Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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NOVE~ G-CSF R~l~ AGONIS~S
The present application claims priority under 35 USC
119(e) of United States provisional application Serial No.
60/004,382 filed October 05, 1995.
Field of the Invention
The present invention relates to human G-CSF receptor
agonists with activity on hematopoietic cell differentiation
and expansion.
Backaround of the Invention
The human blood-forming (hematopoietic) system
replaces a variety of white blood cells (including
neutrophils, macrophages, and basophils/mast cells), red
blood cells (erythrocytes) and clot-forming cells
(megakaryocytes/platelets). The hematopoietic systems of
the average male has been estimated to produce on the order
of 4.5 x 1011 granulocytes and erythrocytes every year,
which is e~uivalent to an annual replacement of total body
weight (De-~ter et al., BioEssays, 2;154-158, 1985).
It is believed that small amounts of certain
hematopoietic growth factors account for the differentiation
of a small number of progenitor "stem cells" into the
variety of blood cell lines, for the tremendous
proliferation of those lines, and for the ultimate
differentiation of mature blood cells from those lines.
Because the hematopoietic growth factors are present in
extremely small amounts, the detection and identification of
these factors has relied upon an array of assays which as
yet only distinguish among the different factors on the
basis of stimulative effects on cultured cells under
artificial conditions.
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U.S. Patent 4,999,291 discloses DNA and methods ~or
making G-CSF the disclosure of which is incorporated herein
by reference in it entirety.
U.S. Patent 4,810,643 relates to DNA and methods of
making G-CSF and Cys to Ser substitution variants of G-CSF.
Kuga et al. (~iochem. + Biophys . Res . Comm. 159:103-
111, 1988) made a series of G-CSF variants to partially
define the structure-function relationship. Kuga et al.
found that internal and C-terminal deletions abolished
acti~vity, while M-terminal deletions of up to 11 amino acids
and amino acid substitutions at positions 1, 2 and 3 were
active.
Watanabe et aL. (Anal. Biochem. 195:38-44, 1991) made a
variant to study G-CSF receptor binding in which amino acids
1 and 3 were changed to Tyr for radioiodination of the
protein. Watanabe et al. found this Tyrl, Tyr3 G-CSF variant
to be active.
WO 95/27732 describes, but does not show that the
molecule has biological activity, a circularly permuted G-
CSF ligand with a breakpoint at positions 68/69 creating a
circularly permuted G-CSF ligand with a new N-terminus at
the original position 69 of G-CSF and a new C-terminus at
the original position 68 of G-CSF. WO 95/27732 also
discloses circularly permuted GM-CSF, IL-2 and IL-4.
Rearr~naement of Protein Seauences
In evolution, rearrangements of DNA seauences serve an
important role in generating a diversity of protein
structure and function. Gene duplication and exon shuffling
provide an important mechanism to rapidly generate diversity
-
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and thereby provide organisms with a competitive advantage,
especially since the basal mutation rate ~s low (Doolittle,
Protein Science 1:191-200, 1992).
The development of recom~inant DNA methods has made it
possible to study the effects of sequence transposition on
protein folding, structure and function. The approach used
in creating new sequences resembles that of naturally
occurring pairs of proteins that are related by linear
reorganization of their amino acid sequences (Cunningham, et
al., Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222, 1979;
Teather & Erfle, J. Bacteriol. 172: 3837-3841, 1990;
Schimming et al., ~ur. J. Biochem. 204: 13-19, 1992;
Yamiuchi and Min~mikawa, FEBS Lett. 260:127-130, 1991:
MacGregor et al., FEBS Let t . 378:263-266, 1996). The first
in vitro application of this type of rearrangement to
proteins was described by Goldenberg and Creighton (~. Mol.
Biol . 165:407-413, 1983). A new N-terminus is selected at
an internal site (breakpoint) of the original sequence, the
new seque~ce having the same order of amino acids as the
original from the breakpoint until it reaches an amino acid
that is at or near the original C-terminus. At this point
the new sequence is joined, either directly or through an
additional portion of sequence (linker), to an amino acid
that is at or near the original N-terminus, and the new
sequence continues with the same sequence as the original
until it reaches a point that is at or near the amino acid
that was N-term~n~l to the breakpoint site of the original
sequence, this residue forming the new C-terminus of the
chain.
This approach has been applied to proteins which range
in size from 58 to 462 amino acids (Goldenberg & Creighton,
~. Mol . Biol . 165:407-413, 1983; Li ~ Coffino, Mol . Cell .
Biol . 13:2377-2383, 1993). The proteins examined have
represent~l a broad range of structural classes, including
proteins that contain predom~n~ntly a-helix (interleukin-4;
-
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Kreitman et al., Cytokine 7:311-318, 1995), ~sheet
(interleukin-li Horlick et al., Protein Eng. 5:427-431,
1992), or mixtures of the two (yeast phosphoribosyl
anthranilate isomerase; Luger et al., Science 243:206-210,
1989). Broad categories o~ protein function are represented
in these sequence reorganization studies:
~nzyme3
T4 lysozy~-e Zhang et al., Biochemistry
32:12311-12318 (1993); Zhang et
al., Nature Struct, Biol. 1:434-438
(1995)
dihydrofolate Buchwalder et al., Biochemistry
reductase 31:1621-1630 (1994); Protasova et
al., Prot. Eng. 7 :1373-1377 (1995)
ribonuclease T1 Mullins et al., ~. Am. Chem. Soc.
116:5529-5533 (1994); Garrett et al.,
Protein Science 5:204-211 (1996)
Bacillus ~-glucanse Hahn et al., Proc. Natl. Acad. Sci.
U.S.A. 91:10417-10421 (1994)
aspartate Yang ~ Schachman, Proc. Natl. Acad.
transcarbamoylase Sci. U.S.A. 90:11980-11984 (1993)
phosphoribosyl Luger et al., Science 243:206-210
anthranilate (1989); Luger et al., Prot. Eng.
isomerase 3:249-258 (1990)
pepsin/pepsinosen Lin et al., Protein Science 4:159-
166 (1995)
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glyceraldehyde-3- Vignais et al., Prote n Science
phosphate dehydro- 4:994-1000 (1995)
genase
ornithine Li & Coffino, Mol. Cell. Biol.
decarboxylase 13:2377-2383 (1993)
yeast Ritco-Vonsovici et al., Biochemistry
phosphoglycerate 34:16543-16551 (1995)
dehydrogenase
Enzyme Inhibitor
basic pancreatic Goldenberg & Creighton, J. Mol.
trypsin inhibitor Biol. 165:407-413 (1983)
Cytok; no~
interleuki~ Horlick et al., Protein Eng. 5:427-
431 (1992)
interleukin-4 Kreitman et al., Cytokine 7:311-
318 (1995)
Tyrosine Kinase
Recognition n- ~; n
a-spectrin SH3 Viguera, et al., J.
domain Mol . Biol . 247:670-681 (1995)
Transmembrane
Protein
omp A Koebnik & Kramer, J. Mol. Biol.
250:617-626 (1995)
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~h; ~Q~iC Protein
interleukin-4- Kreitman et al., Proc. Natl. Acad.
Pseudomonas Sci. U.S.A. 91:6889-6893 ~1994).
exotoxin fusion
molecule
The results of these studies have been highly variable.
In many cases substantially lower activity, solubility or
thermodynamic stability were observed (E. coli dihydrofolate
reductase, aspartate transcarbamoylase, phosphoribosyl
anthranil~te isomerase, glyceraldehyde-3-phosphate
dehydrogenase, ornithine decarboxylase, omp A, yeast
phosphoglycerate dehydrogenase). In other cases, the
sequence rearranged protein appeared to have many nearly
identical properties as its natural counterpart (basic
pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease Tl,
Bacillus ~-glucanase, interleukin-l~, ~spectrin SH3 domain,
pepsinogen, interleukin-4). In exceptional cases, an
unexpected improvement over some properties of the natural
se~uence was observed, e.g., the solubility and refolding
rate ~or rearranged ~spectrin SH3 domain sequences, and the
receptor affinity and anti-tumor activity of transposed
interleukin-4- Pseudomonas exotoxin fusion molecule (Kreitman
et al., Proc. Natl. Acad. Sci. U.S.A. 91: 6889-6893, 1994;
Kreitman et al., Cancer Res. 55:3357-3363, 1995).
The ~rimary motivation for these types of studies has
been to study the role of short-range and long-range
interactions in protein folding and stability. Se~uence
rearrangements of this type convert a subset of interactions
that are long-range in the original sequence into short-
range interactions in the new se~uence, and vice versa. The
fact that many of these sequence rearrangements are able to
attain a conformation with at least some activity is
persuasive evidence that protein folding occurs by multiple
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folding pathways (Viguera, et al., .J. Mol . Biol . 247:670-
681, 1995). In the case of the SH3 domain of ~spectrin,
choosing new termini at locations that corresponded to
-hairpin turns resulted in proteins with slightly less
stability, but which were nevertheless able to fold.
The positions of the internal breakpoints used in the
studies cited here are found exclusively on the surface of
proteins, and are distributed throughout the linear sequence
without any obvious bias towards the ends or the middle (the
variation in the relative distance ~rom the original N-
terminus to the breakpoint is ca. 10 to 80% of the total
sequence length). The linkers connecting the original N- and
C-termini in these studies have ranged from 0 to 9 residues.
In one case (Yang & Schachman, Proc. Natl . Acad . Sci . U. S.A.
90:11980-11984, 1993), a portion of sequence has been
deleted from the original C-t~rm~ n~l segment, and the
connection made from the truncated C-terminus to the
original ~-terminus. Flexible hydrophilic residues such as
Gly and Ser are frequently used in the linkers. Viguera, et
al.(~. Mol. Biol. 247:670-681, 1995) compared joining the
original N- and C- termini with 3- or 4-residue linkers; the
3-residue linker was less thermodynamically stable.
Protasova et al. ( Protein Eng. 7:1373-1377, 1994) used 3- or
5-residue linkers in connecting the original N-termini of E.
coli dihydrofolate reductase; only the 3-residue linker
produced protein in good yield.
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Sllmm~rv of the Invention
The modified human G-CSF receptor agonists of the
present invention can be represented by the Formula:
X1-(L)a-X2
wherein;
a is 0 or li
xl is a peptide comprising an amino acid sequence
corresponding to the sequence of residues n+1 through J;
x2 is a peptide comprising an amino acid sequence
corresponding to the sequence of residues 1 through n;
n is an integer ranging from 1 to J-1; and
L is a linker.
In the formula above the constituent amino acids
residues of human G-CSF are numbered sequentially 1 through
J from the amino to the carboxyl terminus. A pair of
adjacent amino acids within this protein may be numbered n
and n+1 respectively where n is an integer ranging from 1 to
J-1. The ~esidue n+1 becomes the new N-terminus of the new
G-CSF receptor agonist and the residue n becomes the new C-
terminus of the new G-CSF receptor agonist.
The present invention relates to novel G-CSF receptor
agonists of the following formula:
Xaa Xaa Xaa Gly Pro Ala Ser Ser Leu Pro Gln Ser Xaa
Leu Leu Xaa Xaa Xaa Glu Gln Val Xaa Lys Xaa Gln Gly Xaa Gly
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Ala Xaa Leu Gln Glu Xaa Leu Xaa Ala Thr Tyr Lys Leu Xaa Xaa
Xaa Glu Xaa Xaa Val Xaa Xaa Gly His Ser Xaa Gly Ile Pro Trp
~ 5
Ala Pro Leu Ser Ser Xaa Pro Ser Xaa Ala Leu Xaa Leu Ala Gly
Xaa Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu
go 100
Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu
110
Xaa Thr Leu Gln Xaa Asp Val Ala Asp Phe Ala Xaa Thr Ile Trp
120 130
Gln Gln Met Glu Xaa Xaa Gly Met Ala Pro Ala Leu Gln Pro Thr
140
Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Xaa Gln Xaa Xaa Ala
150 160
Gly Gly Val Leu Val Ala Ser Xaa Leu Gln Xaa Phe Leu Xaa Xaa
170
Ser Tyr Arg Val Leu Xaa Xaa Leu Ala Gln Pro (SEQ ID NO:1)
wherein
Xaa at position 1 is Thr, Ser, Arg, Tyr or Gly;
Xaa at position 2 is Pro or Leu;
Xaa at position 3 is Leu, Arg, Tyr or Ser;
Xaa at position 13 is Phe, Ser, His, Thr or Pro;
Xaa at position 16 is Lys, Pro, Ser, Thr or His;
Xaa at position 17 is Cys, Ser, Gly, Ala, Ile, Tyr or Arg;
Xaa at position 18 is Leu, Thr, Pro, His, Ile or Cys;
Xaa at position 22 is Arg, Tyr, Ser, Thr or Ala;
Xaa at position 24 is Ile, Pro, Tyr or Leu;
Xaa at position 27 is Asp, or Gly;
Xaa at position 30 is Ala, Ile, Leu or Gly;
Xaa at position 34 is Lys or Ser;
Xaa at posltion 36 is Cys or Ser;
Xaa at position 42 is Cys or Ser;
Xaa at position 43 is His, Thr, Gly, Val, Lys, Trp, Ala,
Arg, Cys, or Leu;
Xaa at position 44 is Pro, Gly, Arg, Asp, Val, Ala, His,
Trp, Gln, or Thr;
Xaa at position 46 is Glu, Arg, Phe, Arg, Ile or Ala;
Xaa at position 47 is Leu or Thr;
Xaa at position 49 is Leu, Phe, Arg or Ser;
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Xaa at position 50 is Leu, Ile, His, Pro or Tyr;
Xaa at position 54 is Leu or His;
Xaa at position 64 is Cys or Ser;
Xaa at position 67 is Gln, Lys, Leu or Cys;
Xaa at position 70 is Gln, Pro, Leu, Arg or Ser;
Xaa at position 74 is Cys or Ser;
Xaa at po~ition 104 is Asp, Gly or Val;
Xaa at position 108 is Leu, Ala, Val, Arg, Trp, Gln or Gly;
Xaa at position 115 is Thr, His, Leu or Ala;
Xaa at position 120 is Gln, Gly, Arg, Lys or His
Xaa at position 123 is Glu, Arg, Phe or Thr
Xaa at position 144 is Phe, His, Arg, Pro, Leu, Gln or Glu;
Xaa at position 146 is Arg or Gln;
Xaa at position 147 is Arg or Gln;
Xaa at position 156 is His, Gly or Ser;
Xaa at position 159 is Ser, Arg, Thr, Tyr, Val or Gly;
Xaa at position 162 is Glu, Leu, Gly or Trp;
Xaa at position 163 is Val, Gly, Arg or Ala;
Xaa at position 169 is Arg, Ser, Leu, Arg or Cys;
Xaa at position 170 is His, Arg or Ser;
wherein optionally 1-11 amino acids from the N-terminus and
1-5 from the C-terminus can be deleted; and
wherein the N-terminus is joined to the C-terminus directly
or through a linker capable of joining the N-terminus to the
C-terminus and having new C- and N-termini at amino acids;
38-39 62-63 123-124
39-40 63-64 124-125
40-41 64-65 125-126
41-42 65-66 126-127
42-43 66-67 128-129
~3-44 67-68 128-129
45-46 68-69 129-130
48-49 69-70 130-131
49-50 70-71 131-132
52-53 71-72 132-133
53-54 91-92 133-134
54-55 92-93 134-135
55-56 93-94 135-136
56-57 94-95 136-137
57-58 95-96 137-138
58-59 96-97 138-139
59-60 97-98 139-140
60-61 98-99 140-141
61-62 99-100 141-142
or 142-143.
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11
The G-CSF receptor agonists of the present invention
may contain amino acid substitutions, deletions and/or
insertions and may also have amino acid deletions at
either/or both the N- and C- termini.
The more preferred breakpoints at which new C-terminus
and N-terminus can be made are; 38-39, 39-40, 40-41, 41-42,
48-49, 53-54, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 60-
61, 61-62, 62-63, 64-65, 65-66, 66-67, 67-68, 68-69, 69-70,
96-97, 12~-126, 126-127, 127-128, 128-129, 129-130, 130-131,
131-132, 132-133, 133-134, 134-135, 135-136, 136-137, 137-
138, 138-139, 139-140, 140-141 and 141-142.
The most preferred breakpoints at which new C-terminus
and N-terminus can be made are; 38-39, 48-49, 96-97, lZ5-
126, 132-133 and 141-142.
A preferred embodiment o~ the present invention the
linker (L) joining the N-terminus to the C-terminus is a
polypeptide selected from the group consisting of:
GlyGlyGlySer (SEQ ID NO:2);
GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:61);
GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID NO:62);
SerGlyGlySerGlyGlySer (SEQ ID NO:63);
GluPheGlyAsnMet (SEQ ID NO:64);
GluPheGlyGlyAsnMet (SEQ ID NO:65);
GluPheGlyGlyAsnGlyGlyAsnMet (SEQ ID NO:66); and
GlyGlySerAspMetAlaGly (SEQ ID NO:67).
The present invention also encompasses recombinant
human G-CSF receptor agonists co-administered or
sequentially with one or more additional colony stimulating
factors (CSF) including, cytokines, lymphokines,
~ 35 interleukins, hematopoietic growth factors which include but
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12
are not limited to GM-CSF, c-mpl ligand (also known as TPO
or MGDF), M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-
3, IL-5, I~ 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,
IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell
growth factor, B-cell differentiation factor, eosinophil
differentiation factor and stem cell factor (SCF) also known
as steel factor or c-kit ligand (herein collectively
referred to as "colony stimulating factors" or "CSF"). These
co-administered mixtures may be characterized by having the
usual activity of both of the peptides or the mixture may be
further characterized by having a biological or
physiological activity greater than simply the additive
function of the presence of the G-CSF receptor agonists or
the second colony stimulating factor alone. The co-
administration may also provide an enhanced effect on the
activity or an activity different from that expected by the
presence of the G-CSF ligand or the second colony
stimulating factor. The co-administration may also have an
improved activity profile which may include reduction of
undesirable biological activities associated with native
human G-CSF. In addition to the list above, IL-3 variants
taught in WO 94/12639 and WO 94/12638 can be co-administered
with the polypeptides of the present invention.
In addition, it is envisioned that in vitro uses would
include the ability to stimulate bone marrow and blood cell
activation and growth before the expanded cells are infused
into patients
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13
Brief DescriDtion of the Fiaures
Figure 1 schematically illustrates the sequence
rearrangement o~ a protein. The N-terminus (N) and the C-
terminus (C) of the native protein are joined through a
linker, or joined directly. The protein is opened at a
breakpoint creating a new N-terminus (new N) and a new C-
terminus (new-C) resulting in a protein with a new linear
amino acid sequence. A rearranged molecule may be
synthesized de novo as linear molecule and not go through
the steps of joining the original N-terminus and the C-
terminus and opening of the protein at the breakpoint.
Figure 2 shows a schematic of Method I, for creating
new proteins in which the original N-terminus and C-terminus
of the native protein are joined with a linker and different
N-terminus and C-terminus of the protein are created. In the
example shown the sequence rearrangement results in a new
gene encoding a protein with a new N-terminus created at
amino acid 97 of the original protein, the original C-
terminus (a.a. 174) joined to the amino acid 11 (a.a. 1- 10
are deleted) through a linker region and a new C-terminus
created at amino acid 96 of the original sequence.
Figure 3 shows a schematic of Method II, for creating
new proteins in which the original N-terminus and C-terminus
of the native protein are joined without a linker and
different N-terminus and C-terminus of the protein are
created. In the example shown the sequence rearrangement
results in a new gene encoding a protein with a new N-
terminus created at amino acid 97 of the original protein,
the original C-terminus (a.a. 174) joined to the original N-
terminus and a new C-terminus created at amino acid 96 of
the original sequence.
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14
Figure 4 shows a schematic of Method III, for creating
new proteins in which the original N-terminus and C-terminus
of the native protein are joined with a linker and different
N-terminus and C-terminus of the protein are created. In the
example shown the sequence rearrangement results in a new
gene encoding a protein with a new N-terminus created at
amino acid 97 of the original protein, the original C-
terminus (a.a. 174) joined to amino acid 1 through a linker
region and a new C-terminus created at amino acid 96 of the
original sequence.
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Detailed ~escri~tion of the Invention
Receptor agonists of the present invention may be
useful in the treatment of diseases characterized by
decreased levels of granulocytes of the hematopoietic
system.
A G-CSF receptor agonist may be useful in the treatment
or prevention of neutropenia. Many drugs may cause bone
marrow suppression or hematopoietic deficiencies. Examples
of such drugs are AZT, DDI, alkylating agents and anti-
metabolites used in chemotherapy, antibiotics such as
chloramphenicol, penicillin, gancyclovir, daunomycin and
sulfa drugs, phenothiazones, tranquilizers such as
meprobamate, analgesics such as aminopyrine and dipyrone,
anti-convulsants such as phenytoin or carbamazepine,
antithyroids such as propylthiouracil and methimazole and
diuretics. G-CSF receptor agonists may be useful in
preventing or treating the bone marrow su~pression or
hematopoietic de~iciencies which often occur in patients
treated with these drugs.
Hematopoietic deficiencies may also occur as a result
of viral, microbial or parasitic infections and as a result
of treatment for renal disease or renal failure, e.g.,
dialysis. The present peptide may be useful in treating
such hematopoietic deficiency.
Another aspect of the present invention provides
plasmid DNA vectors for use in the method of expression of
these novel G-CSF receptor agonists. These vectors contain
the novel DNA sequences described above which code for the
novel polypeptides of the invention. Appropriate vectors
which can transform host cells capable of expressing the G-
CSF receptor agonists include expression vectors comprising
nucleotide sequences coding for the G-CSF receptor agonists
joined to transcriptional and translational regulatory
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sequences which are selected according to the host cells
used. Vectors incorporating modified sequences as described
above are included in the present invention and are useful
in the production of the modified G-CSF receptor agonist
polypeptides. The vector employed in the method also
contains selected regulatory se~uences in operative
association with the DNA coding se~uences of the invention
and capable of directing the replication and expression
thereof in selected host cells.
As another aspect of the present invention, there is
provided a novel method for producing the novel family of
human G-CSF receptor agonists. The method of the present
invention involves culturing suitable cells or cell line,
which has been transformed with a vector containing a DNA
se~uence coding for expression of the novel G-CSF receptor
agonist polypeptide. Suitable cells or cell lines may
include various strains of bacteria such as E. coli, yeast,
mammalian cells, or insect cells may be utilized as host
cells in the method of the present invention.
Other aspects of the present invention are methods and
therapeutic compositions for treating the conditions
referred to above. Such compositions comprise a
therapeutically effective amount of one or more of the G-CSF
receptor agonists of the present invention in a mixture with
a pharmaceutically acceptable carrier. This composition can
be administered either parenterally, intravenously or
subcutaneously. When administered, the therapeutic
composition for use in this invention is preferably in the
form of a pyrogen-free, parenterally acceptable a~ueous
solution. The preparation of such a parenterally acceptable
protein solution, having due regard to pH, isotonicity,
stability and the like, is within the skill of the art.
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The dosage regimen involved in a metnod for treating
the above-described conditions will be determined by the
attending physician considering various factors which modify
the action of drugs, e.g. the condition, body weight, sex
and diet of the patient, the severity of any infection, time
of administration and other clinical factors. Generally, a
daily regimen may be in the range of 0.5 - 150 ~Lg/kg of non-
glycosylated G-CSF receptor agonists protein per kilogram of
body weight. Dosages would be adjusted relative to the
activity of a given receptor agonist and it would not be
unreasonable to note that dosage regimens may include doses
as low as 0.1 microgram and as high as 1 milligram per
kilogram of body weight per day. In addition, there may
exist specific clrcumstances where dosages of G-CSF receptor
agonist would be adjusted higher or lower than the range of
0.5 - 150 micrograms per kilogram of body weight. These
include co-administration with other CSF or growth factors;
co-administration with chemotherapeutic drugs and/or
radiation; the use of glycosylated G-CSF receptor agonists;
and various patient-related issues mentioned earlier in this
section. As indicated above, the therapeutic method and
compositions may also include co-administration with other
human factors. A non-exclusive list of other appropriate
hematopoietins, CSFs and interleukins for simultaneous or
serial co-administration with the polypeptides of the
present invention includes GM-CSF, c-mpl ligand (also known
as TPO or MGDF), M-CSF, erythropoietin (EPO), IL-1, IL-4,
IL-2, IL-3, IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-
12, IL-13, IL-15, LIF, flt3/flk2 ligand, human growth
hormone, B-cell growth factor, B-cell differentiation
factor, eosinophil differentiation factor and stem cell
factor (SCF) also known as steel factor or c-kit ligand
(herein collectively referred to as "colony stimulating
factors"), or combinations thereof. In addition to the list
~ 35 above, IL-3 variants taught in WO 94/12639 and WO 94/12638
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18
can be co-administered with the polypeptides of the present
invention.
The G-CSF receptor agonists of the present invention
may be useful in the mobilization of hematopoietic
progenitors and stem cells in peripheral blood. Peripheral
blood derived progenitors have been shown to be ef~ective in
reconstituting patients in the setting of autologous marrow
transplantation. Hematopoietic growth fac~ors, including G-
CSF and GM-CSF, have been shown to enhance the number of
circulating progenitors and stem cells in the peripheral
blood. This has simplified the procedure for peripheral stem
cell collection and dramatically decreased the cost of the
procedure by decreasing the number of pheresis required. The
G-CSF receptor agonist of the present invention may be
useful in mobilization of stem cells and further enhance the
efficacy of peripheral stem cell transplantation.
The G-CSF receptor agonists of the present invention
may also be useful in the ex vivo expansion of hematopoietic
progenitors. Colony stimulating factors (CSFs), such as G-
CSF, have been administered alone, co-administered with
other CSFs, or in combination with bone marrow transplants
subse~uent to high dose chemotherapy to treat the
neutropenia and which is often the result of such treatment.
However the period of severe neutropenia may not be totally
eliminated. The myeloid lineage, which is comprised of
monocytes (macrophages), granulocytes (including
neutrophils) and megakaryocytes, is critical in preventing
infections and bleeding which can be life-threatening.
Neutropenia may also be the result of disease, genetic
disorders, drugs, toxins, radiation and many therapeutic
treatments such as conventional oncology therapy.
Bone marrow transplants have been used to treat this
patient population. However, several problems are associated
CA 02234042 1998-04-06
WO 97/12977 PCTAJS96/15935
19
with the use of bone marrow to reconstitute a compromised
hematopoietic system including: 1) the number of stem cells
in bone marrow or other tissues, such as spleen or
peripheral blood, is limited, 2) Graft versus Host Disease,
3) graft rejection and 4) possible contamination with tumor
cells. Stem cells and progenitor cells make up a very small
percentage o~ the nucleated cells in the bone marrow, spleen
and peripheral blood. It is clear that a dose response
exists such that a greater number of multipotential
hematopoietic progenitors will enhance hematopoietic
recovery. Therefore, the in vitro expansion of stem cells
should enhance hematopoietic recovery and patient survival.
Bone marrow from an allogeneic donor has been used to
provide bone marrow for transplant. However, Graft Versus
Host Disease and graft rejection limit bone marrow
transplantation even in recipients with HLA-matched sibling
donors. An alternative to allogeneic bone marrow transplants
is autologous bone marrow transplants. In autologous bone
marrow tra:lsplants, some of the patient~s own marrow is
harvested prior to myeloablative therapy, e.g. high dose
chemotherapy, and is transplanted back into the patient
afterwards. Autologous transplants eliminate the risk of
Graft Versus Host Disease and graft rejection. However,
autologous bone marrow transplants still present problems in
terms of the limited number of stems cells in the marrow and
possible contamination with tumor cells. The limited number
of multipotential hematopoietic progenitors may be overcome
by ex-vivo expansion of the multipotential hematopoietic
progenitors. In addition, stem cells can be specifically
isolated based on the presence of specific surface antigens
such as CD34+ in order to decrease tumor -ell contamination
of the marrow graft.
The f~llowing patents contain further details on
~ 35 separating stem cells, CD34+ cells, culturing the cells with
CA 02234042 l998-04-06
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hematopoietic factors, the use of the cells for the
treatment of patients with hematopoietic disorders and the
use of hematopoietic factors for cell expansion and gene
therapy.
5,061,620 relates to compositions comprising human
hematopoietic stem cells provided by separating the stem
cells from dedicated cells.
5,199,942 describes a method for autologous hematopoietic
cell transplantation comprising: (1) obtaining hematopoietic
progenitor cells from a patient; (2) ex-vivo expansion of
cells with a growth factor selected from the group
consisting of IL-3, flt3 ligand, c-kit ligand, GM CSF, IL-1,
GM-CSF/IL-3 fusion protein and combinations thereof; (3)
administerlng cellular preparation to a patient.
5,240,856 relates to a cell separator that includes an
apparatus for automatically controlling the cell separation
process.
WO 91/16116 describes devices and methods for selectively
isolating and separating target cells from a mixture of
cells.
WO 91/18972 describes methods for in vitro culturing of bone
marrow, by incubating suspension of bone marrow cells, using
a hollow fiber bioreactor.
WO 92/18615 relates to a process for maintaining and
expanding bone marrow cells, in a culture medium containing
speci~ic mixtures o~ cytokines, ~or use in transplants.
WO 93/08268 describes a method for selectively expanding
stem cells, comprising the steps of (a) separating CD34~
CA 02234042 1998-04-06
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21
stem cells ~rom other cells and (b) incubating the separated
cells in a selective medium, such that the stem cells are
selectively expanded.
WO 93/18136 describes a process for in vitro support of
mammalian cells derived from peripheral blood.
WO 93/18648 relates to a composition comprising human
neutrophil precursor cells with a high content of
myeloblasts and promyelocytes for treating genetic or
acquired neutropenia.
WO 94/08039 describes a method o~ enrichment ~or human
hematopoietic stem cells by selection for cells which
express c-kit protein.
WO 94/11493 describes a stem cell population that are CD34+
and small in size, which are isolated using a counterflow
elutriation method.
WO 94/27698 relates to a method combining immunoaffinity
separation and continuous flow centrifugal separation for
the selective separation of a nucleated heterogeneous cell
population from a heterogeneous cell mixture.
WO 94/25848 describes a cell separation apparatus for
collection and manipulation of target cells.
The long term culturing of highly enriched CD34+ precursors
of hematopoietic progenitor cells from human bone marrow in
cultures containing IL-1~, IL-3, IL-6 or GM-CSF is
discussed in Brandt et al (J. Clin. Invest . 86:932-941,
1990 ) .
CA 02234042 1998-04-06
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One aspect of the present invention provides a method
for selective ex-vivo expansion of stem cells. The term
"stem cell" refers to the multipotential hematopoietic cells
as well as early myeloid progenitor and precursors cells
which can be isolated from bone marrow, spleen or peripheral
blood. The term "expansion" refers to the proliferation and
differentiation of the cells The present invention provides
a method ~or selective ex-vivo expansion of stem cells,
comprising the steps of; (a) separating stem cells from
other cells, (b) culturing the separated stem cells with a
selective medium which contains a G-CSF receptor agonist and
optionally a second colony stimulating factor, and (c)
harvesting the cultured stems cells. Stem cells, as well as
committed progenitor cells destined to become neutrophils,
erythrocytes, platelets, etc., may be distinguished from
most other cells by the presence or absence of particular
progenitor marker antigens, such as CD34, that are present
on the surface of these cells and/or by morphological
characteristics. The phenotype for a highly enriched human
stem cell fraction is reported as CD34+, Thy-1+ and lin-,
but it is to be understood that the present invention is not
limited to the expansion of this stem cell population. The
CD34+ enriched human stem cell fraction can be separated by
a number of reported methods, including affinity columns or
~5 beads, magnetic beads or flow cytometry using antibodies
directed to surface antigens such as the CD34+. Further,
physical separation methods such as counterflow elutriation
may be used to enrich hematopoietic progenitors. The CD34+
progenitors are heterogeneous, and may be divided into
3 0 several sub-populations characterized by the presence or
absence of co-expression of different lineage associated
cell surface associated molecules. The most immature
progenitor cells do not express any known lineage associated
markers, such as HLA-DR or CD38, but they may express
CD90 (thy-1). Other surface antigens such ~s CD33, CD38,
CA 02234042 1998-04-06
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23
CD41, CD71, HLA-DR or c-kit can also be used to selectively
isolate hematopoietic progenitors. The separated cells can
be incubated in selected medium in a culture flask, sterile
bag or in hollow fibers. Various colony stimulating factors
may be utilized in order to selectively expand cells.
Representative factors that have been utilized for ex-vivo
expansion of bone marrow include, c-kit ligand, IL-3, G-CSF,
GM-CSF, IL-l, IL-6, IL-ll, flt-3 ligand or combinations
thereof. The proliferation of the stem cells can be
monitored by enumerating the number of stem cells and other
cells, by standard techniques (e.g. hemacytometer, CFU,
LTCIC) or by flow cytometry prior and subsequent to
incubation.
Seve~al methods for ex-vivo expansion of stem cells
have been reported utilizing a number of selection methods
and expansion using various colony stimulating ~actors
including c-kit ligand (Brandt et al., Blood 83:1507-1514,
1994; McKenna et al., Blood 86:3413-3420, 1995), IL-3
(Brandt et al., Blood 83:1507-1514, 1994; Sato et al., Blood
82:3600-3609, 1993), G-CSF (Sato et al., Blood 82:3600-3609,
1993), GM-CSF (Sato et al., Blood 82:3600-3609, 1993), IL-l
(Muench et al., Blood 81:3463-3473, 1993), IL-6 (Sato et
al., Blood 82 :3600-3609, 1993), IL-ll (Lemoli et al., Exp.
Hem. 21:1668-1672, 1993; Sato et al., Blood 82:3600-3609,
1993), flt-3 ligand (McKenna et al., Blood 86 :3413 3420,
1995) and/or combinations thereof (Brandt et al., Blood
83:1507 1514, 1994; Haylock et al., Blood 80:1405-1412,
1992, Koller et al., Biotechnology 11:358-363, 1993; Lemoli
et al., Exp. Hem. 21:1668-1672, 1993), McKenna et al., Blood
86:3413-3420, 1995; Muench et al., Blood 81:3463-3473, 1993;
Patchen et al., Biotherapy 7:13-26, 1994; Sato et al., Blood
82:3600-3609, 1993; Smith et al., Exp. Hem. 21:870-877,
1993; Steen et al., Stem Cells 12:214-224, 1994; Tsujino et
al., Exp. Hem. 21:1379-1386, 1993). Among the individual
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24
colony stimulating factors, hIL-3 has been shown to be one
of the most potent in expanding peripheral blood CD34+ cells
(Sato et al., Blood 82:3600-3609, 1993; Kobayashi et al.,
Blood 73:1836-1841, 1989). However, no single factor has
been shown to be as effective as the comblnation of multiple
factors. The present invention provides methods for ex vivo
expansion ~hat utilize novel G-CSF receptor agonists.
Another aspect of the invention provides methods of
sustaining and/or expanding hematopoietic precursor cells
which includes inoculating the cells into a culture vessel
which contains a culture medium that has been conditioned by
exposure to a stromal cell line such as HS-5 (WO 96/02662,
Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has
been supplemented with a G-CSF receptor agonist of the
present invention.
Another projected clinical use of growth factors has
been in the in vitro activation of hematopoietic progenitors
and stem cells for gene therapy. Due to the long life-span
of hematopoietic progenitor cells and the distribution of
their daughter cells throughout the entire body,
hematopoietic progenitor cells are good candidates for ex
vivo gene transfection. In order to have the gene of
interest incorporated into the genome of the hematopoietic
progenitor or stem cell one needs to stimulate cell division
and DNA replication. Hematopoietic stem cells cycle at a
very low frequency which means that growth factors may be
useful to promote gene transduction and thereby enhance the
clinical prospects for gene therapy. Potential applications
of gene therapy (review Crystal, Science 270:404-410, 1995)
include; 1) the treatment of many congenital metabolic
disorders and immunodeficiencies (Kay and Woo, Trends Genet .
10:253-257, 1994), 2) neurological disorders (Friedmann,
Trends Genet. 10:210-214, 1994), 3) cancer (Culver and
CA 02234042 1998-04-06
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Blaese, Trends Genet. 10:174-178, 1994) and 4) in~ectious
diseases (Gilboa and Smith, Trends Genet. 10:139-144, 1994).
There are a variety of methods, known to those with
skill in the art, for introducing genetic material into a
host cell. ~ number of vectors, both viral and non-viral
have been developed for transferring therapeutic genes into
primary cells. Viral based vectors include; 1) replication
deficient recombinant retrovirus (Boris-Lawrie and Temin,
Curr. Opin. Genet. Dev. 3:102-109, 1993; Boris-Lawrie and
Temin, Annal. New York Acad. Sci. 716:59-71, 1994; Miller,
Current Top. Microbiol. Immunol. 158:1-24, 1992) and
replication-deficient recombinant adenovirus (Berkner,
BioTechni~ues 6:616-629, 1988; Berkner, Current Top.
Microbiol. Immunol. 158:39-66, 1992; srody and Crystal,
Annal. New York Acad. Sci. 716:90-103, 1994). Non-viral
based vectors include protein/DMA complexes (Cristiano et
al., PNAS USA. 90:2122-2126, 1993; Curiel et al., PNAS USA
88:8850-8854, 1991; Curiel, Annal. New York Acad. Sci.
716:36-58, 1994), electroporation and liposome mediated
delivery such as cationic liposomes (Farhood et al., Annal.
New York Acad. ~ci. 716:23-35, 1994).
The present invention provides an improvement to the
existing methods of expanding hematopoietic cells, into
which new genetic material has been introduced, in that it
provides methods utilizing G-CSF receptor agonists that may
have improved biological activity and/or physical
properties.
Determination of the Linker
The length of the amino acid sequence of the linker
r can be selected empirically or with guidance ~rom structural
information, or by using a combination of the two
approaches.
CA 02234042 1998-04-06
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26
When no structural information is available, a small
series of linkers can be prepared for testing using a design
whose length is varied in order to span a range from 0 to 50
A and whose seguence is chosen in order to be consistent
with surface exposure (hydrophilicity, Hopp & Woods, Mol.
Immunol. 20: 483-489, 1983; Kyte ~ Doolittle, ~. Mol. Biol.
157:105-132, 1982; solvent exposed surface area, Lee ~
Richards, ~. Mol. Biol. 55:379-400, 1971) and the ability to
adopt the necessary conformation without deranging the
configuration of the c-mpl receptor agonist
(conformationally flexible; Karplus & Schulz,
Naturwissenschaften 72:212-213, (1985). Assuming an average
of translation of 2.0 to 3.8 A per residue, this would mean
the length to test would be between 0 to 30 residues, with 0
to 15 residues being the preferred range. Exemplary of such
an empirical series would be to construct linkers using a
cassette sequence such as Gly-Gly-Gly-Ser (SEQ ID NO:2)
repeated n times, where n is 1, 2, 3 or 4. Those skilled in
the art will recognize that there are many such seguences
that vary n length or composition that can serve as linkers
with the primary consideration being that they be neither
excessively long nor short (cf., Sandhu, Critical Rev.
Biotech. 12: 437-462, 1992); if they are too long, entropy
effects will likely destabilize the three-~im~n~ional fold,
and may also make folding kinetically impractical, and if
they are too short, they will likely destabilize the
molecule because of torsional or steric strain.
Those skilled in the analysis of protein structural
information will recognize that using the distance between
the chain ends, defined as the distance between the c-alpha
carbons, can be used to define the length of the seguence to
be used, or at least to limit the number of possibilities
that must be tested in an empirical selection of linkers.
They will .1lso recognize that it is sometimes the case that
CA 02234042 1998-04-06
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27
the positions of the ends of the polypeptide chain are ill-
defined in structural models derived ~rom x-ray di~fraction
or nuclear magnetic resonance spectroscopy data, and that
when true, this situation will therefore need to be taken
into account in order to properly estimate the length of the
linker required. From those residues whose positions are
well defined are selected two residues that are close in
sequence to the chain ends, and the distance between their
c-alpha carbons is used to calculate an approximate length
for a linker between them. Using the calculated length as a
guide, linkers with a range of number of residues
(calculated using 2 to 3.8A per residue) are then selected.
These linkers may be composed of the original sequence,
shortened or lengthened as necessary, and when lengthened
the additional residues may be chosen to be flexible and
hydrophilic as described above; or optionally the original
se~uence may be substituted for using a series of linkers,
one example being the Gly-Gly~Gly-Ser (SEQ ID NO:2) cassette
approach mentioned above; or optionally a combination of the
original se~uence and new sequence having the appropriate
total length may be used.
~etermination of the Amino and Carboxyl Termini of G-CSF
Rece~tor Aaonists
Sequences of G-CSF receptor agonists capable of
folding to biologically active states can be prepared by
appropriate selection of the beginning (amino terminus) and
ending (carboxyl terminus) positions from within the
original polypeptide chain while using the linker seguence
as described above. Amino and carboxyl termini are selected
from within a common stretch of sequence, referred to as a
breakpoint region, using the guidelines described below. A
novel amino acid se~uence is thus generated by selecting
CA 02234042 1998-04-06
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amino and carboxyl termini from within the same breakpoint
region. I.n many cases the selection of the new termini will
be such that the original position of the carboxyl terminus
immediately preceded that of the amino terminus. However,
those skilled in the art will recognize that selections of
termini anywhere within the region may function, and that
these will effectively lead to either deletions or additions
to the amino or carboxyl portions of the new sequence.
It is a central tenet of molecular biology that the
primary amino acid sequence of a protein dictates folding to
the three-~im~nsional structure necessary for expression of
its biological function. Methods are known to those skilled
in the art to obtain and interpret three-~ n~ional
structural information using x-ray diffra_tion of single
protein crystals or nuclear magnetic resonance spectroscopy
of protein solutions. Examples of structural information
that are r.~levant to the identification of breakpoint
regions include the location and type of protein secondary
structure (alpha and 3-10 helices, parallel and anti-
parallel beta sheets, chain reversals and turns, and loops;Kabsch & Sander, Biopolymers 22: 2577-2637, 1983; the degree
of solvent exposure of amino acid residues, the extent and
type of interactions of residues with one another (Chothia,
Ann . Rev. Biochem. 53:537-572; 1984) and the static and
dynamic distribution of conformations along the polypeptide
chain (Alber & Mathews, Methods Enzymol. 154: 511-533,
1987). In some cases additional information is known about
solvent exposure of residues; one example is a site of post-
translational attachment of carbohydrate ~hich is
necessarily on the surface of the protein. When
experimental structural information is not available, or is
not feasible to obtain, methods are also available to
analyze the primary amino acid sequence in order to make
predictions of protein tertiary and secondary structure,
solvent accessibility and the occurrence of turns and loops.
CA 02234042 1998-04-06
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29
Biochemical methods are also sometimes applicable for
empirically determining surface exposure when direct
structural methods are not feasible; for example, using the
identification of sites of chain scission following limited
proteolysis in order to infer surface exposure (Gentile &
Salvatore, Eur. ~. Biochem. 218:603-621, 1993)
Thus using either the experimentally derived structural
informaticn or predictive methods (e.g., Srinivisan ~ Rose
Proteins: Struct., Funct . & Genetics, 22: 81-99, 1995) the
parental amino acid sequence is inspected to classify
regions according to whether or not they are integral to the
maintenance of secondary and tertiary structure. The
occurrence of sequences within regions that are known to be
involved in periodic secondary structure (alpha and 3-10
helices, parallel and anti-parallel beta sheets) are regions
that should be avoided. Similarly, regions of amino acid
sequence that are observed or predicted to have a low degree
of solvent exposure are more likely to be part of the so-
called hydrophobic core of the protein and should also be
avoided for selection of amino and carboxyl t~rmi ni . In
contrast, those regions that are known or predicted to be in
surface turns or loops, and especially those regions that
are known not to be required for biological activity, are
the preferred sites for location of the extremes of the
polypeptide chain. Continuous stretches of amino acid
sequence that are preferred based on the above criteria are
referred to as a breakpoint region.
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TART ~
OT.IGONUCL~OTIDES
L-llstart.seq GCTCTGAGAG CCGCCAGAGC CGCCAGAGGG
CTGCGCAAGG TGGCGTAGAA CGCG
(SEQ ID NO:3)
L-llstop.seq CAGCCCTCTG GCGGCTCTGG CGGCTCTCAG
AGCTTCCTGC TCAA~l~Cl~ AGAG
(SEQ ID NO:4)
BlstartP.seq GGGCTGCGCA AGGTGGCG (SEQ ID NO:5)
blstopP.seq ACACCATTGG GCCCTGCCAG C (SEQ ID NO:6)
39start.seq GATCGACCAT GGCTTACAAG CTGTGCCACC CC
(SEQ ID NO:7)
38stop.Seq CGATCGAAGC TTATTAGGTG GCACACAGCT TCTCCT
(SEQ ID NO:8)
97start.seq GATCGACCAT GGCTCCCGAG TTGGGTCCCA CC
(SEQ ID NO:9)
96stop.Seq CGATCGAAGC TTATTAGGAT ATCCCTTCCA GGGCCT
(SEQ ID NO:10)
126start.seq GATCGACCAT GGCTATGGCC CCTGCCCTGC AG
(SEQ ID NO:ll)
125stop.Seq CGATCGAAGC TTATTATCCC AGTTCTTCCA TCTGCT
(SEQ ID NO:12)
133start.seq GATCGACCAT GGCTACCCAG GGTGCCATGC CG
(SEQ ID NO:13)
132stop.seq CGATCGAAGC TTATTAGGGC TGCAGGGCAG GGGCCA
(SEQ ID NO:14)
142start.seq GATCGACCAT GGCTTCTGCT TTCCAGCGCC GG
(SEQ ID NO:15)
141stop.Seq CGATCGAAGC TTATTAGGCG AAGGCCGGCA TGGCAC
(SEQ ID NO:16)
96for.Seq ATATCCATGG CTCCGGAACT GGGTCCAACT CTG
(SEQ ID NO:17)
96rev.Seq ACCTCCAGGA AGCTCTGCAG ATGG
(SEQ ID NO:18)
CA 02234042 1998-04-06
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125for.seq TATATCCATG GCTATGGCTC CAGCTCTGCA
ACCAACTCAA GGTGCAATGC CAGCATTTGC ATCTG
(SEQ ID NO:19)
125rev.seq GATGGCTAGC AACCAGAACA CCACCTGCAC
GACGTTGAAA AGCAGATGCA AATGCTGGCA TTG
(SEQ ID MO:20)
132for.seq TATATCCATG GCTACTCAAG GTGCTATGCC
AGCTTTTGCT TCTGCTTTTC AACGTCG
(SEQ ID NO:21)
132rev seq GCAGATGGCT AGCAACCAGA ACACCACCTG
CACGACGTTG AAAAGCAGAA GCAAAAGC
(SEQ ID NO:22)
141for.seq CATGGCTTCT GCTTTTCAAC GTCGTGCAGG
TGGTGTTCTG GTTG (SEQ ID NO:23)
141rev.seq CTAGCAACCA GAACACCACC TGCACGACGT
TGAAAAGCAG AAGC (SEQ ID NO:24)
49start.seq GATCGACCAT GGCTCTGCTC GGACACTCTC TG
(SEQ ID NO:68)
48stop.seq CGATCGAAGC TTATTACACC AGCTCCTCGG GGTGGC
(SEQ ID NO:69)
77start.seq GATCGACCAT GGCTCAACTC CATAGCGGCC TT
(SEQ ID MO:70)
76stop.seq CGATCGAAGC TTATTAGCTC AAGCAGCCTG CCAGCT
(SEQ ID NO:71)
82start.seq GATCGACCAT GGCT~ C CTCTACCAGG GG
(SEQ ID NO:72)
81stop.seq CGATCGAAGC TTATTAGCCG CTATGGAGTT GGCTCA
(SEQ ID NO:73)
84start.seq GATCGACCAT GGCTCTCTAC CAGGGGCTCC TG
(SEQ ID NO:74)
83stop.seq CGATCGAAGC TTATTAGAAA AGGCCGCTAT GGAGTT
(SEQ ID NO:75)
91start.seq GATCGACCAT GGCTGCCCTG GAAGGGATAT CC
(SEQ ID NO:76)
90stop.seq CGATCGAAGC TTATTACTGC AGGAGCCCCT GGTAGA
- (SEQ ID NO:77)
CA 02234042 1998-04-06
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32
112start.seq GATCGACCAT GGCTGACTTT GCCACCACCA TC
(SEQ ID NO:78)
lllstop.seq CGATCGAAGC TTATTAGGCG ACGTCCAGCT GCAGTG
(SEQ ID NO:79)
117start.seq GATCGACCAT GGCTATCTGG CAGCAGATGG AA
(SEQ ID NO:80)
116stop.seq CGATCGAAGC TTATTAGGTG GTGGCAAAGT CGGCGA
(SEQ ID NO:81)
ll9start.seq GATCGACCAT GGCTCAGCAG ATGGAAGAAC TG
(SEQ ID NO:82)
118stop.seq CGATCGAAGC TTATTACCAG ATGGTGGTGG CAAAGT
(SEQ ID NO:83)
Z4849at.for CATGGCTTTG TTAGGACATT CTTTAGGTAT
TCCATGGGCT CCTCTGAGCT (SEQ ID NO:84)
Z4849at.rev CAGAGGAGCC CATGGAATAC CTAAAGAATG
TCCTAACAAA GC (SEQ ID NO:85)
CA 02234042 1998-04-06
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33
TABT.E 2
DNA seauences
pMON3485.Se~
1 ATGGCTTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC
51 TCTGGGCATC CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC
101 AGCTGGCAGG CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG
151 GGGCTCCTGC AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT
201 GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC
251 AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC
301 ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG GGGTCCTGGT
351 TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTCGTACCGC GTTCTACGCC
401 ACCTTGCGCA GCCCTCTGGC GGCTCTGGCG GCTCTCAGAG CTTCCTGCTC
451 AAGTCTTTAG AGCAAGTGAG GAAGATCCAG GGCGATGGCG CAGCGCTCCA
501 GGAGAAGCTG TGTGCCACCT AATAA (SEQ ID NO:25)
pMON3486.Se~
1 ATGGCTCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC TGGACGTCGC
51 CGACTTTGCC ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC
101 CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC
151 CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT
201 GGAGC.'GTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC TCTGGCGGCT
251 CTGGCGGCTC TCAGAGCTTC CTGCTCAAGT CTTTAGAGCA AGTGAGGAAG
301 ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA
351 GCTGTGCCAC CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC
401 CCTGGGCTCC CCTGAGCTCC TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC
451 TGCTTGAGCC AACTCCATAG CGGCCTTTTC CTCTACCAGG GGCTCCTGCA
501 GGCCCTGGAA GGGATATCCT AATAA (SEQ ID NO:26)
pMON3487.Seq
1 ATGGCTATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT
51 CGCCTCTGCT TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC
101 TGCAGAGCTT CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG
151 CCCTCTGGCG GCTCTGGCGG CTCTCAGAGC TTCCTGCTCA A~l~l"l"l~AGA
201 GCAAGTGAGG AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT
251 GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC
301 TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT
351 GCAGCTGGCA GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC
401 AGGGGCTCCT GCAGGCCCTG GAAGGGATAT CCCCCGAGTT GGGTCCCACC
451 TTGGACACAC TGCAGCTGGA CGTCGCCGAC TTTGCCACCA CCATCTGGCA
501 GCAGATGGAA GAACTGGGAT AATAA (SEQ ID NO:27)
pMON3488.Seq
CA 02234042 1998-04-06
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34
1 ATGGCTACCC AGGGTGCCAT GCCGGCCTTC GCCTCTGCTT TCCAGCGCCG
51 GGCAGGAGGG GTCCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT
101 CGTACCGCGT TCTACGCCAC CTTGCGCAGC CCTCTGGCGG CTCTGGCGGC
151 TCTCAGAGCT TCCTGCTCAA GTCTTTAGAG CAAGTGAGGA AGATCCAGGG
201 CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC AAGCTGTGCC
251 ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT
301 CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG
351 CCAACTCCAT AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG
401 AAGGGATATC CCCCGAGTTG GGTCCCACCT TGGACACACT GCAGCTGGAC
451 GTCGCCGACT TTGCCACCAC CATCTGGCAG CAG~TGGAAG AACTGGGAAT
501 GGCCCCTGCC CTGCAGCCCT AATAA (SEQ ID NO:28)
pMON3489.Seq
1 ATGGCTTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA
51 TCTGCAGAGC TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC
101 AGCCCTCTGG CGGCTCTGGC GGCTCTCAGA GCTTCCTGCT CAAGl~'l'~ A
151 GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT
201 GTGTGCCACC TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC
251 ACTCTCTGGG CATCCCCTGG GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC
301 CTGCAGCTGG CAGGCTGCTT GAGCCAACTC CATAGCGGCC TTTTCCTCTA
351 CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA
401 CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG
451 CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG
501 TGCCATGCCG GCCTTCGCCT AATAA (SEQ ID NO:29)
pMON3490.seq
1 ATGGCTTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC
51 TCTGGGCATC CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC
101 AGCTGGCAGG CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG
151 GGGCTCCTGC AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT
201 GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC
251 AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC
301 ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG GGGTCCTGGT
351 TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTCGTACCGC GTTCTACGCC
401 ACCTTGCGCA GCCCACACCA TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC
451 TTCCTGCTCA A~TC~ AGA GCAAGTGAGA AAGATCCAGG GCGATGGCGC
501 AGCGCTCCAG GAGAAGCTGT GTGCCACCTA ATAA (SEQ ID NO:30)
pMON3491.se~
1 ATGGCTCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC TGGACGTCGC
51 CGACTTTGCC ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC
101 CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC
151 CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT
201 GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC ACACCATTGG
251 GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA
301 GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC
CA 02234042 1998-04-06
W O 97/12977 PCT~US96/15935
351 CACCTACAAG CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC
401 TGGGCPTCCC CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG
451 CTGGCAGGCT GCTTGAGCCA ACTCCATAGC GGCCTTTTCC TCTACCAGGG
501 GCTCCTGCAG GCCCTGGAAG GGATATCCTA ATAA (SEQ ID NO:31)
pMON3492.seq
1 ATGGCTATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT
51 CGCCTCTGCT TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC
101 TGCAGAGCTT CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG
151 CCCACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA
201 ~ AGAG CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG
251 AGAAGCTGTG TGCCACCTAC AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG
301 CTCGGACACT CTCTGGGCAT CCCCTGGGCT CCCCTGAGCT CCTGCCCCAG
351 CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT AGCGGCCTTT
401 TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG
451 GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC
501 CATCTGGCAG CAGATGGAAG AACTGGGATA ATAA (SEQ ID NO:32)
pMON3493.seq
1 ATGGCTACCC AGGGTGCCAT GCCGGCCTTC GCCTCTGCTT TCCAGCGCCG
51 GGCAGG~GGG GTCCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT
101 CGTACCGCGT TCTACGCCAC CTTGCGCAGC CCACACCATT GGGCCCTGCC
151 AGCTCCCTGC CCCAGAGCTT CCTGCTCAAG TCTTTAGAGC AAGTGAGAAA
201 GATCCAGGGC GATGGCGCAG CGCTCCAGGA GAAGCTGTGT GCCACCTACA
251 AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC
301 CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG
351 CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC
401 AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT GGACACACTG
451 CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA
501 ACTGGGAATG GCCCCTGCCC TGCAGCCCTA ATAA (SEQ ID NO:33)
pMON3494.seq
1 ATGGCTTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA
51 TCTGCAGAGC TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC
101 AGCCCACACC ATTGGGCCCT GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC
151 AA~'l~ll~ AG AGCAAGTGAG AAAGATCCAG GGCGATGGCG CAGCGCTCCA
201 GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG GAGCTGGTGC
251 TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC
301 AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT
351 TTTCCTCTAC CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT
401 TGGGTCCCAC CTTGGACACA CTGCAGCTGG ACGTCGCCGA CTTTGCCACC
451 ACCATCTGGC AGCAGATGGA AGAACTGGGA ATGGCCCCTG CCCTGCAGCC
501 CACCCAGGGT GCCATGCCGG CCTTCGCCTA ATAA (SEQ ID NO:34)
pMON25181.seq
CA 02234042 1998-04-06
W O 97/12977 PCT~US96/15935
36
1 ATGGCTCCGG AACTGGGTCC AACTCTGGAC ACACTGCAGC TGGACGTCGC
51 CGACTTTGCC ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC
101 CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC
151 CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT
201 GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC ACACCATTGG
251 GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA
301 GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC
351 CACCTACAAG CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC
401 TGGGCATCCC CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG
451 CTGGCAGGCT GCTTGAGCCA ACTCCATAGC GGCCTTTTCC TCTACCAGGG
501 GCTCCTGCAG GCCCTGGAAG GGATATCCTA A (SEQ ID NO:35)
pMON25182.seq
1 ATGGCTATGG CTCCAGCTCT GCAACCAACT CAAGGTGCAA TGCCAGCATT
51 TGCATCTGCT TTTCAACGTC GTGCAGGTGG TGTTCTGGTT GCTAGCCATC
101 TGCAGAGCTT CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG
151 CCCACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA
201 ~l~C'~ AGAG CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG
251 AGAAGCTGTG TGCCACCTAC AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG
301 CTCGGACACT CTCTGGGCAT CCCCTGGGCT CCCCTGAGCT CCTGCCCCAG
351 CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT AGCGGCCTTT
401 TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG
451 GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC
501 CATCTGGCAG CAGATGGAAG AACTGGGATA A (SEQ ID NO:36)
pMON25183.seq
1 ATGGCTACTC AAGGTGCTAT GCCAGCTTTT G~ GCTT TTCAACGTCG
51 TGCAGGTGGT GTTCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT
101 CGTACCGCGT TCTACGCCAC CTTGCGCAGC CCACACCATT GGGCCCTGCC
151 AGCTCCCTGC CCCAGAGCTT CCTGCTCAAG TCTTTAGAGC AAGTGAGAAA
201 GATCCAGGGC GATGGCGCAG CGCTCCAGGA GAAGCl~ GCCACCTACA
251 AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC
301 CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG
351 CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC
401 AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT GGACACACTG
451 CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA
501 ACTGGGAATG GCCCCTGCCC TGCAGCCCTA A (SEQ ID NO:37)
pMON25184.seq
1 ATGGCTTCTG C~ CAACG TCGTGCAGGT GGTG~ ClGG TTGCTAGCCA
51 TCTGCAGAGC TTCCTGGAGG TGTCGTACCG C~T~l~C~l~ACGC CACCTTGCGC
101 AGCCCACACC ATTGGGCCCT GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC
151 AAGTCTTTAG AGCAAGTGAG AAAGATCCAG GGCGATGGCG CAGCGCTCCA
201 GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG GAGCTGGTGC
251 TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC
CA 02234042 1998-04-06
W O 97/12977 PCT~US96/15935
37
301 AGCC~GGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT
351 TTTCC'CTAC CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT
401 TGGGTCCCAC CTTGGACACA CTGCAGCTGG ACGTCGCCGA CTTTGCCACC
451 ACCATCTGGC AGCAGATGGA AGAACTGGGA ATGGCCCCTG CCCTGCAGCC
501 CACCCAGGGT GCCATGCCGG CCTTCGCCTA A (SEQ ID NO:38)
pMON25185.seq
101 ATGGCTCCGG AACTGGGTCC AACTCTGGAC ACACTGCAGC TGGACGTCGC
51 CGACTTTGCC ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC
101 CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC
151 CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT
201 GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC TCTGGCGGCT
251 CTGGCGGCTC TCAGAGCTTC CTGCTCAAGT CTTTAGAGCA AGTGAGAAAG
301 ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA
351 GCTGTGCCAC CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC
401 CCTGGGCTCC CCTGAGCTCC TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC
451 TGCTTGAGCC AACTCCATAG CGGC~'l"l~ C CTCTACCAGG GGCTCCTGCA
501 GGCCCTGGAA GGGATATCCT AA (SEQ ID MO:~9)
pMON25186.seq
251 ATGGCTATGG CTCCAGCTCT GCAACCAACT CAAGGTGCAA TGCCAGCATT
51 TGCATCTGCT TTTCAACGTC GTGCAGGTGG T~ ~GTT GCTAGCCATC
101 TGCAGAGCTT CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG
151 CCCTCTGGCG GCTCTGGCGG CTCTCAGAGC TTCCTGCTCA AG~l~C~lTAGA
201 GCAAGTGAGA AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT
30251 GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC
301 TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT
351 GCAGCTGGCA GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC
401 AGGGGCTCCT GCAGGCCCTG GAAGGGATAT CCCCCGAGTT GGGTCCCACC
451 TTGGACACAC TGCAGCTGGA CGTCGCCGAC TTTGCCACCA CCATCTGGCA
35501 GCAGATGGAA GAACTGGGAT AA (SEQ ID MO:40)
pMON25187.se~
401 ATGGCTACTC AAGGTGCTAT GCCAGCTTTT GCTTCTGCTT TTCAACGTCG
51 TGCAGGTGGT GTTCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT
101 CGTACCGCGT TCTACGCCAC CTTGCGCAGC CCTCTGGCGG CTCTGGCGGC
151 TCTCAGAGCT TCCTGCTCAA GTCTTTAGAG CAAGTGAGAA AGATCCAGGG
201 CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC AAGCTGTGCC
45251 ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT
301 CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG
351 CCAACTCCAT AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG
401 AAGGGATATC CCCCGAGTTG GGTCCCACCT TGGACACACT GCAGCTGGAC
451 GTCGCCGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGAAT
501 GGCCCCTGCC CTGCAGCCCT AA (SEQ ID NO:41)
CA 02234042 1998-04-06
W O 97/12977 PCTAJS96/15935
38
pMON25188.seq
1 ATGGCTTCTG CTTTTCAACG TCGTGCAGGT GGTGTTCTGG TTGCTAGCCA
51 TCTGCAGAGC TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC
101 AGCCCTCTGG CGGCTCTGGC GGCTCTCAGA GCTTCCTGCT CAAGTCTTTA
151 GAGCAAGTGA GAAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT
201 GTGTGCCACC TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC
251 ACTCTCTGGG CATCCCCTGG GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC
301 CTGCAGCTGG CAGGCTGCTT GAGCCAACTC CATAGCGGCC TTTTCCTCTA
351 CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA
401 CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG
451 CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG
501 TGCCATGCCG GCCTTCGCCT AA (SEQ ID NO:42)
pMON3460.seq
1 ATGGCTCTGC TCGGACACTC TCTGGGCATC CCCTGGGCTC CCCTGAGCTC
51 CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC CAACTCCATA
101 GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA AGGGATATCC
151 CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT
201 TGCCACCACC ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC
251 TGCAGCCCAC CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC
301 CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT
351 GTCGTACCGC GTTCTACGCC ACCTTGCGCA GCCCACACCA TTGGGCCCTG
401 CCAGCTCCCT GCCCCAGAGC TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA
451 AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT GTGCCACCTA
501 CAAGCTGTGC CACCCCGAGG AGCTGGTGTA ATAA (SEQ ID NO:86)
pMON3461.seq
1 ATGGCrCAAC TCCATAGCGG CCTTTTCCTC TACCAGGGGC TCCTGCAGGC
51 CCTGGAAGGG ATATCCCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC
101 TGGACGTCGC CGACTTTGCC ACCACCATCT GGCAGCAGAT GGAAGAACTG
151 GGAATGGCCC CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC
201 CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC
251 AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC
301 ACACCATTGG GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC
351 TTTAGAGCAA GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA
401 AG~l~G~l~G~l~GC CACCTACAAG CTGTGCCACC CCGAGGAGCT GGTGCTGCTC
451 GGACACTCTC TGGGCATCCC CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA
501 GGCCCTGCAG CTGGCAGGCT GCTTGAGCTA ATAA (SEQ ID NO:87)
pMON3462.seq
1 ATGGCTCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC
51 CCCCGAGTTG GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT
101 TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGAAT GGCCCCTGCC
151 CTGCAGCCCA CCCAGGGTGC CATGCCGGCC TTC~-CCTCTG CTTTCCAGCG
201 CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC TTCCTGGAGG
CA 02234042 1998-04-06
W O 97/12977 PCTAUS96/15935
39
251 TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCACACC ATTGGGCCCT
3 01 GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG
3 51 AAAGATCCAG GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT
401 ACAAGCTGTG CCACCCCGAG GAGCTGGTGC TGCTCGGACA CTCTCTGGGC
451 ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC AGCCAGGCCC TGCAGCTGGC
501 AGGCTGCTTG AGCCAACTCC ATAGCGGCTA ATAA (SEQ ID NO: 8 8 )
pMON3463 . seq
1 ATGGCTCTCT ACCAGGGGCT CCTGCAGGCC CTGGAAGGGA TATCCCCCGA
51 GTTGGGTCCC ACCTTGGACA CACTGCAGCT GGACGTCGCC GACTTTGCCA
101 CCACCATCTG GCAGCAGATG GAAGAACTGG GAATGGCCCC TGCCCTGCAG
151 CCCACCCAGG GTGCCATGCC GGCCTTCGCC TCTGCTTTCC AGCGCCGGGC
201 AGGAGGGGTC CTGGTTGCTA GCCATCTGCA GAGCTTCCTG GAGGTGTCGT
251 ACCGCGTTCT ACGCCACCTT GCGCAGCCCA CACCATTGGG CCCTGCCAGC
301 TCCCTGCCCC AGAGCTTCCT GCTCAAGTCT TTAGAGCAAG TGAGAAAGAT
3 51 CCAGGGCGAT GGCGCAGCGC TCCAGGAGAA GCTGTGTGCC ACCTACAAGC
401 TGTGC':~CCC CGAGGAGCTG GTGCTGCTCG GACACTCTCT GGGCATCCCC
451 TGGGCTCCCC TGAGCTCCTG CCCCAGCCAG GCCCTGCAGC TGGCAGGCTG
501 CTTGAGCCAA CTCCATAGCG GC~ll~ A ATAA (SEQ ID NO: 8 9 )
pMON3464 . seq
1 ATGGCTGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA CCTTGGACAC
51 ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG CAGCAGATGG
101 AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG
151 GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG
201 CCATCTGCAG AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG
251 CGCAGCCCAC ACCATTGGGC CCTGCCAGCT CCCTGCCCCA GAGCTTCCTG
301 CTCAAGTCTT TAGAGCAAGT GAGAAAGATC CAGGGCGATG GCGCAGCGCT
351 CCAGGAGAAG CTGTGTGCCA CCTACAAGCT GTGCCACCCC GAGGAGCTGG
401 TGCTGCTCGG ACACTCTCTG GGCATCCCCT GGGCTCCCCT GAGCTCCTGC
3 5 451 CCCAGCCAGG CCCTGCAGCT GGCAGGCTGC TTGAGCCAAC TCCATAGCGG
501 C~ CCTC TACCAGGGGC TCCTGCAGTA ATAA (SEQ ID NO: 9 0 )
pMON3 4 6 5.s eq
1 ATGGCTGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGAAT
51 GGCCC'TGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC TTCGCCTCTG
101 CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC
151 TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCACACC
201 ATTGGGCCCT GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AA~'l~C~l~l'l'AG
251 AGCAAGTGAG AAAGATCCAG GGCGATGGCG CAGCGCTCCA GGAGAAGCTG
3 01 TGTGCCACCT ACAAGCTGTG CCACCCCGAG GAGCTGGTGC TGCTCGGACA
351 CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC AGCCAGGCCC
4 01 TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT TTTCCTCTAC
451 CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC
501 CTTGGACACA CTGCAGCTGG ACGTCGCCTA ATAA (SEQ ID NO:91)
CA 02234042 l998-04-06
W O 97/12977 PCT~US96/15935
pMON 3466.seq
1 ATGGCTATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA
551 GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG
101 CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT GGAGGTGTCG
151 TACCGCGTTC TACGCCACCT TGCGCAGCCC ACACCATTGG GCCCTGCCAG
201 CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA GTGAGAAAGA
251 TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC CACCTACAAG
10301 CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC
351 CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT
401 GCTTGAGCCA ACTCCATAGC GGCCTTTTCC TCTACCAGGG GCTCCTGCAG
451 GCCCTGGAAG GGATATCCCC CGAGTTGGGT CCCACCTTGG ACACACTGCA
501 GCTGGACGTC GCCGACTTTG CCACCACCTA ATAA (SEQ ID NO:92)
pMON3 4 67.seq
1 ATGGCTCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC
2051 CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG
101 GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTCGTACCGC
151 GTTCTACGCC ACCTTGCGCA GCCCACACCA TTGGGCCCTG CCAGCTCCCT
201 GCCCCAGAGC TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA AAGATCCAGG
251 GCGATGGCGC AGCGCTCCAG GAGAAGCTGT GTGCCACCTA CAAGCTGTGC
2 5 301 CACCCCGAGG AGCTGGTGCT GCTCGGACAC TCTCTGGGCA TCCCCTGGGC
351 TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA GGCTGCTTGA
401 GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GCAGGCCCTG
451 GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA
501 CGTCGCCGAC TTTGCCACCA CCATCTGGTA ATAA (SEQ ID NO:9 3)
pMON3499.seq
1 ATGGCTTTGT TAGGACATTC TTTAGGTATT CCATGGGCTC CTCTGAGCTC
3551 CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC CAACTCCATA
101 GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA AGGGATATCC
151 CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT
201 TGCCACCACC ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC
251 TGCAGCCCAC CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC
40301 CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT
3 51 GTCGTACCGC GTTCTACGCC ACCTTGCGCA GCCCACACCA TTGGGCCCTG
401 CCAGCTCCCT GCCCCAGAGC TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA
451 AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT GTGCCACCTA
501 CAAGCTGTGC CACCCCGAGG AGCTGGTGTA ATAA (SEQ ID MO:94)
CA 02234042 1998-04-06
W O 97/12977 PCT~US96/15935
41
TABT.~. 3
PROTEIN SEO~ENCES
pMON3485.Pep
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser
Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala
Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu-Phe
Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu
Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe
Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln
Pro Ser Glv Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu Lys Ser
Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln
Glu Lys Leu Cys Ala Thr (SEQ ID NO:43)
pMON3486.Pep
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu
Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu
3 0 Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro
Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln
Ala Leu Glu Gly Ile Ser (SEQ ID NO:44)
pMON3487.Pep
Met Ala Pr.~ Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe
Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
Leu Ala Gln Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu
Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala
Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro
Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala
Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys
Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu
Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp
Gln Met Glu Glu Leu Gly ( SEQ ID NO:45)
pMON3488.Pep
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Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg
Ala Gly Gl-~ Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu
Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Ser Gly Gly
Ser Gly Gly Ser Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val
Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys
Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly
His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser
Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly
Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gln Pro (SEQ ID NO:46)
pMON3489.Pep
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu
Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu
Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro
Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
Ser Gln Le~ His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln
Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr
Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln
Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly
Ala Met Pro Ala Phe Ala (SEQ ID NO:47)
pMON3490.Pep
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser
Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala
Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe
Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu
Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe
Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln
Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala
Ala Leu Gln Glu Lys Leu Cys Ala Thr (SEQ ID NO:48)
pMON3491.Pep
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
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43
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
5 Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala
Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly
Leu Leu Gln Ala Leu Glu Gly Ile Ser (SEQ ID NO:49)
pMON3492.Pep
Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe
Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
Leu Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln
Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly
Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu
Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile
Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu
Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln
Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro
Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr
Ile Trp Gln Gln Met Glu Glu Leu Gly (SEQ ID NO:50)
pMON3493.Pep
Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg
Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu
Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro Leu
Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu
Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu
Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val
Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu
Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu
Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu
Leu Gly Met Ala Pro Ala Leu Gln Pro (SEQ ID NO:51)
pMON3494.Pep
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
5 0 Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala
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Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly
Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr
Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile
Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro
Thr Gln Gly Ala Met Pro Ala Phe Ala (SEQ ID NO:52)
pMON25181.pep
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
Phe Leu Leu Lys Ser Leu Glu Gln Val Ary Lys Ile Gln Gly Asp
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala
Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly
Leu Leu Gl n Ala Leu Glu Gly Ile Ser (SEQ ID NO:53)
pMON25182.pep
Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe
Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
Leu Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln
Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly
Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu
Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile
Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu
Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln
Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro
Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr
Ile Trp Gln Gln Met Glu Glu Leu Gly ( SEQ ID NO:54)
pMON25183.pep
Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg
Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu
Val Ser Ty-~ Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro Leu
Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu
Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu
Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val
Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu
Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu
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Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu
Leu Gly Met Ala Pro Ala Leu Gln Pro ( SEQ ID NO: 55)
pMON25184.pep
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Se~ Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala
Gly Cys Leu Ser Gln Leu HiS Ser Gly Leu Phe Leu Tyr Gln Gly
Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr
Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile
Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro
Thr Gln Gly Ala Met Pro Ala Phe Ala ( SEQ ID NO: 56)
pMON25185.pep
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu
Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu
Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro
Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln
Ala Leu Glu Gly Ile Ser ( SEQ ID NO: 57)
pMON25186.pep
Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe
Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
Leu Ala Gln Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu
Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala
Ala Leu Gln Glu Lys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu
Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser
Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala
Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu
Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met
Glu Glu Leu Gly ( SEQ ID NO: 58)
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46
pMON25187.pep
Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg
Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu
Val Ser Tyr Ar~ Val Leu Arg His Leu Ala Gln Pro Ser Gly Gly
Ser Gly Gly Ser Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val
Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys
Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly
His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser
10 Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly
Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met GLu Glu Leu Gly Met
Ala Pro Ala Leu Gln Pro (SEQ ID NO:59)
pMON25188.pep
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu
Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu
Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro
Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln
Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr
Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln
Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly
Ala Met Pro Ala Phe Ala (SEQ ID NO:60)
pMON3460.Pep
Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu L~u Gln Ala Leu Glu
Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu
Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu
Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro
Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val
Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu
Arg His Leu Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu
Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile
Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr
Lys Leu Cys His Pro Glu Glu Leu Val (SEQ ID NO:95)
pMON3461.Pep
Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala
Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu
Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met
,
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Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala
Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val
Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg
Val Leu Arg His Leu Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser
Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg
Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala
Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His
Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln
Ala Leu Gln Leu Ala Gly Cys Leu Ser ( SEQ ID NO: 9 6 )
3462. Pep
Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Tt~r Tyr Lys Leu Cys
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala
Gly Cys Leu Ser Gln Leu His Ser Gly (SEQ ID NO:97)
3463.Pep
Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu
Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe
Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln
Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala
Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro
Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala
Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys
Leu Ser Gln Leu His Ser Gly Leu Phe ( SEQ ID NO: 98 )
3464. Pep
Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr
Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln
Met Glu Gl.l Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly
Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly
Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr
Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro Leu Gly Pro Ala
Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val
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48
Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys
Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly
His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser
Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly
Leu Phe Leu Tyr Gln Gly Leu Leu Gln ( SEQ ID NO: 99)
3465. Pep
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Se.~ Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala
Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly
Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr
Leu Asp Thr Leu Gln Leu Asp Val Ala ( SEQ ID NO: 100)
3466. Pep
Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln
Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg
Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu
Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro
Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser
Leu Glu Gln Val Arg Lys Ile Gln Gly Asp G~ y Ala Ala Leu Gln
Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu
Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln
Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu
Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln
Leu Asp Val Ala Asp Phe Ala Thr Thr (SEQ ID NO:101)
3467. Pep
Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr
Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala
Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val
Ser ~yr Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro Leu Gly
Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu
Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys
Leu Cys Ala Thr ~yr Lys Leu Cys His Pro Glu Glu Leu Val Leu
Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys
Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp
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49
Val Ala AS~? Phe Ala Thr Thr Ile Trp (SEQ ID NO:102)
3499.Pep
Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu
Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu
Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu
Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro
Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val
Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu
Arg His Leu Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu
Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile
Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr
Lys Leu Cys His Pro Glu Glu Leu Val (SEQ ID NO:103)
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Materials and Methods
Recombinant DNA methods
Unless noted otherwise, all specialty chemicals were
obtained from Sigma Co., (St. Louis, MO). Restriction
endonuclea~es and T4 DNA ligase were obtained from New
England Biolabs (Beverly, MA) or Boehringer Mannheim
(Indianapolis, IN).
Tr~nsformation of E. coli strains
E. coli strains, such as DH5~M (Life Technologies,
Gaithersburg, MD) and TG1 (Amersham Corp., Arlington
Heights, IL) are used for transformation of ligation
reactions and are the source of plasmid DNA for transfecting
mammalian cells. E. coli strains, such as MON105 and JM101,
can be used for expressing the G-CSF receptor agonist of the
present invention in the cytoplasm or periplasmic space.
MON105 ATCC#55204: F-, lamda-,IN(rrnD, rrE)1, rpoD+, rpoH358
DH5a~: F-, phi80dlacZdeltaM15, delta(lacZYA-argF)U169,
deoR, recA1, endA1, hsdR17(rk-,mk+), phoA, supE441amda-,
thi-1, gyrA96, relA1
TG1: delta(lac-pro), supE, thi-1, hsdD5/F'(traD36, proA+B+,
lacIq, lacZdeltaM15)
DH5a~ Subcloning efficiency cells are purchased as
competent cells and are ready for transformation using the
manufacturer's protocol, while both E. coli strains TG1 and
MON105 are rendered competent to take up DNA using a CaC12
method. Typically, 20 to 50 mL of cells ~re grown in LB
35 medium (1% Bacto-tryptone, 0.5~ Bacto-yeast extract, 150 mM
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NaCl) to a density of approximately 1.0 optical density unit
at 600 nanometers (OD600) as measured by a Baush & Lomb
Spectronic spectrophotometer (Rochester, NY). The cells are
collected by centrifugation and resuspended in one-fifth
culture volume of CaC12 solution (50 mM CaCl2, 10 mM Tris-
Cl, pH7.4) and are held at 4'C for 30 minutes. The cells
are again collected by centrifugation and resuspended in
one-tenth culture volume of CaC12 solution. Ligated DNA is
added to 0.2mL of these cells, and the samples are held at
4 C for 1 hour. The samples are shifted to 42'C for two
minutes and lmL of LB is added prior to shaking the samples
at 37'C for one hour. Cells from these samples are spread
on plates 'LB medium plus 1.5% Bacto-agar) containing either
ampicillin (100 micrograms/mL, ug/mL) when selecting for
ampicillin-resistant transformants, or spectinomycin (75
ug/mL) when selecting for spectinomycin-resistant
transformants. The plates are incubated overnight at 37'C.
Single colonies are picked, grown in LB supplemented with
appropriate antibiotic for 6-16 hours at 37'C with shaking.
Colonies are picked and inoculated into LB plus
appropriate antibiotic (100 ug/mL ampicillin or 75 ug/mL
spectinomycin) and are grown at 37~C while shaking. Before
harvesting the cultures, 1 ul of cells are analyzed by PCR
for the presence of a G-CSF gene. The PCR is carried out
using a combination of primers that anneal to the G-CSF gene
and/or vector. After the PCR is complete, loading dye is
added to the sample followed by electrophoresis as described
earlier. ~ gene has been ligated to the vector when a PCR
product of the expected size is observed.
Methods for creation of aenes with new N-terminus/C-terminus
Method I. Creation of genes with new N-terminus/C-terminus
which contain a linker region.
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Genes with new N-terminus/C-terminus which contain a
linker region separating the original C-terminus and N-
terminus can be made essentially following the method
described in L. S. Mullins, et al ~. Am. Chem. Soc. 116,
5529-5533 '1994). Multiple steps of polymerase chain
reaction (PCR) amplifications are used to rearrange the DNA
sequence encoding the primary amino acid sequence of the
protein. The steps are illustrated in Figure 2.
In the first step, the primer set ("new start" and
"linker start") is used to create and amplify, from the
original gene sequence, the DNA fragment ("Fragment Start")
that contains the sequence encoding the new N-terminal
portion of the new protein followed by the linker that
connects the C-terminal and N-terminal ends of the original
protein. In the second step, the primer set ("new stop" and
"linker stop") is used to create and ampl;fy, from the
original gene sequence, the DMA fragment ("Fragment Stop")
that encodes the same linker as used above, followed by the
new C-terminal portion of the new protein. The "new start"
and "new stop" primers are designed to include the
appropriate restriction enzyme recognition sites which allow
cloning of the new gene into expression plasmids. Typical
PCR conditions are one cycle 95~C melting for two minutes;
25 cycles 94~C denaturation for one minute, 50~C annealing
for one minute and 72~C extension for one minu~e; plus one
cycle 72~C extension for seven minutes. A Perkin Elmer
GeneAmp PCR Core Reagents kit is used. A 100 ul reaction
contains 100 pmole of each primer and one ug of template
DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM
dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2
mM MgC12. PCR reactions are performed in a Model 480 DNA
thermal cycler (Perkin Elmer Corporation, Norwalk, CT).
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"Fragment Start" and "Fragment Stop", which have
complementary sequence in the linker region and the coding
sequence ~or the two amino acids on both sides of the
linker, are joined together in a third PCR step to make the
full-length gene encoding the new protein. The DNA
fragments "Fragment Start" and "Fragment Stop" are resolved
on a 1% TAE gel, stained with ethidium bromide and isolated
using a Qiaex Gel Extraction kit (Qiagen). These fragments
are combined in equimolar quantities, heated at 70~C for ten
minutes and slow cooled to allow annealing through their
shared se~lence in "linker start" and "linker stop". In the
third PCR step, primers "new start~ and "new stop" are added
to the annealed fragments to create and amplify the full-
length new N-terminus/C-terminus gene. Typical PCR
conditions are one cycle 95~C melting for two minutes; 25
cycles 94~C denaturation for one minute, 60~C annealing for
one minute and 72~C extension for one minute; plus one cycle
72~C extension for seven minutes. A Perkin Elmer GeneAmp
PCR Core Reagents kit is used. A 100 ul reaction contains
100 pmole of each primer and approximately 0.5 ug of DNA;
and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP,
200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM
MgCl2. PCR reactions are purified using a Wizard PCR Preps
kit (Promega).
Method II. Creation of genes with new N-terminus/C-terminus
without a linker region.
New N-terminus/C-terminus genes without a linker
joining the original N-terminus and C-terminus can be made
using two steps of PCR amplification and a blunt end
ligation. The steps are illustrated in Figure 3. In the
first step, the primer set ("new start" and "P-bl start") is
used to create and ampli~y, from the original gene sequence,
the DNA ~ragment ("Fragment Start") that contains the
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54
se~uence encoding the new N-terminal portion of the new
protein. In the second step, the primer set ("new stop" and
"P-bl stop") is used to create and amplify, ~rom the
original gene sequence, the DNA fragment ("Fragment Stop")
that contains the se~uence encoding the new C-terminal
portion of the new protein. The "new start" and "new stop"
primers are designed to include appropriate restriction
sites which allow cloning of the new gene into expression
vectors. Typical PCR conditions are one cycle 95~C melting
for two minutes; 25 cycles 94~C denaturation for one minute,
50~C annealing for 45 seconds and 72~C extension for 45
seconds. Deep Vent polymerase (New England Biolabs) is used
to reduce the occurrence of overhangs in conditions
recommended by the manufacturer. The "P-bl start" and "P-bl
stop" primers are phosphorylated at the 5' end to aid in the
subsequent blunt end ligation of "Fragment Start" and
"Fragment Stop" to each other. A 100 ul leaction contained
150 pmole of each primer and one ug of template DNA; and lx
Vent buf~er (New England Biolabs), 300 uM dGTP, 300 uM dATP,
300 uM dTTP, 300 uM dCTP, and 1 unit Deep Vent polymerase.
PCR reactions are performed in a Model 480 DNA thermal
cycler (Perkin Elmer Corporation, Norwalk, CT). PCR
reaction products are purified using a Wizard PCR Preps kit
(Promega).
The primers are designed to include appropriate
restriction enzyme recognition sites which allow for the
cloning of the new gene into expression vectors. Typically
"Fragment Start" is designed to create a NcoI restriction
site , and "Fragment Stop" is designed to create a HindIII
restriction site. Restriction digest reactions are purified
using a Magic DNA Clean-up System kit (Pr~mega). Eragments
Start and Stop are resolved on a 1% TAE gel, stained with
ethidium bromide and isolated using a Qiaex Gel Extraction
kit (Qiagen). These ~ragments are combined with and
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annealed to the ends of the ~ 3800 base pair NcoI/HindIII
vector fragment of pMON3934 by heating at 50~C for ten
minutes and allowed to slow cool. The three fragments are
ligated together using T4 DNA ligase (Boehringer Mannheim).
The result is a plasmid containing the full-length new N-
terminus/C-terminus gene. A portion o~ the ligation reaction
is used to transform E. coli strain DH5~cells (Life
Technologies, Gaithersburg, MD). Plasmid DNA is purified
and sequence confirmed as below.
Method III. Creation of new N-terminus/C-terminus genes by
tandem-duplication method
New N-terminus/C-terminus genes can be made based on
the method described in R. A. Horlick, et al Protein Eng.
5:427-431 (1992). Polymerase chain reaction (PCR)
amplification of the new N-terminus/C-terminus genes is
performed using a tandemly duplicated template DNA. The
steps are illustrated in Figure 4.
The tandemly-duplicated template DNA is created by
cloning and contains two copies of the gene separated by DNA
sequence encoding a linker connecting the original C- and N-
terminal ends of the two copies of the gene. Specific
primer sets are used to create and amplify a full-length new
N terminus~C-terminus gene from the tandemly-duplicated
template DNA. These primers are designed to include
appropriate restriction sites which allow for the cloning of
the new gene into expression vectors. Typical PCR conditions
are one cycle 95~C melting for two minutes; 25 cycles 94~C
denaturation for one minute, 50~C annealing for one minute
and 72~C extension for one minute; plus one cycle 72~C
extension for seven minutes. A Perkin Elmer GeneAmp PCR
Core Reagents kit (Perkin Elmer Corporation, Norwalk, CT) is
35 used. A 100 ul reaction contains 100 pmole of each primer
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and one ug of template DNA; and lx PCR buffer, 200 uM dGTP,
200 uM dAT', 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq
DNA polymerase and 2 mM MgCl2. PCR reactions are performed
in a Model 480 DNA thermal cycler (Perkin Elmer Corporation,
Norwalk, CT). PCR reactions are puri~ied using a Wizard PCR
Preps kit (Promega).
DNA isolation and characterization
Plasmid DNA can be isolated by a number of different
methods and using commercially available kits known to those
skilled in the art. A few such methods are shown herein.
Plasmid DNA is isolated using the Promega Wizard~ Miniprep
kit (Madison, WI), the Qiagen QIAwell Plasmid isolation kits
(Chatsworth, CA) or Qiagen Plasmid Midi kit. These kits
follow the same general procedure for plasmid DNA isolation.
Briefly, c-Lls are pelleted by centrifugation (5000 x g),
plasmid DNA released with sequential NaOH/acid treatment,
and cellular debris is removed by centrifugation (10000 x
g). The supernatant (containing the plasmid DNA) is loaded
onto a column containing a DNA-binding resin, the column is
washed, and plasmid DNA eluted with TE. After screening for
the colonies with the plasmid of interest, the E. coli cells
are inoculated into 50-100 mLs of LB plus appropriate
antibiotic for overnight growth at 37~C in an air incubator
while shaking. The purified plasmid DNA is used for DNA
sequencing, further restriction enzyme digestion, additional
subcloning of DNA fragments and transfection into mammalian,
E. coli or other cells.
Seauence confirmation.
Purif~ed plasmid DNA is resuspended in dH2O and
quantitated by measuring the absorbance at 260/280 nm in a
Bausch and Lomb Spectronic 601 W spectrometer. DNA samples
are sequenced using ABI PRISM~ DyeDeoxy~ terminator
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57
sequencing chemistry (Applied Biosystems Division of Perkin
Elmer Corporation, Lincoln City, CA) kits (Part Number
401388 or 402078) according to the manufacturers suggested
protocol usually modified by the addition of 5% DMSO to the
sequencing mixture. Sequencing reactions are performed in a
Model 480 DNA thermal cycler (Perkin Elmer Corporation,
Norwalk, CT) following the recommended am~lification
conditions. Samples are purified to remove excess dye
termin~tors with Centri-Sep~ spin columns (Princeton
Separations, Adelphia, NJ) and lyophilized. Fluorescent dye
labeled sequencing reactions are resuspended in deionized
formamide, and sequenced on denaturing 4.75% polyacrylamide-
8M urea gels using an ABI Model 373A automated DNA
sequencer. Overlapping DNA sequence fragments are analyzed
and assembled into master DNA contigs using Se~uencher v2.1
DNA analysis software (Gene Codes Corporation, Ann Arbor,
MI).
~x~ression of G-CSF rece~tor aaonists in mammalian cells
Mammalian Cell Transfection/Production of Conditioned Media
The BHK-21 cell line can be obtained from the ATCC
(Rockville, MD). The cells are cultured in Dulbecco~s
modified Eagle media (DMEM/high-glucose), supplemented to
2mM (mM) L-glutamine and 10% fetal bovine serum (FBS). This
formulation is designated BHK growth media. Selective media
is BHK growth media supplemented with 453 units/mL
hygromycin B (Calbiochem, San Diego, CA). The BHK-21 cell
line was previously stably transfected with the HSV
transactivating protein VP16, which transactivates the IE110
promoter found on the plasmid pMON3359 (See Hippenmeyer et
al., Bio/Technolo~y, pp.1037-1041, 1993). The VP16 protein
drives expression of genes inserted behind the IE110
promoter. BHK-21 cells expressing the transactivating
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protein V~'O are designated BHK-VP16. The plasmid pMON1118
(See Highkin et al., Poultry Sci., 70: 970-981, 1991)
expresses the hygromycin resistance gene from the SV40
promoter. A similar plasmid is available ~rom ATCC, pSV2-
hph.
BHK-VP16 cells are seeded into a 60 millimeter (mm)
tissue culture dish at 3 X 105 cells per dish 24 hours prior
to transfection. Cells are transfected for 16 hours in 3 mL
of "OPTIMEM"~ (Gibco-BRL, Gaithersburg, MD) containing 10
ug of plasmid DNA containing the gene of interest, 3 ug
hygromycin resistance plasmid, pMON1118, and 80 ug of Gibco-
BRL "LIPOFECTAMINE"~ per dish. The media is subsequently
aspirated and replaced with 3 mL of growt. media. At 48
hours post-transfection, media from each dish is collected
and assayed for activity (transient conditioned media). The
cells are r2moved from the dish by trypsin-EDTA, diluted
1:10 and transferred to 100 mm tissue culture dishes
containing 10 mL of selective media. After approximately 7
days in selective media, resistant cells grow into colonies
several millimeters in diameter. The colonies are removed
from the dish with filter paper (cut to approximately the
same size as the colonies and soaked in trypsin/EDTA) and
transferred to individual wells of a 24 well plate
con~aining 1 mL of selective media. After the clones are
arown to confluence, the conditioned media is re-assayed,
and positive clones are expanded into growth media.
E~ression of G-CSF rece~tor aaonists in ~. coli
E. coli strain MON105 or JM101 harboring the plasmid of
interest are grown at 37~C in M9 plus casamino acids medium
with shaking in a air incubator Model G2S from New Brunswick
Scientific (Edison, New Jersey). Growth is monitored at
OD600 until it reaches a value of 1, at which time nalidixic
acid (10 milligrams/mL) in 0.1 N NaOH is added to a final
=
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59
concentration of 50 ~g/mL. The cultures are then shaken at
37~C for three to four additional hours. A high degree of
aeration is maintained throughout culture period in order to
achieve maximal production of the desired gene product. The
cells are examined under a light microsccpe for the presence
of inclusion bodies (IB). One mL aliquots of the culture
are removed ~or analysis of protein content by boiling the
pelleted cêlls, treating them with reducing buffer and
electrophoresis via SDS-PAGE (see Maniatis et al. Molecular
Cloning: A Laboratory Manual, 1982). The culture is
centrifuged (5000 x g) to pellet the cells.
Inclusion Bodv pre~aration, Extraction Re~oldina, Dialysis,
D~E Chromatoara~hv, and Characterization of the ~-CSF
rece~tor aaonists which accllmlllate as inclusion bodies in E~
col i .
Isolation of Inclusion Bodies:
The cell pellet from a 330 mL E. col 7 culture is
resuspended in 15 mL of sonication buffer (10 mM 2-amino-2-
(hydroxymethyl) 1,3-propanediol hydrochloride (Tris-HCl), pH
8.0 + 1 mM ethylenediaminetetraacetic acid (EDTA)). These
resuspended cells are sonicated using the microtip probe of
a Sonicator Cell Disruptor (Model W-375, Heat Systems-
Ultrasonics, Inc., Farmingdale, New York). Three rounds of
sonication in sonication buffer followed by centrifugation
are employed to disrupt the cells and wash the inclusion
bodies (IB). The first round of sonication is a 3 minute
burst followed by a 1 minute burst, and the final two rounds
of sonication are for 1 minute each
- Extraction and refolding of proteins from inclusion body
pellets:
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Follc~ing the final centrifugation step, the IB pellet
is resuspended in 10 mL of 50 mM Tris-HCl, pH 9.5, 8 M urea
and 5 mM dithiothreitol (DTT) and stirred at room
tempera~ure for approximately 45 minutes to allow for
denaturation of the expressed protein.
The extraction solution is transferred to a beaker
containing 70 mL of 5mM Tris-HCl, pH 9.5 and 2.3 M urea and
gently stirred while exposed to air at 4~C for 18 to 48
hours to allow the proteins to refold. Refolding is
monitored by analysis on a Vydac (Hesperia, Ca.) C18
reversed phase high pressure liquid chromatography (RP-HPLC)
column (0.46x25 cm). A linear gradient of 40% to 65%
acetonitrile, containing 0.1% trifluoroacetic acid (TFA), is
employed to monitor the refold. This gradient is developed
over 30 minutes at a flow rate of 1.5 mL per minute.
Denatured !;roteins generally elute later in the gradient
than the refolded proteins.
Puri~ication:
Following the refold, contaminating E. coli proteins
are removed by acid precipitation. The pH of the re~old
solution is titrated to between pH 5.0 and pH 5.2 using 15%
(v/v) acetic acid (HOAc). This solution is stirred at 4~C
for 2 hours and then centrifuged for 20 minutes at 12,000 x
g to pellet any insoluble protein.
The supernatant from the acid precipitation step is
dialyzed using a Spectra/Por 3 membrane with a molecular
weight cut off (MWCO) of 3,500 daltons. The dialysis is
against 2 changes of 4 liters (a 50-fold excess) of 10mM
Tris-HCl, ~?H 8.0 for a total of 18 hours. Dialysis lowers
the sample conductivity and removes urea prior to DEAE
chromatography. The sample is then centrifuged (20 minutes
at 12,000 x g) to pellet any insoluble protein following
dialysis.
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61
A Bio-Rad Bio-Scale DEAE2 column (7 x 52 mm) is used
for ion exchange chromatography. The column is e~uilibrated
in a buffer containing lOmM Tris-HCl, pH 8Ø The protein is
eluted using a 0-to-500 mM sodium chloride (NaCl) gradient,
in equilibration buffer, over 45 column volumes. A flow
rate of 1 mL per minute is used throughou~ the run. Column
fractions (2 mL per fraction) are collected across the
gradient and analyzed by RP HPLC on a Vydac (Hesperia, Ca.)
C18 column (0.46 x 25 cm). A linear gradient of 40~ to 65%
acetonitrile, containing 0.1% trifluoroacetic acid (TFA), is
employed. This gradient is developed over 30 minutes at a
flow rate of 1.5 mL per minute. Pooled fractions are then
dialyzed against 2 changes of 4 liters (50-to-500-fold
excess) of 10 mM ammonium acetate (NH4Ac), pH 4.0 for a
total of 18 hours. Dialysis is performed using a
Spectra/Por 3 membrane with a MWCO of 3,500 daltons.
Finally, the sample is sterile filtered using a 0.22~m
syringe filter (~Star LB syringe filter, Costar, Cambridge,
Ma.), and stored at 4~C.
In some cases the folded proteins can be affinity
purified using affinity reagents such as mAbs or receptor
subunits attached to a suitable matrix. Alternatively, (or
in addition) purification can be accomplished using any of a
variety of chromatographic methods such as: ion exchange,
gel filtration or hydrophobic chromatography or reversed
phase HPLC.
These and other protein purification methods are
described in detail in Methods in Enzymology, Volume 182
'Guide to Protein Purification' edited by Murray Deutscher,
Academic Press, San Diego, CA (1990).
- Protein Characterization:
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62
The purified protein is analyzed by RP-HPLC,
electrospray mass spectrometry, and SDS-PAGE. The protein
~uantitation is done by amino acid composition, RP-HPLC, and
Bradford protein determination. In some cases tryptic
peptide mapping is performed in conjunction with
electrospray mass spectrometry to confirm the identity of
the protein.
A~r Proliferation Assay
The factor-dependent cell line AML 193 was obtained
from the American Type Culture Collection (ATCC, Rockville,
MD). This cell line, established from a patient with acute
myelogenous leukemia, is a growth factor dependent cell line
which disp~ayed enhanced growth in GM-CSF supplemented
medium (Lange, B., et al., Blood 70: 192, 1987; Valtieri,
M., et al., ~. Immunol . 138:4042, 1987). The ability o~ AML
193 cells to proliferate in the presence of human IL-3 has
also been documented. (Santoli, D., et al., J. Immunol .
139: 348, 1987). A cell line variant was used, AML 193 1.3,
which was adapted for long term growth in IL-3 by washing
out the growth factors and starving the cytokine dependent
AML 193 cells for growth factors for 24 hours. The cells
are then replated at lx105 cells/well in a 24 well plate in
media containing 100 U/mL IL-3. It took approximately 2
months for the cells to grow rapidly in IL-3. These cells
are maintained as AML 193 1.3 thereafter by supplementing
tissue culture medium (see below) with human IL-3.
AML 193 1.3 cells are washed 6 times in cold Hanks
balanced salt solution (HBSS, Gibco, Grand Island, NY) by
centrifuging cell suspensions at 250 x g for 10 minutes
followed by decantation of the supernatant. Pelleted cells
are resuspended in HBSS and the procedure is repeated until
six wash cycles are completed. Cells washed six times by
this procedure are resuspended in tissue culture medium at a
density ranging from 2 x 105 to 5 x 105 viable cells/mL.
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This medium is prepared by supplementing Iscove's modified
Dulbecco's Medium (IMDM, Hazelton, Lenexa, KS) with albumin,
transferrin, lipids and 2-mercaptoethanol. Bovine albumin
(soehringer-Mannheim, Indianapolis, IN) i~ added at 500
~g/mL; human transferrin (Boehringer-Mannheim, Indianapolis,
IN) is added at 100 ~g/mL; soybean lipid (Boehringer-
Mannheim, lndianapolis, IN) is added at 50 ~g/mL; and 2-
mercaptoethanol (Sigma, St. Louis, MO) is added at 5 x 10- 5
M.
Serial dilutions of G-CSF receptor agonist proteins
are made in triplicate series in tissue culture medium
supplemented as stated above in 96 well Costar 3596 tissue
- culture plates. Each well contained 50 ~1 of medium
containing G-CSF receptor agonist proteins once serial
dilutions are completed. Control wells contained tissue
culture medium alone (negative control). AML 193 1.3 cell
suspensions prepared as above are added to each well by
pipetting 50 ~1 (2.5 x 104 cells) into each well. Tissue
culture plates are incubated at 37~C with 5% CO2 in
humidified air for 3 days. On day 3, 0.5 ~Ci 3H-thymidine
(2 Ci/mM, New England Nuclear, Boston, MA) is added in 50 ~l
of tissue culture medium. Cultures are incubated at 37~C
with 5% CO2 in humidified air for 18-24 hours. Cellular DNA
is harvested onto glass filter mats (Pharmacia LKB,
Gaithersburg, MD) using a TOMTEC cell harvester (TOMTEC,
Orange, CT) which utilized a water wash cycle followed by a
70~ ethanol wash cycle. Filter mats are allowed to air dry
and then placed into sample bags to which scintillation
fluid (Scintiverse II, Fisher Scientific, St. Louis, MO or
BetaPlate Scintillation Fluid, Pharmacia LKB, Gaithersburg,
MD) is added. Beta emissions of samples from individual
tissue culture wells are counted in a LKB BetaPlate model
- 1205 scintillation counter (Pharmacia LKB, Gaithersburg, MD)
and data is expressed as counts per minute of 3H-thymidine
incorporated into cells from each tissue culture well.
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64
Activity of each G-CSF receptor agonist proteins preparation
is quantitated by measuring cell proliferation (3H-thymidine
incorporation) induced by graded concentrations of G-CSF
receptor ayonist. Typically, concentration ranges from 0.05
pM - 105 pM are quantitated in these assays. Activity is
determined by measuring the dose of G-CSF receptor agonist
protein which provides 50% o~ maximal proliferation (ECso =
0.5 x (maximum average counts per minute of 3H-thymidine
incorporated per well among triplicate cultures of all
concentrations of G-CSF receptor agonists tested -
background proliferation measured by 3H-thymidine
incorporation observed in triplicate cultures lacking any
factor). This ECso value is also equivalent to 1 unit of
bioactivity. Every assay is performed with native
interleukin-3 and G-CSF as reference standards so that
relative activity levels could be assigned.
Typically, the G-CSF receptor agonist proteins were
tested in a concentration range of 2000 pM to 0.06 pM
titrated in serial 2 fold dilutions.
Activity for each sample was determined by the
concentration which gave 50% of the maximal response by
fitting a four-parameter logistic model to the data. It was
observed that the upper plateau (maximal response) for the
sample and the standard with which it was compared did not
differ. Therefore relative potency calculation for each
sample was determined from EC50 estimations for the sample
and the standard as indicated above.
Other in vltro cell hased ~rol;feration assays
Other in vitro cell based proliferation assays, known
to those skilled in the art, may also be useful to determine
the activity of the G-CSF receptor agonists in a similar
manner as described in the AML 193.1.3 cell proliferation
assay.
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Transfected cell lines:
Cell lines, such as BHK or the murine pro B cell line
Baf/3, can be transfected with a colony stimulating factor
receptor, .uch as the human G-CSF receptor which the cell
line does not have. These transfected cell lines can be used
to determine the activity of the ligand of which the
receptor has been transfected.
E~MPT,~. 1
Construction o~ pMON3485
The new N-terminus/C-terminus gene in pMON3485 was
created using Method I as described in Materials and
Methods. Fragment Start was created and amplified from G-
CSF Ser17 se~uence in pMON13037 using the primer set, 39
start (SEQ ID NO:7) and L-11 start (SEQ ID NO:3). Fragment
Stop was created and amplified from G-CSF Ser17 se~uence in
the plasmid, pMON13037 (WO 95/21254), using the primer set,
38 stop (SEQ ID NO:8) and L-11 stop (SEQ ID NO:4). The
full-length new N terminus/C-terminus G-CSF Ser17 gene was
created and amplified from the annealed Fragments Start and
Stop using the primers 39 start (SEQ ID NO:7) and 38 stop
(SEQ ID NO:8).
The resulting DNA fragment which contains the new gene
was digested with restriction endonucleases NcoI and HindIII
and purified using a Magic DNA Clean-up System kit (Promega,
Madison, WI). The plasmid, pMON3934 (derivative of
pMON33S9), was digested with restriction endonucleases
HindIII and NcoI, resulting in an approximately 3800 base
pair vector fragment, and gel-purified. The purified
restriction fragments were combined and ligated using T4 DNA
~ 35 ligase. A portion of the ligation reaction was used to
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transform E. coli strain DH5a cells (Life Technologies,
Gaithersburg, MD). Transformant bacteria were selected on
ampicillin-containing plates. Plasmid DMA was isolated and
sequenced to confirm the correct insert. The resulting
plasmid was designated pMON3485.
BHK cells were transfected with the plasmid, pMoN3485,
for protein expression and bioassay.
The plasmid, pMON3485 containing the gene sequence of
(SEQ ID NO:25), encodes the following amino acid sequence:
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser
Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala
Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe
Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu
Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe
Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln
Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu Lys Ser
Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln
Glu Lys Leu Cys Ala Thr (SEQ ID NO:43)
EXAMPLE 2
Construction of ~MON3486
The new N-terminus/C-terminus gene in pMON3486 was
created using Method I as described in Materials and
Methods. Fragment Start was created and amplified from G-
CSF Serl7 sequence in the plasmid, pMON13037, using the
primer set, 97 start (SEQ ID NO:9) and L-ll start (SEQ ID
NO:3). Fragment Stop was created and amplified from G-CSF
Serl7 sequence in pMON13037 using the primer set, 96 stop
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67
(SEQ ID NO:10) and L-11 stop (SEQ ID NO:4). The full-length
new N terminus/C-terminus G-CSF Ser17 gene was created and
amplified from the annealed Fragments Start and Stop using
the primers 97 start (SEQ ID NO:9) and 96 stop (SEQ ID
NO:10).
The resulting DNA ~ragment which contains the new gene
was digested with restriction endonucleases NcoI and HindIII
and gel-purified using a Magic DNA Clean-up System kit. The
plasmid, pMoN3934, was digested with restriction
endonucleases HindIII and NcoI, resulting in an
approximately 3800 base pair vector fragment, and gel-
puri~ied. The purified restriction fragments were combined
and ligated using T4 DNA ligase. A portion of the ligation
reaction was used to trans~orm E. coli strain DH5a cells.
Transformant bacteria were selected on ampicillin-containing
plates. Plasmid DNA was isolated and sequenced to con~irm
the correct insert. The resulting plasmid was designated
pMON3486.
BHK cells were transfected with the plasmid, pMON3486,
for protein expression and bioassay.
The plasmid, pMON3486 containing the gene sequence of
(SEQ ID NO:26), encodes the following amino acid sequence:
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu
Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu
Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro
Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
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68
Ser Gln Leu His Ser Gly Leu Phe Leu ~yr Gln Gly Leu Leu Gln
Ala Leu Glu Gly Ile Ser (SEQ ID NO:44)
.~PLE 3
Construction of ~MON3487
The new N-terminus/C-terminus gene in pMON3487 was
created using Method I as described in Materials and
Methods. Fragment Start was created and ampli~ied from G-
CSF Ser17 sequence in the plasmid, pMON13037, using the
primer set, 126 start (SEQ ID NO:11) and L-11 start (SEQ ID
NO:3). Fragment Stop was created and amplified from G-CSF
Ser17 sequence in pMON13037 using the primer set, 125 stop
(SEQ ID NO:12) and L-11 stop (SEQ ID NO:4). The full-length
new N terminus/C-terminus G-CSF Ser17 gene was created and
amplified from the annealed Fragments Start and Stop using
the primers 126 start (SEQ ID NO:11) and 125 stop (SEQ ID
NO:12).
The resulting DNA fragment which contains the new gene
was digested with restriction endonucleases NcoI and HindIII
and purified using a Magic DNA Clean-up System kit. The
plasmid, pMON3934, was digested with restriction
endonucleases HindIII and NcoI, resulting in an
approximately 3800 base pair vector ~ragment, and gel-
purified. The purified restriction fragments were combined
and ligated using T4 DNA ligase. A portion of the ligation
reaction was used to transform E. coli strain DH5a cells.
Transformant bacteria were selected on ampicillin-containing
plates. Plasmid DNA was isolated and sequenced to confirm
the correct insert. The resulting plasmid was designated
pMON3487.
BHK cells were transfected with the plasmid, pMON3487,
for protein expression and bioassay.
-
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69
The plasmid, pMON3487 containing the gene sequence of
(SEQ ID NO:27), encodes the following amino acid se~uence:
Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe
Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
Leu Ala Gln Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu
Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala
Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro
Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala
Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys
Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu
Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp
Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln
Gln Met Glu Glu Leu Gly (SEQ ID MO:45)
~MPLE 4
Co~truction of ~MON3488
The new N-terminus/C-terminus gene in pMON3488 was
created using Method I as described in Materials and
Methods. Fragment Start was created and amplified from G-
CSF Serl7 sequence in the plasmid, pMON13037, using the
primer set, 133 start (SEQ ID NO:13) and L-ll start (SEQ ID
NO:3). Fragment Stop was created and amplified from G-CSF
Serl7 se~uence in the plasmid, pMON13037 using the primer
set, 132 stop (SEQ ID NO:14) and L-ll stop (SEQ ID NO:4).
The full-length new N terminus/C-terminus G-CSF Serl7 gene
was created and amplified ~rom the annealed Fragments Start
and Stop using the primers 133 start (SEQ ID NO:13) and 132
stop (SEQ ID NO:14).
The resulting DNA ~ragment which contains the new gene
was digested with restriction endonucleases NcoI and HindIII
and purified using a Magic DNA Clean-up System kit. The
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plasmid, pMOM3934, was digested with restriction
endonucleases HindIII and NcoI, resulting in an
approximately 3800 base pair vector fragment, and gel-
purified. The purified restriction fragments were combined
and ligated using T4 DMA ligase. A portion of the ligation
reaction was used to transform E. coli strain DH5acells.
Transformant bacteria were selected on ampicillin-containing
plates. Plasmid DNA was isolated and sequenced to confirm
the correct insert. The resulting plasmid was designated
pMOM3488.
BHK cells were transfected with the plasmid, pMON3488,
for protein expression and bioassay.
The plasmid, pMON3488 containing the gene sequence of
(SEQ ID MO:28), encodes the following amino acid se~uence:
Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg
Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu
Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Ser Gly Gly
Ser Gly Gly Ser Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val
Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys
Ala Thr Ty~ Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly
His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser
Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly
Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gln Pro (SEQ ID MO:46)
E~MPT,~ 5
Construction of ~MOM3489
The new M-terminus/C-terminus gene in pMOM3489 was
created using Method I as described in Materials and
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71
Methods. Fragment Start was created and amplified from G-
CSF Ser17 se~uence in the plasmid, pMON13037, using the
primer set, ~42 start (SEQ ID MO:15) and L-11 start (SEQ ID
NO:3). Fragment Stop was created and amplified from G-CSF
Ser17 sequence in pMON13037 using the primer set, 141 stop
(SEQ ID NO:16) and L-11 stop (SEQ ID NO:4). The full-length
new N terminus/C-terminus G-CSF Ser17 gene was created and
amplified from the annealed Fragments Start and Stop using
the primers 142 start (SEQ ID NO:15) and 141 stop (SEQ ID
NO:16).
The resulting DNA fragment which contains the new gene
was digested with restriction endonucleases NcoI and HindIII
and purified using a Magic DNA Clean-up System kit. The
plasmid, pMON3934, was digested with restriction
endonucleases HindIII and NcoI, resulting in an
approximately 3800 base pair vector fragment, and gel-
purified. The purified restriction fragments were combined
and ligated using T4 DNA ligase. A portion of the ligation
reaction was used to transform E. coli strain DH5acells.
Transformant bacteria were selected on ampicillin-containing
plates. Plasmid DNA was isolated and se~uenced to confirm
the correct insert. The resulting plasmid was designated
pMON3489.
BHK cells were transfected with the plasmid, pMON3489,
for protein expression and bioassay.
The plasmid, pMON3489 containing the gene se~uence of
(SEQ ID NO:29), encodes the following amino acid se~uence:
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu
Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu
Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro
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Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Beu
Ser Gln Leu His Ser Gly Leu Phe Leu ~yr Gln Gly Leu Leu Gln
Ala Leu Gl~3 Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr
Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln
Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly
Ala Met Pro Ala Phe Ala (SEQ ID NO:47)
~MPLE 6
Construction of pMON3490
The new N-terminus/C-terminus gene in pMON3490 was
created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G-
CSF sequence in the plasmid, pMON13037, using the primer
set, 39 start (SEQ ID NO:7) and P-bl start (SEQ ID NO:5).
Fragment Stop was created and amplified from G-CSF Ser17
sequence in pMON13037 using the primer set, 38 stop (SEQ ID
NO:8) and P-bl stop (SEQ ID MO:6). Fragment Start was
digested with restriction endonuclease NcoI, and Fragment
Stop was digested with restriction endonuclease HindIII.
After purification, the digested Fragments Start and Stop
were combined with and ligated to the approximately 3800
base pair NcoI-HindIII vector fragment of pMON3934.
Transformant bacteria were selected on ampicillin-containing
plates. Plasmid DNA was isolated and se~uenced to confirm
the correct insert. The resulting plasmid was designated
pMON3490.
BHK ceLls were transfected with the plasmid, pMON3490,
for protein expression and bioassay.
The plasmid, pMON3490 containing the gene sequence of
(SEQ ID NO:30), encodes the following amino acid sequence:
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73
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser
Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala
Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe
Leu ~yr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu
Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe
Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln
Pro Thr Pr-o Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala
Ala Leu Gln Glu Lys Leu Cys Ala Thr (SEQ ID NO:48)
~MP~E 7
Construction of ~MON3491
The new N-terminus/C-terminus gene in pMON3491 was
created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G-
CSF sequence in the plasmid, pMON13037, using the primer
set, 97 start (SEQ ID MO:9) and P-bl start (SEQ ID NO:5).
Fragment Stop was created and ampli~ied from G-CSF Serl7
sequence in pMON13037 using the primer set, 96 stop (SEQ ID
NO:10) and P-bl stop (SEQ ID NO:6). Fragment Start was
digested with restriction endonuclease NcoI, and Fragment
Stop was digested with restriction endonuclease HindIII.
After purification, the digested Fragments Start and Stop
were combined with and ligated to the approximately 3800
base pair NcoI-HindIII vector fragment of pMON3934. A
portion of the ligation reaction was used to transform E.
coli strain DH5a cells. Transformant bacteria were
selected on ampicillin-containing plates. Plasmid DNA was
isolated and sequenced to confirm the correct insert. The
resulting plasmid was designated pMON3491.
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74
BHK cells were transfected with the plasmid, pMON3491,
for protei. expression and bioassay.
The plasmid, pMON3491 containing the gene sequence of
(SEQ ID NO:31), encodes the following amino acid sequence:
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala
Gly Cys L~u Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly
Leu Leu Gln Ala Leu Glu Gly Ile Ser (SEQ ID NO:49)
~MPT.~ 8
Construction of ~MON3492
The new N-terminus/C-terminus gene in pMON3492 was
created using Method II as described in Materials and
Methods. Fragment Start was created and ampli~ied ~ro~ G-
CSF sequence in the plasmid, pMON13037, using the primer
set, 126 start (SEQ ID NO:11) and P-bl start (SEQ ID NO:5).
Fragment Stop was created and amplified from G-CSF Ser17
sequence in pMON13037 using the primer set, 125 stop (SEQ ID
NO:12) and P-bl stop (SEQ ID NO:6). Fragment Start was
digested w.~th restriction endonuclease NcoI, and Fragment
Stop was digested with restriction endonuclease HindIII.
After purification, the digested Fragments Start and Stop
were combined with and ligated to the approximately 3800
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base pair NcoI-HindIII vector fragment of pMOM3934. A
portion of the ligation reaction was used to transform E.
coli strain DH5a cells. Transformant bacteria were
selected on ampicillin-containing plates. Plasmid DNA was
isolated and sequenced to confirm the correct insert. The
resulting plasmid was designated pMON3492.
BHK cells were trans~ected with the plasmid, pMON3492,
for protein expression and bioassay.
The plasmid, pMON3492 containing the gene sequence of
(SEQ ID NO:32), encodes the following amino acid sequence:
Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe
Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
Leu Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln
Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly
Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu
Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile
Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu
Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln
Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro
Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr
Ile Trp Gln Gln Met Glu Glu Leu Gly (SEQ ID NO:50)
~X~MpT,~ 9
Construction of ~MON3493
The new N-terminus/C-terminus gene in pMON3493 was
created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G-
CSF se~uence in the plasmid, pMON13Q37, using the primer
set, 133 start (SEQ ID NO:13) and P-bl start (SEQ ID NO:5).
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76
Fragment Stop was created and amplified from G-CSF Ser17
sequence in pMON13037 using the primer set, 132 stop (SEQ ID
NO:14) and P-bl stop (SEQ ID NO:6). Fragment Start was
digested with restriction endonuclease NcoI, and Fragment
Stop was digested with restriction endonuclease HindIII.
After purification, the digested Fragments Start and Stop
were combined with and ligated to the approximately 3800
base pair NcoI-HindIII vector fragment of pMON3934. A
portion of the ligation reaction was used to transform E.
coli strain DH5a cells. Transformant bacteria were
selected on ampicillin-containing plates. Plasmid DNA was
isolated and sequenced to confirm the correct insert. The
resulting plasmid was designated pMON3493.
BHK cells were transfected with the plasmid, pMON3493,
for proteil expression and bioassay.
The plasmid, pMON3493 containing the gene sequence of
(SEQ ID NO:33), encodes the following amino acid se~uence:
Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg
Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu
Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro Leu
Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu
Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu
Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val
Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu
Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu
Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu
Leu Gly Met Ala Pro Ala Leu Gln Pro (SEQ ID MO:51)
~MPLE 10
~onstruc~ion cf ~MON3494
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The new N-terminus/C-terminus gene in pMON3494 was
created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G-
CSF sequence in the plasmid, pMON13037, using the primer
set, 142 start (SEQ ID NO:15) and P-bl start (SEQ ID NO:5).
Fragment Stop was created and amplified ~rom G-CSF Ser17
se~uence i~ pMON13037 using the primer set, 141 stop (SEQ ID
NO:16) and P-bl stop (SEQ ID NO:6). Fragment Start was
digested with restriction endonuclease NcoI, and Fragment
Stop was digested with restriction endonuclease HindIII.
After purification, the digested Fragments Start and Stop
were combined with and ligated to the approximately 3800
base pair NcoI-HindIII vector fragment of pMON3934. A
portion o~ the ligation reaction was used to trans~orm E.
coli strain DH5~ cells. Trans~ormant bacteria were
selected on ampicillin-containing plates. Plasmid DMA was
isolated and se~uenced to confirm the correct insert. The
resulting plasmid was designated pMON3494.
BHK cells were transfected with the plasmid, pMON3494,
for protein expression and bioassay.
The plasmid, pMON3494 containing the gene sequence o~
(SEQ ID NO:34), encodes the ~ollowing amino acid sequence:
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala
Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly
Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr
Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile
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Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro
Thr Gln Gly Ala Met Pro Ala Phe Ala (SEQ ID NO:52)
~mnles 11-20
The genes encoding the G-CSF receptor agonists of
Examples 1-10 were excised from the BHK vectors as a
NcoI/HindIII fragment and ligated with the ~ 3630 base pair
NcoI/HindIII vector fragment of pMOM2341 (WO 94/12638). The
resulting plasmids (Examples 11-20) are indicated in Table
4. The plasmids were transformed into E. coli strain JM101
cells and expression of the G-CSF receptor agonist protein
was evaluated. The proteins expressed are the same as those
expressed in the parental BHK expression vector except the
proteins were immediately preceded by a Methionine-Alanine
dipeptide and the Methionine is processed of~ by methionine
aminopeptidase. Overnight growths of cells (20 Klett units)
were inoculated in 10mL of m; ni m~l M9 medium supplemented
with vitamin Bl and trace minerals and incubated with
shaking at 37~C until initial Klett readings of ~120 units
were obtained. At 120 Klett units 50uL of 10mg/mL nalidixic
acid was added. Four hours post-induction, a lml aliquot was
removed for protein expression analysis by SDS-PAGE. Cells
were also examined using light microscopy for the presence
of inclusion bodies. Only pMON3450 and pMON3455 had
significant expression levels of the G-CSF receptor agonist
protein. In an effort to improve expression levels of G-CSF
receptor agonists, the 5' end of the genes were re-
engineered to incorporate AT-rich codons and E. coli
preferred codons between the uni~ue NcoI and NheI
restriction endonuclease recognition sites (Examples 21-28).
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7 TABLE 4
. coli expression plasmids
Resulting Parental
Example # E. coli Breakpoint Linker BHK plasmid
expression pMON~
plasmid
pMON#
Example 11 pMON3450 38/39zero pMON3490
Example 12 pMON3455 38/39~1-10 pMON3485
Example 13 pMON3451 96/97zero pMON3491
Example 14 pMON3456 96/97~1-10 pMON3486
Example 15 pMON3452 125/126zero pMON3492
Example 16 pMON3457 125/126~1-10 pMON3487
Example 17 pMON3453 132/133zero pMON3493
Example 18 pMON3458 132/133~1-10 pMON3488
Example 19 pMON3454 141/142zero pMoN3494
Example 20 pMON3459 141/142~1-10 pMON3489
Ex~mnle 21
Constructi.,n of ~MON25184
The complementary pair of synthetic oligomers,
141for.seq (SEQ ID NO:23) and 141rev.se~ (SEQ ID NO:24),
(Midland Certified Reagent Co., Midland TX) were annealed by
heating 2ug of each synthetic oligomer in a 20ul reaction
mixture containing 20mM Tris-HCl (7.5), 10mM MgCl2 , and
50mM NaC1, at 80~C ~or 5 minutes, and allowing the mixture
to slowly cool to ambient temperature (approximately 45
minutes). When properly annealed the oligomers create an
NcoI site at the 5' end and a NheI site at the 3' end.
Approximately 15 ng of the annealed oligomer pair was
ligated with the gel-purified 4120 base pair NcoI/NheI
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vector fra3ment of pMOM3454 (~molar ratio of 10:1). The
resulting gene, had seven codon changes at the 5' end of the
gene. The ligation reaction was used to transform E. coli
strain DH5~ and the desired codon changes were confirmed by
3NA sequence analysis. The resulting plasmid was designated
pMON25184. Plasmid, pMON25184 containing the gene sequence
of (SEQ ID NO:38), DNA was retransformed into E. coli strain
JM101 cells for protein expression. The protein expressed is
the same as that expressed ~rom pMON3454.
~nle 22
Construction of pMON25188
The complementary pair of synthetic oligomers,
141for.seq (SEQ ID NO:23) and 141rev.seq (SEQ ID NO:24),
(Midland Certified Reagent Co., Midland TX) were annealed by
heating 2ug of each synthetic oligomer in a 20ul reaction
mixture containing 20mM Tris-HCl (7.5), 10mM MgCl2 , and
50mM NaCl, at 80~C for 5 minutes, and allowing the mixture
to slowly cool to ambient temperature (approximately 45
minutes). When properly annealed the oligomers create an
NcoI site at the 5' end and a NheI site at the 3' end.
Approximately 15ng of the annealed oligomer pair was ligated
with the ~ 4110 base pair NcoI/NheI gel-purified pMON3459
(~molar ratio of 10:1). The ligation mixture was used to
transform E. coli strain DH5a and the desired codon changes
were confirmed by DNA sequence analysis. The resulting
plasmid was designated pMON25188. The resulting gene, had
seven codon changes at the 5~ end of the gene. Plasmid,
pMON25188 containing the gene se~uence o~ (SEQ ID NO:42),
DNA was retransformed into E. coli strain JM101 cells for
protein expression. The protein expressed is the same as
that expressed from pMON3459.
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81
~x~m~le 23
"
Construction o~ ~MON25183
-
pMON25183 was constructed using an overlapping PCR
primer method. The synthetic oligomers, 132for.seq (SEQ ID
NO:321 and 132rev.seq (SEQ ID NO:22), encode the NcoI and
NheI restriction recognition se~uence, respectively.
Ampli~ied DNA was generated by the DNA polymerase chain
amplification method using the PCR Optimizer Kit
(Invitrogen). The PCR reactions were performed using the
manufacturer's recommended conditions using 5X buf~er B
(300mM Tris-HC1 pH8.5, 75 mM (NH4)2S04, 10mM MgCl2) for
seven cycles consisting of 94~C for 1', 65~C for 2', and
15 72~C for 2', ~ollowed by 20 cycles o~ 94~C for 1', and 72~C
for 3', and a final cycle of 7 minutes at 72~C using a
Perkin Elmer Model 480 DMA thermal cycler (Perkin Elmer).
The reaction product was desalted using Centri-Sep spin
columns (Princeton Separations) following the manufacturer's
recommended protocol, digested with NcoI/NheI, and gel
purified from TAE-agarose gels using Gene Clean (Bio 101)
and the DNA product was eluted in dH2O The purified PCR
product was ligated with the ~ 4090 base pair NcoI/NheI
pMON3453 vector fragment. Positive clones containing the
AT-rich replacement insert were identified as described in
Example 21. The resulting plasmid was designated pMON25183.
The resulting gene, had 14 codon changes at the 5' end of
the gene. Plasmid, pMON25183 containing the gene se~uence
of (SEQ ID NO:37), DNA was retransformed into E. coli strain
JM101 cells for protein expression. The protein expressed is
the same as that expressed from pMON3453.
~x~mnle 24
35 Constructio~ of ~MON25187
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82
pMON25187 was constructed using an overlapping PCR
primer method. The synthetic oligomers, 132for.seq (SEQ ID
NO:21) and 132rev.seq (SEQ ID NO:22), encode the NcoI and
NheI restriction recognition sequence, respectively.
Amplified DNA was generated by the DNA polymerase chain
amplification method using the PCR Optimizer Kit
(Invitrogen). The PCR reactions were performed using the
manufacturer's recommended conditions, in 5X buffer B for
seven cycles consisting of 94~C for 1', 65~C for 2', and
72~C for 2', followed by 20 cycles of 94~C for 1', and 72~C
for 3', and a final cycle of 7 minutes at 72~C using a
Perkin Elmer Model 480 DNA thermal cycler (Perkin Elmer).
The reaction product was desalted using Centri-Sep spin
columns (P-:inceton Separations) following the manufacturer's
recommended protocol, digested with NcoI/NheI, and gel
purified from TAE-agarose gels using Gene Clean (Bio 101)
and the DNA product was eluted in dH2O. The purified PCR
product was ligated with the ~ 4080 base pair NcoI/NheI
pMON3458 vector fragment. Positive clones containing the
AT-rich replacement insert were identified as described in
Example 21. The resulting plasmid was designated pMON25187.
The resulting gene, had 14 codon changes at the 5' end of
the gene. Plasmid, pMON25187 containing the gene sequence
of (SEQ ID NO:41), DNA was retransformed into E. coli strain
JM101 cells for protein expression. The protein expressed is
the same as that expressed from pMON3458.
~xam~le 25
Construction of ~MON25182
pMON25182 was constructed using the overlapping PCR
primer approach described in Example 23 The synthetic
oligomer primers 125for.seq (SEQ ID NO:l9) and 125rev.seq
CA 02234042 1998-04-06
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83
(SEQ ID NO:20) were used in the PCR reaction. The PCR
reaction conditions were identical to those used in Example
23 except the annealing temperature for the first seven
cycles was 60~C. The purified PCR product was ligated with
~ 4070 base pair NcoI/NheI pMON3452 vector fragment.
Positive clones containing the AT-rich replacement insert
were ident fied as described in Example 21. The resulting
plasmid was designated pMON25182. The resulting gene, had
19 codon changes at the 5' end of the gene. Plasmid,
pMON25182 containing the gene sequence of (SEQ ID NO:36),
DNA was retransformed into E. coli strain JM101 cells ~or
protein expression. The protein expressed is the same as
that expressed from pMON3452.
~xam~le 26
Construction of ~MON25186
pMON25186 was constructed using the overlapping PCR
primer approach described in Example 23. The synthetic
oligomer primers 125for.seq (SEQ ID NO:l9) and 125rev.seq
(SEQ ID NO:20) were used in the PCR reaction. The PCR
reaction conditions were identical to those used in Example
23 except the annealing temperature for the first seven
cycles was 60~C. The purified PCR product was ligated with
the ~ 4060 base pair NcoI/NheI pMON3457 vector fragment.
Positive clones containing the AT-rich replacement insert
were identified as described in Example 21. The resulting
plasmid was designated pMON25186. The resulting gene, had
19 codon changes at the 5' end of the gene. Plasmid,
pMON25186 containing the gene sequence of (SEQ ID NO:40),
DNA was retransformed into E. coli strain JM101 celIs for
~ protein expression. The protein expressed is the same as
that expressed from pMON3457.
- 35
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84
~xam~les 27
Cons~ruction of ~MON25181
5 pMON25181 was constructed using PCR to amplify a DNA
fragment from pMON3451 as the template using the oligomers
96for.seq (SEQ ID NO:17) and 96rev.seq (SEQ ID NO:18). The
oligomer 96for.seq was designed to create six codon changes.
The PCR reaction conditions were the same as described in
10 Example 25, except 10ng of pMOM3451 plasmid DNA was added.
The purified PCR product was ligated with the ~ 3980 base
pair NcoI/NheI pMON3451 vector fragment. Positive clones
containing the AT-rich replacement insert were identified as
described in Example 21. The resulting plasmid was
15 designatec ~MOM25181. The resulting gene, had 6 codon
changes at the 5' end of the gene. Plasmid, pMON25181
containing the gene sequence of (SEQ ID NO:35), DNA was
retransformed into ~. coli strain JM101 cells for protein
expression. The protein expressed is the same as that
expressed from pMON3451.
~n les 28
Co~struction of pMON25185~5
pMON25185 was constructed using PCR to amplify a DNA
fragment from pMON3451 as the template using the oligomers
96for.seq (SEQ ID NO:17) and 96rev.seq (SEQ ID NO:18). The
oligomer 9697for.seg was designed to create six codon
changes. T~D PCR reaction conditions were the same as
described in Example 25, except 10ng of pMON3456 plasmid DNA
was added. The purified PCR product was ligated with the ~
3970 base pair NcoI/NheI pMON3456 vector fragment. Positive
clones containing the AT-rich replacement insert were
identified as described in Example 21. The resulting
-
CA 02234042 1998-04-06
W O 97/12977 PCT~US96/15935
plasmid was designated pMON25185. The resulting gene, had 6
codon changes at the 5' end of the gene. Plasmid, pMON25185
containing the gene sequence of (SEQ ID NO:39), DNA was
retransformed into E. coli strain JM101 cells for protein
expression. The protein expressed is the same as that
expressed from pMON3456.
~X~PT,~ 29
The G-CSF amino acid substitution variants of the
present invention were made using PCR mutagenesis techniques
as described in WO 94/12639 and WO 94/12638. These and other
variants (i.e. amino acid substitutions, insertions or
deletions and N-terminal or C-terminal extensions) could
also be made, by one skilled in the art, using a variety of
other methods including synthetic gene assembly or site-
directed mutagenesis (see ~aylor et al., ~ucl. Acids Res.,
13:7864-8785, 1985; Kunkel et al., Proc. Natl. Acad. sci.
USA, 82:488-492, 1985; Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, MY, 1989, WO 94/12639 and WO
94/12638). These substitutions can be made one at a time or
in combination with other amino acid substitutions, and/or
deletions, and/or insertions and/or extensions. After
sequence verification of the changes, the plasmid DNA can be
transfected into an appropriate mammalian cell, insect cell
or bacterial strain such as E. coli for production. Known
variants of G-CSF, which are active, include substitutions
at positions 1 (Thr to Ser, Arg or Gly, 2 (Pro to Leu), 3
(Leu to Arg or Ser) and 17 (Cys to Ser) and deletions of
amino acids 1-11 (Kuga et al. Biochemicla and Biophysical
Research Comm. 159:103-111, 1989). It is understood that
these G-CSF amino acid substitution variants could serve as
the template sequence for the rearrangement of the amino
acid sequence as described in the other examples.
CA 02234042 1998-04-06
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86
Bioactivity determination of G-CSF amino acid substitution
variants.
The G-CSF amino acid substitution variants were assayed
in the Baf/3 cell line, transfected with the human G-CSF
receptor, proliferation assay to determine their bioactivity
relative to native G-CSF. The G-CSF variants tested and
their relative bioactivity are shown in Table 5. A "+"
indicates that the activity was comparable to native G-CSF
and "-" indicates that the activity was significantly
decreased or not detected.
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87
TART,T~'. 5
CET~T~ PROT.IFF~RATION ACTIVITY OF G-CSF VARIANTS IN BAF/3 C~T,T.
LINE TRANSFECTED WITH THE HUMAN G-CSF RECEPTOR
aa positi~ native aa mutant aa activity *
13 Phe Ser
13 Phe His
13 Phe Thr
13 Phe Pro
16 Lys Pro
16 Lys Ser
16 Lys Thr
16 Lys His
18 Leu Pro
18 Leu Thr
18 Leu His
18 Leu Cys
18 Leu Ile
19 Glu Ala
19 Glu Thr
19 Glu Arg
19 Glu Pro
19 Glu Leu
19 Glu Gly
19 Glu Ser
22 Arg Tyr
22 Arg Ser
22 Arg Ala
22 Arg Val
22 Arg Thr
24 Ile Pro
24 Ile Leu
24 Ile Tvr
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WO 97/12977 PCT~US96/15935
88
TABLE 5 cont.
aa position native aa mutant aa activity
27 Asp Gly +
Ala Ile +
Ala Leu +
34 Lys Ser +
43 His Gly +
43 His Thr +
43 His Val +
43 His Lys +
43 His Trp +
43 His Ala +
43 His Arg +
43 His Cys +
43 His Leu +
44 Pro Arg +
44 Pro Asp +
44 Pro Val +
44 Pro Ala +
44 Pro His +
44 Pro Gln +
44 Pro Trp +
44 Pro Gly +
44 Pro Thr +
46 Glu Ala +
46 Glu Arg +
46 Glu Phe +
46 Glu Ile +
47 Leu Thr +
49 Leu Phe +
49 Leu Arg +
49 Leu Ser +
CA 02234042 1998-04-06
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W O 97/12977 89
TABLE 5 cont.
aa position native aa mutant aa activitv *
Leu His +
Leu Pro +
51 Gly Ser +
51 Gly Met +
54 Leu His +
67 Gln Lys +
67 Gln Leu +
67 Gln Cys +
67 Gln Lys +
Gln Pro +
Gln Leu +
Gln Arg +
Gln Ser +
104 Asp Gly +
104 Asp Val +
108 Leu Ala +
108 Leu Val +
108 Leu Arg +
108 Leu Gly +
108 Leu Trp +
108 Leu Gln +
115 Thr His +
115 Thr Leu +
115 Thr Ala +
115 Thr Ile +
120 Gln Gly +
120 Gln Arg +
120 Gln Lys +
120 Gln His +
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TABLE 5 cont.
aa positionnative aa mutant aa activitv *
123 Glu Arg +
123 Glu Phe +
123 Glu Thr +
144 Phe His +
144 Phe Arg +
144 Phe Pro +
144 Phe Leu +
144 Phe Glu +
146 Arg Gln +
147 Arg Gln +
156 His Asp
156 His Ser +
156 His Gly +
159 Ser Arg +
159 Ser Thr +
159 Ser Tyr +
159 Ser Val +
159 Ser Gly +
162 Glu Gly
162 Glu Trp +
162 Glu Leu +
163 Val Arg +
163 Val Ala +
163 Val Gly +
165 Tyr Cys not determined
169 Ser Leu +
169 Ser Cys +
169 Ser Arg +
170 His Arg +
170 His Ser +
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91
Ex~MT,PL~ 30-37
Examples 30-37 were made in a similar manner as
described in Example 6 using the plasmid pMON13037 as the
template and the oligonucleotide primers indicated in Table
6. The res-~lting gene and the designated plasmid pMON # and
the protein encoded are indicated in Table 6.
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92
TABLE 6
Example breakpoint primers resulting gene resulting
protein
30 48/49 49start
(SEQ ID No:68)pMON3460 (SEQ ID N3:95)
48stop(SEQ ID NO:86)
(SEQ ID No:69)
31 76/77 77start
(SEQ ID NO:70)pMON3461 (SEQ ID NO:96)
76stop(SEQ ID NO:87)
(SEQ ID NO:71)
32 81/82 82start
(SEQ ID NO:72)pMON3462 (SEQ ID NO:97)
81stop(SEQ ID NO:88)
(SEQ ID NO:73)
33 83/84 84start
(SEQ ID NO:74)pMON3463 (SEQ ID NO:98)
83stop(SEQ ID NO:88)
(SEQ ID NO:75)
34 90/91 91start
(SEQ ID NO:76)pMON3464 (SEQ ID NO:99)
90stop(SEQ ID NO:89)
(SEQ ID NO:77)
35111/112 112start pMON3465
(SEQ ID NO:78)(SEQ ID NO:90) (SEQ ID NO:100)
lllstop
(SEQ ID NO:79)
36116/117 117start
(SEQ ID NO:80)pMoN3466 (SEQ ID NO:101)
116stop(SEQ ID NO:91)
(SEQ ID NO:81)
37118/119 ll9start
(SEQ ID NO:82)pMON3467 (SEQ ID NO:102)
118stop(SEQ ID NO:92)
(SEQ ID No:83)
The G-CSF receptor agonist genes in pMON3640, pMON3461,
pMON3462, pMON3463, pMON3464, pMON3465, pMoN3466 and
pMON3467 were transferred to an E. coli expression vector,
pMON2341, as an NcoI/HindIII restriction ~ragment, resulting
CA 02234042 1998-04-06
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93
in the plasmids pMON3468, pMON3469, pMON3470, pMON3471,
pMON3472, pMON3473, pMON3474 and pMON3498 respectively.
~Mprl~ 38
The plasmid, pMON3468, resulted in low expression
levels in E. coli of the desired G~CSF receptor agonist. The
5' end of the gene was redesigned to use codon selection
that was AT rich to increase expression levels. The
oligonucleotides, Z4849AT.for (SEQ ID NO:84) and Z4849AT.rev
(SEQ ID NO:85), were used to re-engineer the gene. The
resulting plasmid, pMON3499, containing the gene (SEQ ID
NO:94) encodes the G-CSF receptor agonist of (SEQ ID
NO:103).
E~MPT~ 39
The G-CSF receptor agonists were assayed in the saf/3
cell line, transfected with the human G-CSF receptor,
(Baf/3-G-CSF) proliferation assay to determine their
bioactivity relative to native G-CSF. The activity of the
receptor agonists is shown in Table 7.
CA 02234042 l998-04-06
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94
TABLE 7
G-CSF receptor agonist activity in Baf/3-G-CSF cell
proliferation assay
pMON#breakpoint Expression E. coli EC50 (pM)
refold
native G-CSF 60 pM
pMON25182 125/126 + + 38 pM
pMON25183 132/133 + + 58 pM
pMON25184 141/142 + + 70 pM
pMON25186 125/126 + + 92 pM
pMON25187 132/133 + + 83 pM
pMON25188 141/142 + + 41 pM
pMoN345038/39 + + 121 pM
pMON345538/39 + + 102 pM
pMON349948/49 + + 137 pM
pMON347081/82 + + no
activity
detected
pMON3473111/112 +
-
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Additional techniques for the construction of the
variant gelles, recombinant protein expression , protein
purification, protein characterization, biological activity
determination can be ~ound in WO 94/12639, WO 94/12638, WO
95/20976, WO 95/21197, WO 95/20977, WO 95/21254 which are
hereby incorporated by reference in their entirety.
All references, patents or applications cited herein
are incorporated by reference in their entirety as i~
written herein.
Various other examples will be apparent to the person
skilled in the art after reading the present disclosure
without departing from the spirit and scope of the
invention. It is intended that all such other examples be
included within the scope o~ the appended claims.
CA 02234042 l998-04-06
WO 97/12977 PCTAUS96/15935
~QU ~:N~ ~: LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: G. D. Searle & Co.
(B) STREET: P. O. Box 5110
(C) CITY: Chicago
(D) STATE: Illinois
(E) COUNTRY: United States of America
(F) POSTAL CODE (ZIP): 60680
(G) TELEPHONE: (708)470-6S01
(H) TELEFAX: (708)470-6881
(A) NAME: Monsanto Company
(B) STREET: 800 North Lindbergh Boulevard
(C) CITY: St. Louis
(D) STATE: Missouri
(E) COUNTRY: United States of America
(F) POSTAL CODE (ZIP): 63167
(G) TELEPHONE: (314)647-3131
(H) TELEFAX: (314)694-5435
(ii) TITLE OF INVENTION: G-CSF Receptor Agonists
(iii) NUMBER OF SEQUENCES: 103
(iv) 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 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: US 2907
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/004,382
(B) FILING DATE: 05-OCT-1995
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Modified-site
CA 02234042 1998-04-06
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97
(B) LOCATION:l
(D) OTHER INFORMATION:/note= "Xaa at position 1 is Thr,
Ser, Arg, Tyr or Gly;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:2
(D) OTHER INFORMATION:/note= "Xaa at position 2 is Pro or
Leu;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:3
(D) OTHER INFORMATION:/note= "Xaa at position 3 is Leu,
Arg, Tyr or Ser;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:13
(D) OTHER INFORMATION:/note= "Xaa at position 13 is Phe,
Ser, His, Thr or Pro;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:16
(D) OTHER INFORMATION:/note= "Xaa at position 16 is Lys,
Pro, Ser, thr or Hisi"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 17
(D) OTHER INFORMATION:/note= "Xaa at position 17 is Cys,
Ser, Gly, Ala, Ile, Tyr or Arg;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:18
(D) OTHER INFORMATION:/note= ~Xaa at position 18 is Leu,
Thr, Pro, His, Ile or Cys;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:22
(D) OTHER INFORMATION:/note= "Xaa at position 22 is Arg,
Tyr, Ser, Thr or Ala;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
~ (B) LOCATION:24
(D) OTHER INFORMATION:/note= "Xaa at position 24 is Ile,
Pro, Tyr or Leu;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
CA 02234042 1998-04-06
WO 97/12977 PCT/US96/15935
98
(B) LOCATION:27
(D) OTHER INFORMATION:/note= "Xaa at position 27 is Asp,
or Glyi"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
tB) LOCATION:30
(D) OTHER INFORMATION:/note= "Xaa at position 30 is Ala,
Ile, Leu or Gly;"
(ix~ FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:34
(D) OTHER INFORMATION:/note= "Xaa at position 34 is Lys
or Ser;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:36
(D) OTEIER INFORMATION:/note= "Xaa at position 36 is Cys
or Ser;"
(ix) FEATURE:
(A) NAME/REY: Modified-site
(B) LOCATION:42
(D) OTHER INFORMATION:/note= "Xaa at position 42 is Cys
or Ser;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:43
(D) OTHER INFORMATION:/note= "Xaa at position 43 is His,
Thr, Gly, Val, Lys, Trp, Ala, Arg, Cys, or Leu;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:44
(D) OTHER INFORMATION:/note= "Xaa at position 44 is Pro,
Gly, Arg, Asp, Val, Ala, His, Trp, Gln, or Thr;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:46
(D) OTHER INFORMATION:/note= "Xaa at position 46 is Glu,
Arg, Phe, Arg, Ile or Ala;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:47
(D) OTHER INFORMATION:/note= "Xaa at position 47 is Leu
or Thr;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
CA 02234042 1998-04-06
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99
(B) LOCATION:49
(D) OTHER INFORMATION:/note= "Xaa at position 49 is Leu,
Phe, Arg or Seri"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:50
(D) OTHER INFORMATION:/note= "Xaa at position 50 is Leu,
Ile, His, Pro or Tyr;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:54
(D) OTHER INFORMATION:/note= "Xaa at position 54 is Leu
or His;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:64
(D) OTHER INFORMATION:/note= "Xaa at position 64 is Cys
or Ser;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:67
(D) OTHER INFORMATION:/note= "Xaa at position 67 is Gln,
Lys, Leu or Cys;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
( B ) LOCATION:70
(D) OTHER INFORMATION:/note= "Xaa at position 70 is Gln,
Pro, Leu, Arg or Ser;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:74
(D) OTHER INFORMATION:/note= "Xaa at position 74 is Cys
or Ser;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:104
(D) OTHER INFORMATION:/note= "Xaa at position 104 is Asp,
Gly or Val;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:108
(D) OTHER INFORMATION:/note= "Xaa at position 108 is Leu,
Ala, Val, Arg, Trp, Gln or Gly;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
CA 02234042 1998-04-06
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100
(B) LOCATION:115
(D) OTHER INFORMATION:/note= "Xaa at position 115 is Thr,
His, Leu or Ala;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:120
(D) OTHER INFORMATION:/note= "Xaa at position 120 is Gln,
Gly, Arg, Lys or His"
(ix) FEATURE:
(A) NAME/REY: Modified-site
(B) LOCATION:123
(D) OTHER INFORMATION:Jnote= "Xaa at position 123 is Glu,
Arg, Phe or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:144
(D) OTHER INFORMATION:/note= "Xaa at position 144 is Phe,
His, Arg, Pro, Leu, Gln or Glu; n
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:146
(D) OTHER INFORMATION:/note= "Xaa at positionl46 is Arg
or Gln;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:147
(D) OTHER INFORMATION:/note= "Xaa ap position 147 is Arg
or Gln;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:156
(D) OTHER INFORMATION:/note= "Xaa at position 156 is His,
Gly or Ser;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:159
(D) OTHER INFORMATION:/note= "Xaa at position 159 is Ser,
Arg, Thr, Tyr, Val or Gly;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:162
(D) OTHER INFORMATION:/note= "Xaa at position 162 is Glu,
Leu, Gly or Trp;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
CA 02234042 1998-04-06
W O 97112977 PCT~US96/15935
101
(B) LOCATION:163
(D) OTHER INFORMATION:/note= "Xaa at position 163 is Val,
Gly, Arg or Ala;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:169
(D) OTHER INFORMATION:/note= "Xaa at position 169 is Arg,
Ser, Leu, Arg or Cys;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:170
(D) OTHER INFORMATION:/note= "Xaa at position 170 is His,
Arg or Ser;"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Xaa Xaa Xaa Gly Pro Ala Ser Ser Leu Pro Gln Ser Xaa Leu Leu Xaa
1 5 10 15
Xaa Xaa Glu Gln Val Xaa Lys Xaa Gln Gly Xaa Gly Ala Xaa Leu Gln
Glu Xaa Leu Xaa Ala Thr Tyr Lys Leu Xaa Xaa Xaa Glu Xaa Xaa Val
Xaa Xaa Gly His Ser Xaa Gly Ile Pro Trp Ala Pro Leu Ser Ser Xaa
Pro Ser Xaa Ala Leu Xaa Leu Ala Gly Xaa Leu Ser Gln Leu His Ser
Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser
Pro Glu Leu Gly Pro Thr Leu Xaa Thr Leu Gln Xaa Asp Val Ala Asp
100 10S 110
Phe Ala Xaa Thr Ile Trp Gln Gln Met Glu Xaa Xaa Gly Met Ala Pro
115 120 125
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Xaa
130 135 140
Gln Xaa Xaa Ala Gly Gly Val Leu Val Ala Ser Xaa Leu Gln Xaa Phe
145 150 155 160
Leu Xaa Xaa Ser Tyr Arg Val Leu Xaa Xaa Leu Ala Gln Pro
165 170
(2) INFORMATION FOR SEQ ID NO: 2:
CA 02234042 1998-04-06
WO 97/12977 PCT~US96/15935
102
(i) ~Q~ CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Gly Gly Gly Ser
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GCTCTGAGAG CCGCCAGAGC CGCCAGAGGG CTGCGCAAGG TGGCGTAGAA CGCG
54
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STR~Nn~N~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) n
(xi) ~Qu~ DESCRIPTION: SEQ ID NO: 4:
CAGCC~l~lG G~GG~ ~G CGGCTCTCAG AG~l~lC~l~GC TCAAGTCTTT AGAG
54
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103
(2~ INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = ~DNA (synthetic)"
(xi) ~ U~N~ DESCRIPTION: SEQ ID NO: 5:
GGGCTGCGCA AGGTGGCG
18
(2) INFORMATION FOR SEQ ID NO: 6:
QU~N~ CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
ACACCATTGG GCCCTGCCAG C
21
(2) INFORMATION FOR SEQ ID NO: 7:
(i) ~h~U~N~: CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRAN~N~SS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
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104
GATCGACCAT GGCTTACAAG CTGTGCCACC CC
32
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) s~Q~N~ DESCRIPTION: SEQ ID NO: 8:
CGATCGAAGC TTATTAGGTG GCACACAGCT TCTCCT
36
(2) INFORMATION FOR SEQ ID NO: 9:
(i) ~:Qu~N~: CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRAN~N~SS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GATCGACCAT GGCTCCCGAG TTGGGTCCCA CC
32
(2) INFORMATION FOR SEQ ID NO: 10:
Qu~N-~ CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STR~Nn~n~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
CA 02234042 1998-04-06
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105
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 10:
CGATCGAAGC TTATTAGGAT ATCCCTTCCA GGGCCT
36
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GATCGACCAT GGCTATGGCC CCTGCCCTGC AG
32
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STR~NnF.nNF..SS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CGATCGAAGC TTATTATCCC A~~ lCCA TCTGCT
36
(2~ INFORMATION FOR SEQ ID NO: 13:
Qu~N-~: CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
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106
(xi) ~:Q~:N-~ DESCRIPTION: SEQ ID NO: 13:
GATCGACCAT GGCTACCCAG GGTGCCATGC CG
32
(2) INFORMATION FOR SEQ ID NO: 14:
(i) ~U~N~: CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
CGATCGAAGC TTATTAGGGC TGCAGGGCAG GGGCCA
36
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STR~Nn~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GATCGACCAT GG~ lGCT TTCCAGCGCC GG
32
(2) INFORMATION FOR SEQ ID NO: 16:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02234042 1998-04-06
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107
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
CGATCGAAGC TTATTAGGCG AAGGCCGGCA TGGCAC
36
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
ATATCCATGG CTCCGGAACT GGGTCCAACT CTG
33
(2) INFORMATION FOR SEQ ID NO: 18:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) ~:Q~N~ DESCRIPTION: SEQ ID NO: 18:
ACCTCCAGGA AGCTCTGCAG ATGG
24
(2) INFORMATION FOR SEQ ID NO: 19:
(i) ~UU~N-~ CHARACTERISTICS:
(A) LENGTH: 65 base pairs
CA 02234042 1998-04-06
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108
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(Xi) ~QU~:N~ DESCRIPTION: SEQ ID NO: l9:
TATATCCATG GCTATGGCTC CAGCTCTGCA ACCAACTCAA GGTGCAATGC CAGCATTTGC
ATCTG
(2) INFOR~ATION FOR SEQ ID NO: 20:
(i) ~QD~N~ CHARACTERISTICS:
(A) LENGTH: 63 base pairs
(B) TYPE: nucleic acid
(C) STRANn~nM~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) ~U~N~ DESCRIPTION: SEQ ID NO: 20:
GATGGCTAGC AACCAGAACA CCACCTGCAC GACGTTGAAA AGCAGATGCA AATGCTGGCA
TTG
63
(2) INFORMATION FOR SEQ ID NO: 2l:
( i ) ~QU~N~'~ CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANn~nN~CS single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)"
CA 02234042 1998-04-06
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109
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
TATATCCATG GCTACTCAAG GTGCTATGCC AGCTTTTGCT TCTGCTTTTC AACGTCG
57
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
(C) STR~Nn~nN~.ss: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GCAGATGGCT AGCAACCAGA ACACCACCTG CACGACGTTG AAAAGCAGAA GCAAAAGC
58
~ (2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
CATGGCTTCT GCTTTTCAAC GTCGTGCAGG ~lG~ lG GTTG
44
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STR~Mn~nN~ss single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
CA 02234042 1998-04-06
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110
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
CTAGCAACCA GAACACCACC TGCACGACGT TGAAAAGCAG AAGC
44
(2) INFORMATION FOR SEQ ID NO: 25:
ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 525 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
ATGGCTTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC
CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC
120
CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA AGGGATATCC
~80
CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC
2~0
ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC
300
ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT
360
CTGCAGAGCT TCCTGGAGGT GTCGTACCGC GTTCTACGCC ACCTTGCGCA GCCCTCTGGC
420
GG~ ~GCG GCTCTCAGAG ~ C~l~G~l~C AAGTCTTTAG AGCAAGTGAG GAAGATCCAG
480
GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT AATAA
525
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
CA 02234042 1998-04-06
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111
~A) LENGTH: 525 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
ATGGCTCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC TGGACGTCGC CGACTTTGCC
ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG
120
GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT
180
AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC
240
TCTGGCGGCT CTGGCGGCTC TCAGAGCTTC CTGCTCAAGT CTTTAGAGCA AGTGAGGAAG
300
ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA GCTGTGCCAC
360
CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC CCTGGGCTCC CCTGAGCTCC
420
TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC TGCTTGAGCC AACTCCATAG CGGC~ lC
480
CTCTACCAGG GGCTCCTGCA GGCCCTGGAA GGGATATCCT AATAA
525
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 525 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
CA 02234042 1998-04-06
W O 97/12977 PCTAUS96/15935
112
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
ATGGCTATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT CGCCTCTGCT
TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC TGCAGAGCTT CCTGGAGGTG
120
TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCCTCTGGCG GCTCTGGCGG CTCTCAGAGC
180
C~lG~l~A A~ AGA GCAAGTGAGG AAGATCCAGG GCGATGGCGC AGCGCTCCAG
240
GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC
300
GGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA
360
GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGG~l~CCT GCAGGCC~
420
GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA CGTCGCCGAC
480
TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGGGAT AATAA
525
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 525 ~ase pairs
(B) TYPE: nucleic acid
(C) STF~NnF.nN~.sS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
ATGGCTACCC AGGGTGCCAT GCCGGCCTTC GCCTCTGCTT TCCAGCGCCG GGCAGGAGGG
GTCCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT CGTACCGCGT TCTACGCCAC
120
CTTGCGCAGC CCTCTGGCGG CTCTGGCGGC TCTCAGAGCT TCCTGCTCAA GTCTTTAGAG
180
CA 02234042 1998-04-06
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113
CAAGTGAGGA AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC
240
AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT
300
CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT
360
AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG
g20
GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG
480
CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCT AATAA
525
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 525 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
ATGGCTTCTG CTTTCCAGCG CCGGGCAGGA GGG~l~C~l~GG TTGCTAGCCA TCTGCAGAGC
C~lGGAGG ~ CG~l~ACCG C~'l"l'~'l'ACGC CACCTTGCGC AG~C~ GG CGG~ ~GC
120
GG~~ ~AGA GCTTCCTGCT CAA~ A GAGCAAGTGA GGAAGATCCA GGGCGATGGC
180
GCAGCGCTCC AGGAGAAGCT GTGTGCCACC TACAAGCTGT GCCACCCCGA GGAGCTGGTG
240
G~lCGGAC A~ lGGG CATCCCCTGG G~l~CCC~l~GA GCTCCTGCCC CAGCCAGGCC
300
CTGCAGCTGG CAGGCTGCTT GAGCCAACTC CATAGCGGCC TTTTCCTCTA CCAGGGGCTC
360
CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA CCTTGGACAC ACTGCAGCTG
420
CA 02234042 1998-04-06
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114
GACGTCGCCG ACTTTGCCAC CACCATCTGG CAGCAGATGG AAGAACTGGG AATGGCCCCT
480
GCCCTGCAGC CCACCCAGGG TGCCATGCCG GCCTTCGCCT AATAA r
525
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B~ TYPE: nucleic acid
(C) STR~Mn~n~SS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
ATGGCTTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC
CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC
12b
CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA AGGGATATCC
180
CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC
240
ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC
300
ATGCCGGCCT TCGC~~ GC TTTCCAGCGC CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT
360
CTGCAGAGCT TCCTGGAGGT GTCGTACCGC GTTCTACGCC ACCTTGCGCA GCCCACACCA
420
TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC TTCCTGCTCA A~ AGA GCAAGTGAGA
480
AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT GTGCCACCTA ATAA
534
(2) INFORMATION FOR SEQ ID NO: 3l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
CA 02234042 l998-04-06
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115
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
ATGGCTCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC TGGACGTCGC CGACTTTGCC
ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG
120
GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT C~lG~l~l~G~l
180
AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC
240
ACACCATTGG GCCCTGCCAG ~CC~l~GCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA
300
GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AG~ GC CACCTACAAG
360
CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC CTGGGCTCCC
420
CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT GCTTGAGCCA ACTCCATAGC
480
GGC~~ CC TCTACCAGGG GCTCCTGCAG GCCCTGGAAG GGATATCCTA ATAA
534
t2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
- (A) DESCRIPTION: /desc = "DNA (Synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
CA 02234042 l998-04-06
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ll6
ATGGCTATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT CGC~
TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC TGCAGAGCTT CCTGGAGGTG
120
TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCCACACCAT TGGGCCCTGC CAG~lCC~lG
180
CCCCAGAGCT TCCTGCTCAA ~'l'-"l"l"l'AGAG CAAGTGAGAA AGATCCAGGG CGATGGCGCA240
GCGCTCCAGG AGAAGCTGTG TGCCACCTAC AAG~ GCC ACCCCGAGGA GCTGGTGCTG
300
CTCGGACACT CTCTGGGCAT CCCCTGGGCT CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG
360
CAGCTGGCAG G~l~G~ lGAG CCAACTCCAT AGCGGCCTTT TCCTCTACCA GGGGCTCCTG
420
CAGGCCCTGG AAGGGATATC CCCCGAGTTG GGTCCCACCT TGGACACACT GCAGCTGGAC
480
GTCGCCGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGATA ATAA
534
(2) INFORMATION FOR SEQ ID NO 33
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 534 base pairs
(B) TYPE nucleic acid
(C) sTR~Nn~n~s single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = "DNA (synthetic)"
(xi) ~Q~ DESCRIPTION SEQ ID NO 33
ATGGCTACCC AGGGTGCCAT G~CGGC~ C GCCTCTGCTT TCCAGCGCCG GGCAGGAGGG
~lC~TG~ G CTAGCCATCT GCAGAGCTTC CTGGAGGTGT CGTACCGCGT TCTACGCCAC
120
CTTGCGCAGC CCACACCATT GGGCCCTGCC AGCTCCCTGC CCCAGAGCTT CCTGCTCAAG
180
CA 02234042 1998-04-06
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117
TCTTTAGAGC AAGTGAGAAA GATCCAGGGC GATGGCGCAG CGCTCCAGGA GAAGCTGTGT
240
GCCACCTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC
300
CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC
360
CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA AGGGATATCC
420
CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC
480
ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCTA ATAA
534
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) ~'~QU~:N~'~ DESCRIPTION: SEQ ID NO: 34:
ATGGCTTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC
TTCCTGGAGG ~l~G~lC~l~ACCG C~'l"l'~'l'ACGC CAC~ll~CGC AGCCCACACC ATTGGGCCCT
120
GCCAGCTCCC TGCCCCAGAG ~ C~l~G~l~C AA~l~ AG AGCAAGTGAG AAAGATCCAG
180
GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG
240
GAGCTGGTGC TGCTCGGACA ~L~ GGGC ATCCCCTGGG CTCCCCTGAG ~'l~C~l~G~CCC
300
AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT TTTCCTCTAC
360
CAGGGG~l~CC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTTGGACACA
420
CA 02234042 1998-04-06
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118
CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA AGAACTGGGA
480
ATGGCCCCTG CCCTGCAGCC CACCCAGGGT GCCATGCCGG CCTTCGCCTA ATAA
534
(2) INFORMATION FOR SEQ ID NO: 35:
Qu~N~'~ CHARACTERISTICS:
(A) LENGTH: 531 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
ATGGCTCCGG AA~l~GG~l~C~' AACTCTGGAC ACACTGCAGC TGGACGTCGC CGACTTTGCC
ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG
120
GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT
180
AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC
240
ACACCATTGG GCCCTGCCAG ~l~CC~l~GCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA
300
GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGC~ GC CACCTACAAG
360
CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC CTGGGCTCCC
420
CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT GCTTGAGCCA ACTCCATAGC
480
GGCCTTTTCC TCTACCAGGG GCTCCTGCAG GCCCTGGAAG GGATATCCTA A
S31
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 531 base pairs
CA 02234042 l998-04-06
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119
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
ATGGCTATGG CTCCAGCTCT GCAACCAACT CAAGGTGCAA TGCCAGCATT TGCATCTGCT
TTTCAACGTC GTGCAGGTGG ~ GGTT GCTAGCCATC TGCAGAGCTT CCTGGAGGTG
120
TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCCACACCAT TGGGCCCTGC CAGCTCCCTG
180
CCCCAGAGCT TCCTGCTCAA ~ AGAG CAAGTGAGAA AGATCCAGGG CGATGGCGCA
240
GCG~l~C~AGG AGAAGCTGTG TGCCACCTAC AAG~ GCC ACCCCGAGGA GCTGGTGCTG
300
CTCGGACACT CTCTGGGCAT CCCCTGGGCT CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG
360
CAGCTGGCAG GCTGCTTGAG CCAACTCCAT AGCGGCCTTT TCCTCTACCA GGGGCTCCTG
420
CAGGCCCTGG AAGGGATATC CCCCGAGTTG GGTCCCACCT TGGACACACT GCAGCTGGAC
480
GTCGCCGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGATA A
531
(2) INFORMATION FOR SEQ ID NO: 37:
(i) ~Q~N~ CHARACTERISTICS:
(A) LENGTH: 531 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
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ATGGCTACTC AAGGTGCTAT GCCAGCTTTT GCTTCTGCTT TTCAACGTCG TGCAGGTGGT
G~ G CTAGCCATCT GCAGAGCTTC CTGGAGGTGT CGTACCGCGT TCTACGCCAC
120
CTTGCGCAGC CCACACCATT GGGCC~lGCC AGCTCCCTGC CCCAGAGCTT CCTGCTCAAG
180
TCTTTAGAGC AAGTGAGAAA GATCCAGGGC GATGGCGCAG CGCTCCAGGA GAAG~
240
GCCACCTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC
300
CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG ~l~G~ GAGC
360
CAACTCCATA GCGGCCTTTT CCTCTACCAG GGG~l~C~l~GC AGGCCCTGGA AGGGATATCC
420
CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC
480
ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCTA A
531
(2) INFORMATION FOR SEQ ID NO: 38:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 531 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
ATGGCTTCTG CTTTTCAACG ~l~C~l~GCAGGT G~~ GG TTGCTAGCCA TCTGCAGAGC
C~l~GGAGG ~l~lC~l'ACCG CGTTCTACGC CACCTTGCGC AGCCCACACC ATTGGGCCCT
~20
GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG AAAGATCCAG
180
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GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG
240
GAGCTGGTGC TGCTCGGACA ~ GGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC
r 300
AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT ~ lC~l~ AC
360
CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTTGGACACA
420
CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA AGAACTGGGA
480
ATGGCCC~lG CCCTGCAGCC CACCCAGGGT GCCATGCCGG C~11'~C~'1'A A
531
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
ATGGCTCCGG AACTGGGTCC AACTCTGGAC ACACTGCAGC TGGACGTCGC CGACTTTGCC
ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG
120
GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT
180
AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC
240
TCTGGCGGCT CTGGCGGCTC TCAGAGCTTC CTGCTCAAGT CTTTAGAGCA AGTGAGAAAG
300
ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAG~L~ G CCACCTACAA GCTGTGCCAC
360
CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC CCTGGGCTCC CCTGAGCTCC
420
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TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC TGCTTGAGCC AACTCCATAG CGG~ lC
480
CTCTACCAGG GGCTCCTGCA GGCCCTGGAA GGGATATCCT AA
522
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 5 ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
ATGGCTATGG CTCCAGCTCT GCAACCAACT CAAGGTGCAA TGCCAGCATT TGCATCTGCT
TTTCAACGTC GTGCAGGTGG ~ G~ GCTAGCCATC TGCAGAGCTT CCTGGAGGTG
120
TCGTACCGCG TTCTACGCCA CCTTGCGCAG CC~ GGCG GCTCTGGCGG CTCTCAGAGC
180
TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA AAGATCCAGG GCGATGGCGC AGCGCTCCAG
240
GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC
300
GGGCA ~l~CCC~l~GGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA
360
GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GCAGGCCCTG
420
GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA CGTCGCCGAC
480
TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGGGAT AA
522
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 base pairs
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123
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
ATGGCTACTC AAGGTGCTAT GCCAGCTTTT GCTTCTGCTT TTCAACGTCG TGCAGGTGGT
G~ G CTAGCCATCT GCAGAGCTTC CTGGAGGTGT CGTACCGCGT TCTACGCCAC
120
CTTGCGCAGC CCTCTGGCGG CTCTGGCGGC TCTCAGAGCT TCCTGCTCAA ~ AGAG
180
CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC
240
AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT
300
CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT
360
AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG
420
GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG
480
CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCT AA
522
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 5 ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
- (A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
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ATGGCTTCTG CTTTTCAACG TCGTGCAGGT G~ GG TTGCTAGCCA TCTGCAGAGC
TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCTCTGG CGGCTCTGGC
120
GGCTCTCAGA G~ C~l~G~l~ CAAGTCTTTA GAGCAAGTGA GAAAGATCCA GGGCGATGGC
180
GCAGCGCTCC AGGAGAAGCT GTGTGCCACC TACAAGCTGT GCCACCCCGA GGAG~lGG~l~G
240
~lG~l~GGAC A~~ GGG CATCCCCTGG GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC
300
CTGCAGCTGG CAGGCTGCTT GAGCCAACTC CATAGCGGCC TTTTCCTCTA CCAGGGGCTC
360
CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA CCTTGGACAC ACTGCAGCTG
420
GACGTCGCCG ACTTTGCCAC CACCATCTGG CAGCAGATGG AAGAACTGGG AATGGCCCCT
480
GCCCTGCAGC CCACCCAGGG TGCCATGCCG GCCTTCGCCT AA
522
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: 5 ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu
1 5 10 15
Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln
Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln
Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr
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Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp
Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln
Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly
100 105 110
Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg
115 120 125
Val Leu Arg His Leu Ala Gln Pro Ser Gly Gly Ser Gly Gly Ser Gln
130 135 140
Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
145 150 155 160
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr
165 170
(2) INFORMATION FOR SEQ ID NO: 44:
(i) ~Q~ CHARACTERI ST ICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acid
( C ) STRANnF:n1~F:~:S: sinyle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp
1 5 10 15
Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
Ala Leu Gln Pro Thr Gln Gly Ala Net Pro Ala Phe Ala Ser Ala Phe
Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Ser Gly
Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val
90 95
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~rg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala
100 105 110
Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser
115 120 125
Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu
130 135 140
Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr
145 150 155 160
Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser
165 170
(2) INFORMATION FOR SEQ ID NO: 45:
(i) ~UU~:N~ CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) ~r:Qu~N~ DESCRIPTION: SEQ ID NO: 45:
Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala
1 5 10 15
Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser
Tyr Arg Val Leu Arg His Leu Ala Gln Pro Ser Gly Gly Ser Gly Gly
Ser Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln
Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu
Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly
100 105 110
Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu
115 120 125
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Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr
130 135 140
Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met
145 150 155 160
Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro
165 170
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
TGGAATAAAA AAGAGAGAAG GAAAAGGATA GAAGAAGGGG GGGGAAGGGA GAAAAGGCAA
TTCGGAGGTA ACGAAGAAGC GGTGGGAAGG GGTATGAAAA AAATTTGGTG GGTAAAAG
118
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu
1 5 10 15
Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln
Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu Lys Ser Leu
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Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys
Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu
Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser
Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu
100 105 110
Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu
115 120 125
Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala
130 135 140
Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu
145 150 155 160
Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
165 170
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STR~Nn~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu
1 5 10 15
Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln
Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln
Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr
Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp
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Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln
Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly
100 105 110
Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg
115 120 125
Val Leu Arg His Leu Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser
130 135 140
Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile
145 150 155 160
Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr
165 170
(2) INFORMATION FOR SEQ ID NO: 49:
(i) ~Qu ~:N~ CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xi) ~yU~N~ DESCRIPTION: SEQ ID NO: 49:
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp
l 5 10 15
Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe
Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro
Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu
Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys
100 105 110
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Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu
115 120 125
Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser
130 135 140
Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu
145 150 155 160
Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser
165 170
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
1 5 10 15
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu
Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln
Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu
Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu
Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu
Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
100 105 110
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
115 120 125
Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile
130 135 140
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Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
145 150 155 160
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly
165 170
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala
1 5 10 15
Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser
Tyr Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro Leu Gly Pro Ala
Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg
Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu
Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln
100 105 110
Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln
115 120 125
Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr
130 135 140
Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp
145 150 155 160
Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro
165 170
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(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu
1 5 10 15
Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln
Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu
Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu
Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu
Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
100 105 110
Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile
115 120 125
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
130 135 140
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala
145 150 155 160
Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
165 170
(2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xi) ~:QU~N~: DESCRIPTION: SEQ ID NO: 53:
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp
1 5 10 15
Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe
Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro
Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu
Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys
100 105 110
Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu
115 120 125
Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser
130 135 140
Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu
145 150 155 160
Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser
165 170
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 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: 54:
Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala ~et Pro Ala Phe Ala
1 5 10 15
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu
Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln
Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu
Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu
Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu
Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
100 105 110
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
115 120 125
Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile
130 135 140
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
145 150 155 160
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly
165 170
(2) INFORMATION FOR SEQ ID NO: 55:
(i) ~Qu~N-~ CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) ~hQU~N~ DESCRIPTION: SEQ ID NO: 55:
Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala
1 5 10 15
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~ly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser
Tyr Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro Leu Gly Pro Ala
Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg
Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu
Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln
100 105 110
Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln
115 120 125
Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr
130 135 140
Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp
145 150 155 160
Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro
165 170
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu
1 5 10 15
Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln
Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu
~0 45
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Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu
Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu
Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
100 105 110
Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile
115 120 125
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
130 135 140
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala
145 150 155 160
Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
165 170
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp
1 5 10 15
Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe
Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Ser Gly
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Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val
Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala
100 105 110
Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser
115 120 125
Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu
130 135 1~0
Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr
145 150 155 160
Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser
165 170
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 169 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
1 5 10 15
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu
Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln
Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu Lys Ser Leu
Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys
Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His
Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala
100 105 110
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Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu
115 120 125
Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu ~eu Gly
130 135 140
Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr
145 150 155 160
Ile Trp Gln Gln Met Glu Glu Leu Gly
165
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acid
(C) STRAN~N~:SS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala
1 5 10 15
Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser
Tyr Arg Val Leu Arg His Leu Ala Gln Pro Ser Gly Gly Ser Gly Gly
Ser Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln
Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu
Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly
100 105 110
Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu
115 120 125
Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr
130 135 140
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Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met
145 150 155 160
7 Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro
165 170
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 171 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Ser Ala Phe Gln Ary Arg Ala Gly Gly Val Leu Val Ala Ser His Leu
1 5 10 15
Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln
Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu Lys Ser Leu
Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys
Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu
Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser
Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu
100 105 110
Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu
115 120 125
Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala
130 135 140
Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu
145 150 155 160
Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
165 170
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(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
Gly Gly Gly Ser Gly Gly Gly Ser
1 5
(2) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STR~Nn~nM~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~Q~N~ DESCRIPTION: SEQ ID NO: 62:
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
Ser Gly Gly Ser Gly Gly Ser
1 5
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(2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
Glu Phe Gly Asn Met
l 5
(2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
Glu Phe Gly Gly Asn Met
l 5
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
Glu Phe Gly Gly Asn Gly Gly Asn Met
l 5
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(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:
Gly Gly Ser Asp Met Ala Gly
l 5
(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
GATCGACCAT GGCTCTGCTC GGACACTCTC TG
32
(2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
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CGATCGAAGC TTATTACACC AGCTCCTCGG GGTGGC
36
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:
GATCGACCAT GGCTCAACTC CATAGCGGCC TT
32
(2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
CGATCGAAGC TTATTAGCTC AAGCAGCCTG CCAGCT
36
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
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(xi) SEQUENCE DESCRIPTION SEQ ID NO 72
GATCGACCAT GG~ llC CTCTACCAGG GG
32
(2) INFORMATION FOR SEQ ID NO 73
Qu~C~ CHARACTERISTICS
(A) LENGTH 36 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = "DNA (synthetic)"
(Xi) S~:Q~N~ DESCRIPTION SEQ ID NO 73
CGATCGAAGC TTATTAGCCG CTATGGAGTT GGCTCA
36
(2) INFORMATION FOR SEQ ID NO 74
( i ) ShQU~N~ CHARACTERISTICS
(A) LENGTH 32 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = "DNA (synthetic)"
(xi) ~Qu~ DESCRIPTION SEQ ID NO 74
GATCGACCAT GG~l~ AC CAGGGGCTCC TG
32
(2) INFORMATION FOR SEQ ID NO 75
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 36 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = "DNA (synthetic)"
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
CGATCGAAGC TTATTAGAAA AGGCCGCTAT GGAGTT
36
(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
GATCGACCAT GGCTGCCCTG GAAGGGATAT CC
32
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
CGATCGAAGC TTATTACTGC AGGAGCCCCT GGTAGA
36
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) ~Q~N~ DESCRIPTION: SEQ ID NO: 78:
GATCGACCAT GGCTGACTTT GCCACCACCA TC
32
(2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
CGATCGAAGC TTATTAGGCG ACGTCCAGCT GCAGTG
36
(2) INFORMATION FOR SEO ID NO: 80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
GATCGACCAT GGCTATCTGG CAGCAGATGG AA
32
(2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
CGATCGAAGC TTATTAGGTG GTGGCAAAGT CGGCGA
36
(2) INFORMATION FOR SEQ ID NO: 82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
GATCGACCAT GGCTCAGCAG ATGGAAGAAC TG
32
(2) INFORMATION FOR SEQ ID NO: 83:
( i ) ~ ~0 U ~N~ ~: CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
CGATCGAAGC TTATTACCAG ATGGTGGTGG CAAAGT
36
(2) INFORMATION FOR SEQ ID NO: 84:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:
CATGGCTTTG TTAGGACATT CTTTAGGTAT TCCATGGGCT CCTCTGAGCT
(2) INFORMATION FOR SEQ ID NO: 85:
ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:
CAGAGGAGCC CATGGAATAC CTAAAGAATG TCCTAACAAA
(2) INFORMATION FOR SEQ ID NO: 86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) ~OLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
,,
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:
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ATGGCTCTGC TCGGACACTC TCTGGGCATC CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC
CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG
- 120
GGGCTCCTGC AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT GGACACACTG
180
CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA ACTGGGAATG
240
GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC
300
CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTCGTACCGC
360
GTTCTACGCC ACCTTGCGCA GCCCACACCA TTGGGCCCTG CCAGCTCCCT GCCC~A~C
420
TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA AAGATCCAGG GCGATGGCGC AGCGCTCCAG
480
GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AG~T~~ A ATAA
534
(2) INFORMATION FOR SEQ ID NO: 87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) ~Qu~N~: DESCRIPTION: SEQ ID NO: 87:
ATGGCTCAAC TCCATAGCGG CCTTTTCCTC TACCAGGGGC TCCTGCAGGC CCTGGAAGGG
ATATCCCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC TGGACGTCGC CGACTTTGCC
120
ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG
180
GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT
240
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AGCCATCTGC AGA~ C~l' GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC
300
ACACCATTGG GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA
360
GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AG~ ~C CACCTACAAG
420
CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC CTGGGCTCCC
480
CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT GCTTGAGCTA ATAA
534
(2) INFORMATION FOR SEQ ID NO: 88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRAMn~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(Xi) ~:OU~N~ DESCRIPTION: SEQ ID NO: 88:
ATGGCTCTTT ~l~C~~ ACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG
GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG
120
CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC
180
TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC
240
~ ~GAGG T~l~ AccG C~ ACGC CACCTTGCGC AGCCCACACC ATTGGGCCCT
300
GCCAGCTCCC TGCCCCAGAG ~llC~lG~l~C AA~ AG AGCAAGTGAG AAAGATCCAG
360
GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG
420
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GAGCTGGTGC TGCTCGGACA ~ l~GGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC
480
AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCTA ATAA
534
(2) INFORMATION FOR SEQ ID NO: 89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:
ATGGCTCTCT ACCAGGGGCT CCTGCAGGCC CTGGAAGGGA TATCCCCCGA GTTGGGTCCC
ACCTTGGACA CACTGCAGCT GGACGTCGCC GACTTTGCCA CCACCATCTG GCAGCAGATG
120
GAAGAACTGG GAATGGCCCC TGCCCTGCAG CCCACCCAGG GTGCCATGCC GGC~ CGCC
180
TCTGCTTTCC AGCGCCGGGC AGGAGGGGTC ~l~G~ GCTA GCCATCTGCA GAG~ C~
240
GAG~l~TC~l~ ACCGCGTTCT ACGCCACCTT GCGCAGCCCA CACCATTGGG CCCTGCCAGC
300
TCCCTGCCCC AGAGCTTCCT GCTCAAGTCT TTAGAGCAAG TGAGAAAGAT CCAGGGCGAT
360
GGCGCAGCGC TCCAGGAGAA G~ ~l~CC ACCTACAAGC TGTGCCACCC CGAGGAGCTG
420
GTGCTGCTCG GACACTCTCT GGGCATCCCC TGGGCTCCCC TGAGCTCCTG CCCCAGCCAG
480
GCCCTGCAGC TGGCAGGCTG CTTGAGCCAA CTCCATAGCG GC~ A ATAA
f 534
(2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
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(C) STRANDEDNESS: single
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)~ -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:
ATGGCTGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA CCTTGGACAC ACTGCAGCTG
GACGTCGCCG ACTTTGCCAC CACCATCTGG CAGCAGATGG AAGAACTGGG AATGGCCCCT
120
GCCCTGCAGC CCACCCAGGG TGCCATGCCG GCCTTCGCCT ~l~C~ CCA GCGCCGGGCA
180
GGAGGGGTCC TGGTTGCTAG CCATCTGCAG AGCTTCCTGG AG~~ C~l~A CCG~l~ lA
240
CGCCACCTTG CGCAGCCCAC ACCATTGGGC CCTGCCAGCT CCCTGCCCCA GAGCTTCCTG
300
CTCAAGTCTT TAGAGCAAGT GAGAAAGATC CAGGGCGATG GCGCAGCGCT CCAGGAGAAG
360
CTGTGTGCCA CCTACAAGCT GTGCCACCCC GAGGAGCTGG TGCTGCTCGG ACACTCTCTG
420
GGCATCCCCT GGGCTCCCCT GAG~TC~l~GC CCCAGCCAGG CCCTGCAGCT GGCAGGCTGC
480
TTGAGCCAAC TCCATAGCGG C~~ llCCTC TACCAGGGGC TCCTGCAGTA ATAA
534
(2) INFORMATION FOR SEQ ID NO: 9l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STR~Nn~N~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9l:
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ATGGCTGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGAAT GGCCCCTGCC
CTGCAGCCCA CCCAGGGTGC CATGCCGGCC TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA
120
GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC TTCCTGGAGG ~ C~lACCG CGTTCTACGC
180
CACCTTGCGC AGCCCACACC ATTGGGCCCT GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC
240
AA~ AG AGCAAGTGAG AAAGATCCAG GGCGATGGCG CAGCGCTCCA GGAGAAGCTG
300
TGTGCCACCT ACAAGCTGTG CCACCCCGAG GAGCTGGTGC TGCTCGGACA ~~ GGGC
360
ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG
420
AGCCAACTCC ATAGCGGCCT TTTCCTCTAC CAGGGGCTCC TGCAGGCCCT GGAAGGGATA
480
TCCCCCGAGT TGGGTCCCAC CTTGGACACA CTGCAGCTGG ACGTCGCCTA ATAA
534
(2) INFORMATION FOR SEQ ID NO: 92:
(i) ~u~: CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)~'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:
ATGGCTATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG
GGTGCCATGC CGGCCTTCGC ~l~lG~ lC CAGCGCCGGG CAGGAGGGGT ~lG~l"lG~
120
AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC
180
ACACCATTGG GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA
240
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GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AG~ GC CACCTACAAG
300
CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC CTGGGCTCCC
360
CTGAGCTCCT GCCCC'AGCCA GGCCCTGCAG CTGGCAGGCT GCTTGAGCCA ACTCCATAGC
420
GGC~'l"l"l"lCC TCTACCAGGG GCTCCTGCAG GCCCTGGAAG GGATATCCCC CGAGTTGGGT
480
CCCACCTTGG ACACACTGCA GCTGGACGTC GCCGACTTTG CCACCACCTA ATAA
534
(2) INFORMATION FOR SEQ ID NO: 93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: tdesc = "DNA (synthetic)"
(xi) SEQUE~CE DESCRIPTION: SEQ ID NO: 93:
ATGGCTCAGC AGATGGAAGA ACTGGGAATG GCCC~l~CCC TGCAGCCCAC CCAGGGTGCC
ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT
120
CTGCAGAGCT TCCTGGAGGT GTCGTACCGC GTTCTACGCC ACCTTGCGCA GCCCACACCA
180
TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA
240
AAGATCCAGG GCGATGGCGC AGCG~l~CCAG GAGAAGCTGT GTGCCACCTA CAAGCTGTGC
300
CACCCCGAGG AGCTGGTGCT GCTCGGACAC TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC
360
TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA GG~l~G~ GA GCCAACTCCA TAGCGGCCTT
420
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TTCCTCTACC AGGGGCTCCT GCAGGCCCTG GAAGGGATAT CCCCCGAGTT GGGTCCCACC
r 480
TTGGACACAC TGCAGCTGGA CGTCGCCGAC TTTGCCACCA CCATCTGGTA ATAA
_ 534
(2) INFORMATION FOR SEQ ID NO: 94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
ATGGCTTTGT TAGGACATTC TTTAGGTATT CCATGGGCTC CTCTGAGCTC CTGCCCCAGC
CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG
120
GGGCTCCTGC AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT GGACACACTG
180
CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA ACTGGGAATG
240
GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC
300
CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTCGTACCGC
360
GTTCTACGCC ACCTTGCGCA GCCCACACCA TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC
420
TTCCTGCTCA A~ AGA GCAAGTGAGA AAGATCCAGG GCGATGGCGC AGCGCTCCAG
480
GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGTA ATAA
534
(2) INFORMATION FOR SEQ ID NO: 95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:
Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys
1 5 10 15
Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser
Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp
Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe
Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe
100 105 110
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro
115 120 125
Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu
130 135 140
Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys
145 150 155 160
Leu Cys Ala Thr Tyr Lys Leu Cys ~is Pro Glu Glu Leu Val
165 170
(2) INFORMATION FOR SEQ ID NO: 96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single r
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:
_ Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu
1 5 10 15
Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu
Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu
Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe
Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala
Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
100 105 110
Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
115 120 125
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
130 135 140
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
145 150 155 160
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser
165 170
(2) INFORMATION FOR SEQ ID NO: 97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) ~U~N~ DESCRIPTION: SEQ ID NO: 97:
Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro
l 5 10 15
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~lu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe
Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala
Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln
Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu
Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro Leu
Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu
100 105 110
Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu
115 120 125
Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly
130 135 140
His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln
145 150 155 160
Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly
165 170
(2) INFORMATION FOR SEQ ID NO: 98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANn~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98:
Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu
1 5 10 15
Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr
Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln
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Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg
_ Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val
Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro Leu Gly Pro
Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val
100 105 110
Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala
llS 120 125
Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser
130 135 140
Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu
145 150 155 ~ 160
Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe
165 170
(2) INFORMATION FOR SEQ ID NO: 99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
txi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:
Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu
1 5 10 15
Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu
Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro
Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala
Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
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Leu Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly
100 105 110
Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro
115 120 125
Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro
130 135 140
Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser
145 150 155 160
Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln
165 170
(2) INFORMATION FOR SEQ ID NO: 100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala
1 5 10 15
Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser
Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Thr
Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser
Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu
Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu
100 105 110
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Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro
115 120 125
Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly
130 135 140
Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro
145 150 155 160
Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
165 170
(2) INFORMATION FOR SEQ ID NO: 101:
;Qu~ CHAP~ACTERISTICS:
( A ) LENGTH: 174 amino ac i ds
(B) TYPE: amino acid
( C ) STRANDEDNES S: s ingl e
( D ) TO POLOGY: l i ne ar
( ii ) MOLECULE TYPE: protein
(Xi) ~ U~;N~; DESCRIPTION: SEQ ID NO: 101:
Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro
Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala
Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser
Tyr Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro Leu Gly Pro Ala
Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg
Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu
100 105 110
Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln
; 115 120 125
Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln
130 135 140
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Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr
145 150 155 160
Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr
165 170
(2~ INFORMATION FOR SEQ ID NO: 102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) ~:Qu~NC~ DESCRIPTION: SEQ ID NO: 102:
Gln Gln Met Glu Glu Leu Gly ~et Ala Pro Ala Leu Gln Pro Thr Gln
1 5 10 15
Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly
Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg
Val Leu Arg His Leu Ala Gln Pro Thr Pro Leu Gly Pro Ala Ser Ser
Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile
Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys
Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile
100 105 110
Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala
115 120 125
Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu
130 135 140
Leu Gln Ala Leu Glu Gly Ile Ser Pro G1U Leu Gly Pro Thr Leu Asp
145 150 155 160
Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp
165 170
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(2) INFORMATION FOR SEQ ID NO: 103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:
Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys
1 5 10 15
Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser
Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp
Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe
Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe
100 105 110
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Thr Pro
115 120 125
Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu
130 135 140
Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys
1~5 150 155 160
Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val
165 170
