAG TGTGAAAGCA GAA.ATTACTA TAAACATTGG
1751 ATTATTGAAA ATATCTTGGG A.AAAGCCAGT CTTTCCAGAG AATAACCTTC
1801 A.ATTCCAGAT TCGCTATGGT TTAAGTGGAA AAGAAGTACA ATGGAAGATG
1851 TATGAGGTTT ATGATGCAAA ATCAAAATCT GTCAGTCTCC CAGTTCCAGA
1901 CTTGTGTGCA GTCTATGCTG TTCAGGTGCG CTGTAAGAGG CTAGATGGAC
1951 TGGGATATTG GAGTAATTGG AGCAATCCAG CCTACACAGT TGTCATGGAT
2001 ATA.AAAGTTC CTATGAGAGG ACCTGA.ATTT TGGAGAATAA TTAATGGAGA
2051 TACTATGA.AA AAGGAGA.AAA ATGTCACTTT ACTTTGGAAG CCCCTGATGA
2101 AAAATGACTC ATTGTGCAGT GTTCAGAGAT ATGTGATAAA CCATCATACT
2151 TCCTGCAATG GAACATGGTC AGAAGATGTG GGAAATCACA CGAAATTCAC
2201 TTTCCTGTGG ACAGAGCAAG CACATACTGT TACGGTTCTG GCCATCAATT
2251 CAATTGGTGC TTCTGTTGCA AATTTTAATT TAACCTTTTC ATGGCCTATG
2301 AGCAAAGTAA ATATCGTGCA GTCACTCAGT GCTTATCCTT TAAACAGCAG
2351 TTGTGTGATT GTTTCCTGGA TACTATCACC CAGTGATTAC AAGCTAATGT
2401 ATTTTATTAT TGAGTGGAAA AATCTTAATG AAGATGGTGA A.ATAAAATGG
2451 CTTAGAATCT CTTCATCTGT TAAGAAGTAT TATATCCATG GTA.AGTTTAC
~ 50
2501 TATACTT
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While the present invention has been described
in terms of preferred embodiments, it is understood that
variations and modifications will occur to those skilled
in the art. Therefore, it is intended that the appended
claims cover all such equivalent variations which come
within the scope of the invention as claimed.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: CHANG, MING-SHI
WELCHER, ANDREW A.
FLETCHER, FREDERICK A.
(ii) TITLE OF INVENTION: OB PROTEIN RECEPTOR AND RELATED
COMPOSITIONS AND METHODS
(iii) NUMBER OF SEQUENCES: 33
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Amgen Inc.
(B) STREET: 1840 Dehavilland Drive
(C) CITY: Thousand Oaks
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 91320
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Pessin, Karol M.
(C) REFERENCE/DOCKET NUMBER: A-382-A
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 965 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Met Ile Cys Gln Lys Phe Cys Val Val Leu Leu His Trp Glu Phe Ile
1 5 10 15
Tyr Val Ile Thr Ala Phe Asn Leu Ser Tyr Pro Ile Thr Pro Trp Arg
20 25 30
Phe Lys Leu Ser Cys Met Pro Pro Asn Ser Thr Tyr Asp Tyr Phe Leu
35 40 45
Leu Pro Ala Gly Leu Ser Lys Asn Thr Ser Asn Ser Asn Gly His Tyr
50 55 60
Glu Thr Ala Val Glu Pro Lys Phe Asn Ser Ser Gly Thr His Phe Ser
65 70 75 80
Asn Leu Ser Lys Thr Thr Phe His Cys Cys Phe Arg Ser Glu Gln Asp
85 90 95
Arg Asn Cys Ser Leu Cys Ala Asp Asn Ile Glu Gly Lys Thr Phe Val
100 105 110
Ser Thr Val Asn Ser Leu Val Phe Gln Gln Ile Asp Ala Asn Trp Asn
115 120 125
Ile Gin Cys Trp Leu Lys Gly Asp Leu Lys Leu Phe Ile Cys Tyr Val
130 135 140
Glu Ser Leu Phe Lys Asn Leu Phe Arg Asn Tyr Asn Tyr Lys Val His
145 150 155 160
Leu Leu Tyr Val Leu Pro Glu Val Leu Glu Asp Ser Pro Leu Val Pro
165 170 175
Gln Lys Gly Ser Phe Gln Met Val His Cys Asn Cys Ser Val His Glu
180 185 190
Cys Cys Glu Cys Leu Val Pro Val Pro Thr Ala Lys Leu Asn Asp Thr
195 200 205
Leu Leu Met Cys Leu Lys Ile Thr Ser G1y Gly Val Ile Phe Gln Ser
210 215 220
Pro Leu Met Ser Val Gln Pro Ile Asn Met Val Lys Pro Asp Pro Pro
225 230 235 240
Leu Gly Leu His Met Glu Ile Thr Asp Asp Gly Asn Leu Lys Ile Ser
245 250 255
Trp Ser Ser Pro Pro Leu Val Pro Phe Pro Leu Gln Tyr Gln Val Lys
260 265 270
___
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Tyr Ser Glu Asn Ser Thr Thr Val Ile Arg Glu Ala Asp Lys Ile Val
275 280 285
Ser Ala Thr Ser Leu Leu Val Asp Ser Ile Leu Pro Gly Ser Ser Tyr
290 295 300
Glu Val Gin Val Arg Gly Lys Arg Leu Asp Gly Pro Gly Ile Trp Ser
305 310 315 320
Asp Trp Ser Thr Pro Arg Val Phe Thr Thr Gln Asp Val Ile Tyr Phe
325 330 335
Pro Pro Lys 11e Leu Thr Ser Val Gly Ser Asn Val Ser Phe His Cys
340 345 350
Ile Tyr Lys Lys Glu Asn Lys Ile Val Pro Ser Lys Glu Ile Val Trp
355 360 365
Trp Met Asn Leu Ala Glu Lys Ile Pro Gln Ser Gln Tyr Asp Val Val
370 375 380
Ser Asp His Val Ser Lys Val Thr Phe Phe Asn Leu Asn Glu Thr Lys
385 390 395 400
Pro Arg Gly Lys Phe Thr Tyr Asp Ala Val Tyr Cys Cys Asn Glu His
405 410 415
Glu Cys His His Arg Tyr Ala Glu Leu Tyr Vai Ile Asp Val Asn Ile
420 425 430
Asn Ile Ser Cys Glu Thr Asp Gly Tyr Leu Thr Lys Met Thr Cys Arg
435 440 445
Trp Ser Thr Ser Thr I1e Gln Ser Leu Ala Glu Ser Thr Leu Gln Leu
450 455 460
Arg Tyr His Arg Ser Ser Leu Tyr Cys Ser Asp Ile Pro Ser Ile His
465 470 475 480
Pro Ile Ser Glu Pro Lys Asp Cys Tyr Leu Gln Ser Asp Giy Phe Tyr
485 490 495
Glu Cys Ile Phe Gin Pro Ile Phe Leu Leu Ser Gly Tyr Thr Met Trp
500 505 510
Ile Arg Ile Asn His Ser Leu Giy Ser Leu Asp Ser Pro Pro Thr Cys
515 520 525
5 0 Val Leu Pro Asp Ser Val Val Lys Pro Leu Pro Pro Ser Ser Val Lys
530 535 540
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Ala Glu Ile Thr Ile Asn Ile Gly Leu Leu Lys Ile Ser Trp Glu Lys
545 550 555 560
Pro Val Phe Pro Glu Asn Asn Leu Gln Phe Gin Ile Arg Tyr Gly Leu
565 570 575
Ser Gly Lys Glu Val Gin Trp Lys Met Tyr Glu Val Tyr Asp Ala Lys
580 585 590
Ser Lys Ser Val Ser Leu Pro Val Pro Asp Leu Cys Ala Val Tyr Ala
595 600 605
Val Gln Val Arg Cys Lys Arg Leu Asp Gly Leu Gly Tyr Trp Ser Asn
610 615 620
Trp Ser Asn Pro Ala Tyr Thr Val Val Met Asp Ile Lys Val Pro Met
625 630 635 640
Arg Gly Pro Glu Phe Trp Arg Ile Ile Asn Gly Asp Thr Met Lys Lys
645 650 655
Glu Lys Asn Val Thr Leu Leu Trp Lys Pro Leu Met Lys Asn Asp Ser
660 665 670
Leu Cys Ser Val Gln Arg Tyr Val Ile Asn His His Thr Ser Cys Asn
675 680 685
Gly Thr Trp Ser Glu Asp Val Gly Asn His Thr Lys Phe Thr Phe Leu
690 695 700
Trp Thr Glu Gln Ala His Thr Val Thr Val Leu Ala Ile Asn Ser Ile
705 710 715 720
Gly Ala Ser Val Ala Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser
725 730 735
Lys Val Asn Ile Val Gln Ser Leu Ser Ala Tyr Pro Leu Asn Ser Ser
740 745 750
Cys Val Ile Val Ser Trp Ile Leu Ser Pro Ser Asp Tyr Lys Leu Met
755 760 765
Tyr Phe Ile Ile Glu Trp Lys Asn Leu Asn Glu Asp Gly Glu Ile Lys
770 775 780
Trp Leu Arg Ile Ser Ser Ser Val Lys Lys Tyr Tyr Ile His Asp His
785 790 795 800
Phe Ile Pro Ile Giu Lys Tyr Gln Phe Ser Leu Tyr Pro I1e Phe Met
805 810 815
Glu Gly Val Gly Lys Pro Lys Ile Ile Asn Ser Phe Thr Gln Asp Asp
820 825 830
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Ile Glu Lys His Gin Ser Asp Ala Gly Leu Tyr Val Ile Val Pro Val
835 840 845
Ile Ile Ser Ser Ser Ile Leu Leu Leu Gly Thr Leu Leu Ile Ser His
850 855 860
Gln Arg Met Lys Lys Leu Phe Trp Glu Asp Val Pro Asn Pro Lys Asn
865 870 875 880
Cys Ser Trp Ala Gin Gly Leu Asn Phe Gln Lys Arg Thr Asp Ile Leu
885 890 895
Ser Leu Ile Met Ile Thr Thr Asp Glu Pro Asn Val Pro Thr Ser Gln
900 905 910
Gin Ser Ile Glu Tyr Lys Ile Phe Thr Phe Arg Arg Gly Ala Asn Leu
915 920 925
Lys Lys Ile Gln Leu Asn Phe Glu Leu Thr Tyr Gly Gly Leu Cys Phe
930 935 940
Arg Thr Asn Arg Cys Val Asn Leu Gly Ser Lys Cys Arg Phe Glu Ser
945 950 955 960
Ser Leu Asp Val Leu
965
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3193 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CCGCCGCCAT CTCTGCCTTC GGTCGAGTTG GACCCCCGGA TCAAGGTGTA CTTCTCTGAA 60
GTAAGATGAT TTGTCAAAAA TTCTGTGTGG TTTTGTTACA TTGGGAATTT ATTTATGTGA 120
TAACTGCGTT TAACTTGTCA TATCCAATTA CTCCTTGGAG ATTTAAGTTG TCTTGCATGC 180
CACCAAATTC AACCTATGAC TACTTCCTTT TGCCTGCTGG ACTCTCAAAG AATACTTCAA 240
ATTCGAATGG ACATTATGAG ACAGCTGTTG AACCTAAGTT TAATTCAAGT GGTACTCACT 300
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TTTCTAACTT ATCCAAAACA ACTTTCCACT GTTGCTTTCG GAGTGAGCAA GATAGAAACT 360
GCTCCTTATG TGCAGACAAC ATTGAAGGAA AGACATTTGT TTCAACAGTA AATTCTTTAG 420
TTTTTCAACA AATAGATGCA AACTGGAACA TACAGTGCTG GCTAAAAGGA GACTTAAAAT 480
TATTCATCTG TTATGTGGAG TCATTATTTA AGAATCTATT CAGGAATTAT AACTATAAGG 540
TCCATCTTTT ATATGTTCTG CCTGAAGTGT TAGAAGATTC ACCTCTGGTT CCCCAAAAAG 600
GCAGTTTTCA GATGGTTCAC TGCAATTGCA GTGTTCATGA ATGTTGTGAA TGTCTTGTGC 660
CTGTGCCAAC AGCCAAACTC AACGACACTC TCCTTATGTG TTTGAAAATC ACATCTGGTG 720
GAGTAATTTT CCAGTCACCT CTAATGTCAG TTCAGCCCAT AAATATGGTG AAGCCTGATC 780
CACCATTAGG TTTGCATATG GAAATCACAG ATGATGGTAA TTTAAAGATT TCTTGGTCCA 840
GCCCACCATT GGTACCATTT CCACTTCAAT ATCAAGTGAA ATATTCAGAG AATTCTACAA 900
CAGTTATCAG AGAAGCTGAC AAGATTGTCT CAGCTACATC CCTGCTAGTA GACAGTATAC 960
TTCCTGGGTC TTCGTATGAG GTTCAGGTGA GGGGCAAGAG ACTGGATGGC CCAGGAATCT 1020
GGAGTGACTG GAGTACTCCT CGTGTCTTTA CCACACAAGA TGTCATATAC TTTCCACCTA 1080
AAATTCTGAC AAGTGTTGGG TCTAATGTTT CTTTTCACTG CATCTATAAG AAGGAAAACA 1140
AGATTGTTCC CTCAAAAGAG ATTGTTTGGT GGATGAATTT AGCTGAGAAA ATTCCTCAAA 1200
GCCAGTATGA TGTTGTGAGT GATCATGTTA GCAAAGTTAC TTTTTTCAAT CTGAATGAAA 1260
CCAAACCTCG AGGAAAGTTT ACCTATGATG CAGTGTACTG CTGCAATGAA CATGAATGCC 1320
ATCATCGCTA TGCTGAATTA TATGTGATTG ATGTCAATAT CAATATCTCA TGTGAAACTG 1380
ATGGGTACTT AACTAAAATG ACTTGCAGAT GGTCAACCAG TACAATCCAG TCACTTGCGG 1440
AAAGCACTTT GCAATTGAGG TATCATAGGA GCAGCCTTTA CTGTTCTGAT ATTCCATCTA 1500
TTCATCCCAT ATCTGAGCCC AAAGATTGCT ATTTGCAGAG TGATGGTTTT TATGAATGCA 1560
TTTTCCAGCC AATCTTCCTA TTATCTGGCT ACACAATGTG GATTAGGATC AATCACTCTC 1620
TAGGTTCACT TGACTCTCCA CCAACATGTG TCCTTCCTGA TTCTGTGGTG AAGCCACTGC 1680
CTCCATCCAG TGTGAAAGCA GAAATTACTA TAAACATTGG ATTATTGAAA ATATCTTGGG 1740
AAAAGCCAGT CTTTCCAGAG AATAACCTTC AATTCCAGAT TCGCTATGGT TTAAGTGGAA 1800
AAGAAGTACA ATGGAAGATG TATGAGGTTT ATGATGCAAA ATCAAAATCT GTCAGTCTCC 1860
CAGTTCCAGA CTTGTGTGCA GTCTATGCTG TTCAGGTGCG CTGTAAGAGG CTAGATGGAC 1920 =
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TGGGATATTG GAGTAATTGG AGCAATCCAG CCTACACAGT TGTCATGGAT ATAAAAGTTC 1980
CTATGAGAGG ACCTGAATTT TGGAGAATAA TTAATGGAGA TACTATGAAA AAGGAGAAAA 2040
ATGTCACTTT ACTTTGGAAG CCCCTGATGA AAAATGACTC ATTGTGCAGT GTTCAGAGAT 2100
ATGTGATAAA CCATCATACT TCCTGCAATG GAACATGGTC AGAAGATGTG GGAAATCACA 2160
CGAAATTCAC TTTCCTGTGG ACAGAGCAAG CACATACTGT TACGGTTCTG GCCATCAATT 2220
CAATTGGTGC TTCTGTTGCA AATTTTAATT TAACCTTTTC ATGGCCTATG AGCAAAGTAA 2280
ATATCGTGCA GTCACTCAGT GCTTATCCTT TAAACAGCAG TTGTGTGATT GTTTCCTGGA 2340
TACTATCACC CAGTGATTAC AAGCTAATGT ATTTTATTAT TGAGTGGAAA AATCTTAATG 2400
AAGATGGTGA AATAAAATGG CTTAGAATCT CTTCATCTGT TAAGAAGTAT TATATCCATG 2460
ATCATTTTAT CCCCATTGAG AAGTACCAGT TCAGTCTTTA CCCAATATTT ATGGAAGGAG 2520
TGGGAAAACC AAAGATAATT AATAGTTTCA CTCAAGATGA TATTGAAAAA CACCAGAGTG 2580
ATGCAGGTTT ATATGTAATT GTGCCAGTAA TTATTTCCTC TTCCATCTTA TTGCTTGGAA 2640
CATTATTAAT ATCACACCAA AGAATGAAAA AGCTATTTTG GGAAGATGTT CCGAACCCCA 2700
AGAATTGTTC CTGGGCACAA GGACTTAATT TTCAGAAGAG AACGGACATT CTTTGAAGTC 2760
TAATCATGAT CACTACAGAT GAACCCAATG TGCCAACTTC CCAACAGTCT ATAGAGTATT 2820
AGAAGATTTT TACATTTTGA AGAAGGGGAG CAAATCTAAA AAAAATTCAG TTGAACTTCT 2880
GAGAGTTAAC ATATGGTGGA TTATGTTGAT TTAGAACTTA AAATAGATGT GTAAATTTGG 2940
GTTCAAAATG TAGATTTGAG TCCAGTTTGG ATGTGTGATT AATTTTCAAA TCATCTAAAG 3000
TTTAAAAGTA GTATTCATGA TTTCTGGCTT TTGATTTGCC ATATTCCTGG TCATAAAACA 3060
TTAAGAAAAT TATGGCTGTT GCTGTCATTA CATATCTATT AAATGTCATC AAATATGTAG 3120
TAGACAATTT TGTAATTAGG TGAACTCTAA AACTGCAACA TCTGACAAAT TGCTTTAAAA 3180
ATACAATGAT TAT 3193
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 995 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met I1e Cys Gln Lys Phe Cys Val Val Leu Leu His Trp Glu Phe Ile
1 5 10 15
Tyr Val Ile Thr Ala Phe Asn Leu Ser Tyr Pro Ile Thr Pro Trp Arg
25 30
Phe Lys Leu Ser Cys Met Pro Pro Asn Ser Thr Tyr Asp Tyr Phe Leu
15 35 40 45
Leu Pro Ala Gly Leu Ser Lys Asn Thr Ser Asn Ser Asn Gly His Tyr
50 55 60
20 Giu Thr Ala Val Glu Pro Lys Phe Asn Ser Ser Gly Thr His Phe Ser
65 70 75 80
Asn Leu Ser Lys Thr Thr Phe His Cys Cys Phe Arg Ser Glu Gln Asp
85 90 95
Arg Asn Cys Ser Leu Cys Ala Asp Asn Ile Glu Gly Lys Thr Phe Val
100 105 110
Ser Thr Val Asn Ser Leu Val Phe Gln Gln Ile Asp Ala Asn Trp Asn
115 120 125
Ile Gin Cys Trp Leu Lys Gly Asp Leu Lys Leu Phe Ile Cys Tyr Val
130 135 140
Glu Ser Leu Phe Lys Asn Leu Phe Arg Asn Tyr Asn Tyr Lys Val His
145 150 155 160
Leu Leu Tyr Val Leu Pro Glu Val Leu Glu Asp Ser Pro Leu Val Pro
165 170 175
Gln Lys Giy Ser Phe Gin Met Val His Cys Asn Cys Ser Val His Glu
180 185 190
Cys Cys Glu Cys Leu Val Pro Val Pro Thr Ala Lys Leu Asn Asp Thr
195 200 205
Leu Leu Met Cys Leu Lys I1e Thr Ser Giy Gly Val Ile Phe Gln Ser
210 215 220
Pro Leu Met Ser Val Gln Pro Ile Asn Met Val Lys Pro Asp Pro Pro
225 230 235 240
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Leu Gly Leu His Met Glu Ile Thr Asp Asp Gly Asn Leu Lys Ile Ser
245 250 255
Trp Ser Ser Pro Pro Leu Val Pro Phe Pro Leu Gln Tyr Gln Val Lys
260 265 270
Tyr Ser Glu Asn Ser Thr Thr Val Ile Arg Glu Ala Asp Lys Ile Val
275 280 285
Ser Ala Thr Ser Leu Leu Val Asp Ser Ile Leu Pro Gly Ser Ser Tyr
290 295 300
Glu Val Gln Val Arg Gly Lys Arg Leu Asp Gly Pro Gly Ile Trp Ser
305 310 315 320
Asp Trp Ser Thr Pro Arg Val Phe Thr Thr Gln Asp Val Ile Tyr Phe
325 330 335
Pro Pro Lys Ile Leu Thr Ser Val Gly Ser Asn Val Ser Phe His Cys
340 345 350
Ile Tyr Lys Lys Glu Asn Lys Ile Val Pro Ser Lys Giu Ile Val Trp
355 360 365
Trp Met Asn Leu Ala Glu Lys Ile Pro Gln Ser Gln Tyr Asp Val Val
370 375 380
Ser Asp His Val Ser Lys Vai Thr Phe Phe Asn Leu Asn Glu Thr Lys
385 390 395 400
Pro Arg Gly Lys Phe Thr Tyr Asp Ala Val Tyr Cys Cys Asn Giu His
405 410 415
Glu Cys His His Arg Tyr Ala Glu Leu Tyr Val Ile Asp Val Asn Ile
420 425 430
Asn Ile Ser Cys Glu Thr Asp Gly Tyr Leu Thr Lys Met Thr Cys Arg
435 440 445
Trp Ser Thr Ser Thr Ile Gin Ser Leu Ala Glu Ser Thr Leu Gln Leu
450 455 460
Arg Tyr His Arg Ser Ser Leu Tyr Cys Ser Asp Ile Pro Ser Ile His
465 470 475 480
Pro Ile Ser Glu Pro Lys Asp Cys Tyr Leu Gin Ser Asp Gly Phe Tyr
485 490 495
Glu Cys Ile Phe Gin Pro Ile Phe Leu Leu Ser Gly Tyr Thr Met Trp
500 505 510
Ile Arg Ile Asn His Ser Leu Gly Ser Leu Asp Ser Pro Pro Thr Cys
515 520 525
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Val Leu Pro Asp Ser Val Val Lys Pro Leu Pro Pro Ser Ser Val Lys
530 535 540
Ala Glu Ile Thr Ile Asn Ile Gly Leu Leu Lys Ile Ser Trp Glu Lys
545 550 555 560
Pro Val Phe Pro Glu Asn Asn Leu Gin Phe Gln Ile Arg Tyr Gly Leu
565 570 575
Ser Gly Lys Glu Val Gin Trp Lys Met Tyr Glu Val Tyr Asp Ala Lys
580 585 590
Ser Lys Ser Val Ser Leu Pro Va1 Pro Asp Leu Cys Ala Val Tyr Ala
595 600 605
Val Gin Val Arg Cys Lys Arg Leu Asp Gly Leu Gly Tyr Trp Ser Asn
610 615 620
Trp Ser Asn Pro Ala Tyr Thr Val Val Met Asp Ile Lys Val Pro Met
625 630 635 640
Arg Gly Pro Glu Phe Trp Arg Ile Ile Asn Gly Asp Thr Met Lys Lys
645 650 655
Glu Lys Asn Val Thr Leu Leu Trp Lys Pro Leu Met Lys Asn Asp Ser
660 665 670
Leu Cys Ser Val Gln Arg Tyr Val Ile Asn His His Thr Ser Cys Asn
675 680 685
Gly Thr Trp Ser Glu Asp Val Gly Asn His Thr Lys Phe Thr Phe Leu
690 695 700
Trp Thr Glu Gln Ala His Thr Val Thr Val Leu Ala Ile Asn Ser Ile
705 710 715 720
Gly Ala Ser Val Ala Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser
725 730 735
Lys Val Asn Ile Val Gin Ser Leu Ser Ala Tyr Pro Leu Asn Ser Ser
740 745 750
Cys Vai Ile Val Ser Trp Ile Leu Ser Pro Ser Asp Tyr Lys Leu Met
755 760 765
Tyr Phe Ile Ile Glu Trp Lys Asn Leu Asn Glu Asp Giy Glu Ile Lys
770 775 780
Trp Leu Arg Ile Ser Ser Ser Val Lys Lys Tyr Tyr Ile His Asp His
785 790 795 800
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Phe Ile Pro Ile Glu Lys Tyr Gln Phe Ser Leu Tyr Pro Ile Phe Met
805 810 815
Glu Gly Val Gly Lys Pro Lys Ile Ile Asn Ser Phe Thr Gln Asp Asp
820 825 830
+ I1e Glu Lys His Gln Ser Asp Ala Gly Leu Tyr Val Ile Val Pro Val
835 840 845
Ile Ile Ser Ser Ser Ile Leu Leu Leu Gly Thr Leu Leu Ile Ser His
850 855 860
Gln Arg Met Lys Lys Leu Phe Trp Glu Asp Val Pro Asn Pro Lys Asn
865 870 875 880
Cys Ser Trp Ala Gln Gly Leu Asn Phe Gln Lys Lys Arg Leu Ser Ile
885 890 895
Phe Leu Ser Ser Ile Gln His Gin His Val Val Leu Phe Phe Trp Ser
900 905 910
Leu Lys Gln Phe Gln Lys Ile Ser Val Leu Ile His His Gly Lys Ile
915 920 925
Lys Met Arg Cys Gln Gln Leu Trp Ser Leu Tyr Phe Gln Gln Gln I1e
930 935 940
Leu Lys Arg Val Leu Phe Val Leu Val Thr Ser Ser. Thr Val Leu Thr
945 950 955 960
Ser Leu Arg Leu Arg Val Leu Arg Pro Met Arg Thr Lys Ala Arg Asp
965 970 975
Asn Pro Leu Leu Asn Thr Pro Arg Ser Ala Thr Leu Asn Gln Val Lys
980 985 990
Leu Val Lys
995
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3063 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CCGCCGCCAT CTCTGCCTTC GGTCGAGTTG GACCCCCGGA TCAAGGTGTA CTTCTCTGAA 60
'rJ GTAAGATGAT TTGTCAAAAA TTCTGTGTGG TTTTGTTACA TTGGGAATTT ATTTATGTGA 120
TAACTGCGTT TAACTTGTCA TATCCAATTA CTCCTTGGAG ATTTAAGTTG TCTTGCATGC 180
CACCAAATTC AACCTATGAC TACTTCCTTT TGCCTGCTGG ACTCTCAAAG AATACTTCAA 240
ATTCGAATGG ACATTATGAG ACAGCTGTTG AACCTAAGTT TAATTCAAGT GGTACTCACT 300
TTTCTAACTT ATCCAAAACA ACTTTCCACT GTTGCTTTCG GAGTGAGCAA GATAGAAACT 360
-GCTCCTTATG TGCAGACAAC ATTGAAGGAA AGACATTTGT TTCAACAGTA AATTCTTTAG 420
TTTTTCAACA AATAGATGCA AACTGGAACA TACAGTGCTG GCTAAAAGGA GACTTAAAAT 480
TATTCATCTG TTATGTGGAG TCATTATTTA AGAATCTATT CAGGAATTAT AACTATAAGG 540
TCCATCTTTT ATATGTTCTG CCTGAAGTGT TAGAAGATTC ACCTCTGGTT CCCCAAAAAG 600
GCAGTTTTCA GATGGTTCAC TGCAATTGCA GTGTTCATGA ATGTTGTGAA TGTCTTGTGC 660
CTGTGCCAAC AGCCAAACTC AACGACACTC TCCTTATGTG TTTGAAAATC ACATCTGGTG 720
GAGTAATTTT CCAGTCACCT CTAATGTCAG TTCAGCCCAT AAATATGGTG AAGCCTGATC 780
CACCATTAGG TTTGCATATG GAAATCACAG ATGATGGTAA TTTAAAGATT TCTTGGTCCA 840
GCCCACCATT GGTACCATTT CCACTTCAAT ATCAAGTGAA ATATTCAGAG AATTCTACAA 900
CAGTTATCAG AGAAGCTGAC AAGATTGTCT CAGCTACATC CCTGCTAGTA GACAGTATAC 960
TTCCTGGGTC TTCGTATGAG GTTCAGGTGA GGGGCAAGAG ACTGGATGGC CCAGGAATCT 1020
GGAGTGACTG GAGTACTCCT CGTGTCTTTA CCACACAAGA TGTCATATAC TTTCCACCTA 1080
AAATTCTGAC AAGTGTTGGG TCTAATGTTT CTTTTCACTG CATCTATAAG AAGGAAAACA 1140
AGATTGTTCC CTCAAAAGAG ATTGTTTGGT GGATGAATTT AGCTGAGAAA ATTCCTCAAA 1200
GCCAGTATGA TGTTGTGAGT GATCATGTTA GCAAAGTTAC TTTTTTCAAT CTGAATGAAA 1260
CCAAACCTCG AGGAAAGTTT ACCTATGATG CAGTGTACTG CTGCAATGAA CATGAATGCC 1320
ATCATCGCTA TGCTGAATTA TATGTGATTG ATGTCAATAT CAATATCTCA TGTGAAACTG 1380
ATGGGTACTT AACTAAAATG ACTTGCAGAT GGTCAACCAG TACAATCCAG TCACTTGCGG 1440
AAAGCACTTT GCAATTGAGG TATCATAGGA GCAGCCTTTA CTGTTCTGAT ATTCCATCTA 1500
TTCATCCCAT ATCTGAGCCC AAAGATTGCT ATTTGCAGAG TGATGGTTTT TATGAATGCA 1560
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TTTTCCAGCC AATCTTCCTA TTATCTGGCT ACACAATGTG GATTAGGATC AATCACTCTC 1620
TAGGTTCACT TGACTCTCCA CCAACATGTG TCCTTCCTGA TTCTGTGGTG AAGCCACTGC 1680
CTCCATCCAG TGTGAAAGCA GAAATTACTA TAAACATTGG ATTATTGAAA ATATCTTGGG 1740
AAAAGCCAGT CTTTCCAGAG AATAACCTTC AATTCCAGAT TCGCTATGGT TTAAGTGGAA 1800
AAGAAGTACA ATGGAAGATG TATGAGGTTT ATGATGCAAA ATCAAAATCT GTCAGTCTCC 1860
CAGTTCCAGA CTTGTGTGCA GTCTATGCTG TTCAGGTGCG CTGTAAGAGG CTAGATGGAC 1920
TGGGATATTG GAGTAATTGG AGCAATCCAG CCTACACAGT TGTCATGGAT ATAAAAGTTC 1980
CTATGAGAGG ACCTGAATTT TGGAGAATAA TTAATGGAGA TACTATGAAA AAGGAGAAAA 2040
ATGTCACTTT ACTTTGGAAG CCCCTGATGA AAAATGACTC ATTGTGCAGT GTTCAGAGAT 2100
ATGTGATAAA CCATCATACT TCCTGCAATG GAACATGGTC AGAAGATGTG GGAAATCACA 2160
CGAAATTCAC TTTCCTGTGG ACAGAGCAAG CACATACTGT TACGGTTCTG GCCATCAATT 2220
CAATTGGTGC TTCTGTTGCA AATTTTAATT TAACCTTTTC ATGGCCTATG AGCAAAGTAA 2280
ATATCGTGCA GTCACTCAGT GCTTATCCTT TAAACAGCAG TTGTGTGATT GTTTCCTGGA 2340
TACTATCACC CAGTGATTAC AAGCTAATGT ATTTTATTAT TGAGTGGAAA AATCTTAATG 2400
AAGATGGTGA AATAAAATGG CTTAGAATCT CTTCATCTGT TAAGAAGTAT TATATCCATG 2460
ATCATTTTAT CCCCATTGAG AAGTACCAGT TCAGTCTTTA CCCAATATTT ATGGAAGGAG 2520
TGGGAAAACC AAAGATAATT AATAGTTTCA CTCAAGATGA TATTGAAAAA CACCAGAGTG 2580
ATGCAGGTTT ATATGTAATT GTGCCAGTAA TTATTTCCTC TTCCATCTTA TTGCTTGGAA 2640
CATTATTAAT ATCACACCAA AGAATGAAAA AGCTATTTTG GGAAGATGTT CCGAACCCCA 2700
AGAATTGTTC CTGGGCACAA GGACTTAATT TTCAGAAGAA ACGTTTGAGC ATCTTTTTAT 2760
CAAGCATACA GCATCAGTGA CATGTGGTCC TCTTCTTTTG GAGCCTGAAA CAATTTCAGA 2820
AGATATCAGT GTTGATACAT CATGGAAAAA TAAAGATGAG ATGATGCCAA CAACTGTGGT 2880
CTCTCTACTT TCAACAACAG ATCTTGAAAA GGGTTCTGTT TGTTTTAGTG ACCAGTTCAA 2940
CAGTGTTAAC TTCTCTGAGG CTGAGGGTAC TGAGGTAACC TATGAGGACG AAAGCCAGAG 3000
ACAACCCTTT GTTAAATACG CCACGCTGAT CAGCAACTCT AAACCAAGTG AAACTGGTGA 3060
AGA 3063
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(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 969 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Ile Cys Gln Lys Phe Cys Val Val Leu Leu His Trp Glu Phe Ile
1 5 10 15
Tyr Val Ile Thr Ala Phe Asn Leu Ser Tyr Pro Ile Thr Pro Trp Arg
20 25 30
Phe Lys Leu Ser Cys Met Pro Pro Asn Ser Thr Tyr Asp Tyr Phe Leu
35 40 45
Leu Pro Ala Gly Leu Ser Lys Asn Thr Ser Asn Ser Asn Gly His Tyr
50 55 60
Glu Thr Ala Val Glu Pro Lys Phe Asn Ser Ser Gly Thr His Phe Ser
65 70 75 80
Asn Leu Ser Lys Thr Thr Phe His Cys Cys Phe Arg Ser Glu Gln Asp
85 90 95
Arg Asn Cys Ser Leu Cys Ala Asp Asn Ile Glu Gly Lys Thr Phe Val
100 105 110
Ser Thr Val Asn Ser Leu Val Phe Gln Gln Ile Asp Ala Asn Trp Asn
115 120 125
Ile Gln Cys Trp Leu Lys Gly Asp Leu Lys Leu Phe Ile Cys Tyr Val
130 135 140
Glu Ser Leu Phe Lys Asn Leu Phe Arg Asn Tyr Asn Tyr Lys Val His
145 150 155 160
Leu Leu Tyr Val Leu Pro Glu Val Leu Glu Asp Ser Pro Leu Val Pro
165 170 175
Gln Lys Gly Ser Phe Gln Met Val His Cys Asn Cys Ser Val His Glu
180 185 190
Cys Cys Giu Cys Leu Val Pro Vai Pro Thr Ala Lys Leu Asn Asp Thr
195 200 205
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Leu Leu Met Cys Leu Lys Ile Thr Ser Gly Gly Val Ile Phe Gin Ser
210 215 220
Pro Leu Met Ser Val Gln Pro Ile Asn Met Val Lys Pro Asp Pro Pro
225 230 235 240
Leu Gly Leu His Met Glu Ile Thr Asp Asp Gly Asn Leu Lys Ile Ser
245 250 255
Trp Ser Ser Pro Pro Leu Vai Pro Phe Pro Leu Gln Tyr Gln Val Lys
260 265 270
Tyr Ser Glu Asn Ser Thr Thr Val Ile Arg Glu Ala Asp Lys Ile Val
275 280 285
Ser Ala Thr Ser Leu Leu Val Asp Ser Ile Leu Pro Gly Ser Ser Tyr
290 295 300
Glu Val Gln Val Arg Gly Lys Arg Leu Asp Gly Pro Gly Ile Trp Ser
305 310 315 320
Asp Trp Ser Thr Pro Arg Val Phe Thr Thr Gln Asp Val Ile Tyr Phe
325 330 335
Pro Pro Lys Ile Leu Thr Ser Val Gly Ser Asn Val Ser Phe His Cys
340 345 350
I1e Tyr Lys Lys Glu Asn Lys Ile Val Pro Ser Lys Glu Ile Val Trp
355 360 365
Trp Met Asn Leu Ala Glu Lys Ile Pro Gln Ser Gln Tyr Asp Val Val
370 375 380
Ser Asp His Val Ser Lys Val Thr Phe Phe Asn Leu Asn Glu Thr Lys
385 390 395 400
Pro Arg Gly Lys Phe Thr Tyr Asp Ala Val Tyr Cys Cys Asn Glu His
405 410 415
Glu Cys His His Arg Tyr Ala Glu Leu Tyr Val Ile Asp Vai Asn Ile
420 425 430
Asn Ile Ser Cys Glu Thr Asp Gly Tyr Leu Thr Lys Met Thr Cys Arg
435 440 445
Trp Ser Thr Ser Thr Ile Gln Ser Leu Ala Glu Ser Thr Leu Gln Leu
450 455 460
Arg Tyr His Arg Ser Ser Leu Tyr Cys Ser Asp Ile Pro Ser Ile His
465 470 475 480
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.Pro Ile Ser Glu Pro Lys Asp Cys Tyr Leu Gin Ser Asp Gly Phe Tyr
485 490 495
Glu Cys Ile Phe Gln Pro I1e Phe Leu Leu Ser G1y Tyr Thr Met Trp
500 505 510
Ile Arg Ile Asn His Ser Leu Gly Ser I4eu Asp Ser Pro Pro Thr Cys
515 520 525
Val Leu Pro Asp Ser Val Val Lys Pro Leu Pro Pro Ser Ser Val Lys
530 535 540
Ala Glu Ile Thr Ile Asn Ile Gly Leu Leu Lys Ile Ser Trp Glu Lys
545 550 555 560
Pro Val Phe Pro Glu Asn Asn Leu Gin Phe G1n Ile Arg Tyr Gly Leu
565 570 575
Ser Gly Lys Glu Val Gln Trp Lys Met Tyr Glu Val Tyr Asp Ala Lys
580 585 590
Ser Lys Ser Val Ser Leu Pro Val Pro Asp Leu Cys Ala Val Tyr Ala
595 600 605
Val Gin Val Arg Cys Lys Arg Leu Asp Gly Leu Gly Tyr Trp Ser Asn
610 615 620
Trp Ser Asn Pro Ala Tyr Thr Val Val Met Asp Ile Lys Val Pro Met
625 630 635 640
Arg Gly Pro Glu Phe Trp Arg Ile Ile Asn Gly Asp Thr Met Lys Lys
645 650 655
Glu Lys Asn Val Thr Leu Leu Trp Lys Pro Leu Met Lys Asn Asp Ser
660 665 670
Leu Cys Ser Val Gln Arg Tyr Val I1e Asn His His Thr Ser Cys Asn
675 680 685
Gly Thr Trp Ser Glu Asp Val Gly Asn His Thr Lys Phe Thr Phe Leu
690 695 700
Trp Thr Glu Gin Ala His Thr Vai Thr Val Leu Ala Ile Asn Ser Ile
705 710 715 720
Gly Ala Ser Val Ala Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser
725 730 735
Lys Val Asn Ile Val Gln Ser Leu Ser Ala Tyr Pro Leu Asn Ser Ser
740 745 750
Cys Val Ile Val Ser Trp Ile Leu Ser Pro Ser Asp Tyr Lys Leu Met
755 760 765
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Tyr Phe Ile Ile Glu Trp Lys Asn Leu Asn Glu Asp Gly Glu Ile Lys
770 775 780
Trp Leu Arg Ile Ser Ser Ser Val Lys Lys Tyr Tyr Ile His Asp His
785 790 795 800
Phe Ile Pro Ile Glu Lys Tyr Gln Phe Ser Leu Tyr Pro Ile Phe Met
805 810 815
Glu Gly Val Gly Lys Pro Lys Ile Ile Asn Ser Phe Thr Gln Asp Asp
820 825 830
Ile Glu Lys His Gln Ser Asp Ala Gly Leu Tyr Val Ile Val Pro Val
835 840 845
Ile Ile Ser Ser Ser Ile Leu Leu Leu Gly Thr Leu Leu Ile Ser His
850 855 860
Gln Arg Met Lys Lys Leu Phe Trp G1u Asp Val Pro Asn Pro Lys Asn
865 870 875 880
Cys Ser Trp Ala Gln Gly Leu Asn Phe Gln Lys Met Leu Glu Gly Ser
885 890 895
Met Phe Val Lys Ser His His His Ser Leu Ile Ser Ser Thr Gln Gly
900 905 910
His Lys His Cys Gly Arg Pro Gln Giy Pro Leu His Arg Lys Thr Arg
915 920 925
Asp Leu Cys Ser Leu Val Tyr Leu Leu Thr Leu Pro Pro Leu Leu Ser
930 935 940
Tyr Asp Pro Ala Lys Ser Pro Ser Val Arg Asn Thr Gln Glu Ser Ile
945 950 955 960
Lys Lys Lys Lys Lys Lys Leu Glu Gly
965
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 969 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
1
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ile Cys Gln Lys Phe Cys Val Val Leu Leu His Trp Glu Phe Ile
1 5 10 15
Tyr Val Ile Thr Ala Phe Asn Leu Ser Tyr Pro Ile Thr Pro Trp Arg
20 25 30
Phe Lys Leu Ser Cys Met Pro Pro Asn Ser Thr Tyr Asp Tyr Phe Leu
35 40 45
Leu Pro Ala Giy Leu Ser Lys Asn Thr Ser Asn Ser Asn Gly His Tyr
50 55 60
Glu Thr Ala Val Glu Pro Lys Phe Asn Ser Ser Gly Thr His Phe Ser
65 70 75 80
Asn Leu Ser Lys Thr Thr Phe His Cys Cys Phe Arg Ser Glu Gln Asp
85 90 95
Arg Asn Cys Ser Leu Cys Ala Asp Asn Ile Glu Gly Lys Thr Phe Val
100 105 110
Ser Thr Val Asn Ser Leu Val Phe Gln Gln Ile Asp Ala Asn Trp Asn
115 120 125
Ile Gln Cys Trp Leu Lys Gly Asp Leu Lys Leu Phe Ile Cys Tyr Val
130 135 140
Glu Ser Leu Phe Lys Asn Leu Phe Arg Asn Tyr Asn Tyr Lys Val His
145 150 155 160
Leu Leu Tyr Val Leu Pro Glu Val Leu Glu Asp Ser Pro Leu Val Pro
165 170 175
Gin Lys Gly Ser Phe Gln Met Val His Cys Asn Cys Ser Val His Glu
180 185 190
Cys Cys Giu Cys Leu Val Pro Val Pro Thr Ala Lys Leu Asn Asp Thr
195 200 205
Leu Leu Met Cys Leu Lys Ile Thr Ser Gly Gly Val Ile Phe Gln Ser
210 215 220
Pro Leu Met Ser Val Gln Pro Ile Asn Met Val Lys Pro Asp Pro Pro
225 230 235 240
Leu Gly Leu His Met Glu Ile Thr Asp Asp Gly Asn Leu Lys Ile Ser
245 250 255
Trp Ser Ser Pro Pro Leu Val Pro Phe Pro Leu Gin Tyr Gln Val Lys
260 265 270
~
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Tyr Ser Glu Asn Ser Thr Thr Val Ile Arg Glu Ala Asp Lys Ile Val
275 280 285
Ser Ala Thr Ser Leu Leu Val Asp Ser Ile Leu Pro Gly Ser Ser Tyr
290 295 300
Glu Val Gln Val Arg Gly Lys Arg Leu Asp Gly Pro Gly Ile Trp Ser
305 310 315 320
Asp Trp Ser Thr Pro Arg Val Phe Thr Thr Gln Asp Val Ile Tyr Phe
325 330 335
Pro Pro Lys Ile Leu Thr Ser Val Gly Ser Asn Val Ser Phe His Cys
340 345 350
Ile Tyr Lys Lys Glu Asn Lys Ile Val Pro Ser Lys Glu Ile Val Trp
355 360 365
Trp Met Asn Leu Ala Glu Lys Ile Pro Gln Ser Gin Tyr Asp Val Val
370 375 380
Ser Asp His Val Ser Lys Val Thr Phe Phe Asn Leu Asn Glu Thr Lys
385 390 395 400
Pro Arg Gly Lys Phe Thr Tyr Asp Ala Val Tyr Cys Cys Asn Glu His
405 410 415
Giu Cys His His Arg Tyr Ala Glu Leu Tyr Val Ile Asp Val Asn Ile
420 425 430
Asn Ile Ser Cys Glu Thr Asp Giy Tyr Leu Thr Lys Met Thr Cys Arg
435 440 445
Trp Ser Thr Ser Thr Ile Gln Ser Leu Ala Glu Ser Thr Leu Gin Leu
450 455 460
Arg Tyr His Arg Ser Ser Leu Tyr Cys Ser Asp Ile Pro Ser Ile His
465 470 475 480
Pro Ile Ser Glu Pro Lys Asp Cys Tyr Leu Gln Ser Asp Gly Phe Tyr
485 490 495
Glu Cys Ile Phe Gln Pro I1e Phe Leu Leu Ser Gly Tyr Thr Met Trp
500 505 510
Ile Arg Ile Asn His Ser Leu Gly Ser Leu Asp Ser Pro Pro Thr Cys
515 520 525
Val Leu Pro Asp Ser Val Val Lys Pro Leu Pro Pro Ser Ser Vai Lys
530 535 540
Ala Glu Ile Thr Ile Asn Ile Gly Leu Leu Lys Ile Ser Trp Glu Lys
545 550 555 560
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Pro Val Phe Pro Glu Asn Asn Leu Gln Phe Gln Ile Arg Tyr Gly Leu
565 570 575
Ser Gly Lys Glu Val Gin Trp Lys Met Tyr Glu Val Tyr Asp Ala Lys
580 585 590
Ser Lys Ser Val Ser Leu Pro Val Pro Asp Leu Cys Ala Val Tyr Ala
595 600 605
Val Gln Val Arg Cys Lys Arg Leu Asp Gly Leu Gly Tyr Trp Ser Asn
610 615 620
Trp Ser Asn Pro Ala Tyr Thr Val Val Met Asp Ile Lys Val Pro Met
625 630 635 640
Arg Gly Pro Glu Phe Trp Arg Ile Ile Asn Gly Asp Thr Met Lys Lys
645 650 655
Glu Lys Asn Val Thr Leu Leu Trp Lys Pro Leu Met Lys Asn Asp Ser
660 665 670
Leu Cys Ser Vai Gin Arg Tyr Vai Ile Asn His His Thr Ser Cys Asn
675 680 685
Gly Thr Trp Ser Glu Asp Val Gly Asn His Thr Lys Phe Thr Phe Leu
690 695 700
Trp Thr Glu Gln Ala His Thr Val Thr Vai Leu Ala Ile Asn Ser Ile
705 710 715 720
Gly Ala Ser Val Ala Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser
725 730 735
Lys Val Asn Ile Val Gln Ser Leu Ser Ala Tyr Pro Leu Asn Ser Ser
740 745 750
Cys Val Ile Val Ser Trp Ile Leu Ser Pro Ser Asp Tyr Lys Leu Met
755 760 765
Tyr Phe Ile Ile Glu Trp Lys Asn Leu Asn Glu Asp Gly Giu Ile Lys
770 775 780
Trp Leu Arg Ile Ser Ser Ser Val Lys Lys Tyr Tyr Ile His Asp His
785 790 795 800
Phe Ile Pro Ile Glu Lys Tyr Gln Phe Ser Leu Tyr Pro Ile Phe Met
805 810 815
Glu Gly Val Gly Lys Pro Lys Ile Ile Asn Ser Phe Thr Gln Asp Asp
820 825 830
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Ile Glu Lys His Gln Ser Asp Ala Gly Leu Tyr Val Ile Val Pro Val
835 840 845
Ile Ile Ser Ser Ser Ile Leu Leu Leu Gly Thr Leu Leu Ile Ser His
850 855 860
Gln Arg Met Lys Lys Leu Phe Trp Glu Asp Val Pro Asn Pro Lys Asn
865 870 875 880
Cys Ser Trp Ala Gln Gly Leu Asn Phe Gin Lys Met Leu Glu Gly Ser
885 890 895
Met Phe Val Lys Ser His His His Ser Leu Ile Ser Ser Thr Gln Gly
900 905 910
His Lys His Cys Gly Arg Pro Gln Gly Pro Leu His Arg Lys Thr Arg
915 920 925
Asp Leu Cys Ser Leu Val Tyr Leu Leu Thr Leu Pro Pro Leu Leu Ser
930 935 940
Tyr Asp Pro Ala Lys Ser Pro Ser Val Arg Asn Thr Gln Glu Ser Ile
945 950 955 960
Lys Lys Lys Lys Lys Lys Leu Glu Gly
965
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1216 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Met Ile Cys Gln Lys Phe Cys Val Vai Leu Leu His Trp Glu Phe Ile
1 5 10 15
Tyr Val Ile Thr Ala Phe Asn Leu Ser Tyr Pro Ile Thr Pro Trp Arg
20 25 30
Phe Lys Leu Ser Cys Met Pro Pro Asn Ser Thr Tyr Asp Tyr Phe Leu
35 40 45
Leu Pro Ala Gly Leu Ser Lys Asn Thr Ser Asn Ser Asn Giy His Tyr
50 55 60
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Glu Thr Ala Val Glu Pro Lys Phe Asn Ser Ser Gly Thr His Phe Ser
65 70 75 80
Asn Leu Ser Lys Thr Thr Phe His Cys Cys Phe Arg Ser Glu Gin Asp
85 90 95
Arg Asn Cys Ser Leu Cys Ala Asp Asn Ile Glu Gly Lys Thr Phe Val
100 105 110
Ser Thr Val Asn Ser Leu Val Phe Gln Gln Ile Asp Ala Asn Trp Asn
115 120 125
Ile Gln Cys Trp Leu Lys Gly Asp Leu Lys Leu Phe Ile Cys Tyr Val
130 135 140
Glu Ser Leu Phe Lys Asn Leu Phe Arg Asn Tyr Asn Tyr Lys Vai His
145 150 155 160
Leu Leu Tyr Val Leu Pro Glu Val Leu Giu Asp Ser Pro Leu Val Pro
165 170 175
Gln Lys Gly Ser Phe Gln Met Val His Cys Asn Cys Ser Val His Glu
180 185 190
Cys Cys Glu Cys Leu Val Pro Val Pro Thr Ala Lys Leu Asn Asp Thr
195 200 205
Leu Leu Met Cys Leu Lys Ile Thr Ser Giy Gly Val Ile Phe Gin Ser
210 215 220
Pro Leu Met Ser Val Gln Pro Ile Asn Met Val Lys Pro Asp Pro Pro
225 230 235 240
Leu Gly Leu His Met Glu Ile Thr Asp Asp Gly Asn Leu Lys Ile Ser
245 250 255
Trp Ser Ser Pro Pro Leu Val Pro Phe Pro Leu Gln Tyr Gln Val Lys
260 265 270
Tyr Ser Glu Asn Ser Thr Thr Val Ile Arg Glu Ala Asp Lys Ile Val
275 280 285
Ser Ala Thr Ser Leu Leu Val Asp Ser Ile Leu Pro Gly Ser Ser Tyr
290 295 300
Glu Val Gln Val Arg Giy Lys Arg Leu Asp G1y Pro Gly Ile Trp Ser
305 310 315 320
Asp Trp Ser Thr Pro Arg Val Phe Thr Thr Gin Asp Val Ile Tyr Phe
325 330 335
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Pro Pro Lys Ile Leu Thr Ser Val Gly Ser Asn Val Ser Phe His Cys
340 345 350
Ile Tyr Lys Lys Glu Asn Lys Ile Val Pro Ser Lys Glu Ile Val Trp
355 360 365
Trp Met Asn Leu Ala Glu Lys Ile Pro Gln Ser Gin Tyr Asp Val Val
370 375 380
Ser Asp His Val Ser Lys Val Thr Phe Phe Asn Leu Asn Glu Thr Lys
385 390 395 400
Pro Arg Gly Lys Phe Thr Tyr Asp Ala Val Tyr Cys Cys Asn Glu His
405 410 415
Glu Cys His His Arg Tyr Ala Glu Leu Tyr Val Ile Asp Val Asn Ile
420 425 430
Asn Ile Ser Cys Glu Thr Asp Gly Tyr Leu Thr Lys Met Thr Cys Arg
435 440 445
Trp Ser Thr Ser Thr Ile Gln Ser Leu Ala Glu Ser Thr Leu Gln Leu
450 455 460
Arg Tyr His Arg Ser Ser Leu Tyr Cys Ser Asp Ile Pro Ser Ile His
465 470 475 480
Pro Ile Ser Glu Pro Lys Asp Cys Tyr Leu Gln Ser Asp Gly Phe Tyr
485 490 495
Glu Cys Ile Phe Gln Pro Ile Phe Leu Leu Ser Gly Tyr Thr Met Trp
500 505 510
Ile Arg Ile Asn His Ser Leu Gly Ser Leu Asp Ser Pro Pro Thr Cys
515 520 525
Val Leu Pro Asp Ser Val Val Lys Pro Leu Pro Pro Ser Ser Val Lys
530 535 540
Ala Glu I1e Thr Ile Asn Ile Gly Leu Leu Lys Ile Ser Trp Glu Lys
545 550 555 560
Pro Val Phe Pro Glu Asn Asn Leu Gln Phe Gln Ile Arg Tyr Gly Leu
565 570 575
Ser Gly Lys Glu Val Gln Trp Lys Met Tyr Giu Val Tyr Asp Ala Lys
580 585 590
Ser Lys Ser Val Ser Leu Pro Val Pro Asp Leu Cys Ala Val Tyr Ala
595 600 605
Val Gln Val Arg Cys Lys Arg Leu Asp Gly Leu Gly Tyr Trp Ser Asn
610 615 620
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Trp Ser Asn Pro Ala Tyr Thr Val Val Met Asp Ile Lys Val Pro Met
625 630 635 640
Arg Gly Pro Glu Phe Trp Arg Ile Ile Asn Gly Asp Thr Met Lys Lys
645 650 655
Glu Lys Asn Val Thr Leu Leu Trp Lys Pro Leu Met Lys Asn Asp Ser
660 665 670
Leu Cys Ser Val Gln Arg Tyr Val Ile Asn His His Thr Ser Cys Asn
675 680 685
Gly Thr Trp Ser Glu Asp Val Gly Asn His Thr Lys Phe Thr Phe Leu
690 695 700
Trp Thr Glu Gln Ala His Thr Val Thr Val Leu Ala Ile Asn Ser Ile
705 710 715 720
Gly Ala Ser Val Ala Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser
725 730 735
Lys Val Asn Ile Val Gln Ser Leu Ser Ala Tyr Pro Leu Asn Ser Ser
740 745 750
Cys Val Ile Val Ser Trp Ile Leu Ser Pro Ser Asp Tyr Lys Leu Met
755 760 765
Tyr Phe Ile Ile Glu Trp Lys Asn Leu Asn Glu Asp Gly Glu Ile Lys
770 775 780
Trp Leu Arg Ile Ser Ser Ser Val Lys Lys Tyr Tyr Ile His Asp His
785 790 795 800
Phe Ile Pro Ile Glu Lys Tyr Gln Phe Ser Leu Tyr Pro Ile Phe Met
805 810 815
Glu Gly Val Gly Lys Pro Lys Ile Ile Asn Ser Phe Thr Gin Asp Asp
820 825 830
Ile Glu Lys His Gln Ser Asp Ala Gly Leu Tyr Val Ile Val Pro Val
835 840 845
Ile I1e Ser Ser Ser Ile Leu Leu Leu Gly Thr Leu Leu Ile Ser His
850 855 860
Gin Arg Met Lys Lys Leu Phe Trp Glu Asp Val Pro Asn Pro Lys Asn
865 870 875 880
Cys Ser Trp Ala Gln Gly Leu Asn Phe Gln Lys Pro Glu Thr Phe Glu 885 890 895
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His Leu Phe Ile Lys His Thr Ala Ser Val Thr Cys Gly Pro Leu Leu
900 905 910
Leu Glu Pro Glu Thr Ile Ser Glu Asp Ile Ser Val Asp Thr Ser Trp
915 920 925
Lys Asn Lys Asp Glu Met Met Pro Thr Thr Val Val Ser Leu Leu Ser
930 935 940
Thr Thr Asp Leu Glu Lys Gly Ser Val Cys Ile Ser Asp Gln Phe Asn
945 950 955 960
Ser Val Asn Phe Ser Glu Ala Glu Gly Thr Glu Val Thr Tyr Glu Asp
965 970 975
Glu Ser Gln Arg Gln Pro Phe Val Lys Tyr Ala Thr Leu Ile Ser Asn
980 985 990
Ser Lys Pro Ser Glu Thr Gly Glu Giu Gin Gly Leu Ile Asn Ser Ser
995 1000 1005
Val Thr Lys Cys Phe Ser Ser Lys Asn Ser Pro Leu Lys Asp Ser Phe
1010 1015 1020
Ser Asn Ser Ser Trp Glu Ile Giu Ala Gin Ala Phe Phe Ile Leu Ser
1025 1030 1035 1040
Asp Gln His Pro Asn Ile Ile Ser Pro His Leu Thr Phe Ser Glu Gly
1.045 1050 1055
Leu Asp Glu Leu Leu Lys Leu Glu Gly Asn Phe Pro Giu Glu Asn Asn
1060 1065 1070
Asp Lys Lys Ser Ile Tyr Tyr Leu Gly Val Thr Ser Ile Lys Lys Arg
1075 1080 1085
Glu Ser Gly Val Leu Leu Thr Asp Lys Ser Arg Val Ser Cys Pro Phe
1090 1095 1100
Pro Ala Pro Cys Leu Phe Thr Asp I1e Arg Val Leu Gln Asp Ser Cys
1105 1110 1115 1120
Ser His Phe Val Glu Asn Asn Ile Asn Leu Gly Thr Ser Ser Lys Lys
1125 1130 1135
Thr Phe Ala Ser Tyr Met Pro Gln Phe Gln Thr Cys Ser Thr Gln Thr
1140 1145 1150
His Lys Ile Met Glu Asn Lys Met Cys Asp Leu Thr Val Phe His Arg
1155 1160 1165
Asn Leu Gin Ile Cys Vai Ile Met Gly Asn Ile Lys Cys Asn Arg Leu
1170 1175 1180
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Leu Trp Val Gly Glu Arg Lys Glu Thr Arg Val Lys Phe Glu Asn Asn
1185 1190 1195 1200
Cys Ser Lys Lys Lys Lys Lys Lys Asn Ser Arg Pro Ala Arg Pro Asp
1205 1210 1215
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3599 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GCGGCCGCCA GTGTGATGGA TATCTGCAGA ATTCGGCTTT CTCTGCCTTC GGTCGAGTTG 60
GACCCCCGGA TCAAGGTGTA CTTCTCTGAA GTAAGATGAT TTGTCAAAAA TTCTGTGTGG 120
TTTTGTTACA TTGGGAATTT ATTTATGTGA TAACTGCGTT TAACTTGTCA TATCCAATTA 180
CTCCTTGGAG ATTTAAGTTG TCTTGCATGC CACCAAATTC AACCTATGAC TACTTCCTTT 240
TGCCTGCTGG GCTCTCAAAG AATACTTCAA ATTCGAATGG ACATTATGAG ACAGCTGTTG 300
AACCTAAGTT TAATTCAAGT GGTACTCACT TTTCTAACTT ATCCAAAACA ACTTTCCACT 360
GTTGCTTTCG GAGTGAGCAA GATAGAAACT GCTCCTTATG TGCAGACAAC ATTGAAGGAA 420
AGACATTTGT TTCAACAGTA AATTCTTTAG TTTTTCAACA AATAGATGCA AACTGGAACA 480
TACAGTGCTG GCTAAAAGGA GACTTAAAAT TATTCATCTG TTATGTGGAG TCATTATTTA 540
AGAATCTATT CAGGAATTAT AACTATAAGG TCCATCTTTT ATATGTTCTG CCTGAAGTGT 600
TAGAAGATTC ACCTCTGGTT CCCCAAAAAG GCAGTTTTCA GATGGTTCAC TGCAATTGCA 660
GTGTTCACGA ATGTTGTGAA TGTCTTGTGC CTGTGCCAAC AGCCAAACTC AACGACACTC 720
TCCTTATGTG TTTGAAAATC ACATCTGGTG GAGTAATTTT CCAGTCACCT CTAATGTCAG 780
TTCAGCCCAT AAATATGGTG AAGCCTGATC CACCATTAGG TTTGCATATG GAAATCACAG 840
ATGATGGTAA TTTAAAGATT TCTTGGTCCA GCCCACCATT GGTACCATTT CCACTTCAAT 900
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ATCAAGTGAA ATATTCAGAG AATTCTACAA CAGTTATCAG AGAAGCTGAC AAGATTGTCT 960
CAGCTACATC CCTGCTAGTA GACAGTATAC TTCCTGGGTC TTCGTATGAG GTTCAGGTGA 1020
GGGGCAAGAG ACTGGATGGC CCAGGAATCT GGAGTGACTG GAGTACTCCT CGTGTCTTTA 1080
CCACACAAGA TGTCATATAC TTTCCACCTA AAATTCTGAC AAGTGTTGGG TCTAATGTTT 1140
CTTTTCACTG CATCTATAAG AAGGAAAACA AGATTGTTCC CTCAAAAGAG ATTGTTTGGT 1200
GGATGAATTT AGCTGAGAAA ATTCCTCAAA GCCAGTATGA TGTTGTGAGT GATCATGTTA 1260
GCAAAGTTAC TTTTTTCAAT CTGAATGAAA CCAAACCTCG AGGAAAGTTT ACCTATGATG 1320
CAGTGTACTG CTGCAATGAA CATGAATGCC ATCATCGCTA TGCTGAATTA TATGTGATTG 1380
ATGTCAATAT CAATATCTCA TGTGAAACTG ATGGGTACTT AACTAAAATG ACTTGCAGAT 1440
GGTCAACCAG TACAATCCAG TCACTTGCGG AAAGCACTTT GCAATTGAGG TATCATAGGA 1500
GCAGCCTTTA CTGTTCTGAT ATTCCATCTA TTCATCCCAT ATCTGAGCCC AAAGATTGCT 1560
ATTTGCAGAG TGATGGTTTT TATGAATGCA TTTTCCAGCC AATCTTCCTA TTATCTGGCT 1620
ACACAATGTG GATTAGGATC AATCACTCTC TAGGTTCACT TGACTCTCCA CCAACATGTG 1680
TCCTTCCTGA TTCTGTGGTG AAGCCACTGC CTCCATCCAG TGTGAAAGCA GAAATTACTA 1740
TAAACATTGG ATTATTGAAA ATATCTTGGG AAAAGCCAGT CTTTCCAGAG AATAACCTTC 1800
AATTCCAGAT TCGCTATGGT TTAAGTGGAA AAGAAGTACA ATGGAAGATG TATGAGGTTT 1860
ATGATGCAAA ATCAAAATCT GTCAGTCTCC CAGTTCCAGA CTTGTGTGCA GTCTATGCTG 1920
TTCAGGTGCG CTGTAAGAGG CTAGATGGAC TGGGATATTG GAGTAATTGG AGCAATCCAG 1980
CCTACACAGT TGTCATGGAT ATAAAAGTTC CTATGAGAGG ACCTGAATTT TGGAGAATAA 2040
TTAATGGAGA TACTATGAAA AAGGAGAAAA ATGTCACTTT ACTTTGGAAG CCCCTGATGA 2100
AAAATGACTC ATTGTGCAGT GTTCAGAGAT ATGTGATAAA CCATCATACT TCCTGCAATG 2160
GAACATGGTC AGAAGATGTG GGAAATCACA CGAAATTCAC TTTCCTGTGG ACAGAGCAAG 2220
CACATACTGT TACGGTTCTG GCCATCAATT CAATTGGTGC TTCTGTTGCA AATTTTAATT 2280
TAACCTTTTC ATGGCCTATG AGCAAAGTAA ATATCGTGCA GTCACTCAGT GCTTATCCTT 2340
TAAACAGCAG TTGTGTGATT GTTTCCTGGA TACTATCACC CAGTGATTAC AAGCTAATGT 2400
ATTTTATTAT TGAGTGGAAA AATCTTAATG AAGATGGTGA AATAAAATGG CTTAGAATCT 2460
CTTCATCTGT TAAGAAGTAT TATATCCATG ATCATTTTAT CCCCATTGAG AAGTACCAGT 2520
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TCAGTCTTTA CCCAATATTT ATGGAAGGAG TGGGAAAACC AAAGATAATT AATAGTTTCA 2580
CTCAAGATGA TATTGAAAAA CACCAGAGTG ATGCAGGTTT ATATGTAATT GTGCCAGTAA 2640
TTATTTCCTC TTCCATCTTA TTGCTTGGAA CATTATTAAT ATCACACCAA AGAATGAAAA 2700 ~
AGCTATTTTG GGAAGATGTT CCGAACCCCA AGAATTGTTC CTGGGCACAA GGACTTAATT 2760
TTCAGAAGCC AGAAACGTTT GAGCATCTTT TTATCAAGCA TACAGCATCA GTGACATGTG 2820
GTCCTCTTCT TTTGGAGCCT GAAACAATTT CAGAAGATAT CAGTGTTGAT ACATCATGGA 2880
AAAATAAAGA TGAGATGATG CCAACAACTG TGGTCTCTCT ACTTTCAACA ACAGATCTTG 2940
AAAAGGGTTC TGTTTGTATT AGTGACCAGT TCAACAGTGT TAACTTCTCT GAGGCTGAGG 3000
GTACTGAGGT AACCTATGAG GACGAAAGCC AGAGACAACC CTTTGTTAAA TACGCCACGC 3060
TGATCAGCAA CTCTAAACCA AGTGAAACTG GTGAAGAACA AGGGCTTATA AATAGTTCAG 3120
TCACCAAGTG CTTCTCTAGC AAAAATTCTC CGTTGAAGGA TTCTTTCTCT AATAGCTCAT 3180
GGGAGATAGA GGCCCAGGCA TTTTTTATAT TATCGGATCA GCATCCCAAC ATAATTTCAC 3240
CACACCTCAC ATTCTCAGAA GGATTGGATG AACTTTTGAA ATTGGAGGGA AATTTCCCTG 3300
AAGAAAATAA TGATAAAAAG TCTATCTATT ATTTAGGGGT CACCTCAATC AAAAAGAGAG 3360
AGAGTGGTGT GCTTTTGACT GACAAGTCAA GGGTATCGTG CCCATTCCCA GCCCCCTGTT 3420
TATTCACGGA CATCAGAGTT CTCCAGGACA GTTGCTCACA CTTTGTAGAA AATAATATCA 3480
ACTTAGGAAC TTCTAGTAAG AAGACTTTTG CATCTTACAT GCCTCAATTC CAAACTTGTT 3540
CTACTCAGAC TCATAAGATC ATGGAAAACA AGATGTGTGA CCTAACTGTG TAATCTAGA 3599
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
NNNNNTACCT TTTCCAG 17
fi
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 839 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Ile Cys Gln Lys Phe Cys Val Val Leu Leu His Trp Glu Phe Ile
1 5 10 15
Tyr Val Ile Thr Ala Phe Asn Leu Ser Tyr Pro Ile Thr Pro Trp Arg
20 25 30
Phe Lys Leu Ser Cys Met Pro Pro Asn Ser Thr Tyr Asp Tyr Phe Leu
40 45
Leu Pro Ala Gly Leu Ser Lys Asn Thr Ser Asn Ser Asn Gly His Tyr
30 50 55 60
Glu Thr Ala Val Glu Pro Lys Phe Asn Ser Ser Gly Thr His Phe Ser
65 70 75 80
35 Asn Leu Ser Lys Thr Thr Phe His Cys Cys Phe Arg Ser Glu Gln Asp
85 90 95
Arg Asn Cys Ser Leu Cys Ala Asp Asn Ile Glu Gly Lys Thr Phe Val
100 105 110
Ser Thr Val Asn Ser Leu Val Phe Gln G1n Ile Asp Ala Asn Trp Asn
115 120 125
Ile Gln Cys Trp Leu Lys Gly Asp Leu Lys Leu Phe Ile Cys Tyr Val
130 135 140
Glu Ser Leu Phe Lys Asn Leu Phe Arg Asn Tyr Asn Tyr Lys Val His
145 150 155 160
Leu Leu Tyr Val Leu Pro Glu Val Leu Glu Asp Ser Pro Leu Val Pro
165 170 175
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Gln Lys Gly Ser Phe Gin Met Val His Cys Asn Cys Ser Val His Glu
180 185 190
Cys Cys Glu Cys Leu Val Pro Val Pro Thr Ala Lys Leu Asn Asp Thr }
195 200 205
Leu Leu Met Cys Leu Lys Ile Thr Ser Gly Gly Val Ile Phe Gln Ser
210 215 220
Pro Leu Met Ser Val Gln Pro Ile Asn Met Val Lys Pro Asp Pro Pro
225 230 235 240
Leu Gly Leu His Met Glu Ile Thr Asp Asp Gly Asn Leu Lys Ile Ser
245 250 255
Trp Ser Ser Pro Pro Leu Val Pro Phe Pro Leu Gln Tyr Gln Vai Lys
260 265 270
Tyr Ser Giu Asn Ser Thr Thr Val Ile Arg Glu Ala Asp Lys Ile Vai
275 280 285
Ser Ala Thr Ser Leu Leu Val Asp Ser Ile Leu Pro Gly Ser Ser Tyr
290 295 300
Glu Val Gln Val Arg Gly Lys Arg Leu Asp Gly Pro Gly Ile Trp Ser
305 310 315 320
Asp Trp Ser Thr Pro Arg Val Phe Thr Thr Gin Asp Val Ile Tyr Phe
325 330 335
Pro Pro Lys Ile Leu Thr Ser Val Gly Ser Asn Val Ser Phe His Cys
340 345 350
Ile Tyr Lys Lys Glu Asn Lys Ile Val Pro Ser Lys Glu Ile Val Trp
355 360 365
Trp Met Asn Leu Ala Glu Lys Ile Pro Gln Ser Gin Tyr Asp Vai Val
370 375 380
Ser Asp His Val Ser Lys Vai Thr Phe Phe Asn Leu Asn Glu Thr Lys
385 390 395 400
Pro Arg Gly Lys Phe Thr Tyr Asp Ala Val Tyr Cys Cys Asn Glu His
405 410 415
Glu Cys His His Arg Tyr Ala Glu Leu Tyr Val Ile Asp Val Asn I1e
420 425 430
Asn Ile Ser Cys Glu Thr Asp Gly Tyr Leu Thr Lys Met Thr Cys Arg
435 440 445
Trp Ser Thr Ser Thr Ile Gln Ser Leu Ala Glu Ser Thr Leu Gln Leu
450 455 460
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Arg Tyr His Arg Ser Ser Leu Tyr Cys Ser Asp Ile Pro Ser Ile His
465 470 475 480
t
Pro Ile Ser Glu Pro Lys Asp Cys Tyr Leu Gln Ser Asp Gly Phe Tyr
485 490 495
Glu Cys Ile Phe Gln Pro Ile Phe Leu Leu Ser Gly Tyr Thr Met Trp
500 505 510
Ile Arg Ile Asn His Ser Leu Gly Ser Leu Asp Ser Pro Pro Thr Cys
515 520 525
Val Leu Pro Asp Ser Val Vai Lys Pro Leu Pro Pro Ser Ser Val Lys
530 535 540
Ala Glu Ile Thr Ile Asn Ile Gly Leu Leu Lys Ile Ser Trp Glu Lys
545 550 555 560
Pro Val Phe Pro Glu Asn Asn Leu Gln Phe Gln Ile Arg Tyr Gly Leu
565 570 575
Ser Gly Lys Giu Val Gln Trp Lys Met Tyr Glu Val Tyr Asp Ala Lys
580 585 590
Ser Lys Ser Vai Ser Leu Pro Vai Pro Asp Leu Cys Ala Val Tyr Ala
595 600 605
Val Gln Val Arg Cys Lys Arg Leu Asp Gly Leu Gly Tyr Trp Ser Asn
610 615 620
Trp Ser Asn Pro Ala Tyr Thr Val Val Met Asp Ile Lys Val Pro Met
625 630 635 640
Arg Gly Pro Glu Phe Trp Arg Ile Ile Asn Gly Asp Thr Met Lys Lys
645 650 655
Giu Lys Asn Val Thr Leu Leu Trp Lys Pro Leu Met Lys Asn Asp Ser
660 665 670
Leu Cys Ser Val Gln Arg Tyr Val Ile Asn His His Thr Ser Cys Asn
675 680 685
Gly Thr Trp Ser Glu Asp Val Gly Asn His Thr Lys Phe Thr Phe Leu
690 695 700
Trp Thr Glu Gln Ala His Thr Val Thr Vai Leu Ala Ile Asn Ser Ile
705 710 715 720
Gly Ala Ser Val Ala Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser
725 730 735
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Lys Val Asn Ile Val Gln Ser Leu Ser Ala Tyr Pro Leu Asn Ser Ser
740 745 750
Cys Val I1e Val Ser Trp Ile Leu Ser Pro Ser Asp Tyr Lys Leu Met
755 760 765
Tyr Phe Ile Ile Glu Trp Lys Asn Leu Asn Glu Asp Gly Glu Ile Lys
770 775 780
Trp Leu Arg Ile Ser Ser Ser Val Lys Lys Tyr Tyr Ile His Asp His
785 790 795 800
Phe Ile Pro Ile Glu Lys Tyr Gln Phe Ser Leu Tyr Pro Ile Phe Met
805 810 815
Glu Gly Val Gly Lys Pro Lys Ile Ile Asn Ser Phe Thr Gln Asp Asp
820 825 830
Ile Glu Lys His Gln Ser Asp
835
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2624 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GCGGCCGCCA GTGTGATGGA TATCTGCAGA ATTCGGCTTT CTCTGCCTTC GGTCGAGTTG 60
GACCCCCGGA TCAAGGTGTA CTTCTCTGAA GTAAGATGAT TTGTCAAAAA TTCTGTGTGG 120
TTTTGTTACA TTGGGAATTT ATTTATGTGA TAACTGCGTT TAACTTGTCA TATCCAATTA 180
CTCCTTGGAG ATTTAAGTTG TCTTGCATGC CACCAAATTC AACCTATGAC TACTTCCTTT 240
TGCCTGCTGG GCTCTCAAAG AATACTTCAA ATTCGAATGG ACATTATGAG ACAGCTGTTG 300
AACCTAAGTT TAATTCAAGT GGTACTCACT TTTCTAACTT ATCCAAAACA ACTTTCCACT 360
GTTGCTTTCG GAGTGAGCAA GATAGAAACT GCTCCTTATG TGCAGACAAC ATTGAAGGAA 420
AGACATTTGT TTCAACAGTA AATTCTTTAG TTTTTCAACA AATAGATGCA AACTGGAACA 480
TACAGTGCTG GCTAAAAGGA GACTTAAAAT TATTCATCTG TTATGTGGAG TCATTATTTA 540
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AGAATCTATT CAGGAATTAT AACTATAAGG TCCATCTTTT ATATGTTCTG CCTGAAGTGT 600
TAGAAGATTC ACCTCTGGTT CCCCAAAAAG GCAGTTTTCA GATGGTTCAC TGCAATTGCA 660
GTGTTCACGA ATGTTGTGAA TGTCTTGTGC CTGTGCCAAC AGCCAAACTC AACGACACTC 720
TCCTTATGTG TTTGAAAATC ACATCTGGTG GAGTAATTTT CCAGTCACCT CTAATGTCAG 780
TTCAGCCCAT AAATATGGTG AAGCCTGATC CACCATTAGG TTTGCATATG GAAATCACAG 840
ATGATGGTAA TTTAAAGATT TCTTGGTCCA GCCCACCATT GGTACCATTT CCACTTCAAT 900
ATCAAGTGAA ATATTCAGAG AATTCTACAA CAGTTATCAG AGAAGCTGAC AAGATTGTCT 960
CAGCTACATC CCTGCTAGTA GACAGTATAC TTCCTGGGTC TTCGTATGAG GTTCAGGTGA 1020
GGGGCAAGAG ACTGGATGGC CCAGGAATCT GGAGTGACTG GAGTACTCCT CGTGTCTTTA 1080
CCACACAAGA TGTCATATAC TTTCCACCTA AAATTCTGAC AAGTGTTGGG TCTAATGTTT 1140
CTTTTCACTG CATCTATAAG AAGGAAAACA AGATTGTTCC CTCAAAAGAG ATTGTTTGGT 1200
GGATGAATTT AGCTGAGAAA ATTCCTCAAA GCCAGTATGA TGTTGTGAGT GATCATGTTA 1260
GCAAAGTTAC TTTTTTCAAT CTGAATGAAA CCAAACCTCG AGGAAAGTTT ACCTATGATG 1320
CAGTGTACTG CTGCAATGAA CATGAATGCC ATCATCGCTA TGCTGAATTA TATGTGATTG 1380
ATGTCAATAT CAATATCTCA TGTGAAACTG ATGGGTACTT AACTAAAATG ACTTGCAGAT 1440
GGTCAACCAG TACAATCCAG TCACTTGCGG AAAGCACTTT GCAATTGAGG TATCATAGGA 1500
GCAGCCTTTA CTGTTCTGAT ATTCCATCTA TTCATCCCAT ATCTGAGCCC AAAGATTGCT 1560
ATTTGCAGAG TGATGGTTTT TATGAATGCA TTTTCCAGCC AATCTTCCTA TTATCTGGCT 1620
ACACAATGTG GATTAGGATC AATCACTCTC TAGGTTCACT TGACTCTCCA CCAACATGTG 1680
TCCTTCCTGA TTCTGTGGTG AAGCCACTGC CTCCATCCAG TGTGAAAGCA GAAATTACTA 1740
TAAACATTGG ATTATTGAAA ATATCTTGGG AAAAGCCAGT CTTTCCAGAG AATAACCTTC 1800
AATTCCAGAT TCGCTATGGT TTAAGTGGAA AAGAAGTACA ATGGAAGATG TATGAGGTTT 1860
ATGATGCAAA ATCAAAATCT GTCAGTCTCC CAGTTCCAGA CTTGTGTGCA GTCTATGCTG 1920
TTCAGGTGCG CTGTAAGAGG CTAGATGGAC TGGGATATTG GAGTAATTGG AGCAATCCAG 1980
CCTACACAGT TGTCATGGAT ATAAAAGTTC CTATGAGAGG ACCTGAATTT TGGAGAATAA 2040
TTAATGGAGA TACTATGAAA AAGGAGAAAA ATGTCACTTT ACTTTGGAAG CCCCTGATGA 2100
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AAAATGACTC ATTGTGCAGT GTTCAGAGAT ATGTGATAAA CCATCATACT TCCTGCAATG 2160
GAACATGGTC AGAAGATGTG GGAAATCACA CGAAATTCAC TTTCCTGTGG ACAGAGCAAG 2220
CACATACTGT TACGGTTCTG GCCATCAATT CAATTGGTGC TTCTGTTGCA AATTTTAATT 2280
TAACCTTTTC ATGGCCTATG AGCAAAGTAA ATATCGTGCA GTCACTCAGT GCTTATCCTT 2340
TAAACAGCAG TTGTGTGATT GTTTCCTGGA TACTATCACC CAGTGATTAC AAGCTAATGT 2400
ATTTTATTAT TGAGTGGAAA AATCTTAATG AAGATGGTGA AATAAAATGG CTTAGAATCT 2460
CTTCATCTGT TAAGAAGTAT TATATCCATG ATCATTTTAT CCCCATTGAG AAGTACCAGT 2520
TCAGTCTTTA CCCAATATTT ATGGAAGGAG TGGGAAAACC AAAGATAATT AATAGTTTCA 2580
CTCAAGATGA TATTGAAAAA CACCAGAGTG ATTGATAAGG ATCC 2624
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2948 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CCATTGAAGT CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGGATTT CCAAAATGTC 60
GTAATAACCC CGCCCCGTTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA 120
TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCTCTAGAAG CTGGGTACCA GCTGCTAGCA 180
AGCTTGCTAG CGGCCGCCAG TGTGATGGAT ATCTGCAGAA TTCGGCTTTC TCTGCCTTCG 240
GTCGAGTTGG ACCCCCGGAT CAAGGTGTAC TTCTCTGAAG TAAGATGATT TGTCAAAAAT 300
TCTGTGTGGT TTTGTTACAT TGGGAATTTA TTTATGTGAT AACTGCGTTT AACTTGTCAT 360
ATCCAATTAC TCCTTGGAGA TTTAAGTTGT CTTGCATGCC ACCAAATTCA ACCTATGACT 420
ACTTCCTTTT GCCTGCTGGG CTCTCAAAGA ATACTTCAAA TTCGAATGGA CATTATGAGA 480
CAGCTGTTGA ACCTAAGTTT AATTCAAGTG GTACTCACTT TTCTAACTTA TCCAAAACAA 540
CTTTCCACTG TTGCTTTCGG AGTGAGCAAG ATAGAAACTG CTCCTTATGT GCAGACAACA 600
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TTGAAGGAAA GACATTTGTT TCAACAGTAA ATTCTTTAGT TTTTCAACAA ATAGATGCAA 660
ACTGGAACAT ACAGTGCTGG CTAAAAGGAG ACTTAAAATT ATTCATCTGT TATGTGGAGT 720
~
CATTATTTAA GAATCTATTC AGGAATTATA ACTATAAGGT CCATCTTTTA TATGTTCTGC 780
=
CTGAAGTGTT AGAAGATTCA CCTCTGGTTC CCCAAAAAGG CAGTTTTCAG ATGGTTCACT 840
GCAATTGCAG TGTTCACGAA TGTTGTGAAT GTCTTGTGCC TGTGCCAACA GCCAAACTCA 900
ACGACACTCT CCTTATGTGT TTGAAAATCA CATCTGGTGG AGTAATTTTC CAGTCACCTC 960
TAATGTCAGT TCAGCCCATA AATATGGTGA AGCCTGATCC ACCATTAGGT TTGCATATGG 1020
AAATCACAGA TGATGGTAAT TTAAAGATTT CTTGGTCCAG CCCACCATTG GTACCATTTC 1080
CACTTCAATA TCAAGTGAAA TATTCAGAGA ATTCTACAAC AGTTATCAGA GAAGCTGACA 1140
AGATTGTCTC AGCTACATCC CTGCTAGTAG ACAGTATACT TCCTGGGTCT TCGTATGAGG 1200
TTCAGGTGAG GGGCAAGAGA CTGGATGGCC CAGGAATCTG GAGTGACTGG AGTACTCCTC 1260
GTGTCTTTAC CACACAAGAT GTCATATACT TTCCACCTAA AATTCTGACA AGTGTTGGGT 1320
CTAATGTTTC TTTTCACTGC ATCTATAAGA AGGAAAACAA GATTGTTCCC TCAAAAGAGA 1380
TTGTTTGGTG GATGAATTTA GCTGAGAAAA TTCCTCAAAG CCAGTATGAT GTTGTGAGTG 1440
ATCATGTTAG CAAAGTTACT TTTTTCAATC TGAATGAAAC CAAACCTCGA GGAAAGTTTA 1500
CCTATGATGC AGTGTACTGC TGCAATGAAC ATGAATGCCA TCATCGCTAT GCTGAATTAT 1560
ATGTGATTGA TGTCAATATC AATATCTCAT GTGAAACTGA TGGGTACTTA ACTAAAATGA 1620
CTTGCAGATG GTCAACCAGT ACAATCCAGT CACTTGCGGA AAGCACTTTG CAATTGAGGT 1680
ATCATAGGAG CAGCCTTTAC TGTTCTGATA TTCCATCTAT TCATCCCATA TCTGAGCCCA 1740
AAGATTGCTA TTTGCAGAGT GATGGTTTTT ATGAATGCAT TTTCCAGCCA ATCTTCCTAT 1800
TATCTGGCTA CACAATGTGG ATTAGGATCA ATCACTCTCT AGGTTCACTT GACTCTCCAC 1860
CAACATGTGT CCTTCCTGAT TCTGTGGTGA AGCCACTGCC TCCATCCAGT GTGAAAGCAG 1920
AAATTACTAT AAACATTGGA TTATTGAAAA TATCTTGGGA AAAGCCAGTC TTTCCAGAGA 1980
ATAACCTTCA ATTCCAGATT CGCTATGGTT TAAGTGGAAA AGAAGTACAA TGGAAGATGT 2040
ATGAGGTTTA TGATGCAAAA TCAAAATCTG TCAGTCTCCC AGTTCCAGAC TTGTGTGCAG 2100
TCTATGCTGT TCAGGTGCGC TGTAAGAGGC TAGATGGACT GGGATATTGG AGTAATTGGA 2160
GCAATCCAGC CTACACAGTT GTCATGGATA TAAAAGTTCC TATGAGAGGA CCTGAATTTT 2220
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GGAGAATAAT TAATGGAGAT ACTATGAAAA AGGAGAAAAA TGTCACTTTA CTTTGGAAGC 2280
CCCTGATGAA AAATGACTCA TTGTGCAGTG TTCAGAGATA TGTGATAAAC CATCATACTT 2340
-
CCTGCAATGG AACATGGTCA GAAGATGTGG GAAATCACAC GAAATTCACT TTCCTGTGGA 2400
CAGAGCAAGC ACATACTGTT ACGGTTCTGG CCATCAATTC AATTGGTGCT TCTGTTGCAA 2460
ATTTTAATTT AACCTTTTCA TGGCCTATGA GCAAAGTAAA TATCGTGCAG TCACTCAGTG 2520
,CTTATCCTTT AAACAGCAGT TGTGTGATTG TTTCCTGGAT ACTATCACCC AGTGATTACA 2580
AGCTAATGTA TTTTATTATT GAGTGGAAAA ATCTTAATGA AGATGGTGAA ATAAAATGGC 2640
TTAGAATCTC TTCATCTGTT AAGAAGTATT ATATCCATGA TCATTTTATC CCCATTGAGA 2700
AGTACCAGTT CAGTCTTTAC CCAATATTTA TGGAAGGAGT GGGAAAACCA AAGATAATTA 2760
ATAGTTTCAC TCAAGATGAT ATTGAAAAAC ACCAGAGTGA TGCAGGTGAC TACAAGGACG 2820
ACGATGACAA GTAGGGATCC AGACATGATA AGATACATTG ATGAGTTTGG ACAACCCACA 2880
ACTAGAATGC AGTGAAAAAA ATGCTTTATT TGTGAAATTT GTGATGCTAT TGCTTTATTT 2940
GTAACCAT 2948
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 804 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
'(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Ile Cys Gin Lys Phe Cys Val Val Leu Leu His Trp Glu Phe Ile
1 5 10 15
Tyr Val Ile Thr Ala Phe Asn Leu Ser Tyr Pro Ile Thr Pro Trp Arg
20 25 30
Phe Lys Leu Ser Cys Met Pro Pro Asn Ser Thr Tyr Asp Tyr Phe Leu
35 40 45
Leu Pro Ala Gly Leu Ser Lys Asn Thr Ser Asn Ser Asn Gly His Tyr
50 55 60
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Glu Thr Ala Val Glu Pro Lys Phe Asn Ser Ser Gly Thr His Phe Ser
65 70 75 80
Asn Leu Ser Lys Thr Thr Phe His Cys Cys Phe Arg Ser Glu Gln Asp
85 90 95
Arg Asn Cys Ser Leu Cys Ala Asp Asn Ile Glu Gly Lys Thr Phe Val
100 105 110
Ser Thr Val Asn Ser Leu Val Phe Gln Gln Ile Asp Ala Asn Trp Asn
115 120 125
Ile Gln Cys Trp Leu Lys Gly Asp Leu Lys Leu Phe Ile Cys Tyr Val
130 135 140
Glu Ser Leu Phe Lys Asn Leu Phe Arg Asn Tyr Asn Tyr Lys Val His
145 150 155 160
Leu Leu Tyr Val Leu Pro Glu Val Leu Glu Asp Ser Pro Leu Val Pro
165 170 175
Gln Lys Gly Ser Phe Gln Met Val His Cys Asn Cys Ser Val His Glu
180 185 190
Cys Cys Glu Cys Leu Val Pro Val Pro Thr Ala Lys Leu Asn Asp Thr
195 200 205
Leu Leu Met Cys Leu Lys Ile Thr Ser Gly Gly Val Ile Phe Gln Ser
210 215 220
Pro Leu Met Ser Val Gln Pro Ile Asn Met Val Lys Pro Asp Pro Pro
225 230 235 240
Leu Gly Leu His Met Glu Ile Thr Asp Asp Gly Asn Leu Lys Ile Ser
245 250 255
Trp Ser Ser Pro Pro Leu Val Pro Phe Pro Leu Gln Tyr Gln Val Lys
260 265 270
Tyr Ser Glu Asn Ser Thr Thr Val Ile Arg Glu Ala Asp Lys Ile Val
275 280 285
Ser Ala Thr Ser Leu Leu Val Asp Ser Ile Leu Pro Gly Ser Ser Tyr
290 295 300
Glu Val Gln Val Arg Gly Lys Arg Leu Asp Gly Pro Gly Ile Trp Ser
305 310 315 320
Asp Trp Ser Thr Pro Arg Val Phe Thr Thr Gln Asp Val Ile Tyr Phe
325 330 335
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Pro Pro Lys Ile Leu Thr Ser Val Gly Ser Asn Val Ser Phe His Cys
340 345 350
Ile Tyr Lys Lys Glu Asn Lys Ile Val Pro Ser Lys Glu Ile Vai Trp ~
355 360 365
Trp Met Asn Leu Ala Glu Lys Ile Pro Gin Ser Gln Tyr Asp Val Val
370 375 380
Ser Asp His Vai Ser Lys Val Thr Phe Phe Asn Leu Asn Glu Thr Lys
385 390 395 400
Pro Arg Gly Lys Phe Thr Tyr Asp Ala Val Tyr Cys Cys Asn Glu His
405 410 415
Glu Cys His His Arg Tyr Ala Glu Leu Tyr Val Ile Asp Val Asn Ile
420 425 430
Asn Ile Ser Cys Glu Thr Asp Gly Tyr Leu Thr Lys Met Thr Cys Arg
435 440 445
Trp Ser Thr Ser Thr Ile Gin Ser Leu Ala Glu Ser Thr Leu Gln Leu
450 455 460
Arg Tyr His Arg Ser Ser Leu Tyr Cys Ser Asp Ile Pro Ser Ile His
465 470 475 480
Pro Ile Ser Glu Pro Lys Asp Cys Tyr Leu Gln Ser Asp Gly Phe Tyr
485 490 495
Giu Cys Ile Phe Gln Pro Ile Phe Leu Leu Ser Gly Tyr Thr Met Trp
500 505 510
Ile Arg Ile Asn His Ser Leu Gly Ser Leu Asp Ser Pro Pro Thr Cys
515 520 525
Val Leu Pro Asp Ser Val Val Lys Pro Leu Pro Pro Ser Ser Val Lys
530 535 540
Ala Glu Ile Thr Ile Asn Ile Gly Leu Leu Lys Ile Ser Trp Glu Lys
545 550 555 560
Pro Vai Phe Pro Glu Asn Asn Leu Gin Phe Gin Ile Arg Tyr Gly Leu
565 570 575
Ser Gly Lys Glu Val Gln Trp Lys Met Tyr Glu Val Tyr Asp Ala Lys
580 585 590
Ser Lys Ser Val Ser Leu Pro Val Pro Asp Leu Cys Ala Val Tyr Ala
595 600 605
Val Gln Val Arg Cys Lys Arg Leu Asp G1y Leu Gly Tyr Trp Ser Asn
610 615 620
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Trp Ser Asn Pro Ala Tyr Thr Val Val Met Asp Ile Lys Val Pro Met
625 630 635 640
Arg Gly Pro Glu Phe Trp Arg I1e Ile Asn Gly Asp Thr Met Lys Lys
645 650 655
Glu Lys Asn Val Thr Leu Leu Trp Lys Pro Leu Met Lys Asn Asp Ser
660 665 670
Leu Cys Ser Val Gln Arg Tyr Val Ile Asn His His Thr Ser Cys Asn
675 680 685
Gly Thr Trp Ser Glu Asp Val Gly Asn His Thr Lys Phe Thr Phe Leu
690 695 700
Trp Thr Glu Gln Ala His Thr Val Thr Val Leu Ala Ile Asn Ser Ile
705 710 715 720
Gly Ala Ser Vai Ala Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser
725 730 735
Lys Val Asn Ile Val Gln Ser Leu Ser Ala Tyr Pro Leu Asn Ser Ser
740 745 750
Cys Val Ile Val Ser Trp Ile Leu Ser Pro Ser Asp Tyr Lys Leu Met
755 760 765
Tyr Phe Ile Ile Glu Trp Lys Asn Leu Asn Glu Asp Gly Glu Ile Lys
770 775 780
Trp Leu Arg Ile Ser Ser Ser Val Lys Lys Tyr Tyr Ile His Gly Lys
785 790 795 800
Phe Thr Ile Leu
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2507 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GCGGCCGCCA GTGTGATGGA TATCTGCAGA ATTCGGCTTT CTCTGCCTTC GGTCGAGTTG 60
GACCCCCGGA TCAAGGTGTA CTTCTCTGAA GTAAGATGAT TTGTCAAAAA TTCTGTGTGG 120
TTTTGTTACA TTGGGAATTT ATTTATGTGA TAACTGCGTT TAACTTGTCA TATCCAATTA 180
CTCCTTGGAG ATTTAAGTTG TCTTGCATGC CACCAAATTC AACCTATGAC TACTTCCTTT 240
TGCCTGCTGG GCTCTCAAAG AATACTTCAA ATTCGAATGG ACATTATGAG ACAGCTGTTG 300
AACCTAAGTT TAATTCAAGT GGTACTCACT TTTCTAACTT ATCCAAAACA ACTTTCCACT 360
GTTGCTTTCG GAGTGAGCAA GATAGAAACT GCTCCTTATG TGCAGACAAC ATTGAAGGAA 420
AGACATTTGT TTCAACAGTA AATTCTTTAG TTTTTCAACA AATAGATGCA AACTGGAACA 480
TACAGTGCTG GCTAAAAGGA GACTTAAAAT TATTCATCTG TTATGTGGAG TCATTATTTA 540
AGAATCTATT CAGGAATTAT AACTATAAGG TCCATCTTTT ATATGTTCTG CCTGAAGTGT 600
TAGAAGATTC ACCTCTGGTT CCCCAAAAAG GCAGTTTTCA GATGGTTCAC TGCAATTGCA 660
GTGTTCACGA ATGTTGTGAA TGTCTTGTGC CTGTGCCAAC AGCCAAACTC AACGACACTC 720
TCCTTATGTG TTTGAAAATC ACATCTGGTG GAGTAATTTT CCAGTCACCT CTAATGTCAG 780
TTCAGCCCAT AAATATGGTG AAGCCTGATC CACCATTAGG TTTGCATATG GAAATCACAG 840
ATGATGGTAA TTTAAAGATT TCTTGGTCCA GCCCACCATT GGTACCATTT CCACTTCAAT 900
ATCAAGTGAA ATATTCAGAG AATTCTACAA CAGTTATCAG AGAAGCTGAC AAGATTGTCT 960
CAGCTACATC CCTGCTAGTA GACAGTATAC TTCCTGGGTC TTCGTATGAG GTTCAGGTGA 1020
GGGGCAAGAG ACTGGATGGC CCAGGAATCT GGAGTGACTG GAGTACTCCT CGTGTCTTTA 1080
CCACACAAGA TGTCATATAC TTTCCACCTA AAATTCTGAC AAGTGTTGGG TCTAATGTTT 1140
CTTTTCACTG CATCTATAAG AAGGAAAACA AGATTGTTCC CTCAAAAGAG ATTGTTTGGT 1200
GGATGAATTT AGCTGAGAAA ATTCCTCAAA GCCAGTATGA TGTTGTGAGT GATCATGTTA 1260
GCAAAGTTAC TTTTTTCAAT CTGAATGAAA CCAAACCTCG AGGAAAGTTT ACCTATGATG 1320
CAGTGTACTG CTGCAATGAA CATGAATGCC ATCATCGCTA TGCTGAATTA TATGTGATTG 1380
ATGTCAATAT CAATATCTCA TGTGAAACTG ATGGGTACTT AACTAAAATG ACTTGCAGAT 1440
GGTCAACCAG TACAATCCAG TCACTTGCGG AAAGCACTTT GCAATTGAGG TATCATAGGA 1500
GCAGCCTTTA CTGTTCTGAT ATTCCATCTA TTCATCCCAT ATCTGAGCCC AAAGATTGCT 1560
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ATTTGCAGAG TGATGGTTTT TATGAATGCA TTTTCCAGCC AATCTTCCTA TTATCTGGCT 1620
$ ACACAATGTG GATTAGGATC AATCACTCTC TAGGTTCACT TGACTCTCCA CCAACATGTG 1680
TCCTTCCTGA TTCTGTGGTG AAGCCACTGC CTCCATCCAG TGTGAAAGCA GAAATTACTA 1740
TAAACATTGG ATTATTGAAA ATATCTTGGG AAAAGCCAGT CTTTCCAGAG AATAACCTTC 1800
AATTCCAGAT TCGCTATGGT TTAAGTGGAA AAGAAGTACA ATGGAAGATG TATGAGGTTT 1860
ATGATGCAAA ATCAAAATCT GTCAGTCTCC CAGTTCCAGA CTTGTGTGCA GTCTATGCTG 1920
TTCAGGTGCG CTGTAAGAGG CTAGATGGAC TGGGATATTG GAGTAATTGG AGCAATCCAG 1980
CCTACACAGT TGTCATGGAT ATAAAAGTTC CTATGAGAGG ACCTGAATTT TGGAGAATAA 2040
TTAATGGAGA TACTATGAAA AAGGAGAAAA ATGTCACTTT ACTTTGGAAG CCCCTGATGA 2100
AAAATGACTC ATTGTGCAGT GTTCAGAGAT ATGTGATAAA CCATCATACT TCCTGCAATG 2160
GAACATGGTC AGAAGATGTG GGAAATCACA CGAAATTCAC TTTCCTGTGG ACAGAGCAAG 2220
CACATACTGT TACGGTTCTG GCCATCAATT CAATTGGTGC TTCTGTTGCA AATTTTAATT 2280
TAACCTTTTC ATGGCCTATG AGCAAAGTAA ATATCGTGCA GTCACTCAGT GCTTATCCTT 2340
TAAACAGCAG TTGTGTGATT GTTTCCTGGA TACTATCACC CAGTGATTAC AAGCTAATGT 2400
ATTTTATTAT TGAGTGGAAA AATCTTAATG AAGATGGTGA AATAAAATGG CTTAGAATCT 2460
CTTCATCTGT TAAGAAGTAT TATATCCATG GTAAGTTTAC TATACTT 2507
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
4 GTAAGTTATT TGNNNNNATA TCCTAACAG 29
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(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs i
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GTAAGCATTA GCNNNNNTTT TAAATTCAG 29
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GTAAGTACCA AANNNNNTTT TCAATATAG 29
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GTAAGTTATG CANNNNNTTT TTCCTTAAG 29
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(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
} (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GTAAGTATAT TTNNNNAATA TTTAACAG 28
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GTAGGTTATG TANNNNNCCC TCATTACAG 29
(2) INFORMATION'FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
GTAAGAAAAC AGNNNNNTGT TTCAAATAG 29
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(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear {
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
GTACGTATTA TTNNNNNTAT CTTTTAAAG 29
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GTATGTCAAG CTNNNNNAAA AATTTCTAG 29
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GTACCTTTTA CTNNNNNCTT ATTTTACAG 29
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(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
( (A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GTCTGCAGAG ATNNNNNGTC ATTTTGCAG 29
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
GTATTCCCAA TTNNNNNTAT TTACTACAG 29
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
GTATTCCCAA TTNNNNNTAT TTACTACAG 29
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(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
GTAAGTTTAC TANNNNNTTT TCTCCTCAG 29
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
GTAAAAATTA TANNNNNTTT CTTTTTCAG 29
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
GTATTGTACT TGNNNNNTAT CCTTTGTAG 29
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(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
GTTGCTTTTT CANNNNNTTA TCTAAACAG 29
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
GTACATTTGT CTNNNNNCTT TTCTTTTAG 29
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
GTATCCAGTG TTNNNNNCTT TTTAAACAG 29
