Note: Descriptions are shown in the official language in which they were submitted.
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IMPROVED BACILLUS THURINGIENSIS TOXIN
BACKGROUND OF THE INVENTION
{i) Field of the Invention
. 5 The present invention provides new improved proteins derived from a
Bacillus
thuringiensis Cry9C crystal protein. In accordance with this invention, amino
acid
positions in a Cry9C protein were identified as involved in insect toxicity.
Further in
accordance with this invention are provided modified Cry9C proteins with
increased
of decreased toxicity to an insect species, and DNA sequences encoding such
modified Cry9C proteins. Plants can be protected from insect damage by
expressing a chimeric gene encoding an improved Cry9C protein with an
increased
toxicity to an insect species.
(ii) Description of Related Art
Bacillus thuringiensis (Bt)-derived proteins are currently widely used to
protect
plants from insects by expression of such proteins in transgenic plants.
Concerns of
insect resistance development and the desire to achieve the optimum toxicity
and
control of additional insect species resulted in efforts to modify existing Bt-
derived
proteins so as to increase their toxicity or alter their mode of action.
Most studies on the mode of action of Bacillus thuringiensJS toxins have
focused on lepidopteran-specific Cry1 insecticidal crystal proteins ("ICPs").
The
following picture has emerged from these studies {Gill et al., 1992, Annu.
Rev.
Entomol. 37, 615-36; Knowles, 1993, BioEssays, 15, 469-476). Following
ingestion
of the crystals by a susceptible insect, they are dissolved in the alkaline
reducing
environment of the insect midgut lumen. The liberated proteins, the protoxins,
are
then proteolytically processed by insect midgut proteases to a protease-
resistant
fragment. This active fragment, the toxin, then passes through the peritrophic
membrane and binds to specific receptors located on the brush border membrane
of
gut epithelial cells. Subsequent to binding, the toxin or part thereof inserts
in the
membrane resulting in the formation of pores. These pores lead to colloid
osmotic
swelling and ultimately lysis of the midgut cells, causing death of the
insect.
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Binding studies have demonstrated that receptor binding is a crucial step in
the mode of action of ICPs (Hofmann et al., 1988, 173, 85-91; Hofmann et al.,
1988,
Proc. Natl. Acad. Sci. USA, 85, 7844-7848; Van Rie et af., 1990, Appl.
Environm.
Microbiol. 56, 1378-85).
The three dimensional structure of two ICPs, Cry3A and the CrylAa toxic
fragment, has been solved (Li et al., 1991, Nature 353, 815-21; Grochulski et
al.,
1994, Journal of Molecular Biology 254, 1-18). The Cry proteins have been
found to
have three structural domains: the N-terminal domain I consists of 7 alpha
helices,
domain II contains three beta-sheets and the C-terminal domain 111 is a beta-
sandwich. Based on this structure, a hypothesis has been formulated regarding
the
structure-function relationships of ICPs. The bundle of long, hydrophobic and
amphipathic helices (domain I) is equipped for pore formation in the insect
membrane, and regions of the three-sheet domain (domain II) are probably
responsible for receptor binding (Li et al, 1991, supra}. The function of
domain 111 is
less clear. When different ICP amino acid sequences are aligned, five
conserved
sequence blocks are evident (Hofte & Whiteley, 1989, Microbiol. Revs. 53, 242-
255).
These conserved blocks are all located in the interior of a structural domain
or at the
interface between domains. The high degree of conservation of these internal
residues implies that homologous proteins would adopt a similar fold {Li et
al., 1991,
supra).
Data from Ahmad et al. (1991, FEMS Microbiol. Lett. 68, 97-104}; Wu et al.
(1992, J. Biol. Chem. 267, 2311-2317) and Gazit et al. (1993, Biochemistry 32,
3429-3436) provide evidence for the function of domain I of ICPs as a pore
formation unit.
Deletions and alanine substitutions in the Cry1 Aa protoxin at a position
predicted to be at or near the second loop of domain fl significantly altered
toxicity
and receptor binding ability (Lu et al., 1993, XXVIth Annual meeting of the
Society
for Invertebrate Pathology, Asheville, USA, Conference book, page 31, Abstract
17).
Smith and Ellar {1992, XXVth Annual meeting of the Society for Invertebrate
Pathology, Heidelberg, Germany, Conference book, page 111, abstract 68)
observed dramatic effects on toxicity towards in vitro insect cell cultures
with mutant
Cry1 C proteins, differing in the amino acid sequence of the predicted loop
regions.
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They formulated the hypothesis that it should be possible to map the putative
receptor binding domain of this toxin and eventually generate toxins with
increased
potency. In some cases however, a contribution to specificity and binding from
domain Ill of the Cry toxin could not be excluded (Schnepf et al., 1990,
supra; Ge et
S al., 1991, J. Biol. Chem. 266, 17954-17958). Furthermore, a recent study
using
hybrid ICPs, constructed by exchanging gene fragments between cry1C and crylE,
has indicated that domain II of Cry1 C is not sufficient to confer the high
activity of
this protein towards Spodoptera exigua and Mamestra brassicae (Schipper et
al.,
1993, Seventh International Conference on Bacillus, Institut Pasteur, July 18-
23,
Abstracts of lectures, p. L69). Site-directed mutagenesis experiments on
CrylAc
indicated that certain amino acids in domain I are important for receptor
binding (Wu
et al., 1992, supra). Rajamohan et al: (1996, J. Biol. Chem. 271, 2390-2396)
explored the role of loop 2 residues in domain I I of the Cry1 Ab protein in
reversible
and irreversible binding to Manduca sexta and Heliothis virescens.
Also, changes outside the 60 kD toxin region of the Bt protoxin were found to
influence toxicity. It was suggested that this may be related to the
activation
processes by the gut juice (Nakamura et al., 1990, Agric. Biol. Chem. 54, 715-
24).
Visser et al. (1993, In "Bacillus thuringiensis, an Environmental Biopesticide
Theory and Practice", pp.71-88, eds.: Entwistle, P.F., Cory, J.S., Bailey,
M.J., and
Higgs, S., John Wiley & Sons, NY) reviewed the domain-function studies with Bt
ICPs and concluded that in general, the function of essential stretches of the
toxic
fragment of Bt ICPs is unknown. From studies of mutant proteins, it was found
that
several amino acid residues from different regions of the toxic fragment,
either
conserved or variable, were shown to affect toxic activity.
Lambert et al. (1996, Appl. & Environm. Microbiol. 62, p. 80-86) and PCT
patent publication WO 94/05771 describe a new Bt protein which is currently
named
cry9Ca1 (abbreviated as Cry9C) (Peferoen et al., 1997, in Advances in Insect
Control: The role of transgenic plants; pp. 21-48, Taylor & Francis Ltd.,
London).
This protein was found to have a broad insect target range within the group of
lepidopteran pest insects making it interesting for insect control
applications in
agriculture.
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De Roeck et al. (1995, the 28th annual meeting of the Society for Invertebrate
Pathology, Cornell University, Ithaca, New York, p. 52) suggests to determine
the
likely position of the binding epitope of the CryIH protein by making Alanine
mutants
so as to allow the determination of the contribution of amino acid positions
in binding
of the CryIH protein to different insects. The CryIH protein is currently
named Cry9C
in the new nomenclature (Crickmore et al., 1995, 28t" annual meeting of the
Society
for Invertebrate Pathology, Cornell University, Ithaca, New York, p.14.). De
Roeck et
al. (1997, the 6th International Conference on Perspectives in Protein
Engineering,
John Innes Centre, Norwich, UK, June 28-July 1, p. 34) determined the likely
position of residues in the loops at the apex of the molecule in domain II of
the
Cry9C protein.
SUMMARY OF THE INVENTION
This invention provides a modified Cry9C protein with an improved toxicity to
an insect species, comprising the amino acid sequence of SEQ ID No. 2 or an
insecticidally-effective fragment thereof, wherein at least one amino acid in
the
following regions in SEQ ID No. 2 is replaced by another amino acid: 313-334,
358-
369, 418-425, 480-492.
This invention further provides improved Cry9C proteins comprising the amino
acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid
position 658, wherein at least one of the amino acids at the following
positions in
SEQ ID No. 2 have been replaced by another amino acid: 313, 316, 317, 318,
319,
321, 323, 325, 329, 330, 368, 369, 418, 420, 421, 422, 480, 481, 483, 484,
485,
487, 488, 490 and 491. Preferred improved Cry9C proteins comprise the amino
acid
sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid
position
658, wherein at least one of the amino acids at the following positions are
replaced
by other amino acids: 316, 317, 319, 321, 329, 330, 369, 422, and 488.
This invention also provides a modified Cry9C protein with improved toxicity
to Ostrinia nubilalis, comprising the amino acid sequence of SEQ ID No. 2 from
amino acid position 1 or 44 to amino acid position 658, wherein at least the
amino
acids at position 488 or at least at positions 364 and 488 are replaced by
other
amino acids, preferably by alanine.
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This invention also provides modified Cry9C proteins with improved toxicity to
Heliothis virescens, comprising the amino acid sequence of SEQ ID No. 2 from
amino acid position 1 or 44 to amino acid position 658, wherein the amino acid
at
position 321 or position 329, is replaced by another amino acid, preferably by
alanine.
This invention further provides modified Cry9C proteins with improved toxicity
to Diatraea grandiosella, comprising the amino acid sequence of SEQ ID No. 2
from
amino acid position 1 or 44 to amino acid position 658, wherein the amino acid
at
any or all of positions 316, 317, 319, 321, 330, 369, or 422 is replaced by
another
amino acid, preferably by alanine.
Further in accordance are provided DNA sequences encoding the modified
Cry9C proteins, and particularly chimeric genes designed for expression in
plants
comprising these DNA sequences.
In another preferred embodiment of this invention, a plant transformed with a
DNA sequence encoding a modified Cry9C protein is provided, so that the plant
acquires increased resistance to insects, particularly a corn plant
transformed with a
modified Cry9C protein yielding increased toxicity towards Heliothis
virescens,
Ostrinia nubilalis, or Diatraea grandiosella insects.
Other objects and advantages of this invention will become evident from the
following description.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
In this invention, certain amino acid residues important for toxicity of the
Cry9C protein have been identified. These amino acid residues can be replaced
by
other amino acids to increase the toxicity to a specific insect species.
The "Cry9C protein", as used herein, refers to an insecticidal protein
characterized by the amino acid sequence of SEQ ID No. 2 or any equivalents
thereof such as the insecticidally effective truncated proteins or the fusion
proteins of
the Cry9C protein described in PCT patent publications WO 94/05771 and WO
94/24264. Particularly preferred Cry9C proteins, in accordance with this
invention,
are proteins containing at least the amino acid sequence of SEQ lD No. 2 from
amino acid position 1 or 44 to amino acid position 658. Throughout the
description
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and the claims, the new nomenclature for Bt crystal proteins as suggested by
Crickmore et al. (1995, 28th annual meeting of the Society for Invertebrate
Pathology, Cornell University, Ithaca, New York, p. 14) and reported in
Peferoen et
al. (1997, in Advances in Insect Control: The role of transgenic plants, pp.
21-48,
Taylor & Francis Ltd., London) has been used.
"Cry9C protein variants", for a particular insect species, are insecticidal
proteins that differ from but are indirectly or directly derived from the
Cry9C protein.
Indeed, several variants of a Bt protein in which some amino acids are changed
into
others without significantly changing activity and/or specificity to a
particular insect
species can be found in nature (Hofte & Whiteley, 1989, supra) or can be made
by
recombinant DNA techniques. Variants of a Cry9C protein, as used herein, also
include proteins containing the specificity- or toxicity-determining domain or
region of
the Cry9C protein, e.g., in a hybrid with another protein, such as another Bt
ICP, a
membrane-permeating protein domain, a cytotoxin or an antibody fragment,
provided that the Cry9C specificity- or toxicity-determining domain or region
contributes to the toxicity or specificity of the hybrid protein. Particularly
preferred
Cry9C protein variants are those proteins comprising the amino acid sequence
of
SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658
wherein
the arginine at position 164 has been replaced by another amino acid,
preferably
alanine or lysine. These variants with a replacement of the arginine at
position 164
in the sequence of SEQ ID No. 2 show a significantly lower susceptibility to
breakdown upon protease treatment, and are named herein the "protease-
resistant
Cry9C variants". Like here for the protease-resistant variants, whenever
reference
to a particular region or position in SEQ ID No. 2 is made, this does not
necessarily
imply that the protein referred to is the full-length protein of SEQ ID No. 2;
this
statement merely refers to the position corresponding to the particular
position in the
reference Cry9C protein in SEQ ID No. 2. Indeed, improved Cry9C proteins of
the
invention can be truncated so that the actual position of an amino acid in
that protein
will differ but nevertheless reference will be made throughout this invention
to the
positions in the full-length reference protein, shown in SEQ ID No. 2.
Following the teachings of this invention, Cry9C proteins or variants thereof
can be modified to have an increased toxicity for an insect species. "Modified
Cry9C
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protein", as used herein, refers to a Cry9C protein or its protease-resistant
variant
wherein amino acids have been modified to analyse the contribution of amino
acid
positions in toxicity, particularly a Cry9C protein or its protease-resistant
variant
wherein amino acids have been modified in the regions at the following
positions in
SEQ ID No. 2: 313-334, 358-369, 418-425, 480-492. "Improved Cry9C protein", in
accordance with this invention, refers to a Cry9C protein or its protease-
resistant
variant wherein at least one amino acid has been replaced, so that the
toxicity of this
improved protein towards an insect species is significantly increased. In a
particularly preferred improved Cry9C protein or its protease-resistant
variant, the at
least one amino acid change is located in domain II of the Cry9C protein,
particularly
in the regions of the Cry9C protein characterized by the following positions
in SEQ ID
No. 2: 313-334, 358-369, 418-425, 480-492. A modified Cry9C protein, differing
in
one amino acid from the native protein or its protease-resistant variant and
being
significantly less toxic towards the target insect, allows the direct
identification of this
amino acid position as involved in toxicity (provided no gross structural
changes are
introduced), and thus has considerable value in improving toxicity. In
accordance
with this invention, the identification of these amino acid positions involved
in toxicity
allows the construction of modified proteins having increased toxicity to the
target
insect by amino acid randomization at these positions. Preferred modified
Cry9C
proteins in accordance with this invention are the modified Cry9C proteins
having
altered toxicity to Ostrinia nubilalis, Heliothis virescens or Diatraea
grandiosella as
shown in Table 1, as well as combinations of those modifications in one
modified
protein.
An example of an improved Cry9C protein in accordance with this invention is
a protein comprising the amino acid sequence of SEQ ID No. 2 from amino acid
position 1 or 44 to amino acid position 658 or 666 wherein an amino acid in at
least
one of the following amino acid positions of SEQ ID No. 2 has been replaced by
another amino acid: 313, 316, 317, 318, 319, 321, 323, 325, 329, 330, 362,
364,
368, 369, 418, 420, 421, 422, 480, 481, 483, 484, 485, 487, 488, 490 and 491;
or an
amino acid position located in the immediate vicinity of any one of these
positions in
the three-dimensional structure of the protein, preferably those amino acids
whose
C-alpha atom is at a maximum distance of about 7 Angstrom from the C-alpha
atom
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of the amino acid listed above. A preferred improved Cry9C protein in
accordance
with this invention is the protein of SEQ ID No. 2 with at least one of the
following
amino acid changes: P316A, A317V, V319A, L321 A, P329A, Y330A, S364A, Y369A,
1422A, and 1488A. "V319A" or "Cry9C(V319A)", as used herein, means a change of
the valine amino acid at position 319 in SEQ ID No. 2 to an alanine amino
acid.
Preferred improved Cry9C proteins also include Cry9C proteins having also the
arginine amino acid at position 164 in SEQ ID No. 2 altered into another amino
acid,
particularly alanine or lysine, to enhance stability upon protease,
particularly trypsin,
cleavage.
A preferred Cry9C protein for the control of Ostrinia nubilalis insects in
accordance with this invention is a protein comprising the amino acid sequence
of
SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658
wherein an
amino acid in at least one of the following amino acid positions in SEQ ID No.
2 has
been replaced by another amino acid: 325, 364, 418, 421, 485, and 488. A
particularly preferred improved Cry9C protein for the control of Ostrinia
nubilalis
insects is a protein comprising the amino acid sequence of SEQ ID No. 2 from
amino
acid position 1 or 44 to amino acid position 658 wherein the amino acids in at
least
position 364 or at least in positions 364 and 488 of SEQ ID No. 2 are replaced
by
another amino acid, particularly alanine.
A preferred Cry9C protein for the control of Heliothis virescens insects in
accordance with this invention is a protein comprising the amino acid sequence
of
SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658
wherein an
amino acid in at least one of the following amino acid positions in SEQ ID No.
2 has
been replaced by another amino acid: 313, 316, 317, 318, 319, 321, 323, 325,
329,
330, 368, 369, 418, 420, 421, 422, 480, 481, 483, 484, 485, 487, 488, 490 and
492,
particularly at least one of the following amino acid positions: 321, 325,
329, 418,
420, and 480. A particularly preferred improved Cry9C protein for the control
of
Heliothis virescens insects is a protein comprising the amino acid sequence of
SEQ
ID No. 2 from amino acid position 1 or 44 to amino acid position 658 wherein
the
amino acids in at least one of the amino acid positions 321 and 329 of SEQ ID
No. 2
are replaced by another amino acid, particularly alanine.
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A preferred Cry9C protein for the control of Diatraea grandiosella insects in
accordance with this invention is a protein comprising the amino acid sequence
of
SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658
wherein an
amino acid in at least one of the following amino acid positions in SEQ ID No.
2 has
been replaced by another amino acid: 316, 317, 319, 321, 325, 330, 369, 421,
422,
480, 483, 484, 485, 487, 488, 490, and 491; particularly at least one of the
following
amino acid positions: 480, 484, 485, 487, and 490. A particularly preferred
improved
Cry9C protein for the control of Diatraea grandiosella insects is a protein
comprising
the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to
amino
acid position 658 wherein the amino acids in at least one of the amino acid
positions
316, 317, 319, 321, 330, 369 and 422 of SEQ ID No. 2 are replaced by another
amino acid, particularly alanine or valine (for 317).
By using DNA sequences encoding improved Cry9C proteins in accordance
with this invention, improved toxicity to a selected insect species can be
obtained
upon expression of such DNA in a transgenic plant.
A "cry9C gene", as used herein, is a DNA sequence comprising a DNA
encoding a Cry9C protein (a coding region), and includes necessary regulatory
sequences so that a Cry9C protein can be expressed in a cell, preferably a
plant or
bacterial cell. A cry9C gene does not necessarily need to be expressed
everywhere
at all times, expression can be periodic (e.g. at certain stages of
development in a
plant) and/or can be spatially restricted (e.g. in certain cells or tissues in
a plant),
mainly depending on the activity of regulatory elements provided in the
chimeric gene
or in the site of insertion in the plant genome. A cry9C gene can be naturally-
occurring or can be a hybrid or synthetic DNA and the regulatory elements can
be
from prokaryotic or eucaryotic origin.
The "modified cry9C gene", as used herein, is a DNA sequence comprising a
DNA encoding a modified Cry9C protein (a modified coding region), and includes
necessary regulatory sequences so that a Cry9C protein can be expressed in a
cell,
preferably a plant or bacterial cell. An example of a modified cry9C coding
region is
the cry9C coding region of SEQ ID No. 3 wherein the valine codon at nucleotide
positions 844-846 of SEQ ID No. 3 has been replaced by an alanine codon.
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"Substantial sequence homology" to a DNA sequence, as used herein, refers
to DNA sequences differing in some, most or. all of their colons from another
DNA
sequence but encoding the same or substantially the same protein. Indeed,
because
of the degeneracy of the genetic code, the colon usage of a particular DNA
coding
region can be substantially modified, e.g., so as to more closely resemble the
colon
usage of the genes in the host cell, without changing the encoded protein.
Changing
the colon usage of a DNA coding region to that of the host cell has been
described
to be desired for gene expression in foreign hosts (e.g. Bennetzen & Hall,
1982, J.
Biol. Chem. 257, 3026-3031.; Itakura, 1977, Science 198, 1056-1063). Colon
usage
tables are available in the literature (Wada et al., 1990, Nucl. Acids Res.
18, 2367-
1411; Murray et al., 1989, Nucl. Acids Res. 17(2), 477-498) and in the major
DNA
sequence databanks (e.g. at EMBL in Heidelberg, Germany). Accordingly,
recombinant or synthetic DNA sequences can be constructed so that the same or
substantially the same proteins with substantially the same insecticidal
activity are
produced (Koziel et al., 1993, Biotechnology 11, 194-200; Perlak et al., 1993,
Plant
Mol. Biol. 22, 313-321 ). A modified cry9C gene has all appropriate control
regions so
that the modified Cry9C protein can be expressed in a host cell, e.g. for
expression in
plants, a plant-expressible promoter and a 3' termination and polyadenylation
region
active in plants.
A "chimeric improved cry9C gene", as used herein, refers to a chimeric gene
comprising a DNA sequence encoding the improved Cry9C protein inserted in
between controlling elements of different origin, e.g. a DNA sequence encoding
the
improved Cry9C protein under the control of a promoter transcribing the DNA in
the
plant cell, and fused to 3' transcription termination sequences active in
plant cells.
Protection of a plant, preferably a corn or cotton plant, against an insect
species which is known to feed on said plant is preferably accomplished by
expressing an improved Cry9C protein in the cells of the plant. This is
preferably
accomplished by expressing a chimeric improved cry9C gene encoding such an
improved Cry9C protein in the cells of a plant, preferably a corn or cotton
plant. An
improved Cry9C protein of this invention preferably only has a small number,
particularly less than 20, more particularly less than 15, preferably less
than 10
amino acids replaced by other amino acids as compared to the Cry9C protein,
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preferably as compared to the region from between amino acid positions 1 and
45 to
amino acid position 658 of the Cry9C protein of SEQ ID No. 2. A significant
increase
in toxicity can already be obtained by replacing only 1 amino acid, but it is
preferred
that more than one amino acid is changed to improve toxicity.
The following steps are followed to construct the new modified Cry9C proteins:
amino acids in domain II of the Cry9C protein from amino acid positions 313-
334,
358-369, 418-425, and 480-492 were chosen for modification, using alanine-
scanning mutagenesis (Cunningham & Wells, 1989, Science 244, 1081-85). In case
the original position is alanine, a substitution by valine is done. These
regions occur
at positions corresponding to the solvent-exposed positions in the loop
between
beta-strands 1 and 2 (comprising alpha-helix 8) and in loop 1 (located between
beta
strands 2 and 3), in loop 2 (located between beta-strands 6 and 7), and in
loop 3
(located between beta-strands 10 and 11 ) in the three-dimensional model of
the
Cry3A protein (Li et al., 1991, supra). To discount any observed lower
toxicity of a
modified Cry9C protein which is due to misfolding or structural distortion,
the
structural stability of mutant ICPs can be analysed by a variety of methods
including
toxicity to another target insect, crystal formation, solubilization,
monoclonal antibody
binding analysis, protease resistance, fluorometric monitoring of unfolding
and
circular dichroism spectrum analysis. In the case of structural distortion, it
is
impossible to determine the functional role of this position by alanine
replacement.
However, a more conservative amino acid substitution may yield a correctly
folded
mutant protein which allows to determine the functional role of this position.
The amino acid positions, identified above, which yield modified proteins with
significantly decreased toxicity ("down-mutants") are randomized. This means
that a
set of 20 different mutants, representing each type of amino acid, is
generated for
each position of interest (the original amino acid and the alanine
substitution function
as a control). This method is further referred to as "amino acid
randomization".
Such mutants may be generated by a variety of methods, e.g. following the PCR
overlap extension method (Ho et al., 1989, Gene 77, 51-59). These mutant
proteins
are then tested in toxicity assays on the target insect. Mutants at each
position which
are more toxic, e.g., yield higher mortality than the wild type protein, are
selected.
Such mutants with improved toxicity are termed "up-mutants". Alternatively, it
is also
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possible to select potential up-mutants on the basis of increased reversible
binding
which can be measured following the procedures of Van Rie et al. (1990, Appl.
Environm. Microbiol. 56, 1378-1385) or Liang et al. (1995, J. Biol. Chem. 270,
24719-24724), which is incorporated herein by reference.
All or some of the "up-mutant" amino acids, identified in step 2, are combined
in a single modified protein. According to additivity principles, mutations in
non-
interacting parts of a protein should combine to give simple additive changes
in the
free energy of binding (Lowman and Wells, 1993, J. Mol. Biol., 234, 564-578).
Increases in toxicity are thus accumulated by combining several single mutants
into
one multiple mutant. Finally a modified protein with improved toxicity is
designed,
which comprises some or all, preferably all, of the up-mutant amino acids
previously
identified.
In accordance with this invention, amino acids of domain II of a Cry9C
protein,
located at the protruding regions of domain II are chosen for modification. By
"protruding regions of domain II", as used herein, are meant the solvent-
exposed
regions organized in loops, alpha helices or beta-strands which are protruding
from
domain II and are located at or towards the apex of the molecule.
This invention is particularly suited for improving the toxicity to an insect
species for which the Cry9C protein has a rather weak toxicity. The toxicity
of this
improved Cry9C protein can be increased by combining amino acid mutations in
the
protein, each yielding an increased toxicity when compared to the amino acid
present
in the native Cry9C protein. Insect species for which improved Cry9C proteins
can
be made also include Spodoptera frugiperda, Heliothis zea, Heliothis armigera,
and
Agrotis ipsilon. Also, this invention is suited to increase toxicity of a
Cry9C protein or
its protease-resistant variant to one insect species and to decrease toxicity
of the
same protein to another insect species by making the proper amino acid
substitutions in the protein. This may be advantageous, e.g., to limit the
likelihood of
insect resistance occurrence to the protein in a particular insect species.
An insecticidally effective part of the modified cry9C gene of this invention
encoding an insecticidally effective portion of the modified Cry9C protein,
can be
made in a conventional manner. An "insecticidally effective part" of the
modified
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cry9C gene refers to a gene comprising a DNA coding region encoding a
polypeptide
with fewer amino acids than the full length modified Cry9C protein but that
still retains
toxicity to insects. A preferred insecticidally effective part of the Cry9C
protein is the
part from amino acid position 1 or 44 to amino acid position 658 in SEQ ID No.
2.
In order to express all or an insecticidally effective part of the improved
cry9C
gene in E. coli, in Bt strains and in plants, suitable restriction sites can
be introduced,
flanking each gene or gene part. This can be done by site-directed
mutagenesis,
using well-known procedures (Stanssens et al., 1989, Nucl. Acids Res. 92,
4441-4454; White et al., 1989, Trends in Genet. 5, 185-189).
In order to improve expression in foreign host cells such as plant cells, it
may
be preferred to alter the improved cry9C coding region or its insecticidally
effective
part to form an equivalent, artificial improved cry9C coding region.
Expression is
improved by selectively inactivating certain cryptic regulatory or processing
elements
present in the native sequence as described in PCT publications WO 91/16432
and
WO 93/09218. This can be done by site-directed mutagenesis or site-directed
intron-
insertion (WO 93/09218), or by introducing overall changes to the codon usage,
e.g.,
adapting the codon usage to that most preferred by the host organism
(publication of
European patent application number ("EP") 0 385 962, EP 0 359 472, publication
of
PCT patent application WO 93/07278, Murray et al., 1989, supra) without
significantly changing, preferably without changing, the encoded amino acid
sequence. Small modifications to a DNA sequence such as described above can be
routinely made by PCR-mediated mutagenesis (Ho et al., 1989, supra; White et
al.,
1989, supra). For major changes to the DNA sequence, DNA synthesis methods are
available in the art (e.g. Davies et al., 1991, Society for Applied
Bacteriology,
Technical Series 28, pp. 351-359). For obtaining enhanced expression in
monocot
plants such as com, a monocot intron can be added to the chimeric improved
cry9C
gene (Callis et al., 1987, Genes & Development 1, 1183-1200; PCT publication
WO
93/07278). Another preferred embodiment of this invention is the expression of
the
improved Cry9C proteins by the method described in PCT patent publication WO
97/49814, which is incorporated herein by reference.
The chimeric improved cry9C gene can be stably inserted in a conventional
manner into the nuclear genome of a single plant cell, and the so-transformed
plant
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WO 99/00407 PCT/EP98/04033
cell can be used in a conventional manner to produce a transformed plant that
is
insect-resistant. Particularly preferred plants in accordance with this
invention are
corn plants. Corn cells can be stably transformed (e.g. by electroporation)
using
wounded or enzyme-degraded intact tissues capable of forming compact
embryogenic callus (such as corn immature embryos), or the embryogenic callus
(such as type I callus in corn) obtained thereof, as described in PCT patent
publication WO 92/09696 or US Patent 5,641,664. Other methods for
transformation
of corn include the methods by Fromm et al. (1990, Bio/Technology 8, 833-839),
Cordon-Kamm et al. (1990, The Plant Cell 2, 603-618) and Ishida et al. (1996,
Nature Biotechnology 14, 745-750).
Alternatively, a disarmed Ti plasmid, containing the insecticidally effective
chimeric improved cry9C gene, in Agrobacterium tumefaciens can be used to
transform the plant cell, preferably the corn or cotton cell, and thereafter,
a
transformed plant can be regenerated from the transformed plant cell using the
procedures described, for example, in EP 0116718, EP 0270822, PCT publication
WO 84/02913 and EP 0242246 (which are also incorporated herein by reference),
and in Could et al. (1991, Plant Physiol. 95, 426-434) or Ishida et al. (1996,
supra),
particularly the method described in PCT publication WO 94/00977. Preferred Ti-
plasmid vectors each contain the insecticidally effective chimeric improved
cry9C
gene between the border sequences, or at least located to the left of the
right border
sequence, of the T-DNA of the Ti-plasmid. Of course, other types of vectors
can be
used to transform the plant cell, using procedures such as direct gene
transfer (as
described, for example in EP 0233247), pollen mediated transformation (as
described, for example in EP 0270356, PCT publication WO 85/01856, and US
Patent 4,684,611 ), plant RNA virus-mediated transformation (as described, for
example in EP 0067553 and US Patent 4,407,956), and liposome-mediated
transformation (as described, for example in US Patent 4,536,475).
A resulting transformed plant, such as a transformed corn or cotton plant, can
be used in a conventional plant breeding scheme to produce more transformed
plants with the same characteristics or to introduce the improved cry9C gene,
or an
insecticidally effective part thereof in other varieties of the same or
related plant
species. Seeds, which are obtained from the transformed plants, contain the
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chimeric improved cry9C gene or its insecticidally effective part as a stable
genomic
insert. Cells of the transformed plant can be. cultured in a conventional
manner to
produce the improved Cry9C protein or insecticidally effective portions
thereof, which
can be recovered for use in conventional insecticide compositions against
insects,
particularly lepidopteran insects {U.S. Patent 5,254,799). Preferred plants in
accordance with this invention, besides corn and cotton, include rice, plants
of the
genus Brassica such as oilseed rape, cauliflower and broccoli, and also
soybean,
tomato, tobacco, potato, eggplant, beet, oat, pepper, gladiolus, dahlia,
chrysanthemum, sorghum, and garden peas.
The improved cry9C coding region or its insecticidally effective part is
inserted
in a plant cell genome so that the inserted coding region is downstream (i.e.,
3') of,
and under the control of, a promoter which can direct the expression of the
gene part
in the plant cell. This is preferably accomplished by inserting the chimeric
improved
cry9C gene or its insecticidally effective part in the plant cell genome.
Preferred
promoters include: the strong constitutive 35S promoters (the "35S promoters")
of
the cauliflower mosaic virus of isolates CM 1841 (Gardner et al., 1981,
Nucleic Acids
Research 9, 2871-2887), CabbB-S (Franck et al., 1980, Cell 27, 285-294) and
CabbB-JI (Hull and Howell, 1987, Virology 86, 482-493); the ubiquitin promoter
(EP
0342926), and the TR1' promoter and the TR2' promoter which drive the
expression
of the 1' and 2' genes, respectively, of the T-DNA (Velten et al., 1984, EMBO
J. 3,
2723-2730). Alternatively, a promoter can be utilized which is not
constitutive but
rather is specific for one or more tissues or organs of the plant, preferably
leaf and
stem tissue, whereby the inserted chimeric improved cry9C gene or its
insecticidally
effective part is expressed only in cells of the specific tissues) or
organ(s). Another
alternative is to use a promoter whose expression is inducible (e.g., by
insect feeding
or by chemical factors). Known wound-induced promoters inducing systemic
expression of their gene product throughout the plant are also of particular
interest.
The improved cry9C coding region, or its insecticidally effective part, is
inserted in the plant genome so that the inserted coding region is upstream
(i.e., 5')
of suitable 3' end transcription regulation signals (i.e., transcript
termination and
polyadenylation signals). Preferred polyadenylation and transcript formation
signals
include those of the 35S gene (Mogen et al., 1990, The Plant Cell 2, 1261-
1272), the
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octopine synthase gene (Gielen et aL, 1984, EMBO J. 3, 835-845) and the T-DNA
gene 7 (Velten and Schell, 1985, Nucl. Acids Res. 13, 6981-6998), which act as
3'-untranslated DNA sequences in transformed plant cells.
The chimeric improved cry9C gene, or its insecticidally effective gene part,
can optionally be inserted in the plant genome as a hybrid gene (EP 0 193 259;
Vaeck et al., 1987, Nature 327, 33-37) under the control of the same promoter
as the
coding region of a selectable marker gene, such as the coding region of the
neo
gene (EP 0 242 236) encoding kanamycin resistance, so that the plant expresses
a
fusion protein.
Preferably, the improved cry9C gene is expressed in a plant in combination
with another insect control protein, e.g., another Bt-derived crystal protein
or an
insecticidal fragment thereof, particularly a Cry1 Ab- or Cry1 B-type protein,
to prevent
or delay the occurrence of insect resistance development (EP 0 408 403).
All or part of the improved cry9C coding region can also be used to transform
bacteria, such as a B, thuringiensis which produces other insecticidal toxins
(Lereclus
et al., 1992, Bio/Technology 10, 418-421; Gelernter & Schwab, 1993, In
Bacillus
thuringiensis, An Environmental Biopesticide: theory and Practice, pp. 89-104,
eds.
Entwistle, P.F., Cory, J.S., Bailey, M.J. and Higgs, S., John Wiley & Sons
Ltd.).
Thereby, a transformed Bt strain is produced which is useful for combating a
wide
spectrum of insect pests or for combating insects in such a manner that insect
resistance development is prevented or delayed (EP 0 408 403). Preferred
promoter
and 3' termination and polyadenylation sequences for the chimeric improved
cry9C
gene are derived from Bacillus thuringiensis genes, such as the native ICP
genes.
Alternatively, the improved coding region of the invention can be inserted and
expressed in endophytic and/or root-colonizing bacteria, such as bacteria of
the
genus Pseudomonas or Clavibacter, e.g., under the control of a Bt ICP gene
promoter and 3' termination sequences. Successful transfer and expression of
ICP
genes into such bacteria has been described by Stock et al. (1990, Can. J.
Microbiol.
36, 879-884), Dimock et at. (1989, In Biotechnology, Biopesticides and Novel
Plant
Pest Resistance Management, eds. Roberts, D.W. & Granados, R.R., pp.88-92,
Boyce Thompson Institute for Plant Research, Ithaca, New York), and Waalwijk
et al.
(1991, FEMS Microbiol. Lett. 77, 257-264). Transformation of bacteria with all
or part
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WO 99/00407 PCT/EP98/04033
of the improved cry9C coding region of the invention, incorporated in a
suitable
cloning vehicle, can be carried out in a conventional manner, preferably using
conventional electroporation techniques as described in Mahillon et al. {1989,
FEMS
Microbiol. Letters 60, 205-210), in PCT patent publication WO 90/06999, Chassy
et
al. (1988, Trends Biotechnol. 6, 303-309) or other methods, e.g., as described
by
~ Lerecfus et al. (1992, Bio/Technology 90, 418).
The improved Cry9C-producing strain can also be transformed with all or an
insecticidally effective part of one or more DNA sequences encoding a Bt
protein or
an insecticidally effective part thereof, such as: a DNA encoding the Bt2 or
Cry1 Ab
protein (US patent 5,254,799; EP 0 193 259) or the Bt109P or Cry3C protein
(PCT
publication WO 91/16433), or another DNA coding for an anti-lepidoptera or an
anti-
Coleoptera protein. Thereby, a transformed Bt strain can be produced which is
useful for combating an even greater variety of insect pests {e.g., Coleoptera
and/or
additional lepidoptera) or for preventing or delaying the development of
insect
resistance.
For the purpose of combating insects by contacting them with the improved
Cry9C protein, e.g. in the form of transformed plants or insecticidal
formulations, any
DNA sequence encoding any of the above described improved Cry9C proteins, can
be used.
The following Examples are offered by way of illustration and not by way of
limitation. The sequence listing referred to in the description and the
Examples is as
follows:
SEQUENCE LISTING
SEQ ID No. 1: Nucleotide sequence of the Bacillus thuringiensis cry9C gene,
showing the coding region and flanking 5' and 3' regions.
SEQ ID No. 2: Amino acid sequence of the full length Bacillus thuringiensis
Cry9C protein.
SEQ ID No. 3: Nucleotide sequence of a codon-optimized DNA sequence
encoding a truncated Cry9C protein wherein the arginine at
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WO 99/00407 PCT/EP98/04033
amino acid position 123 (corresponding to amino acid position
164 in the protein of SEQ ID No. 2) has been replaced by lysine.
SEQ ID No. 4: Amino acid sequence of the modified Cry9C protein encoded by
the DNA of SEQ ID No. 3.
Unless otherwise stated in the Examples, all general materials and methods,
including procedures for making and manipulating recombinant DNA are carried
out
by the standardized procedures as described in volumes 1 and 2 of Ausubel et
al.,
Current Protocols in Molecular Biology, Current Protocols, USA (1994), in
Plant
Molecular Biology Labfax (1993, by R.D.D. Croy, jointly published by BIOS
Scientific
publications Ltd. UK and Blackwell Scientific Publications, UK) and Sambrook
et al.,
Molecular Cloning - A Laboratory Manual, Second Ed., Cold Spring Harbor
Laboratory Press, NY (1989).
EXAMPLES:
1. CONSTRUCTION OF MODIFIED CRY9C PROTEINS
Multiple alignments between Bt crystal protein sequences including the
sequences of Cry9C, Cry3A and Cry1 Aa allowed identification of the amino
acids
located in the expected binding site of the Cry9C domain II. Using known
alignment
programs, 52 amino acid positions were identified for amino acid replacement.
The
amino acids in the Cry9C protein of SEQ ID No. 2 from amino acid positions 313-
334, 358-369, 418-425, 480-492 have been identified to correspond to the
solvent-
accessible regions most likely involved in receptor-binding in the Cry3A
protein, and
these positions in the Cry9C protein were chosen for amino acid modification.
Since
alanine substitution does not alter the main chain of a protein, and does not
impose
extreme electrostatic or steric effects and since it eliminates the side chain
beyond
the beta carbon, each of the amino acids in these identified regions was
changed
into alanine, one by one, using splice overlap extension PCR (Ho et al., 1989,
supra)
on the protease-resistant form of the native cry9C gene wherein the arginine
codon
at position 164 was replaced by an alanine codon. The codon most preferred in
the
cry9C native gene for alanine, GCA, was used for these modifications. When the
original codon encodes alanine, then this is replaced by a valine codon (GTA).
The
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obtained PCR fragments were ligated in pUCl9-derived vectors. If not present,
suitable unique restriction sites were created in the cry9C DNA. All plasmids
containing modified DNA sequences were controlled by sequencing the relevant
portions and were found to be correctly constructed. The modified cry9C genes
were
expressed in transformed WK6 cells. Every mutant protein was expressed in
these
E. coli cells at least twice. Mutants causing problems in expression, probably
caused
by structural changes in these mutants, were discarded. No gross folding
aberrations
of the mutants identified to be involved in toxicity (and fisted in Table 1 )
are found,
e.g., as was evidenced by the similar SDS-PAGE patterns following trypsin
cleavage
or treatment with midgut juice of the insect larvae of solubilized mutant and
Cry9C(R164A) proteins.
2. INSECT TOXICITY OF THE MODIFIED CRY9C PROTEINS
Bio assays on the modified Cry9C proteins obtained in Example 1 were
carried out with first instar larvae of the Southwestern corn borer, Diatraea
grandiosella (family Pyralidae); the European corn borer, 4strinia nubilalis
(family
Pyralidae); and the tobacco budworm, Heliothis virescens (family Noctuidae). A
dilution series of each protein was surface-layered on the artificial diet to
determine'
the LCSO value. The artificial diet consisted of: agar (20 g), water (1,000
ml), corn
flour (96 g, ICN Biochemicals), yeast (30 g), wheat germs (64 g, 1CN
Biochemicals),
wesson salt (7.5 g, ICN Biochemicals), casein (15 g), sorbic acid (2 g),
aureomycin
(0.3 g), nipagin (1 g), wheat germ oil (4 ml), sucrose (15 g), cholesterol (1
g),
ascorbic acid (3.5 g), Vanderzand modified vitamin mix (12 g, ICN
Biochemicals).
Larvae were placed on the diet in multi-well plates, 1 larva per well (2 for
Ostrinia
nubilalis). For each dilution, 24 larvae were tested, and dead and living
larvae were
counted after 5 days. Prior to application, the mutant proteins were digested
with
trypsin to release the toxin fragments. For each mutant protein, the assays
are
repeated at least 5 times, using two different protein preparations. As
control protein,
the trypsin-digested Cry9C(R164A) protein was used. The Cry9C(R164A) protein
has the amino acid sequence of SEQ ID No. 2 wherein the arginine at position
1G4
was replaced by alanine. This protein was found to be more stable than the
wild-type
Cry9C toxin while retaining its toxicity to the test insects (see, e.g., PCT
patent
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WO 99/00407 PCT/EP98/04033
publication WO 94/24264). The LCSO values were calculated with the POLO-
program, which is based on the probit analysis (POLO-PC, LeOra Software, 1119
Shattuck Ave., Berkeley California 94707). The results of these assays for
those
protein mutants which gave an LCSO value that is significantly different from
that of
the control protein in repeated bio assays are summarized in Table 1. It is
clear that
different positions in the Cry9C protein when substituted to alanine cause
increased
toxicity in each of the tested insects.
Binding assays on isolated brush border membrane vesicles of Heliothis
virescens and Ostrinia nubilalis performed as described in Van Rie et al.
(1990, Appl.
Environm. Microbiol. 56, 1378-1385) showed that for all, with the exception of
two, of
the modified Cry9C proteins with altered toxicity, receptor binding is also
altered
(e.g., an observed shift in K~ value), thus confirming that for most amino
acid
residues altered toxicity is due to altered receptor binding. Hence, these
residues
are proper candidates for improvement of toxicity by amino acid randomization
at or
near the identified critical position.
3. COMPETITION BINDING EXPERIMENTS
The Cry9C(R164A) protein was tested in competition binding assays using the
ECL protein biotinylation system (Amersham Life Sciences, Amersham
International
plc., UK) as described by Lambent et al. (1996, supra) to determine if
competition
occurred with other Bt toxins in selected insects. For the assays, 3ng
biotinylated
Cry9C(R164A) protein was added to 30 Ng brush border membrane vesicles in PBS
buffer (comprising 0,1 % BSA) in the presence of a 300-fold excess of non-
biotinylated toxin (homologous competition assays were included in every test
as
control). Repeated competition tests showed that in both Ostrinia nubilaiis
and
Heliothis virescens brush border membranes, there was no detectable
competition in
receptor binding between the (activated) Cry9C(R164A) protein and any one of
the
following (activated) Bt toxins: the Cry1 Aa (Schnepf et al., 1985, J. Biol.
Chem. 260,
6264-6272), CrylAb (Hofte et a1.,1986, Eur. J. Biochem. 161, 271-280), CrylAc
(Adang et al., 1985, Gene 36, 289-300), Cry1 B (Brizzard & Whiteley, 1988,
Nucl.
Acids Res. 16, 4168-4169) and Cry1 C (Honee et al., 1988, Nucl. Acids Res. 16,
6240) toxins. Thus, in these insects the Cry9C(R164A) protein binds to a
different
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receptor than these other Bt toxins. In Diatreae grandiosella competition
assays, it
was found that the Cry9C(R164A) does compete for a receptor site with the Cry1
B
and Cry1 C Bt toxins, but does not compete with any one of the Cry1 Aa, Cry1
Ab, and
Cry1 Ac toxins.
The same results are found for all three insects when testing the Cry9C
~ protein with the amino acid sequence of SEQ ID No. 2 from amino acids 1-658.
Thus, in all these three insects, combination of the Cry9C and a selected non-
competitively binding Bt toxin with good toxicity to the target insect can be
used
simultaneously in order to prevent or delay insect resistance development. In
transgenic corn plants, a particularly interesting combination would be the
Cry9C (or
its protease-resistant variant} and a Cry1 B and/or any of the Cry1 A-type
toxins for
Osfrinia nubilalis control and the Cry9C (or its protease-resistant variant)
and any
one of the Cry1 A-type toxins, preferably a Cry1 Ab-type toxin, for D.
grandiosella
control. For Heliothis virescens control, the Cry9C (or its protease-resistant
variant)
and any of the Cry1 A-type toxins are preferred toxins to be co-expressed.
4. CONSTRUCTION OF IMPROVED CRY9C PROTEINS
The modified position in every mutant protein of Example 2 giving rise to a
significantly decreased or increased toxicity to an insect species is altered
to all other
amino acids and the toxicity is re-evaluated. The amino acids yielding the
highest
toxicity at a particular position are combined to form an improved Cry9C
protein.
Also the alanine mutants yielding an increase in toxicity (up-mutant amino
acid
positions) are included in such combinations to form improved Cry9C proteins
for the
selected insect species. Table 1 indeed shows already two up-mutant proteins
for
every insect tested. Analysis of all these improved Cry9C proteins in the bio
assay
shows that combinations of up-mutant amino acid positions can substantially
increase toxicity of the Cry9C protein towards selected insect species.
5. GENE CONSTRUCTION AND PLANT TRANSFORMATION
A modified DNA sequence encoding a truncated Cry9C(R164K) protein for
expression in corn and cotton plants is shown in SEQ lD No. 3. This DNA
sequence
has an optimized codon usage for plants and encodes an N- and C-terminally
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truncated Cry9C protein wherein an arginine amino acid has been replaced by a
lysine (at position 123 in SEQ iD No. 3). Based on this DNA sequence, DNA
sequences are made encoding the above improved Cry9C proteins and comprising
amino acids 1 to 666 of the Cry9C(R164K) protein. Preferred codons to encode
the
amino acid replacements in the improved Cry9C proteins are those which are
most
preferred by the plant host (see, e.g., Murray, 1989, supra). A chimeric
improved
cry9C gene comprising the 35S promoter and 35S 3' transcription termination
and
polyadenylation signal is constructed by routine molecular biology techniques
as
described in the detailed description.
Corn cells are stably transformed by either Agrobacterium-mediated
transformation (Ishida et al., 1996, supra and U.S. Patent No. 5,591,616) or
by
electroporation using wounded and enzyme-degraded embryogenic callus, as
described in WO 92/09696 or US Patent 5,641,664 (incorporated herein by
reference). The resulting transformed cells are selected by means of the
incorporated selectable marker gene, grown into plants and tested for
susceptibility
towards insects. Corn plants expressing a truncated improved Cry9C(R164K)
protein
wherein the amino acids at positions 364, 488, 319 and 321 have been replaced
into
alanine show a significantly higher protection from Ostrinia nubilalis and
Diatraea
grandiosella damage in comparative tests against corn plants expressing a
truncated
Cry9C(R164K} protein. A positive correlation is found between the level of
expression, as measured by RNA and protein analysis, and the observed
insecticidal
effect.
Cotton cells are stably transformed by Agrobacterium-mediated transformation
(Umbeck et al., 1987, Bio/Technology 5, 263-266; US Patent 5,004,863,
incorporated
herein by reference). The resulting transformed cells are selected by means of
the
incorporated selectable marker gene, grown into plants and tested for
susceptibility
towards insects. Cotton plants expressing the truncated improved Cry9C(R164K,
L321 A, P329A) protein at similar levels than cotton plants expressing the
truncated
Cry9C(R164K) protein show a significantly higher protection from Heliofhis
virescens
damage. A positive correlation is found between the level of expression, as
measured by RNA and protein analysis, and the observed insecticidal effect.
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The examples and embodiments of this invention described herein are only
supplied for illustrative purposes. Many variations and modifications in
accordance
with the present invention are known to the person skilled in the art and are
included
. in this invention and the scope of the claims. For instance, it is possible
to alter,
delete or add some nucleotides or amino acids to certain regions of the DNA or
~ protein sequences of the invention without departing from the invention.
All publications (including patent publications) referred to in this
application are
hereby incorporated by reference, particularly WO 94/05771, WO 94/24264, and
Lambert et al. (1996, supra).
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Table 1: relative toxicity of modified trypsin-digested Cry9C proteins to
different
insects when compared with the Cry9C(R164A) trypsin-digested protein (mutant
'F313A': the Cry9C(R164A) trypsin-digested protein wherein also the
phenylalanine
at position 313 is replaced by alanine; 'down(2x)': mutant protein with a
significantly
lower toxicity (LC50 value about 2 times higher than the control protein), 'up
(2x)':
mutant with a significantly higher toxicity (LC50 value about two times lower
than that
of the control protein), '-': no difference in toxicity found):
mutant H. virescens O. nubilalis D. grandiosella
F313A down {2x) - -
P3i6A - - up (2x)
A317V - - up (2x)
N318A down (2-3x) - -
V319A - - up (3x)
L321 A up (2x) - up (2x)
R323A down (3x) - -
W325A down (4-5x) down (2x) down (2-3x)
P329A up (2x) - -
Y330A - down (1.5x) up (2x)
V362A down (3-4x) - -
S364A - up (2x) -
D368A down (2-3x) - -
Y369A - - up (2x)
R418A down ( 16x) down (2x) -
A420V down (12x) - -
L421 A - down (2x) -
1422A - - up (2x)
F480A down (5x) - down (40x)
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mutant H. virescens O. nubilalis D. grandiosella
Q481 A down (3x) - -
N483A - - down (2x)
Q484A - - down (20x}
A485V down (3x) down (2x) down (20x)
S487A down (2x) - down (20x)
1488A down (2x) up (2-3x) down (5x}
N490A - - down (20x)
A491 V - - down (3x)
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: PLANT GENETIC SYSTEMS N.V.
(B) STREET: Jozef Plateaustraat 22
(C) CITY: Gent
(E) COUNTRY: Belgium
(F) POSTAL CODE (ZIP): B-9000
(G) TELEPHONE: (32)(9)2358411
(H) TELEFAX: (32)(9)2231923
(ii) TITLE OF INVENTION: Improved Bacillus thuringiensis toxin
(iii) NUMBER OF SEQUENCES: 4
(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)
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/884,389
(B) FILING DATE: 27-JUN-1997
(2) INFORMATION FOR SEQ ID N0: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4344 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:668..4141
(D) OTHER INFORMATION:/note= "coding sequence"
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID NO:
1:
GAATTCGAGCTCGGTACCTTTTCAGTGTATCGTTTCCCTTCCATCAGGTTTTCAAATTGA 60
AAAGCCGAATGATTTGAAACTTGTTTACGATGTAAGTCATTTGTCTATGACGAAAGATAC 120
GTGTAAAAAACGTATTGAGATTGATGAATGTGGACAAGTAGAAATTGACTTACAAGTATT 180
AAAGATTAAGGGTGTCCTTTCTTTTATCGGAAATTTCTCTATTGAACCTATTCTGTGTGA 240
AAACATGTATACAACGGTTGATAGAGATCCGTCTATTTCCTTAAGTTTCCAAGATACGGT 300
ATATGTGGACCATATTTTAAAATATAGCGTCCAACAACTACCATATTATGTAATTGATGG 360
TGATCATATTCAAGTACGTGATTTACAAATCAAACTGATGAAAGAGAATCCGCAATCTGC 420
TCAAGTATCAGGTTTGTTTTGTTTTGTATATGAGTAAGAACCGAAGGTTTGTP.AAAAAGA480
AATAGGAATAAATACTATCCATTTTTTCAAGAAATATTTTTTTATTAGAAAGGAATCTTT 540
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CTTACACGGG AAAATCCTAA 600
GATTGAGAGT AAAGATATAT
ATATATAAAT ACAATAAAGA
GTTTGTCAGG ATTTTTGAAA 660
GATATGATAT GAACATGCAC
TAGATTTATA GTATAGGAGG
AAAAAGTATG AATCGAAATA ATCAAAATGA ATATGAAATT ATTGATGCCC 720
CCCATTGTGG
' GTGTCCATCA GATGACGATG TGAGGTATCC TTTGGCAAGT GACCCAAATG 780
CAGCGTTACA
AAATATGAAC TATAAAGATT ACTTACAAAT GACAGATGAG GACTACACTG 840
ATTCTTATAT
AAATCCTAGT TTATCTATTA GTGGTAGAGA TGCAGTTCAG ACTGCGCTTA 900
CTGTTGTTGG
GAGAATACTC GGGGCTTTAG GTGTTCCGTT TTCTGGACAA ATAGTGAGTT 960
TTTATCAATT
CCTTTTAAAT ACACTGTGGC CAGTTAATGA TACAGCTATA TGGGAAGCTT 1020
TCATGCGACA
GGTGGAGGAA CTTGTCAATC AACAAATAAC AGAATTTGCA AGAAATCAGG 1080
CACTTGCAAG
ATTGCAAGGA TTAGGAGACT CTTTTAATGT ATATCAACGT TCCCTTCAAA 1140
ATTGGTTGGC
TGATCGAAAT GATACACGAA ATTTAAGTGT TGTTCGTGCT CAATTTATAG 1200
CTTTAGACCT
TGATTTTGTT AATGCTATTC CATTGTTTGC AGTAAATGGA CAGCAGGTTC 1260
CATTACTGTC
AGTATATGCA CAAGCTGTGA ATTTACATTT GTTATTATTA AAAGATGCAT 1320
CTCTTTTTGG
AGAAGGATGG GGATTCACAC AGGGGGAAAT TTCCACATAT TATGACCGTC 1380
AATTGGAACT
AACCGCTAAG TACACTAATT ACTGTGAAAC TTGGTATAAT ACAGGTTTAG 1440
ATCGTTTAAG
AGGAACAAAT ACTGAAAGTT GGTTAAGATA TCATCAATTC CGTAGAGAAA 1500
TGACTTTAGT
GGTATTAGAT GTTGTGGCGC TATTTCCATA TTATGATGTA CGACTTTATC 1560
CAACGGGATC
AAACCCACAG CTTACACGTG AGGTATATAC AGATCCGATT GTATTTAATC 1620
CACCAGCTAA
TGTTGGACTT TGCCGACGTT GGGGTACTAA TCCCTATAAT ACTTTTTCTG 1680
AGCTCGAAAA
TGCCTTCATT CGCCCACCAC ATCTTTTTGA TAGGCTGAAT AGCTTAACAA 1740
TCAGCAGTAA
TCGATTTCCA GTTTCATCTA ATTTTATGGA TTATTGGTCA GGACATACGT 1800
TACGCCGTAG
TTATCTGAAC GATTCAGCAG TACAAGAAGA TAGTTATGGC CTAATTACAA 1860
CCACAAGAGC
AACAATTAAT CCCGGAGTTG ATGGAACAAA CCGCATAGAG TCAACGGCAG 1920
TAGATTTTCG
TTCTGCATTG ATAGGTATAT ATGGCGTGAA TAGAGCTTCT TTTGTCCCAG 1980
GAGGCTTGTT
TAATGGTACG ACTTCTCCTG CTAATGGAGG ATGTAGAGAT CTCTATGATA 2040
CAAATGATGA
ATTACCACCA GATGAAAGTA CCGGAAGTTC AACCCATAGA CTATCTCATG 2100
TTACCTTTTT
TAGCTTTCAA ACTAATCAGG 2160
CTGGATCTAT AGCTAATGCA
GGAAGTGTAC CTACTTATGT
TTGGACCCGT CGTGATGTGG 2220
ACCTTAATAA TACGATTACC
CCAAATAGAA TTACACAATT
ACCATTGGTA AAGGCATCTG 2280
CACCTGTTTC GGGTACTACG
GTCTTAAAAG GTCCAGGATT
TACAGGAGGG GGTATACTCC 2340
GAAGAACAAC TAATGGCACA
TTTGGAACGT TAAGAGTAAC
GGTTAATTCA CCATTAACAC 2400
AACAATATCG CCTAAGAGTT
CGTTTTGCCT CAACAGGAAA
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TTTCAGTATAAGGGTACTCCGTGGAGGGGTTTCTATCGGTGATGTTAGATTAGGGAGCAC 2460
AATGAACAGAGGGCAGGAACTAACTTACGAATCCTTTTTCACAAGAGAGTTTACTACTAC 2520
TGGTCCGTTCAATCCGCCTTTTACATTTACACAAGCTCAAGAGATTCTAACAGTGAATGC 2580
AGAAGGTGTTAGCACCGGTGGTGAATATTATATAGATAGAATTGAAATTGTCCCTGTGAA 2640
TCCGGCACGAGAAGCGGAAGAGGATTTAGAAGCGGCGAAGAAAGCGGTGGCGAGCTTGTT 2700
TACACGTACAAGGGACGGATTACAGGTAAATGTGACAGATTATCAAGTGGACCAAGCGGC 2760
AAATTTAGTGTCATGCTTATCCGATGAACAATATGGGCATGACAAAAAGATGTTATTGGA 2820
AGCGGTAAGAGCGGCAAAACGCCTCAGCCGCGAACGCAACTTACTTCAAGATCCAGATTT 2880
TAATACAATCAATAGTACAGAAGAGAATGGCTGGAAGGCAAGTAACGGTGTTACTATTAG 2940
CGAGGGCGGTCCATTCTTTAAAGGTCGTGCACTTCAGTTAGCAAGCGCAAGAGAAAATTA 3000
TCCAACATACATTTATCAAAAAGTAGATGCATCGGTGTTAAAGCCTTATACACGCTATAG 3060
ACTAGATGGATTTGTGAAGAGTAGTCAAGATTTAGAAATTGATCTCATCCACCATCATAA 3120
AGTCCATCTTGTAAAAAATGTACCAGATAATTTAGTATCTGATACTTACTCAGATGGTTC 3180
TTGCAGCGGAATCAACCGTTGTGATGAACAGCATCAGGTAGATATGCAGCTAGATGCGGA 3240
GCATCATCCAATGGATTGCTGTGAAGCGGCTCAAACACATGAGTTTTCTTCCTATATTAA 3300
TACAGGGGATCTAAATGCAAGTGTAGATCAGGGCATTTGGGTTGTATTAAAAGTTCGAAC 3360
AACAGATGGGTATGCGACGTTAGGAAATCTTGAATTGGTAGAGGTTGGGCCATTATCGGG 3420
TGAATCTCTAGAACGGGAACAAAGAGATAATGCGAAATGGAATGCAGAGCTAGGAAGAAA 3480
ACGTGCAGAAATAGATCGTGTGTATTTAGCTGCGAAACAAGCAATTAATCATCTGTTTGT 3540
AGACTATCAAGATCAACAATTAAATCCAGAAATTGGGCTAGCAGAAATTAATGAAGCTTC 3600
AAATCTTGTAGAGTCAATTTCGGGTGTATATAGTGATACACTATTACAGATTCCTGGGAT 3660
TAACTACGAAATTTACACAGAGTTATCCGATCGCTTACAACAAGCATCGTATCTGTATAC 3720
GTCTAGAAATGCGGTGCAAAATGGAGACTTTAACAGTGGTCTAGATAGTTGGAATACAAC 3780
TATGGATGCATCGGTTCAGCAAGATGGCAATATGCATTTCTTAGTTCTTTCGCATTGGGA 3840
TGCACAAGTTTCCCAACAATTGAGAGTAAATCCGAATTGTAAGTATGTCTTACGTGTGAC 3900
AGCAAGAAAAGTAGGAGGCGGAGATGGATACGTCACAATCCGAGATGGCGCTCATCACCA 3960
AGAAACTCTTACATTTAATGCATGTGACTACGATGTAAATGGTACGTATGTCAATGACAA 4020
TTCGTATATAACAGAAGAAGTGGTATTCTACCCAGAGACAAAACATATGTGGGTAGAGGT 4080
GAGTGAATCCGAAGGTTCATTCTATATAGACAGTATTGAGTTTATTGAAACACAAGAGTA 4140
GAAGAGGGGGATCCTAACGTATAGCAACTATGAGAGGATACTCCGTACAAACAAAGATTA 4200
1~AAAAAGGTAAAATGAATAGAACCCCCTACTGGTAGAAGGACCGATAGGGGGTTCTTACA 4260
TGAAAAAATGTAGCTGTTTACTAAGGTGTATAAAAAACAGCATATCTGATAGAAP.AAAGT4320
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GAGTACCTTA TAAAGAAAGA ATTC 4344
(2) INFORMATION FOR SEQ ID N0: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1157 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Asn Arg Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Pro His
1 5 10 15
Cys Gly Cys Pro Ser Asp Asp Asp Val Arg Tyr Pro Leu Ala Ser Asp
20 25 30
Pro Asn Ala Ala Leu Gln Asn Met Asn Tyr Lys Asp Tyr Leu Gln Met
35 40 45
Thr Asp Glu Asp Tyr Thr Asp Ser Tyr Ile Asn Pro Ser Leu Ser Ile
50 55 60
Ser Gly Arg Asp Ala Val Gln Thr Ala Leu Thr Val Val Gly Arg Ile
65 70 75 80
Leu Gly Ala Leu Gly Val Pro Phe Ser Gly Gln Ile Val Ser Phe Tyr
85 90 95
Gln Phe Leu Leu Asn Thr Leu Trp Pro Val Asn Asp Thr Ala Ile Trp
100 105 110
Glu Ala Phe Met Arg Gln Val Glu Glu Leu Val Asn Gln Gln Ile Thr
115 120 125
Glu Phe Ala Arg Asn Gln Ala Leu Ala Arg Leu Gln Gly Leu Gly Asp
130 135 140
Ser Phe Asn Val Tyr Gln Arg Ser Leu Gln Asn Trp Leu Ala Asp Arg
145 150 155 160
Asn Asp Thr Arg Asn Leu Ser Val Val Arg Ala Gln Phe Ile Ala Leu
165 170 175
Asp Leu Asp Phe Val Asn Ala Ile Pro Leu Phe Ala Val Asn Gly Gln
180 185 190
Gln Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Val Asn Leu His Leu
195 200 205
Leu Leu Leu Lys Asp Ala Ser Leu Phe Gly Glu Gly Trp Gly Phe Thr
210 215 220
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Gln Gly Glu Ile Ser Thr Tyr Tyr Asp Arg Gln Leu Glu Leu Thr Ala
225 230 235 240
Lys Tyr Thr Asn Tyr Cys Glu Thr Trp Tyr Asn Thr Gly Leu Asp Arg
245 250 255
Leu Arg Gly Thr Asn Thr Glu Ser Trp Leu Arg Tyr His Gln Phe Arg
260 265 270
Arg Glu Met Thr Leu Val Val Leu Asp Val Val Ala Leu Phe Pro Tyr
275 280 285 _
Tyr Asp Val Arg Leu Tyr Pro Thr Gly Ser Asn Pro Gln Leu Thr Arg
290 295 300
Glu Val Tyr Thr Asp Pro Ile Val Phe Asn Pro Pro Ala Asn Val Gly
305 310 315 320
Leu Cys Arg Arg Trp Gly Thr Asn Pro Tyr Asn Thr Phe Ser Glu Leu
325 330 335
Glu Asn Ala Phe Ile Arg Pro Pro His Leu Phe Asp Arg Leu Asn Ser
340 345 350
Leu Thr Ile Ser Ser Asn Arg Phe Pro Val Ser Ser Asn Phe Met Asp
355 360 365
Tyr Trp Ser Gly His Thr Leu Arg Arg Ser Tyr Leu Asn Asp Ser Ala
370 375 380
Val Gln Glu Asp Ser Tyr Gly Leu Ile Thr Thr Thr Arg Ala Thr Ile
385 390 395 400
Asn Pro Gly Val Asp Gly Thr Asn Arg Ile Glu Ser Thr Ala Val Asp
405 410 415
Phe Arg Ser Ala Leu Ile Gly Ile Tyr Gly Val Asn Arg Ala Ser Phe
420 425 430
Val Pro Gly Gly Leu Phe Asn Gly Thr Thr Ser Pro Ala Asn Gly Gly
435 440 445
Cys Arg Asp Leu Tyr Asp Thr Asn Asp Glu Leu Pro Pro Asp Glu Ser
450 455 460
Thr Gly Ser Ser Thr His Arg Leu Ser His Val Thr Phe Phe Ser Phe
465 470 475 480
Gln Thr Asn Gln Ala Gly Ser Ile Ala Asn Ala Gly Ser Val Pro Thr
485 490 495
Tyr Val Trp Thr Arg Arg Asp Val Asp Leu Asn Asn Thr Ile Thr Pro
500 505 510
Asn Arg Ile Thr Gln Leu Pro Leu Val Lys Ala Ser Ala Pro Val Ser
515 520 525
Gly Thr Thr Val Leu Lys Gly Pro Gly Phe Thr Gly Gly Gly Ile Leu
530 535 540
Arg Arg Thr Thr Asn Gly Thr Phe Gly Thr Leu Arg Val Thr Val Asn
545 550 555 560
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Ser Pro Leu Thr Gln Gln Tyr Arg Leu Arg Va1 Arg Phe Ala Ser Thr
565 570 575
Gly Asn Phe Ser hle Arg Val Leu Arg Gly Gly Val Ser Ile Gly Asp
580 585 590
Val Arg Leu Gly Ser Thr Met Asn Arg Gly Gln Glu Leu Thr Tyr Glu
595 600 605
Ser Phe Phe Thr Arg Glu Phe Thr Thr Thr Gly Pro Phe Asn Pro Pro
610 615 620
Phe Thr Phe Thr Gln Ala Gln Glu Ile Leu Thr Val Asn Ala Glu Gly
625 630 635 640
Val Ser Thr Gly Gly Glu Tyr Tyr Ile Asp Arg Ile Glu Ile Val Pro
645 650 655
Val Asn Pro Ala Arg Glu Ala Glu Glu Asp Leu Glu A1a Ala Lys Lys
660 665 670
Ala Val Ala Ser Leu Phe Thr Arg Thr Arg Asp Gly Leu Gln Val Asn
675 680 685
Val Thr Asp Tyr Gln Val Asp Gln Ala Ala Asn Leu Val Ser Cys Leu
690 695 700
Ser Asp Glu Gln Tyr Gly His Asp Lys Lys Met Leu Leu Glu Ala Val
705 710 715 720
Arg Ala Ala Lys Arg Leu Ser Arg Glu Arg Asn Leu Leu Gln Asp Pro
725 730 735
Asp Phe Asn Thr Ile Asn Ser Thr Glu Glu Asn Gly Trp Lys Ala Ser
740 745 750
Asn Gly Val Thr Ile Ser Glu Gly Gly Pro Phe Phe Lys Gly Arg Ala
755 760 765
Leu Gln Leu Ala Ser Ala Arg Glu Asn Tyr Pro Thr Tyr Ile Tyr Gln
770 775 780
Lys Val Asp Ala Ser Val Leu Lys Pro Tyr Thr Arg Tyr Arg Leu Asp
785 790 795 800
Gly Phe Val Lys Ser Ser Gln Asp Leu Glu Ile Asp Leu Ile His His
805 810 815
His Lys Val His Leu Val Lys Asn Val Pro Asp Asn Leu Val Ser Asp
820 825 830
Thr Tyr Ser Asp Gly Ser Cys Ser Gly Ile Asn Arg Cys Asp Glu Gln
835 840 845
His Gln Val Asp Met Gln Leu Asp Ala Glu His His Pro Met Asp Cys
850 855 860
Cys Glu Ala Ala Gln Thr His Glu Phe Ser Ser Tyr Ile Asn Thr Gly
865 870 875 880
Asp Leu Asn Ala Ser Val Asp Gln Gly Ile Trp Val Val Leu Lys Val
885 890 895
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Arg Thr Thr Asp Gly Tyr Ala Thr Leu Gly Asn Leu Glu Leu Val Glu
900 905 910
Val Gly Pro Leu Ser Gly Glu Ser Leu Glu Arg Glu Gln Arg Asp Asn
915 920 925
Ala Lys Trp Asn Ala Glu Leu Gly Arg Lys Arg Ala Glu Ile Asp Arg
930 935 940
Val Tyr Leu Ala Ala Lys Gln Ala Ile Asn His Leu Phe Val Asp Tyr
945 950 955 960 -
Gln Asp Gln Gln Leu Asn Pro Glu Ile Gly Leu Ala Glu Ile Asn Glu
965 970 975
Ala Ser Asn Leu Val Glu Ser Ile Ser Gly Val Tyr Ser Asp Thr Leu
980 985 990
Leu Gln Ile Pro Gly Ile Asn Tyr Glu Ile Tyr Thr Glu Leu Ser Asp
995 1000 1005
Arg Leu Gln Gln Ala Ser Tyr Leu Tyr Thr Ser Arg Asn Ala Val Gln
1010 1015 1020
Asn Gly Asp Phe Asn Ser Gly Leu Asp Ser Trp Asn Thr Thr Met Asp
1025 1030 1035 1040
Ala Ser Val Gln Gln Asp Gly Asn Met His Phe Leu Val Leu Ser His
1045 1050 1055
Trp Asp Ala Gln Val Ser Gln Gln Leu Arg Val Asn Pro Asn Cys Lys
1060 1065 1070
Tyr Val Leu Arg Val Thr Ala Arg Lys Val Gly Gly Gly Asp Gly Tyr
1075 1080 1085
Val Thr Ile Arg Asp Gly Ala His His Gln Glu Thr Leu Thr Phe Asn
1090 1095 1100
Ala Cys Asp Tyr Asp Val Asn Gly Thr Tyr Val Asn Asp Asn Ser Tyr
1105 1110 1115 1120
Ile Thr Glu Glu Val Val Phe Tyr Pro Glu Thr Lys His Met Trp Val
1125 1130 1135
Glu Val Ser Glu Ser Glu Gly Ser Phe Tyr Ile Asp Ser Ile Glu Phe
1140 1145 1150
Ile Glu Thr Gln Glu
1155
(2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1897 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "synthetic DNA"
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(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:13..1890
(D) OTHER INFORMATION:/note = ~"coding sequence"
(xi) SEQUENCE DESCRIPTION:
SEQ ID NO: 3:
GGTACCAAAA CCATGGCTGACTACCTGCAG AGGACTACACCGACAGCTAC 60
ATGACCGACG
ATCAACCCCA GCCTGAGCATCAGCGGTCGCGACGCCGTGCAGACCGCTCTGACCGTGGTG 120
GGTCGCATCC TGGGTGCCCTGGGCGTGCCCTTCAGCGGTCAGATCGTGAGCTTCTACCAG 180
TTCCTGCTGA ACACCCTGTGGCCAGTGAACGACACCGCCATCTGGGAAGCTTTCATGCGC 240
CAGGTGGAGG AGCTGGTGAACCAGCAGATCACCGAGTTCGCTCGCAACCAGGCCCTGGCT 300
CGCCTGCAGG GCCTGGGCGACAGCTTCAACGTGTACCAGCGCAGCCTGCAGAACTGGCTG 360
GCCGACCGCA ACGACACCAAGAACCTGAGCGTGGTGAGGGCCCAGTTCATCGCCCTGGAC 420
CTGGACTTCG TGAACGCCATCCCCCTGTTCGCCGTGAACGGCCAGCAGGTGCCCCTGCTG 480
AGCGTGTACG CCCAGGCCGTGAACCTGCACCTGCTGCTGCTGAAGGATGCATCCCTGTTC 540
GGCGAGGGCT GGGGCTTCACCCAGGGCGAGATCAGCACCTACTACGACCGCCAGCTCGAG 600
CTGACCGCCA AGTACACCAACTACTGCGAGACCTGGTACAACACCGGTCTGGACCGCCTG 660
AGGGGCACCA ACACCGAGAGCTGGCTGCGCTACCACCAGTTCCGCAGGGAGATGACCCTG 720
GTGGTGCTGG ACGTGGTGGCCCTGTTCCCCTACTACGACGTGCGCCTGTACCCCACCGGC 780
AGCAACCCCC AGCTGACACGTGAGGTGTACACCGACCCCATCGTGTTCAACCCACCAGCC 840
AACGTGGGCC TGTGCCGCAGGTGGGGCACCAACCCCTACAACACCTTCAGCGAGCTGGAG 900
AACGCCTTCA TCAGGCCACCCCACCTGTTCGACCGCCTGAACAGCCTGACCATCAGCAGC 960
AATCGATTCC CCGTGAGCAGCAACTTCATGGACTACTGGAGCGGTCACACCCTGCGCAGG 1020
AGCTACCTGA ACGACAGCGCCGTGCAGGAGGACAGCTACGGCCTGATCACCACCACCAGG 1080
GCCACCATCA ACCCAGGCGTGGACGGCACCAACCGCATCGAGAGCACCGCTGTGGACTTC 1140
CGCAGCGCTC TGATCGGCATCTACGGCGTGAACAGGGCCAGCTTCGTGCCAGGTGGCCTG 1200
TTCAACGGCA CCACCAGCCCAGCCAACGGTGGCTGCCGAGATCTGTACGACACCAACGAC 1260
GAGCTGCCAC CCGACGAGAGCACCGGCAGCAGCACCCACCGCCTGAGCCACGTCACCTTC 1320
TTCAGCTTCC AGACCAACCAGGCTGGCAGCATCGCCAACGCTGGCAGCGTGCCCACCTAC 1380
GTGTGGACCA GGAGGGACGTGGACCTGAACAACACCATCACCCCCAACCGCATCACCCAG 1440
CTGCCCCTGG TGAAGGCCAGCGCTCCCGTGAGCGGCACCACCGTGCTGAAGGGTCCAGGC 1500
TTCACCGGTG GCGGTATACTGCGCAGGACCACCAACGGCACCTTCGGCACCCTGCGCGTG 1560
ACCGTGAATT CCCCACTGACCCAGCAGTACCGCCTGCGCGTGCGCTTCGCCAGCACCGGC 1620
AACTTCAGCA TCCGCGTGCT GAGGGGTGGCGTGAGCATCG 1680
GCGACGTGCG
CCTGGGCAGC
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ACCATGAACA GGGGCCAGGA GCTGACCTAC GAGAGCTTCT TCACCCGCGA GTTCACCACC 1740
ACCGGTCCCT TCAACCCACC CTTCACCTTC ACCCAGGCCC AGGAGATCCT GACCGTGAAC 1800
GCCGAGGGCG TGAGCACCGG TGGCGAGTAC TACATCGACC GCATCGAGAT CGTGCCCGTG 1860
AACCCAGCTC GCGAGGCCGA GGAGGACTGA GGCTAGC 1897
(2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 625 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:
Met Ala Asp Tyr Leu Gln Met Thr Asp Glu Asp Tyr Thr Asp Ser Tyr
1 5 10 15
Ile Asn Pro Ser Leu Ser Ile Ser Gly Arg Asp Ala Val Gln Thr Ala
20 25 30
Leu Thr Val Val Gly Arg Ile Leu Gly Ala Leu Gly Val Pro Phe Ser
35 40 45
Gly Gln Ile Val Ser Phe Tyr Gln Phe Leu Leu Asn Thr Leu Trp Pro
50 55 60
Val Asn Asp Thr Ala Ile Trp Glu Ala Phe Met Arg Gln Val Glu Glu
65 70 75 80
Leu Val Asn Gln Gln Ile Thr Glu Phe Ala Arg Asn Gln Ala Leu Ala
85 90 95
Arg Leu Gln Gly Leu Gly Asp Ser Phe Asn Val Tyr Gln Arg Ser Leu
100 105 110
Gln Asn Trp Leu Ala Asp Arg Asn Asp Thr Lys Asn Leu Ser Val Val
115 120 125
Arg Ala Gln Phe Ile Ala Leu Asp Leu Asp Phe Val Asn Ala Ile Pro
130 135 140
Leu Phe Ala Val Asn Gly Gln Gln Val Pro Leu Leu Ser Val Tyr Ala
145 150 155 160
Gln Ala Val Asn Leu His Leu Leu Leu Leu Lys Asp Ala Ser Leu Phe
165 170 175
Gly Glu Gly Trp Gly Phe Thr Gln Gly Glu Ile Ser Thr Tyr Tyr Asp
180 185 190
Arg Gln Leu Glu Leu Thr Ala Lys Tyr Thr Asn Tyr Cys Glu Thr Trp
195 200 205
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Tyr Asn Thr Gly Leu Asp Arg Leu Arg Gly Thr Asn Thr Glu Ser Trp
210 215 220
Leu Arg Tyr His Gln Phe Arg Arg Glu Met Thr Leu Val Val Leu Asp
225 230 235 240
Val Val Ala Leu Phe Pro Tyr Tyr Asp Val Arg Leu Tyr Pro Thr Gly
245 250 255
Ser Asn Pro Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Ile Val Phe
260 265 270
Asn Pro Pro Ala Asn Val Gly Leu Cys Arg Arg Trp Gly Thr Asn Pro
275 280 285
Tyr Asn Thr Phe Ser Glu Leu Glu Asn Ala Phe Ile Arg Pro Pro His
290 295 300
Leu Phe Asp Arg Leu Asn Ser Leu Thr Ile Ser Ser Asn Arg Phe Pro
305 310 315 320
Val Ser Ser Asn Phe Met Asp Tyr Trp Ser Gly His Thr Leu Arg Arg
325 330 335
Ser Tyr Leu Asn Asp Ser Ala Val Gln Glu Asp Ser Tyr Gly Leu Ile
340 345 350
Thr Thr Thr Arg Ala Thr Ile Asn Pro Gly Val Asp Gly Thr Asn Arg
355 360 365
Ile Glu Ser Thr Ala Val Asp Phe Arg Ser Ala Leu Ile Gly Ile Tyr
370 375 380
Gly Val Asn Arg Ala Ser Phe Val Pro Gly Gly Leu Phe Asn Gly Thr
385 390 395 400
Thr Ser Pro Ala Asn Gly Gly Cys Arg Asp Leu Tyr Asp Thr Asn Asp
405 410 415
Glu Leu Pro Pro Asp Glu Ser Thr Gly Ser Ser Thr His Arg Leu Sex
420 425 430
His Val Thr Phe Phe Ser Phe Gln Thr Asn Gln Ala Gly Ser Ile Ala
435 440 445
Asn Ala Gly Ser Val Pro Thr Tyr Val Trp Thr Arg Arg Asp Val Asp
450 455 460
Leu Asn Asn Thr Ile Thr Pro Asn Arg Ile Thr Gln Leu Pro Leu Val
465 470 475 480
Lys Ala Ser Ala Pro Val Ser Gly Thr Thr Val Leu Lys Gly Pro Gly
485 490 495
Phe Thr Gly Gly Gly Ile Leu Arg Arg Thr Thr Asn Gly Thr Phe Gly
500 505 510
Thr Leu Arg Val Thr Val Asn Ser Pro Leu Thr Gln Gln Tyr Arg Leu
515 520 525
Arg Val Arg Phe Ala Ser Thr Gly Asn Phe Ser Ile Arg Val Leu Arg
530 535 540
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Gly Gly Val Ser Ile Gly Asp Val Arg Leu Gly Ser Thr Met Asn Arg
545 550 555 560
Gly Gln Glu Leu Thr Tyr Glu Ser Phe Phe Thr Arg Glu Phe Thr Thr
565 570 575
Thr Gly Pro Phe Asn Pro Pro Phe Thr Phe Thr Gln Ala Gln Glu Ile
580 585 590
Leu Thr Val Asn Ala Glu Gly Val Ser Thr Gly Gly Glu Tyr Tyr Ile
595 600 605 -
Asp Arg Ile Glu Ile Val Pro Val Asn Pro Ala Arg Glu Ala Glu Glu
610 615 620
Asp
625
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