U.S. patent application number 10/809953 was filed with the patent office on 2004-09-16 for prevention of bt resistance development.
This patent application is currently assigned to Bayer BioScience N.V.. Invention is credited to Botterman, Johan, Joos, Henk, Van Mellaert, Herman, Van Rie, Jeroen.
Application Number | 20040181825 10/809953 |
Document ID | / |
Family ID | 34119899 |
Filed Date | 2004-09-16 |
United States Patent
Application |
20040181825 |
Kind Code |
A1 |
Van Mellaert, Herman ; et
al. |
September 16, 2004 |
Prevention of Bt resistance development
Abstract
Plants made resistant to insects by transforming their nuclear
genome with two or more DNA sequences, each encoding a different
non-competitively binding B. thuringiensis protoxin or insecticidal
part thereof, preferably the toxin thereof.
Inventors: |
Van Mellaert, Herman;
(Leuven, BE) ; Botterman, Johan; (Zevergem-De
Pinte, BE) ; Van Rie, Jeroen; (Eeklo, BE) ;
Joos, Henk; (Aalter, BE) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
Bayer BioScience N.V.
|
Family ID: |
34119899 |
Appl. No.: |
10/809953 |
Filed: |
March 26, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10809953 |
Mar 26, 2004 |
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09661016 |
Sep 13, 2000 |
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09661016 |
Sep 13, 2000 |
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09176320 |
Oct 22, 1998 |
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6172281 |
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09176320 |
Oct 22, 1998 |
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08465609 |
Jun 5, 1995 |
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5866784 |
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08465609 |
Jun 5, 1995 |
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08173274 |
Dec 23, 1993 |
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08173274 |
Dec 23, 1993 |
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07640400 |
Jan 22, 1991 |
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Current U.S.
Class: |
800/279 ;
435/419 |
Current CPC
Class: |
C07K 2319/00 20130101;
Y02A 40/146 20180101; C07K 14/325 20130101; C12N 1/205 20210501;
C12N 15/8286 20130101; C12R 2001/075 20210501 |
Class at
Publication: |
800/279 ;
435/419 |
International
Class: |
A01H 001/00; C12N
015/82; C12N 005/04 |
Foreign Application Data
Date |
Code |
Application Number |
May 31, 1989 |
GB |
89401499.2 |
May 30, 1990 |
WO |
PCT/EP90/00905 |
Claims
1. A cell of a plant, characterized by: at least two B.
thuringiensis ICP genes stably inserted into the genome of said
plant; each of said genes encoding a different non-competitively
binding ICP for an insect species; whereby at least two different
ICPs can be produced by said cell which do not bind competitively
to the brush border membrane of the columnar midgut epithelial cell
of said insect species.
2. The cell of claim 1 wherein at least one marker gene, encoding a
protein or polypeptide which renders said cell easily
distinguishable from cells which do not contain said protein or
polypeptide, is in the same genetic locus as at least one of said
ICP genes.
3. The cell of claim 1 or 2, wherein each of said ICP genes is
under the control of a separate promoter capable of directing gene
expression in said cell and is provided with a separate signal for
3' end formation and within a same transcriptional unit.
4. The cell of claim 2 or 3, in which said marker DNA is under the
control of a separate promoter capable of directing gene expression
in said plant cell and is provided with a signal for 3' end
formation within a same transcriptional unit.
5. The cell of claim 1 or 2, wherein said ICP genes are within a
same transcriptional unit and under the control of a single
promoter.
6. The cell of claim 5, wherein said marker gene is fused with at
least one of said ICP genes and is within said same transcriptional
unit and under the control of said promoter.
7. The cell of claim 5 or 6, wherein a DNA fragment, encoding a
protease-sensitive or -cleavable amino acid sequence, is in said
same transcriptional unit as said ICP genes and intercalated in
frame between said ICP genes.
8. The cell of claim 5 or 6, wherein said ICP genes are combined in
a dicistronic unit comprising an intergenic DNA sequence which
allows reinitiation of translation and is in said same
transcriptional unit as said ICP genes and intercalated between
said ICP genes.
9. The cell of anyone of claims 1 to 8, wherein said ICP genes are
genes encoding insecticidal proteins having activity against
Lepidoptera species and are particularly the following genes: bt2
and/or bt73 and/or bt4 and/or bt14 and/or bt15 and/or bt18.
10. The cell of any of claims 1 to 8, wherein said ICP genes are
genes encoding insecticidal proteins having activity against a
Coleoptera species and are particularly the following genes: bt13
and/or bt21 and/or bt22.
11. The cell of any of claims 2 to 10 wherein said marker DNA is:
an herbicide resistance gene, particularly a sfr or sfrv gene; a
gene encoding a modified target enzyme for a herbicide having a
lower affinity for the herbicide, particularly a modified 5-EPSP as
a target for glyphosate or a modified glutamine synthetase as a
target for a GS inhibitor; or an antibiotic resistance gene,
particularly NPTII.
12. The cell of any of claims 3 to 6, wherein said promoter is: a
constitutive promoter, particularly a 35S promoter or a 35S3
promoter; a PNOS promoter; a POCS promoter; a wound-inducible
promoter, particularly a TR1' or TR2' promoter; a promoter which
directs gene expression selectively in plant tissue having
photosynthetic activity, particularly a SSU promoter; or a
tissue-specific promoter, particularly a tuber-specific promoter, a
stem-specific promoter or a seed-specific promoter.
13. A vector suitable for transforming a cell of a plant,
particularly a plant capable of being infected with Agrobacterium,
comprising said ICP genes of any of claims 1 to 12.
14. A process for producing a plant having improved insect
resistance and having said ICP genes of anyone of claims 1 to 12
stably integrated into the nuclear genome of their cells,
characterized by the non-biological steps of transforming a cell of
said plant by introducing said ICP genes into the nuclear genome of
said cell and regenerating said plant and reproduction material
from said cell.
15. A plant cell culture, consisting of the plant cells of anyone
of claims 1 to 12.
16. A plant, consisting of the plant cells of anyone of claims 1 to
12.
17. Brassica, tomato, potato, tobacco, cotton or lettuce consisting
of the plant cells of anyone of claims 1 to 12, wherein said ICP
genes comprise one of the following pairs of genes: bt2 and bt18 or
bt73 and bt15 or bt2 and bt18 or bt2 and bt14 or bt2 and bt4 or
bt15 and bt18 or bt14 and bt15 or bt4 and bt15 or bt13 and bt21 or
bt21 and bt22 or bt13 and bt22.
18. The cell of anyone of claims 1-12, made by a process as
described hereinabove.
19. A method for rendering a plant resistant to an insect species
by transforming the plant with said ICP genes of anyone of claims
1-12.
Description
[0001] This invention relates to plant cells and plants, the
genomes of which are transformed to contain at least two genes,
each coding for a different non-competitively binding Bacillus
thuringiensis ("B. thuringiensis" or "Bt") insecticidal crystal
protein ("ICP"). for a specific target insect species, preferably
belonging to the order of Lepidoptera or Coleoptera. Such
transformed plants have advantages over plants transformed with a
single B. thuringiensis ICP gene, especially with respect to the
prevention of resistance development in the target insect species
against the at least two B. thuringiensis ICPs, expressed in such
plants.
[0002] This invention also relates to a process for the production
of such transgenic plants, taking into account the competitive and
non-competitive binding properties of the at least two B.
thuringiensis ICPs in the target insect species' midgut.
Simultaneous expression in plants of the at least two genes, each
coding for a different non-competitively binding B. thuringiensis
ICP in plants, is particularly useful to prevent or delay
resistance development of insects against the at least two B.
thuringiensis ICPs expressed in the plants.
[0003] This invention further relates to a process for the
construction of novel plant expression vectors and to the novel
plant expression vectors themselves, which contain the at least two
B. thuringiensis ICP genes encoding the at least two
non-competitively binding B. thuringiensis ICPS. Such vectors allow
integration and coordinate expression of the at least two B.
thuringiensis ICP genes in plants.
BACKGROUND OF THE INVENTION
[0004] Since the development and the widespread use of chemical
insecticides, the occurrence of resistant insect strains has been
an important problem. Development of insecticide resistance is a
phenomenon dependent on biochemical, physiological, genetic and
ecological mechanisms. Currently, insect resistance has been
reported against all major classes of chemical insecticides
including chlorinated hydrocarbons, organophosphates, carbamates,
and pyrethroid compounds (Brattsten et al., 1986).
[0005] In contrast to the rapid development of insect resistance to
synthetic insecticides, development of insect resistance to
bacterial insecticides such as B. thuringiensis sprays has evolved
slowly despite many years of use (Brattsten et al., 1986). The
spore forming gram-positive bacterium B. thuringiensis produces a
parasporal crystal which is composed of crystal proteins (ICPs)
having insecticidal activity. Important factors decreasing the
probability of emergence of resistant insect strains in the field
against B. thuringiensis sprays are: firstly the short half-life of
B. thuringiensis sprays after foliar application; secondly the fact
that commercial B. thuringiensis preparations often consist of a
mixture of several insecticidal factors including spores, ICPs and
eventually beta-exotoxins (Shields, 1987); and thirdly the
transitory nature of plant-pest interactions. Many successful field
trials have shown that commercial preparations of a B.
thuringiensis containing its spore-crystal complex, effectively
control lepidopterous pests in agriculture and forestry (Krieg and
Langenbruch, 1981). B. thuringiensis is at present the most widely
used pathogen for microbial control of insect pests.
[0006] Various laboratory studies, in which selection against B.
thuringiensis was applied over several generations of insects, have
confirmed that resistance against B. thuringiensis is seldom
obtained. However, it should be emphasized that the laboratory
conditions represented rather low selection pressure
conditions.
[0007] For example, Goldman et al. (1986) have applied selection
with B. thuringiensis israelensis toxin over 14 generations of
Aedes aegypti and found only a marginal decrease in sensitivity.
The lack of any observable trend toward decreasing susceptibility
in the selected strains may be a reflection of the low selection
pressure (LC.sub.50) carried out over a limited number of
generations. However, it should be pointed out that Georghiou et
al. (In: Insecticide Resistance in Mosquitoes : Research on new
chemicals and techniques for management. In "Mosquito Control
Research, Annual Report 1983, University of California.") with
Culex quinquefasciatus obtained an 11-fold increase in resistance
to B. thuringiensis israelensis after 32 generations at LC.sub.95
selection presssure.
[0008] McGaughey (1985) reported that the grain storage pest Plodia
interpunctella developed resistance to the spore-crystal complex of
B. thuringiensis; after 15 generations of selection with the Indian
meal moth, Plodia interpunctella, using a commercial B.
thuringiensis HD-1 preparation ("Dipel", Abbott Laboratories, North
Chicago, Ill. 60064, USA), a 100-fold decrease in B. thuringiensis
sensitivity was reported. Each of the colonies was cultured for
several generations on a diet treated with a constant B.
thuringiensis dosage which was expected to produce 70-90% larval
mortality. Under these high selection presssure conditions, insect
resistance to B. thuringiensis increased rapidly. More recently,
development of resistance against B. thuringiensis is also reported
for the almond moth, Cadra cautella (McGaughey and Bebman, 1988).
Resistance was stable when selection was discontinued and was
inherited as a recessive trait (McGaughey and Beeman, 1988). The
mechanism of insect resistance to B. thuringiensis toxins of Plodia
interpunctella and Cadra cautella has not been elucidated.
[0009] The main cause of B. thuringiensis resistance development in
both reported cases involving grain storage was the environmental
conditions prevailing during the grain storage. Under the
conditions in both cases, the environment was relatively stable, so
B. thuringiensis degradation was slow and permitted successive
generations of the pest to breed in the continuous presence of the
microbial insecticide. The speed at which Plodia developed
resistance to B. thuringiensis in one study suggests that it could
do so within one single storage season in the bins of treated
grain.
[0010] Although insect resistance development against B.
thuringiensis has mostly been observed in laboratory and pilot
scale studies, very recent indications of B. thuringiensis
resistance development in Plutella xylostella populations in the
(cabbage) field have been reported (Kirsch and Schmutterer, 1988).
A number of factors have led to a continuous exposure of P.
xylostella to B. thuringiensis in a relatively small geographic
area. This and the short generation cycle of P. xylostella have
seemingly led to an enormous selection pressure resulting in
decreased susceptibility and increased resistance to B.
thuringiensis.
[0011] A procedure for expressing a B. thuringiensis ICP gene in
plants in order to render the plants insect-resistant (European
patent publication ("EP") 0193259 [which is incorporated herein by
reference]; Vaeck et al., 1987; Barton et al., 1987; Fischhoff et
al., 1987) provides an entirely new approach to insect control in
agriculture which is at the same time safe, environmentally
attractive and cost-effective. An important determinant for the
success of this approach will be whether insects will be able to
develop resistance to B. thuringiensis ICPs expressed in transgenic
plants (Vaeck et al., 1987; Barton et al., 1987; Fischhoff et al.,
1987). In contrast with a foliar application, after which B.
thuringiensis ICPs are rapidly degraded, the transgenic plants will
exert a continuous selection pressure. It is clear from laboratory
selection experiments that a continuous selection pressure has led
to adaptation to B. thuringiensis and its components in several
insect species. In this regard, it should be pointed out that the
conditions in the laboratory which resulted in the development of
insect-resistance to B. thuringiensis are very similar to the
situation with transgenic plants which produce B. thuringiensis
ICPs and provide a continuous selection pressure on insect
populations feeding on the plants. Mathematical models of selection
pressure predict that, if engineered insect-resistant plants become
a permanent part of their environment, resistance development in
insects will emerge rapidly (Gould, 1988). Thus, the chances for
the development of insect resistance to B. thuringiensis in
transgenic plants may be considerably increased as compared to the
field application of B. thuringiensis sprays. A Heliothis virescens
strain has been reported that is 20 times more resistant to B.
thuringiensis HD-1 ICP produced by transgenic Pseudomonas
fluorescens and 6 times more resistant to the pure ICP (Stone et
al., 1989). Furthermore, the monetary and human costs of resistance
are difficult to assess, but loss of pesticide effectiveness
invariably entails increased application frequencies and dosages
and, finally, more expensive replacement compounds as new
pesticides become more difficult to discover and develop.
[0012] Therefore, it would be desirable to develop means for
delaying or even preventing the evolution of resistance to B.
thuringiensis.
[0013] B. thuringiensis strains, active against Lepidoptera
(Dulmage et al., 1981), Diptera (Goldberg and Margalit, 1977) and
Coleoptera (Krieg et al., 1983), have been described. It has become
clear that there is a substantial heterogeneity among ICPs from
different strains active against Lepidoptera, as well as among ICPs
from strains active against Coleoptera (Hofte and Whiteley, 1989).
An overview of the different B. thuringiensis ICP genes, that have
been characterized, is given in Table 2 (which follows the Examples
herein).
[0014] Most of the anti-Lepidopteran B. thuringiensis (e.g., Bt3,
Bt2, Bt73, Bt14, Bt15, Bt4, Bt18) ICP genes encode 130 to 140 kDa
protoxins which dissolve in the alkaline environment of an insect's
midgut and are proteolytically activated into an active toxin of
60-65 kDa. These ICPs are related and can be recognized as members
of the same family based on sequence homologies. The sequence
divergence however is substantial, and the insecticidal spectrum,
among the order Lepidoptera, may be substantially different (Hofte
et al., 1988).
[0015] The P2 toxin gene and the cry B2 gene are different from the
above-mentioned genes in that they do not encode high molecular
weight protoxins but rather toxins of around 70 kDa (Donovan et
al., 1988 and Widner and Whiteley, 1989, respectively).
[0016] It has recently become clear that heterogeneity exists also
in the anti-Coleopteran toxin gene family. Whereas several
previously reported toxin gene sequences from different B.
thuringiensis isolates with anti-Coleopteran activity were
identical (EP 0149162 and 0202739), the sequences and structure of
bt21 and bt22 are substantially divergent (European patent
application ("EPA") 89400428.2).
[0017] While the insecticidal spectra of B. thuringiensis ICPs are
different, the major pathway of their toxic action is believed to
be common. All B. thuringiensis ICPs, for which the mechanism of
action has been studied in any detail, interact with the midgut
epithelium of sensitive species and cause lysis of the epithelial
cells (Knowles and Ellar, 1986) due to the fact that the
permeability characteristics of the brush border membrane and the
osmotic balance over this membrane are perturbed. In the pathway of
toxic action of B. thuringiensis ICPs, the binding of the toxin to
receptor sites on the brush border membrane of these cells is an
important feature (Hofmann et al., 1988b). The toxin binding sites
in the midgut can be regarded as an ICP-receptor since toxin is
bound in a saturable way and with high affinity (Hofmann et al.,
1988a).
[0018] Although this outline of the mode of action of B.
thuringiensis ICPs is generally accepted, it remains a matter of
discussion what the essential determinants) are for the differences
in their insecticidal spectra. Haider et al. (1986) emphasize the
importance of specific proteases in the insect midgut. Hofmann et
al. (1988b) indicate that receptor binding is a prerequisite for
toxic activity and describe that Pieris brassicae has two distinct
receptor populations for two toxins. Other authors have suggested
that differences in the environment of the midgut (e.g., pH of the
midgut) might be crucial.
SUMMARY OF THE INVENTION
[0019] In accordance with this invention, a plant is provided
having, stably integrated into its genome, at least two B.
thuringiensis ICP genes encoding at least two non-competitively
binding insecticidal B. thuringiensis ICPs, preferably the active
toxins thereof, against a specific target insect, preferably
against a Lepidoptera or Coleoptera. Such a plant is characterized
by the simultaneous expression of the at least two
non-competitively binding B. thuringiensis ICPs.
[0020] Also in accordance with this invention, at least two ICP
genes, particularly two genes or parts thereof coding for two
non-competitively binding anti-Lepidopteran or anti-Coleopteran B.
thuringiensis ICPs, are cloned into a plant expression vector.
Plant cells transformed with this vector are characterized by the
simultaneous expression of the at least two B. thuringiensis ICP
genes. The resulting transformed plant cell can be used to produce
a transformed plant in which the plant cells: 1. contain the at
least two B. thuringiensis ICP genes or parts thereof encoding at
least two non-competitively binding anti-Lepidopteran or
anti-Coleopteran B. thuringiensis ICPs as a stable insert into
their genome; and 2. express the genes simultaneously, thereby
conferring on the plant improved resistance to at least one target
species of insect, so as to prevent or delay development of
resistance to B. thuringiensis of the at least one target species
of insect feeding on the transformed plant.
[0021] Further in accordance with this invention, plant expression
vectors are provided which allow integration and simultaneous
expression of at least two B. thuringiensis ICP genes in a plant
cell and which comprise one or more chimeric genes, each containing
in the same transcriptional unit: a promoter which functions in the
plant cell to direct the synthesis of mRNA encoded by one of the
ICP genes; one or more different ICP genes, each encoding a
non-competitively binding B. thuringiensis ICP; preferably a marker
gene; a 3' non-translated DNA sequence which functions in the plant
cell for 3' end formation and the addition of polyadenylate
nucleotides to the 3' end of the mRNA; and optionally a DNA
sequence encoding a protease-sensitive protein part between any two
ICP genes.
DETAILED DESCRIPTION OF THE INVENTION
[0022] Definitions
[0023] As used herein, "B. thuringiensis ICP" (or "ICP") should be
understood as an intact protein or a part thereof which has
insecticidal activity and which can be produced in nature by B.
thuringiensis. An ICP can be a protoxin, as well as an active toxin
or another insecticidal truncated part of a protoxin which need not
be crystalline and which need not be a naturally occurring protein.
In this regard, an ICP can be a chimaeric toxin encoded by the
combination of two variable regions of two different ICP genes as
disclosed in EP 0228838.
[0024] As used herein, "protoxin" should be understood as the
primary translation product of a full-length gene encoding an
ICP.
[0025] As used herein, "toxin", "toxic core" or "active toxin"
should all be understood as a part of a protoxin which can be
obtained by protease (e.g., by trypsin) cleavage and has
insecticidal activity.
[0026] As used herein, "gene" should be understood as a full-length
DNA sequence encoding a protein (e.g., such as is found in nature),
as well as a truncated fragment thereof encoding at least the
active part (i.e., toxin) of the protein encoded by the full-length
DNA sequence, preferably encoding just the active part of the
protein encoded by the full-length DNA sequence. A gene can be
naturally occurring or synthetic.
[0027] As used herein, "truncated B. thuringiensis gene" should be
understood as a fragment of a full-length B. thuringiensis gene
which still encodes at least the toxic part of the B. thuringiensis
ICP, preferentially the toxin.
[0028] As used herein, "marker gene" should be understood as a gene
encoding a selectable marker (e.g., encoding antibiotic resistance)
or a screenable marker (e.g., encoding a gene product which allows
the quantitative analysis of transgenic plants).
[0029] Two ICPs are said to be "competitively binding ICPs" for a
target insect species when one ICP competes for all. ICP receptors
of the other ICP, which receptors are present in the brush border
membrane of the midgut of the target insect species.
[0030] Two ICPs are said to be "non-competitively binding ICPs"
when, for at least one target insect species, the first ICP has at
least one receptor for which the second ICP does not compete and
the second ICP has at least one receptor for which the first ICP
does not compete, which receptors are present in the brush border
membrane of the midgut of the target insect species.
[0031] A "receptor" should be understood as a molecule, to which a
ligand (here a B. thuringiensis ICP, preferably a toxin) can bind
with high affinity (typically a dissociation constant (Kd) between
10.sup.-11 and 10.sup.-6M) and saturability. A determination of
whether two ICPs are competitively or non-competitively binding
ICPs can be made by determining whether: 1. a first ICP competes
for all of the receptors of a second ICP when all the binding sites
of the second ICP with an affinity in the range of about 10.sup.-11
to 10.sup.-6M can be saturated with the first ICP in concentrations
of the first ICP of about 10.sup.-5M or less (e.g., down to about
10.sup.-11M); and 2. the second ICP competes for the all of the
receptors of the first ICP when all the binding sites of the first
ICP with an affinity in the range of about 10.sup.-11 to 10.sup.-6M
can be saturated with the second ICP in concentrations of the
second ICP of about 10.sup.-5M or less.
[0032] General Procedures
[0033] This section describes in broad terms general procedures for
the evaluation and exploitation of at least two B. thuringiensis
ICP genes for prevention of the development, in a target insect, of
a resistance to the B. thuringiensis ICPs expressed in transgenic
plants of this invention. A non-exhaustive list of consecutive
steps in the general procedure follows, after which are described
particular Examples that are based on this methodology and that
illustrate this invention.
[0034] In accordance with this invention, specific B. thuringiensis
ICPs can be isolated in a conventional manner from the respective
strains such as are listed in Table 2 (which follows the Examples).
The ICPs can be used to prepare monoclonal or polyclonal antibodies
specific for these ICPs in a conventional manner (Hofte et al.,
1988).
[0035] The ICP genes can each be isolated from their respective
strains in a conventional manner. Preferably, the ICP genes are
each identified by: digesting total DNA from their respective
strains with suitable restriction enzyme(s); size fractionating the
DNA fragments, so produced, into DNA fractions of 5 to 10 Kb;
ligating such fractions to suitable cloning vectors (e.g.,
pEcoR251, deposited at the Deutsche Sammlung von Mikroorganismen
und Zellculturen ("DSM"), Braunschweig, Federal Republic of
Germany, under accession number no. 4711 on Jul. 13, 1988);
transforming E. coli with the cloning vectors; and screening the
clones with a suitable DNA probe. The DNA probe can be constructed
from a highly conserved region which is commonly present in
different B. thuringiensis genes which encode crystal protoxins
against Coleoptera or Lepidoptera, such as on the basis of an
N-terminal amino acid sequence determined by gas-phase sequencing
of the purified proteins (EPA 88402115.5).
[0036] Alternatively, the desired fragments, prepared from total
DNA of the respective strains, can be ligated in suitable
expression vectors (e.g., a pUC vector (Yanisch-Perron et al.,
1985) with the insert under the control of the lac promoter) and
transformed in E. coli, and the clones can then be screened by
conventional colony immunoprobing methods (French et al., 1986) for
expression of the toxins with monoclonal or polyclonal antibodies
raised against the toxins produced by the strains.
[0037] The isolated B. thuringiensis ICP genes can then be
sequenced in a conventional manner using well-known procedures
(e.g., Maxam and Gilbert, 1980).
[0038] At present, several ICP genes have been cloned from
different subspecies of B. thuringiensis (Table 2). The nucleotide
sequences from several of these B. thuringiensis ICP genes have
been reported. Whereas several sequences are identical or nearly
identical and represent the same gene or slight variants of the
same gene, several sequences display substantial heterogeneity and
show the existence of different B. thuringiensis ICP gene classes.
Several lines of evidence suggest that all these genes specify a
family of related insecticidal proteins. Analysis of the
distribution of B. thuringiensis ICPs in different B. thuringiensis
strains by determining the protein composition of their crystals,
by immunodetection using polyclonal antisera or monoclonals against
purified crystals, or by using gene-specific probes, shows that
subspecies of B. thuringiensis might contain up to three related B.
thuringiensis ICP genes belonging to different classes (Kronstad et
al., 1983).
[0039] To express the isolated and characterized gene in a
heterologous host for purification and characterization of the
recombinant protein, the preferred organism is Escherichia coli. A
number of expression vectors for enhanced expression of
heterologous genes in E. coli have been described (e.g., Remaut et
al., 1981). Usually the gene is cloned under control of a strong
regulatable promoter, such as the lambda pL or pR promoters (e.g.,
Botterman and Zabeau, 1987), the lac promoter (e.g., Fuller, 1982)
or the tac promoter (e.g., De Boer et al., 1983), and provided with
suitable translation initiation sites (e.g., Stanssens et al, 1985
and 1987). Gene cassettes of the B. thuringiensis ICP genes can be
generated by site-directed mutagenesis, for example according to
the procedure described by Stanssens et al. (1985 and 1987). This
allows cassettes to be made comprising, for example, a truncated
ICP gene fragment encoding the toxic core (i.e., toxin) of an ICP
or a hybrid gene encoding the toxic core and a selectable marker
according to the procedures described in EPA 88402241.9.
[0040] The cells of an E. coli culture, which has been induced to
produce a recombinant ICP, are harvested. The method used to induce
the cells to produce the recombinant ICP depends on the choice of
the promoter. For example, the lac promoter (Fuller, 1982) is
induced by isopropyl-B-D-thiogalacto-pyranoside ("IPTG"); the pL
promoter is induced by temperature shock (Bernard et al., 1979).
The recombinant ICP is usually deposited in the cells as insoluble
inclusions (Hsuing and Becker, 1988). The cells are lysed to
liberate the inclusions. The bulk of E. coli proteins is removed in
subsequent washing steps. A semi-purified protoxin pellet is
obtained, from which the protoxin can be dissolved in alkaline
buffer (e.g., Na.sub.2CO.sub.3, pH 10). The procedure for the ICP
Bt2, which is also applicable to other recombinant toxins, has been
described by Hofte et al., 1986.
[0041] In accordance with this invention, the binding of various
ICPs to ICP receptors on the brush border membrane of the columnar
midgut epithelial cells of various insect species has been
investigated. The brush border membrane is the primary target of
each ICP, and membrane vesicles, preferentially derived from the
brush border membrane, can be obtained according to Wolfersberger
et al., 1987.
[0042] The binding to ICP receptors of one or more ICPs (e.g., ICP
A, ICP B, etc.) can be characterized by the following steps
(Hofmann et al, 1988b):
[0043] 1. ICP A is labelled with a suitable marker (usually a
radioisotope such as .sup.125I).
[0044] 2. Brush border membranes are incubated with a small amount
(preferably less than 10.sup.-10 M) of labelled ICP A together with
different concentrations of non-labelled ICP A (preferably from
less than 10.sup.-11 to 10.sup.-5 M).
[0045] 3. For all concentrations tested the amount of labelled ICP
A bound to the brush border membranes is measured.
[0046] 4. Mathematical analysis of these data allows one to
calculate various characteristics of the ICP receptor such as the
magnitude of the population of binding sites (Scatchard, 1949).
[0047] 5. Competition by other toxins (e.g. ICP B) is preferably
studied by incubating the same amount of labelled ICP A with brush
border membranes in combination with different amounts of ICP B
(preferentially from 10.sup.-11 to 10.sup.-6 M; and subsequently,
steps 3 and 4 are repeated.
[0048] By this procedure, it has been found, for example, that Bt3
toxin, Bt2 toxin and Bt73 toxin are competitively binding
anti-Lepidopteran ICPs for Manduca sexta and Heliothis virescens
(See example 6 which follows). Various other combinations of toxins
have been found to be non-competitively binding anti-Lepidopteran
or anti-Coleopteran toxins (example 6).
[0049] Although the concept of competitivity versus
non-competitivity of ICP binding does not have any practical
importance by itself, the observation of the non-competitivity of
two B. thuringiensis ICPs, active against the same target insect,
can be put to very significant practical use. This is because a
combination of two non-competitively binding B. thuringiensis ICPs
can be used to prevent development, by a target insect, of
resistance against such B. thuringienis ICPS.
[0050] A selection experiment with M. sexta, using Bt2 toxin, Bt18
toxin, and a mixture of Bt2 and Bt18 toxins, has shown that Bt2 and
Bt18 are two non-competitively binding anti-Lepidopteran toxins.
After 20 generations of selection, a very pronounced reduction in
ICP sensitivity was observed in the selection experiments with Bt2
or Bt18 alone (>100 times). The reduction in sensitivity in the
selection experiment with a Bt2-Bt18 mixture was only marginal (3
times). This demonstrates the unexpected practical advantage of a
simultaneous use of two non-competitively binding ICPs in a
situation which models the high selection pressure which will exist
with the use of transgenic plants transformed with ICP genes. In
this regard, the two resistant strains showed a specific loss in
receptor sites for either the Bt2 or Bt18 toxin. In each case,
receptor sites for the toxin, which was not used for selection,
were not affected or their concentration even increased. Thus, the
Bt2 selected strain retained its Bt18 receptors, and the Bt18
selected strain developed an increased number of Bt2 receptors.
Indeed, the Bt18 selected strain showed an increased sensitivity
for Bt2 along with its increased Bt2 receptor concentration. No
significant changes in receptor sites were found in the strain
selected against the combined toxins. These findings are described
in detail in Example 7 which follows.
[0051] A similar mechanism of resistance to Bt has been observed
with respect to a strain of diamondback moth, Plutella xylostella.
This strain had developed resistance in the field to Dipel which is
a commercial formulation of the Bt HD-1 strain. Crystals of Dipel
comprise a mixture of several BtICPs, similar to the Bt2, Bt3 and
Bt73 proteins which are competitively-binding ICPs. As shown by
both insect bioassays and competitive binding studies using Bt2 and
Bt15, the Dipel-resistant diamondback moth strain is resistant to
Bt2 protoxin and toxin but maintains full sensitivity to Bt15
protoxin and toxin. This finding is relevant to other combinations
of non-competitively binding anti-Lepidopteran or Coleopteran ICPs
which are expected to have the same beneficial effect against their
common target insects.
[0052] Hence, a combination of non-competitively binding ICPs, when
directly expressed in a transgenic plant, offers the substantial
advantage of reducing the chances of development of insect
resistance against the ICPs expressed in the plant. There may be
additional benefits because the combined spectrum of two toxins may
be broader than the spectrum of a single ICP expressed in a plant
(See Examples 8, 9 and 10 which follow).
[0053] If, among two competitively binding ICPs, one has a larger
binding site population than the other against a given target
insect, it will be most advantageous to use the one with the larger
population of binding sites to control the target pest in
combination with the most suitable non-competitively binding B.
thuringiensis ICP. For example, as seen from Example 6, it is
preferred to use Bt73 against Heliothis virescens, rather than Bt2
or Bt3, and it is preferred to use Bt3 against Manduca sexta rather
than Bt2 or Bt73. The selected gene can then be combined with the
best suitable non-competitively binding ICP.
[0054] Previously, plant transformations involved the introduction
of a marker gene together with a single ICP gene, within the same
plasmid, in the plant genome (e.g., Vaeck et al., 1987; Fischoff et
al., 1987). Such chimeric ICP genes usually comprised either all or
part of an ICP gene, preferably a truncated ICP gene fragment
encoding the toxic core, fused to a selectable marker gene, such as
the neo gene coding for neomycin phosphotransferase. The chimeric
ICP gene was placed between the T-DNA border repeats for
Agrobacterium Ti-plasmid mediated transformation (EP 0193259).
[0055] This invention involves the combined expression of two or
even more B. thuringiensis ICP genes in transgenic plants. The
insecticidally effective B. thuringiensis ICP genes, encoding two
non-competitively binding ICPs for a target insect species,
preferably encoding the respective truncated ICP genes, are
inserted in a plant cell genome, preferably in its nuclear genome,
so that the inserted genes are downstream of, and under the control
of, a promoter which can direct the expression of the genes in the
plant cell. This is preferably accomplished by inserting, in the
plant cell genome, one or more chimaeric genes, each containing in
the same transcriptional unit: at least one ICP gene; preferably a
marker gene; and optionally a DNA sequence encoding a protease
(e.g., trypsin)-sensitive or -cleavable protein part intercalated
in frame between any two ICP genes in the chimaeric gene. Each
chimaeric gene also contains at least one promoter which can direct
expression of its ICP gene in the plant cell.
[0056] The selection of suitable promoters for the chimaeric genes
of this invention is not critical. Preferred promoters for such
chimaeric genes include: the strong constitutive 35S promoter
obtained from the cauliflower mosaic virus, isolates CM 1841
(Gardner et al., 1981), CabbB-S (Franck et al., 1980) and CabbB-JI
(Hull and Howell, 1987); the promoter of the nopaline synthetase
gene ("PNOS") of the Ti-plasmid (Herrera-Estrella, 1983); the
promoter of the octopine synthase gene ("POCS" [De Greve et al.,
1982]); and the wound-inducible 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). Alternatively, a promoter can be
utilized which is specific for one or more tissues or organs of the
plant, whereby the inserted genes are expressed only in cells of
the specific tissue(s) or organ(s). Examples of such promoters are
a stem-specific promoter such as the AdoMet-synthetase promoter
(Peleman et al., 1989), a tuber-specific promoter (Rocha-Sosa et
al., 1989), and a seed-specific promoter such as the 2S promoter
(Krebbers et al., 1988). The ICP genes could also be selectively
expressed in the leaves of a plant (e.g., potato) by placing the
genes under the control of a light-inducible promoter such as the
promoter of the ribulose-1,5-bisphosphate carboxylase small subunit
gene of the plant itself or of another plant such as pea as
disclosed in EP 0193259. Another alternative is to use a promoter
whose expression is inducible (e.g., by temperature or chemical
factors).
[0057] A 3' non-translated DNA sequence, which functions in plant
cells for 3' end formation and the polyadenylation of the 3' end of
the mRNA sequence encoded by the at least one ICP gene in the plant
cell, also forms part of each such chimeric gene. The selection of
a suitable 3' non-translated DNA sequence is not critical. Examples
are the 3' untranslated end of the octopine synthase gene, the
nopaline synthase gene or the T-DNA gene 7 (Velten and Schell,
1985).
[0058] The selection of marker genes for the chimaeric genes of
this invention also is not critical, and any conventional DNA
sequence can be used which encodes a protein or polypeptide which
renders plant cells, expressing the DNA sequence, readily
distinguishable from plant cells not expressing the DNA sequence
(EP 0344029). The marker gene can be under the control of its own
promoter and have its own 3' non-translated DNA sequence as
disclosed above, provided the marker gene is in the same genetic
locus as the ICP gene(s) which it identifies. The marker gene can
be, for example: a herbicide resistance gene such as the sfr or
sfrv genes (EPA 87400141); a gene encoding a modified target enzyme
for a herbicide having a lower affinity for the herbicide than the
natural (non-modified) target enzyme, such as a modified 5-EPSP as
a target for glyphosate (U.S. Pat. No. 4,535,060; EP 0218571) or a
modified glutamine synthetase as a target for a glutamine
synthetase inhibitor (EP 0240972); or an antibiotic resistance
gene, such as a neo gene (PCT publication WO 84/02913; EP
0193259).
[0059] Using A. tumefaciens Ti vector-mediated plant transformation
methodology, all chimeric genes of this invention can be inserted
into plant cell genomes after the chimaeric genes have been placed
between the T-DNA border repeats of suitable disarmed Ti-plasmid
vectors (Deblaere et al., 1988). This transformation can be carried
out in a conventional manner, for example as described in EP
0116718, PCT publication WO 84/02913 and EPA 87400544.0. The
chimeric genes can also be in non-specific plasmid vectors which
can be used for direct gene transfer (e.g., as described by
Pazkowski et al., 1984; De La Pena et al., 1986). Different
conventional procedures can be followed to obtain a combined
expression of two B.thuringiensis ICP genes in transgenic plants as
summarized below.
[0060] I Chimeric Gene Constructs Whereby Two or More ICP Genes and
a Marker Gene are Transferred to the Plant Genome as a Single Piece
of DNA and Lead to the Insertion in a Single Locus in the
Genome
[0061] Ia The Genes Can Be Engineered in Different Transcriptional
Units Each Under Control of a Distinct Promoter
[0062] To express two or more ICP genes and a marker gene as
separate transcriptional units, several promoter fragments
directing expression in plant cells can be used as described above.
All combinations of the promoters mentioned above in the chimaeric
constructs for one ICP gene are possible. Examples of such
individual chimeric constructs are described for the bt2 gene in EP
0193259, for the bt13 gene in EPA 88402115.5 and for the bt18 gene
in EPA 88402241.9. The ICP gene in each chimeric gene of this
invention can be the intact ICP gene or preferably an
insecticidally-effective part of the intact ICP gene, especially a
truncated gene fragment encoding the toxic core of the ICP. The
individual chimeric genes are cloned in the same plasmid vector
according to standard procedures (e.g., EP 0193259).
[0063] Ib Two Genes (e.g., Either an ICP and a Marker Gene or Two
ICP Genes) or More can be Combined in the Same Transcriptional
Unit.
[0064] To express two or more ICP genes in the same transcriptional
unit, the following cases can be distinguished:
[0065] In a first case, hybrid genes in which the coding region of
one gene is in frame fused with the coding region of another gene
can be placed under the control of a single promoter. Fusions can
be made between either an ICP and a marker gene or between two ICP
genes. An example of an ICP gene-marker gene fusion has been
described in EP 0193259. (i.e., a hybrid truncated bt2-neo gene
encoding a Bt2 toxin-NPTII fusion protein).
[0066] Another possibility is the fusion of two ICP genes. Between
each gene encoding an ICP which still is insecticidally active
(i.e., a toxic part of the protoxin), a gene fragment encoding a
protease (e.g., trypsin)--sensitive protein part should be
included, such as a gene fragment encoding a part of the N-terminal
or C-terminal amino acid sequence of one of the ICPs which is
removed or cleaved upon activation by the midgut enzymes of the
target insect species.
[0067] In a second case, the coding regions of the two respective
ICP genes can be combined in dicistronic units placed under the
control of a promoter. The coding regions of the two ICP genes are
placed after each other with an intergenic sequence of defined
length. A single messenger RNA molecule is generated, leading to
the translation into two separate gene products. Based on a
modified scanning model (Kozak, 1987), the concept of reinitiation
of translation has been accepted provided that a termination codon
in frame with the upstream ATG precedes the downstream ATG.
Experimental data also demonstrated that the plant translational
machinery is able to synthesize several polypeptides from a
polycistronic mRNA (Angenon et al., 1989).
[0068] II Chimeric Constructs With One or More ICP Genes That Are
Transferred to the Genome of a Plant Already Transformed With a One
or More ICP Genes
[0069] Several genes can be introduced into a plant cell during
sequential transformation steps (retransformation), provided that
an alternative system to select transformants is available for the
second round of transformation. This retransformation leads to the
combined expression of ICP genes which are introduced at multiple
loci in the genome. Preferably, two different selectable marker
genes are used in the two consecutive transformation steps. The
first marker is used for selection of transformed cells in the
first transformation, while the second marker is used for selection
of transformants in the second round of transformation. Sequential
transformation steps using kanamycin and hygromycin have been
described, for example by Sandler et al. (1988) and Delauney et al.
(1988).
[0070] III Chimeric Constructs With One or More ICP Genes, That Are
Separately Transferred to the Nuclear Genome of Separate Plants in
Independent Transformation Events and are Subsequently Combined in
a Single Plant Genome Through Crosses.
[0071] The first plant should be a plant transformed with a first
ICP gene or an F1 plant derived herefrom through selfing
(preferably an F1 plant which is homozygous for the ICP gene). The
second plant should be a plant transformed with a second ICP gene
or an F1 plant derived herefrom through selfing (preferably an F1
plant which is homozygous for the second ICP gene). Selection
methods can be applied to the plants obtained from this cross in
order to select those plants having the two ICP genes present in
their genome (e.g., Southern blotting) and expressing the two ICPs
(e.g., separate ELISA detection of the immunologically different
ICPs). This is a useful strategy to produce hybrid varieties from
two parental lines, each transformed with a different ICP gene, as
well as to produce inbred lines containing two different ICP genes
through crossing of two independent transformants (or their F1
selfed offspring) from the same inbred line.
[0072] IV Chimeric Constructs With One or More ICP Genes Separately
Transferred to the Genome of a Single Plant in the Same
Transformation Experiment Leading to the Insertion of the
Respective Chimeric Genes at Multiple Loci.
[0073] Cotransformation involves the simultaneous transformation of
a plant with two different expression vectors, one containing a
first ICP gene, the second containing a second ICP gene. Along with
each ICP gene, a different marker gene can be used, and selection
can be made with the two markers simultaneously. Alternatively, a
single marker can be used, and a sufficiently large number of
selected plants can be screened in order to find those plants
having the two ICP genes (e.g., by Southern blotting) and
expressing the two proteins (e.g., by means of ELISA).
Cotransformation with more than one T-DNA can be accomplished by
using simultaneously two different strains of Agrobacterium, each
with a different Ti-plasmid (Depicker et al., 1985) or with one
strain of Agrobacterium containing two T-DNAs on separate plasmids
(de Framond et al., 1986). Direct gene transfer, using a mixture of
two plasmids, can also be employed to cotransform plant cells with
a selectable and a non-selectable gene (Schocher et al., 1986).
[0074] The transgenic plant obtained can be used in further plant
breeding schemes. The transformed plant can be selfed to obtain a
plant which is homozygous for the inserted genes. If the plant is
an inbred line, this homozygous plant can be used to produce seeds
directly or as a parental line for a hybrid variety. The gene can
also be crossed into open pollinated populations or other inbred
lines of the same plant using conventional plant breeding
approaches.
[0075] Of course other plant transformation methods can be used and
are within the scope of the invention as long as they result is a
plant which expresses two or more non-competitively binding ICPs.
In this regard, this invention is not limited to the use of
Agrobacterium Ti-plasmids for transforming plant cells with genes
encoding non-competitively binding ICPs. Other known methods for
plant cell transformations, such as electroporation or by the use
of a vector system based on plant viruses or pollen, can be used
for transforming monocotyledonous and dicotyledonous plants in
order to obtain plants which express two non-competitively binding
ICPs. Furthermore, DNA sequences encoding two non-competitively
binding ICPs other than those disclosed herein can be used for
transforming plants. Also, each of the ICP genes, described herein,
can be encoded by equivalent DNA sequences, taking into
consideration the degeneracy of the genetic code. Also, equivalent
ICPs with only a few amino acids changed, such as would be obtained
through mutations in the ICP gene, can also be used, provided they
encode a protein with essentially the same characteristics (e.g.,
insecticidal activity and receptor binding).
[0076] The following Examples illustrate the invention. Those
skilled in the art will, however, recognize that other combinations
of two or more non-competitively binding B. thuringiensis ICP genes
can be used to transform plants in accordance with this invention
in order to prevent the development, in a target insect, of
resistance to B. thuringiensis ICPs expressed in the transformed
plants. Unless otherwise indicated, all procedures for making and
manipulating DNA were carried out by the standardized procedures
described in Maniatis et al, Molecular Cloning--A Laboratory
Manual, Cold Spring Harbor Laboratory (1982).
EXAMPLE 1
Collection of Genes
[0077] The collection of anti-Lepidopteran and anti-Coleopteran Bt
genes encoding ICPs, which are the subject of the Examples, is
described in Table 2 (following the Examples). References for the
respective genes are indicated in Table 2. The origin, the
isolation and characterization of the Bt genes, which have not been
published, are described below. Bt strains, such as strains HD-1,
HD-68, HD-110, and HD-73, are publicly available from the
Agricultural Research Culture Collection, Northern Regional
Research Laboratory, U.S. Dept. of Agriculture, Peoria, Ill. 61604,
U.S.A.
[0078] bt3
[0079] gene: From B. thuringiensis var. kurstaki HD-1, the ICP was
cloned. Characterization of this gene revealed an open reading
frame of 3528 bp which encodes a protoxin of 133 kDa. This gene was
identical to the one described by Schnepf et al. (1985).
[0080] bt73
[0081] gene: From B. thuringiensis var HD-73. The ICP gene was
cloned as described by Adang et al. (1985).
[0082] bt4
[0083] gene: A genomic library was prepared from total DNA of
strain B. thuringiensis aizawai HD-68. Using the 1.1 kb internal
HindIII fragment of the bt2 gene as a probe, a gene designated bt4
was isolated. Characterization of this gene revealed an open
reading frame of 3495 bp which encodes a protoxin of 132 kDa and a
trypsin activated toxin fragment of 60 kDa. This (insect
controlling protein) gene differs from previously identified genes
and was also found in several other strains of subspecies aizawai
and entomocidus including HD-110. FIG. 13 shows the nucleotide
sequence and deduced amino acid sequence of the open reading frame
("ORF") of the bt4 gene extending from nucleotide 264 to nucleotide
3761.
[0084] bt14 and bt15
[0085] genes: A genomic library was prepared from total DNA of
strain B. thuringiensis var. entomocidus HD-110 by partial Sau3A
digest of the total DNA and cloning in the vector pEcoR251
(deposited at DSM under accession number 4711). Using monoclonal
antibodies (Hofte et al., 1988), at least three structurally
distinct ICPs were identified in crystals of B. thuringiensis
entomocidus HD-110. These monoclonal antibodies were used to clone
the three different ICP genes from this B. thuringiensis strain.
One of these genes is the bt4 gene as described above.
[0086] The second gene was called "bt15". FIG. 14 shows the
nucleotide sequence and deduced amino acid sequence of the ORF of
the bt15 gene, isolated from HD-110, extending from nucleotide 234
to nucleotide 3803. The Shine and Dalgarno sequence, preceding the
initiation codon is underlined. This gene has an open reading frame
of 3567 bp which encodes a protoxin of 135 kDa and a 63 kDa toxin
fragment. A similar gene has been described by Honee et al. 1988,
isolated from B. thuringiensis entomocidus 60.5. The bt15 gene
differs from the published sequence at three positions: an Ala
codon (GCA) is present instead of an Arg codon (CGA) at position
925 and a consecution of a Thr-His codon (ACGCAT) is present
instead of a Thr-Asp codon (ACCGAT) at position 1400. (The numbers
of the positions are according to Honnee et al., 1988). Another
similar gene has been described in EP 0295156, isolated from B.
thuringiensis aizawai 7-29 and entomocidus 6-01. The bt15 gene is
different from this published nucleotide sequence at three
different places : 1) a Glu codon (GAA) instead of an Ala codon
(GCA) at position 700; 2) the sequence TGG, CCA, GCG, CCA instead
of TGC, CAG, CGC, CAC, CAT at position 1456 and 3) an Arg codon
(CGT) instead of an Ala codon (GCG) at position 2654. (The numbers
of the positions are according to EP 0295156).
[0087] The third gene isolated was called "bt14". It has an open
reading frame of 3621 bp which encodes a 137 kDa protoxin and a 66
kDa activated toxin fragment. A similar gene has been cloned from
B. thuringiensis HD-2 (Brizzard and Whiteley, 1988). The bt14 gene
differs from the published nucleotide sequence by two nucleotide
substitutions: a T instead of a C at position 126, and a C instead
of a T at position 448 (the numbers of the positions are according
to Brizzard and Whiteley, 1988). In the first case, the Ile codon
(ATT or ATC) is conserved whereas in the second case the Tyr codon
(TAT) is converted to a His codon (CAC).
[0088] bt2
[0089] gene: The bt2 gene was cloned as described in EP
0193259.
[0090] bt18
[0091] gene: Cloning of the bt18 gene was performed as described in
EPA 88402241.9.
[0092] bt13
[0093] gene: The bt13 gene was cloned as described in EPA
88402115.5.
[0094] bt21 and bt22
[0095] genes: These genes, encoding Coleopteran-active ICPs, were
cloned as described in EPA 89400428.2.
EXAMPLE 2
Construction of Gene Cassettes and Expression of Bt Genes In E.
coli
[0096] 1) bt2, bt18: the construction of bt2 and bt18 gene
cassettes has been previously described in EPA 86300291.1 and
88402241.9, respectively. Basically, they comprise a truncated gene
encoding the toxic core and a hybrid gene comprising the truncated
gene fused in frame to the N-terminus of the neo gene. The gene
cassettes are used to transform E. coli to express the Bt2 and Bt18
ICP toxins.
[0097] 2) bt14, bt15: as described in EPA 88402241.9, gene
cassettes for the bt14 and bt15 genes were constructed in order to
express the genes in E. coli and in plants.
[0098] First, a NcoI site was introduced at the N-terminus of the
genes by site-directed mutagenesis.
[0099] In the case of the bt15 gene, the conversion of the TT
nucleotides, immediately in front of the ATG codon, into CC yielded
a NcoI site overlapping with the ATG initiation codon. This site
was introduced using the pMa/c vectors for site-directed
mutagenesis (Stanssens et al., 1987) and a 28-mer oligonucleotide
with the following sequence:
[0100] 5'-CGGAGGTATTCCATGGAGGAAAATAATC-3'.
[0101] This yielded the plasmid pVE29 carrying the N-terminal
fragment of the bt15 gene with a NcoI site at the ATG initiation
codon.
[0102] According to Brizzard and Whiteley (1988), the initiation
codon of the bt14 gene is a TTG codon. Thus, a NcoI site was
created in a like manner at this codon for site directed
mutagenesis using a 34-mer oligonucleotide with the following
sequence:
[0103] 5'-CCTATTTGAAGCCATGGTAACTCCTCCTTTTATG-3'.
[0104] In this case the sequence of the intitiation codon was
converted from ATATTGA to ACCATGG. This yielded the plasmid pHW44
carrying the N-terminal fragment of the bt14 gene with a NcoI site
at the initiation codon.
[0105] In a second step, the genes were reconstructed by ligating
the N-terminal gene fragments with a suitable C-terminal gene
fragment, yielding a bt15 gene and bt14 gene with a NcoI site at
the ATG initiation codon.
[0106] To express the bt14 and bt15 genes encoding the protoxin in
E. coli, the following constructs were made: pOH50 containing the
bt15 gene under the control of the lac promoter; and pHW67
containing the bt14 gene under the control of the tac promoter.
Induction of a culture of the E. coli strain WK6 carrying the
respective plasmids with IPTG yielded an overproduced protein
(Fuller, 1982).
[0107] The active toxic fragments of the Bt15 and Bt14 protoxins
comprise 63 and 60 kDa trypsin digest products respectively.
Instead of expressing the whole bt15 or bt14 gene, it is also
possible to express a toxin-encoding gene fragment or derivative
thereof in plants. To this end, truncated bt14 and bt15 gene
fragments were constructed. In order to be able to select
transgenic plants producing the ICP gene products, hybrid genes of
the truncated gene fragments were also made with the neo gene
encoding a selectable marker as described in EP 0193259.
[0108] By comparison of the nucleotide sequence of the bt4, bt14
and bt15 genes, respectively, with the bt2 and bt18 genes,
respectively, the BclI site could be identified as a suitable site
localized downstream of the coding sequence encoding the toxin gene
fragment. To construct a truncated gene fragment and a hybrid gene
of the truncated gene fragment with the neo gene, the filled BclI
site was ligated to the filled EcoRI site of pLKM91 (Hofte et al.,
1986) and the filled HindIII site of pLK94 respectively (Botterman
and Zabeau, 1987). pLKM91 carries a 5' truncated neo gene fragment
which codes for an enzymatically active C-terminal gene fragment of
the neo gene, and pLK94 contains translation stop codons in three
reading frames. This yielded the following plasmids which are then
used to transform E. coli to express the ICP genes: pHW71 carrying
a truncated bt14-neo hybrid gene; pHW72 carrying a truncated bt14
gene; pVE34 carrying a truncated bt15-neo hybrid gene; and pVE35
carrying a truncated bt15 gene.
[0109] In a similar way as described for the bt14 and bt15 genes,
gene cassettes are constructed for the bt3 and bt4 genes which are
then expressed in E. coli.
EXAMPLE 3
Purification of Recombiant ICPs
[0110] The ICPs expressed in E. coli in Example 2 are purified by
the method (described for recombinant Bt2 protoxin) by Hofte et al.
(1986).
EXAMPLE 4
Purification of Toxins
[0111] Solubilized protoxins of Bt2, Bt3, Bt73, Bt4, Bt14, Bt15,
Bt18, Bt13, Bt21and Bt22 (in Na.sub.2CO.sub.3 50 mM, DTT 10 mM
pH=10) are dialyzed against 0.5% (NH.sub.4).sub.2CO.sub.3 at pH 8
and treated with trypsin (trypsin/protoxin=1/20 w/w) for 2 h at
37.degree. C. The activated toxin is chromatographically purified
(Mono-Q column on FPLC) as described by Hofmann et al.(1988b).
EXAMPLE 5
Determination of the Insecticidal Spectrum
[0112] The ICP protoxins and toxins of Examples 3 and 4 are
evaluated for their insecticidal activity. Each protoxin is
dissolved in alkaline buffer containing a reducing agent
(Na.sub.2CO.sub.3 50 mM, DTT 10 mM pH=10), and each toxin is used
as soluble protein directly from FPLC. Protein concentrations are
determined. Subsequently, dilutions of the resulting protoxin or
toxin solution are prepared in PBS buffer pH=7.4 containing 0.15M
NaCl and 0.1% bovine serum albumin ("BSA").
[0113] The artificial medium for insect culture, described by Bell
and Joachim (1976) for Manduca sexta, is poured in appropriate
receptacles and allowed to solidify. Subsequently a quantity of the
(pro)toxin dilutions is applied on this medium, and the water is
allowed to evaporate under a laminar flow. This results in a medium
with a certain quantity (in the range of 0.1 to 10000 ng/cm2) of
toxin coated on its surface. For example, for the Bt2 toxin,
typical dilutions for a toxicity test on Manduca sexta are 1, 5,
25, 125 and 625 ng/cm2. First instar larvae of Manduca sexta are
then applied on the coated medium, and growth and mortality are
assessed after 6 days. Mortality increases with dosage. Dose
response data is analysed in probit analysis (Finney, 1962), and
the data are best summarized by an LD.sub.50 value which is the
amount of toxin which kills 50% of the insects. The LD.sub.50 for
Bt2 toxin against Manduca sexta is around 20 ng/cm2.
[0114] Similar assays are carried out for other insect species
using a suitable diet or by applying the ICPs on leaves for
insects, for which no artificial diet is used.
EXAMPLE 6
Binding Studies
[0115] Toxins
[0116] All protoxins and their toxic fragments were purified
according to the methods described for the Bt2 protoxin and toxin
in Hofte et al. (1986) and EP 0193259. The activated and purified
toxins are further referred to as the Bt2, Bt3, Bt73, Bt4, Bt14,
Bt15, Bt18, Bt13, Bt21 and Bt22 toxins.
[0117] By way of example for the Bt73 toxin, it has been shown that
B. thuringiensis var. kurstaki HD73 produces a protein of 133 kDa
encoded by a 6.6 kb type gene. A culture of this strain was grown
as described by Mahillon and Delcour (1984). The autolysed culture
was spun down (20 minutes at 4500 rpm in a HB4 rotor) and washed
with a buffer containing 20 mM Tris, 100 mM NaCl and 0.05% Triton
X-100, pH 8. The final pellet was resuspended in this buffer (4 ml
buffer for 100 ml culture). This solution was then layered onto a
linear Urograffin gradient (60-70%) which was centrifuged in a SW
28 rotor for 90 minutes at 18000 rpm. Crystals were collected and
stored at -20.degree. C. until further use. Activation was
performed according to Hofte et al. (1986). The purified toxin is
further referred to as the Bt73 toxin.
[0118] Iodination of ICPs
[0119] Iodination of Bt2, Bt3, and Bt73 toxins was performed using
the Chloramin-T method (Hunter and Greenwood, 1962). 1 mCi
.sup.125I-NaI and 20 to 37.5 ug Chloramin-T in NaCl/P.sub.i were
added to 50 ug of purified toxin. After gentle shaking for 60
seconds, the reaction was stopped by adding 53 ug of potassium
metabisulfite in H.sub.2O. The whole mixture was loaded on a PD 10
Sephadex G-25M gelfiltration column to remove free iodine. A
subsequent run on a Biogel P-60 column was carried out in order to
increase the purity.
[0120] Alternatively, toxins were labeled using the Iodogen method.
Iodogen (Pierce) was dissolved in chloroform at 0.1 mg/ml. 100 ul
of this solution was pipetted into a disposable glass vessel and
dried under a stream of nitrogen gas. The vessel was rinsed with
Tris buffer (20 mM Tris, pH 8.65 with 0.15M NaCl). 50 ug of toxin
(in Tris buffer) was incubated with 1 mCi of .sup.125I-NaI in the
tube for 10 minutes. The reaction was then stopped by the addition
of 1 M NaI (one fourth of the sample volume). The sample was
immediately loaded onto a PD10 Sephadex G-25M column and later on a
Biogel P-60 column to remove free iodine and possible degradation
products.
[0121] Other toxins were iodinated using one of the above mentioned
procedures.
[0122] Determination of Specific Activity of Iodinated Toxin
[0123] Specific activity of iodinated Bt2, Bt3, and Bt73 toxin
samples was determined using a "sandwich" ELISA technique according
to Voller, Bidwell and Barlett (1976). Primary antibody was a
polyclonal antiserum raised against Bt2 toxin, and the secondary
antibody was a monoclonal antibody 4D6.
[0124] The conjugate used was alkaline phosphatase coupled to
anti-mouse IgG. The reaction intensity of a standard dilution
series of unlabeled toxin and dilutions of the iodinated toxin
sample (in NaCl/P.sub.i--0.1% BSA) was measured. Linear regression
calculations yielded the protein content of the radioactive toxin
sample. The samples with the highest specific activities were used
in the binding assays. Specific activities were 59400, 33000 and
19800 Ci/mole (on reference date) for Bt73 toxin (labeled according
to Iodogen procedure), Bt2 toxin (Chloramin-T method) and Bt3 toxin
(Iodogen method) respectively.
[0125] Specific activities of other toxins were determined using a
similar approach. Specific monoclonal and polyclonal antibodies for
each of these toxins were raised and applied in ELISA.
[0126] Preparation of Brush Border Membrane Vesicles
[0127] Brush border membrane vesicles ("BBMV") from Manduca sexta,
Heliothis virescens, Plutella xylostella, Phthorimaea operculella,
Spodoptera exigua, Spodoptera littoralis, Plodia interpunctella,
Mamestra brassicae, Pieris brassicae and Leptinotarsa decemlineata
were prepared according to the method of Wolfersberger et al.
(1987). This is a differential centrifugation method that makes use
of the higher density of negative electrostatic charges on luminal
than on basolateral membranes to separate these fractions.
[0128] Binding Assay
[0129] Duplicate samples of .sup.125I-labeled toxin, either alone
or in combination with varying amounts of unlabeled toxin, were
incubated at the appropriate temperature with brush border membrane
vesicles in a total volume of 100 ul of Tris buffer (Tris 10 mM,
150 nM NaCl, pH 7.4). All buffers contained 0.1% BSA. The
incubation temperature was 20 C. Ultrafiltration through Whatman
GF/F glass fiber filters was used to separate bound from free
toxin. Each filter was rapidly washed with 5 ml of ice-cold buffer
(NaCl/P.sub.i--0.1% BSA). The radioactivity of the filter was
measured in a gammacounter (1275 Minigamma, LKB). Binding data were
analyzed using the LIGAND computer program. This program calculates
the bound concentration of ligand as a function of the total
concentration of ligand, given the affinity (Ka or its inverse
Kd=1/Ka, the dissociation constant) and the total concentration of
receptors or binding site concentration (R.sub.t).
[0130] Determination of Protein Concentration
[0131] Protein concentrations of purified Bt2, Bt3, Bt73 and Bt15
toxins were calculated from the OD at 280 nm (measured with a
Uvikon 810 P, Kontron Instruments spectrofotometer). The protein
content of solutions of other toxins and of brush border membrane
vesicles (BBMV) as measured according to Bradford (1976).
[0132] Binding of Bt2, Bt3 and Bt73 Toxins to BBMV of Manduca sexta
and Heliothis virescens: an Example of 3 Competitively Binding
Lepidopteran ICPs.
[0133] Bt2, Bt3 and Bt73 toxins are toxic to both Manduca sexta and
Heliothis virescens: LC50 values for Manduca sexta are respectively
17.70, 20.20 and 9.00 ng/cm2; for Heliothis virescens the LC50's
are 7.16, 90.00 and 1.60 ng/cm2.
[0134] Labelled toxin, either Bt3 (0.8 nM) or Bt2 (1.05 nM) or Bt73
(1.05 nM), was incubated with BBMV in a volume of 0.1 ml. BBMV
protein concentrations were 100 ug/ml for M. sexta and for Bt2-H.
virescens, for Bt3-H. virescens 150 and for Bt73-H. virescens 50
ug/ml. The labelled toxin was combined with varying amounts of an
unlabeled toxin (competitor). After a 30 min. incubation, bound and
free toxins were separated through filtration.
[0135] FIGS. 1-3 show the percentages binding of respectively
labelled Bt2, Bt3 and Bt73 toxins as a function of the
concentration of competitor for Manduca sexta. FIGS. 4-6 show these
data for Heliothis virescens. The amount bound in the absence of
competitor is always taken as 100% binding. FIGS. 1-6 show the
binding of .sup.125I-labeled toxins to M. sexta (in FIGS. 1, 2 and
3) and H. virescens (in FIGS. 4, 5 and 6) brush border membrane
vesicles. Vesicles were incubated with labeled toxin [in FIGS. 1
and 4: .sup.125I-Bt2-toxin (1.05 nM); in FIGS. 2 and 5:
.sup.125I-Bt3-toxin (0.8 nM); in FIGS. 3 and 6:
.sup.125I-Bt73-toxin (1.05 nM)] in the presence of increasing
concentrations of Bt2 toxin (*), Bt3 toxin (.circle-solid.) or Bt73
toxin (.tangle-solidup.). Binding is expressed as percentage of the
amount bound upon incubation with labeled toxin alone. On M. sexta
vesicles, these amounts were 1820, 601 and 2383 cpm, and on H.
virescens vesicles 1775, 472 and 6608 cpm for .sup.125I-Bt2-, Bt3-
and Bt73-toxin, respectively. Non-specific binding was not
substracted. Data were analyzed with the LIGAND computer program.
Each point is the mean of a duplicate sample.
[0136] FIG. 1: shows the binding of .sup.125I Bt2 toxin to M. sexta
BBMV
[0137] FIG. 2: shows the binding of .sup.125I Bt3 toxin to M. sexta
BBMV
[0138] FIG. 3: shows the binding of .sup.125I Bt73 toxin to M.
sexta BBMV
[0139] FIG. 4: shows the binding of .sup.125I Bt2 toxin to H.
virescens BBMV
[0140] FIG. 5: shows the binding of .sup.125I Bt3 toxin to H.
virescens BBMV
[0141] FIG. 6: shows the binding of .sup.125I Bt73 toxin to H.
virescens BBMV
[0142] The conclusions from FIGS. 1-6 are that Bt2 and Bt3, Bt3 and
Bt73, and Bt2 and Bt73 are competitively-binding ICP's both for
Manduca sexta and for Heliothis virescens. Indeed Bt3 competes for
the entire population of receptor sites of Bt2. in Manduca sexta
(FIG. 1): the % labelled Bt2 bound in the presence of 100 nM Bt3 is
equal to the % Bt2 bound with 100 nM of Bt2 itself. The opposite is
not true: in the presence of 100 nM Bt2 the % of labelled Bt3 is
not reduced to the same level as with 100 nM of Bt3 (FIG. 2).
[0143] A similar reasoning is followed to observe competitivity of
other toxin combinations: Bt3 competes for the entire population of
receptor sites of Bt73 (FIG. 3) in M. sexta; the opposite is not
true (FIG. 2); Bt2 and Bt73 compete for the entire population of
each other's binding sites in M. sexta (FIGS. 1 and 3).
[0144] In Heliothis virescens: Bt2 competes for the entire
population of receptor sites of Bt3 (FIG. 5); Bt73 competes for the
entire population of receptor sites of Bt3 (FIG. 5); Bt73 competes
for the entire population of receptor sites of Bt2 (FIG. 4); but
the opposite statements are not true (FIGS. 4, 5 and 6).
[0145] The same data can be used in mathematical analysis (e.g.,
Scatchard analysis according to Scatchard, 1949; analysis with the
LIGAND computer program according to Munson and Rodbard, 1980) to
calculate the dissociation constant (Kd) of the toxin-receptor
complex and the concentration of binding sites (Rt); the results of
these calculations using the LIGAND computer program were the
following:
1 Bt2-M. sexta: Kd = 0.4 nM Rt = 3.4 pmol/mg vesicle protein Bt3-M.
sexta: Kd = 1.5 nM Rt = 9.8 pmol/mg vesicle protein Bt73-M. sexta:
Kd = 0.6 nM Rt = 4.0 pmol/mg vesicle protein Bt2-H. virescens: Kd =
0.6 nM Rt = 9.7 pmol/mg vesicle protein Bt3-H. virescens: Kd = 1.2
nM Rt = 3.7 pmol/mg vesicle protein Bt73-H. virescens: Kd = 0.8 nM
Rt = 19.5 pmol/mg vesicle protein
[0146] These data demonstrate the high affinity receptor binding of
the toxins (Kds in the range of 10.sup.-10 to 10.sup.-9 M.
[0147] Binding of Bt2 and Bt14 Toxins to BBMV of P. brassicae,
Plutella xylostella and Phthorimaea opercullella: an Example Two
Non-Competitively Binding Lepidopteran ICPs
[0148] Bt2 and Bt14 toxins are toxic to P. brassicae (p.b.), P.
xylostella (p.x.) and P. operculella (p.o.) as seen from the table
below.
2 LC.sub.50 of Toxins Bt2 Bt14 P.b. 1.3 2.0 P.x. 6.7 5.4 P.o. 4.20
0.8-4.0
[0149] LC.sub.50 values of solubilized purified Bt2 and Bt14 toxins
for P.x. are expressed as ng protein spotted per cm.sup.2 of
artificial diet. LC.sub.50 values for P.b. are expressed as
ug.sup.2 toxin per ml solution into which leaf discs, fed to first
instar Pb larvae, were dipped. For P.o., LC.sub.50 values are
expressed in ug/ml into which potato chips were dipped prior to
feeding.
[0150] Labelled Bt2 toxin (1.05 nM) or Bt14 toxin (1.4 nM) was
incubated with BBMV from P. brassicae (100 ug protein/ml) in a
volume of 0.1 ml in combination with varying amounts of unlabelled
Bt2 or Bt14. After a 30 min. incubation period at 22.degree. C.,
the bound and free toxins were separated.
[0151] FIGS. 7 and 8 show the binding of .sup.125I-labeled toxins
to P. brassicae brush border membrane vesicles. Vesicles were
incubated with labeled toxin [in FIG. 7: .sup.125I-Bt2-toxin (1.05
nM); in FIG. 8: .sup.125I-Bt14-toxin (1.4 nM)] in the presence of
increasing concentrations of Bt2 toxin (.smallcircle.) or Bt14
toxin (.circle-solid.). Binding is expressed as percentage of the
amount bound upon incubation with labeled toxin alone. Non-specific
binding was not substracted. Data were analyzed with the LIGAND
computer program. Each point is the mean of a duplicate sample.
FIG. 7 shows the binding of labelled Bt2 toxin to P. brassicae
BBMV, and FIG. 8 shows the binding of labelled Bt14 toxin to P.
brassicae BBMV.
[0152] The competition data demonstrate the presence of high
affinity binding sites both for Bt2 and Bt14, as well as the almost
complete absence of competition of Bt14 for the Bt2 binding sites
and of Bt14 for the Bt2 binding sites. This demonstrates that Bt2
and Bt14 are non-competitively binding toxins. Hence they are
useful to prevent the development of Pieris brassicae resistance
against B. thuringiensis ICP's expressed in Brassica sp.
[0153] Calculated Kd and Rt values were from these experiments
were:
[0154] Bt2: Kd=2.8 nM, Rt=12.9 pmol/mg vesicle protein
[0155] Bt14: Kd=8.4 nM, Rt=21.4 pmol/mg vesicle protein.
[0156] Binding of Bt2 and Bt15 toxins to BBMV of M. sexta, M.
brassicae, P. xylostella and P. interpunctella: an Example of Two
Non-Competitively Binding Lepidopteran ICPs
[0157] Bt2 and Bt15 toxins are both toxic to M. sexta (LC50's of 20
and 111 ng/cm2, respectively). They also show activity against M.
brassicae, P. xylostella and P. interpunctella.
[0158] Labelled Bt2 (1.05 nM) or Bt15 (0.7 nM) was incubated with
BBMV from M. sexta (100 ug protein/ ml) in a volume of 0.1 ml in
combination with varying amounts of unlabelled Bt2 or Bt15. After a
30 min. incubation period at 22.degree. C., the bound and free
toxins were separated.
[0159] FIGS. 9-10 show the binding of .sup.125I-labeled toxins to
M. sexta brush border membrane vesicles. Vesicles were incubated
with labeled toxin [in FIG. 9: .sup.125I-Bt2-toxin (1.05 nM); in
FIG. 10: .sup.125I-Bt15-toxin (0.7 nM)] in the presence of
increasing concentrations of Bt2-toxin (.smallcircle.) or
Bt15-toxin (.circle-solid.). Binding is expressed as percentage of
the amount bound upon incubation with labeled toxin alone.
Non-specific binding was not substracted. Data were analyzed with
the LIGAND computer program. Each point is the mean of a duplicate
sample. FIG. 9 shows the data for binding of labelled Bt2 and FIG.
10 shows the binding of labelled Bt15.
[0160] The competition data demonstrate the presence of high
affinity binding sites for both Bt2 and Bt15, as well as the
complete absence of competition of Bt15 for the Bt2 binding sites
and of Bt2 for the Bt15 binding sites. This demonstrates that Bt2
and Bt15 are non-competitively binding toxins. Hence the
combination of Bt2 and Bt15 is useful to prevent the development of
resistance of M. sexta against B. thuringiensis ICP's expressed in
tobacco or other crops in which Manduca sp. are a pest. Calculated
Kd and Rt values are:
[0161] Bt2: Kd=0.4 nM, Rt=3.4 pmol/mg vesicle protein.
[0162] Bt15: Kd=0.3 nM Kd2=2.9 nM, Rt1=5.9 and Rt2=6.7 pmol/mg
vesicle protein (2 distinct high affinity receptor sites are
present).
[0163] Similar studies were performed for M. brassicae, S.
littoralis and P. interpunctella. Although LD50, Kd and Rt values
differed substantially, the essential observation that Bt2 and Bt15
are both toxic and are non-competitively binding toxins was
confirmed in these three insect species. Thus, it is also a useful
toxin combination to prevent resistance of M. brassicae to ICP's or
to prevent resistance of Spodoptera species against ICP's expressed
in any of the crop plants in which Spodoptera species are a
pest.
[0164] Binding of Bt2 and Bt4 Toxins to BBMV of M. sexta: an
Example of Two Non-Competitively Binding Lepidopteran ICPs
[0165] Both Bt2 and Bt4 toxins are toxic to Manduca sexta. LD50
values are 20 and 5.4. ng/cm2, respectively. No mutual competition
of Bt2 for binding of labelled Bt4 and of Bt4 for binding of
labelled Bt2. was observed, demonstrating that Bt2 and Bt4 are
non-competitively binding toxins.
[0166] Binding of Bt15 and Bt18 Toxins to BBMV of S. littoralis: an
Example of Two Non-Competitively Binding Lepidopteran ICPs
[0167] Both Bt15 and Bt18 toxins are toxic to S. littoralis. LD 50
values are 93 and 88 ng toxin/cm.sup.2, respectively. Labelled Bt15
(0.7 nM) or Bt18 (0.9 nM) was incubated with 100 ug of vesicle
protein from S. littoralis in combination with varying amounts of
unlabelled Bt15 or Bt18 toxin. After a 45-min. incubation period,
bound and free toxins were separated. Binding data demonstrate high
affinity binding for both Bt15 and Bt18 to S. littoralis BBMV. As
seen from FIGS. 11 and 12, the entire population of receptor sites
of Bt15 was not saturable with Bt18, nor was the entire population
of receptor sites of Bt18 saturable with Bt15.
[0168] Binding of Bt13 and Bt22 toxins to BBMV of L. decemlineata:
an Example of Two Non-Competitively Binding Coleopteran ICPs.
[0169] Both Bt13 and Bt22 toxins are toxic to L. decemlineata. LD
50 values are 0.8 and 1.1 ug toxin/ml respectively. Labelled Bt13
(1 nM) or Bt22 (0.7 nM) was incubated with 100 ug of vesicle
protein/ml from S. littoralis in combination with varying amounts
of unlabelled Bt13 or Bt22 toxin. After a 45 min. incubation
period, bound and free toxins were separated. Binding data
demonstrate high affinity binding for both Bt13 and Bt22 to S.
littoralis BBMV. The entire population of receptor sites of Bt13
was not saturable with Bt22. Nor was the entire population of
receptor sites of Bt22 saturable with Bt13.
[0170] Binding of Bt2 and Bt18 Toxins to BBMV of M. sexta: an
Example of Two Non-Competitively Binding Lepidopteran ICPs.
[0171] Both Bt2 and Bt18 toxins are toxic to M. sexta, and LD 50
values are 20 to 73 ng toxin/cm.sup.2 respectively. Labelled Bt2
(1.05 nM) or Bt18 (0.7 nM) was incubated with 100 ug/ml of vesicle
protein from M. sexta in combination with varying amounts of
unlabelled Bt2 or Bt18 toxin. After a 45 min. incubation period,
bound and free toxins were separated. Binding data (FIGS. 11-12)
demonstrate high affinity binding for both Bt2 and Bt18 to M. sexta
BBMV. The entire population of receptor sites of Bt2 was not
saturable with Bt18. Nor was the entire population of receptor
sites of Bt18 saturable with Bt2. Calculated Kd and Rt values
are:
[0172] Bt2: Kd=0.4 nM, Rt=3.4 pmol/mg vesicle protein.
[0173] Bt18: Kd1=0.04 nM, Rt1=2.2 pmoles/mg vesicle protein and
Kd2=168 nM Rt2=194 pmoles/mg vesicle protein (2 distinct receptor
sites for Bt18 are present).
[0174] A list of non-competitively binding anti-Lepidopteran ICP
combinations and anti-Coleopteran ICP combinations is given below,
together with their common target insect species in which
non-competitivity has been demonstrated:
[0175] Bt2-Bt15 (Manduca sexta, Plutella xylostella, Pieris
brassicae, Mamestra brassicae, Plodia interpunctella)
[0176] Bt2-Bt18 (Manduca sexta, Spodoptera littoralis)
[0177] Bt2-Bt14 (Pieris brassicae, Plutella xylostella, Phthorimaea
operculella)
[0178] Bt2-Bt4 (Manduca sexta)
[0179] Bt15-Bt18 (Manduca sexta, Spodoptera littoralis)
[0180] Bt14-Bt15 (Pieris brassicae)
[0181] Bt15-Bt4 (Manduca sexta, Spodoptera exigua)
[0182] Bt18-Bt4 (Manduca sexta, Spodoptera littoralis)
[0183] Bt18-Bt14 (Pieris brassicae)
[0184] Bt18-Bt4 (Manduca sexta)
[0185] Bt13-Bt21 (Leptinotarsa decemlineata)
[0186] Bt13-Bt22 (Leptinotarsa decemlineata)
[0187] Bt21-Bt22 (Leptinotarsa decemlineata)
[0188] Of course, this list of specific non-competitively binding
ICP combinations for specific target insect pests is not
exhaustive, and it is believed that other such ICP combinations,
including combinations for yet-to-be discovered ICPs, will be found
using a similar approach for any target insect species. Likewise,
the foregoing list of target insect pests also is not exhaustive,
and it is believed that other target insects pests (as well as the
plants that are to be transformed to prevent their attack by such
pests), against which the specific combinations of ICPs can be used
(e.g., the combination of the Bt2 and Bt14 ICPs in Brassica to
prevent resistance of Pieris brassicae against the ICPs expressed
in the plant), will be found using a similar approach.
EXAMPLE 7
Selection for Resistance of Manduca sexta (tobacco hornworm)
[0189] A selection experiment involves exposing a large number of
larvae to a concentration of a toxin in a diet killing (e.g.,
50-90%) of the larvae. The surviving larvae are again exposed to
toxin concentrations killing a similar proportion of the larvae,
and this process is continued for several generations. The
sensitivity of the larvae to the toxin is investigated after each
four generations of selection.
[0190] Selections for 20 generations of M. sexta were performed
with Bt2 toxin alone, with Bt18 toxin alone and with a 1/4 (by
weight) Bt2/Bt18 mixture. LC50 values of the reference strain for
Bt2, Bt18 and the 1/4 Bt2/Bt18 mixture respectively were the
following:
[0191] 20 ng/cm2, 73 ng/cm2 and 62 ng/cm2 of diet.
[0192] Selection was initiated at concentrations killing around 75%
of the larvae. After 4 generations of selection, survival increased
in both the Bt2 and the Bt18 selection to around 70%, no such
increase was observed in the selection with the combination of Bt2
and Bt18. Dosages were again increased to calculated LC75 values.
This was repeated every 4 generations. The selection process was
thus continued to the 20th generation. Final results were the
following (LC50 of the 20th generation):
[0193] Bt2 selection: LC50 was 6400 ug/g (320 times decreased
sensitivity)
[0194] Bt18 selection: LC50 was 15100 ug/g (207 times decreased
sensitivity)
[0195] Bt2/Bt18 selection: LC50 was 181 ug/g (3 times decreased
sensitivity).
[0196] Thus the decrease in sensitivity was about 100 times slower
in the combined selection experiment.
[0197] Receptor binding in the three selected M. sexta strains was
investigated with Bt2 and Bt18 and compared to those of the
reference M. sexta strain (non-selected strain). Binding
characteristics of the reference strain for the Bt2 and BT18 toxins
were:
[0198] Bt2: Kd=0.4 nM, Rt=3.4 pmol/mg vesicle protein
[0199] Bt18: Kd1=0.04 nM, Rt1=2.2 pmoles/mg vesicle protein and
Kd2=168 nM, Rt2=194 pmoles/mg vesicle protein (2 distinct receptor
sites for Bt18 are present).
[0200] FIGS. 11 and 12 show the binding of .sup.125I-labeled toxins
to M. sexta brush border membrane vesicle. Vesicles were incubated
with labeled toxin [in FIG. 11: .sup.125I-Bt2-toxin (1.05 nM); in
FIG. 12: .sup.125I-Bt18-toxin (0.7 nM)] in the presence of
increasing concentrations of Bt2-toxin (.smallcircle.) or
Bt18-toxin (.circle-solid.). Binding is expressed as percentage of
the amount bound upon incubation with labeled toxin alone.
Non-specific binding was not substracted. Data were analyzed with
the LIGAND computer program. Each point is the mean of a duplicate
sample.
[0201] The Bt2 selected strain showed no detectable high affinity
binding of Bt2 whereas its Bt18 binding characteristics remained
close to the reference strain. (Bt18: Kd1=0.03 nM, Rt1=2.8
pmoles/mg vesicle protein and Kd2=199 nM, Rt2=109 pmoles/mg.
vesicle protein; 2 distinct receptor sites for Bt18 are still
present).
[0202] The Bt18 selected strain lost the high affinity receptor
site for Bt18. The lower affinity site for Bt18 was still present
in lower concentration than in the reference strain (Kd=189 nM,
Rt=43 nM). Bt2 binding site concentration increased markedly
compared to the reference strain (Kd=0.4 nM, Rt=20.8 pmoles/mg
vesicle protein). This strain had a Bt2 sensitivity of LC.sub.50=4
ng/cm.sup.2. Thus, its sensitivity for Bt2 had increased as
compared to the reference strain (LC.sub.50=20 ng/cm.sup.2).
[0203] The Bt2/Bt18 selected strain showed a slight but
statistically non-significant decrease in Bt18 binding site
concentration. (Bt2: Kd=0.4 nM, Rt=3.4 pmol/mg vesicle protein,
Bt18: Kd1=0.04 nM, Rt1=1.0 pmoles/mg vesicle protein and Kd2=168
nM, Rt2=194 pmoles/mg vesicle protein; 2 distinct receptor sites
for Bt18 are present). These data demonstrate that, in the two
selection lines where resistance occurred, the mechanism was
situated at the receptor level. Changes in receptor site are shown
to be the most likely mechanism of resistance to B. thuringiensis
ICPs.
EXAMPLE 8
Mechanism of Resistance of the Diamondback Moth to the Microbial
Insecticide Bacillus thuringiensis
[0204] The mechanism of development of insect resistance to ICPs
has been investigated in a P. xylostella strain ("PxR"). This
insect strain has developed a high level of resistance in the field
against Dipel. Crystals of Dipel preparations contain a mixture of
ICPs such as Bt3, Bt2 and Bt73 ICPs; in Example 6, it has been
shown that these toxins are competitively binding ICPs.
[0205] Resistance to Dipel was confirmed by the toxicity data for
the sensitive strain ("PxS") and for the Dipel-resistant strain
("PxR"). High levels of resistance are also observed for the Bt2
protoxin and toxin as shown in the following table :
3 LC.sub.50 of Strains PxS PxR Bt2 6.7 >1350 Bt15 132.6
120.4
[0206] LC.sub.50 data are expressed as ng protein spotted per
cm.sup.2 of artificial diet.
[0207] However, insect toxicity data show that there is no
resistance to the Bt15 protoxin and Bt15 toxin; this ICP is not
present in Dipel crystals. To investigate whether a change in
toxin-membrane binding was responsible for resistance, receptor
binding studies were performed with .sup.125I-labeled Bt2 toxin and
Bt15 toxin, with BBMV derived from larvae midguts of the PxR and
PxS strains. The results are summarized in Table 1, below.
[0208] Table 1. Binding characteristics of Bt2 and Bt15 toxins to
brush border membrane vesicles from sensitive and resistant P.
xylostella.
4 Rt (pmol/ ICP strain Kd (nM) mg protein) Bt2 toxin PxS 8.1 1.6
PxR no binding detectable Bt15 toxin PxS 1.9 4.2 PxR 3.7 5.8
[0209] Table 1 shows that there was high-affinity saturable binding
of the Bt2 toxin to midgut membranes of the PxS strain, but the PxR
strain showed no detectable level of Bt2 toxin binding. With the
Bt15 toxin, there was significant binding to BBMW of both the PxR
and PxS strains, and values are not significantly different for the
two strains.
[0210] These data show that resistance in P. xylostella is due to
an alteration in toxin-membrane binding. Resistance to the Bt2
toxin and the sensitivity toward the Bt15 toxin of the PxR strain
is reflected by the binding characteristics shown in Table 1.
[0211] Hence, when different non-competitively binding ICPs (i.e.,
Bt2 and Bt15) are available with activity against the same insect
species (e.g., P. xylostella), resistance to one ICP(Bt2) does not
imply resistance against other ICPs (such as Bt15). Thus, ICPs with
different binding properties can be used in combination to delay
development of insect resistance to ICPs.
EXAMPLE 9
Separate Transfer of Two ICP Genes Within Individual
Transcriptional Units to the Genome of Plant Cells
[0212] Two procedures are envisaged for obtaining the combined
expression of two ICP genes, such as the bt2 and bt15 genes in
transgenic plants, such as tomato plants. These procedures are
based on the transfer of two chimeric ICP genes, not linked within
the same DNA fragment, to the genome of a plant of interest.
[0213] A first procedure is based on sequential transformation
steps in which a plant, already transformed with a first chimeric
ICP gene, is retransformed in order to introduce a second ICP gene.
The sequential transformation makes use of two different selectable
marker genes, such as the resistance genes for kanamycin ("km") and
phosphinotricin acetyl transferase ("PPT"), which confers
resistance to phoshinotricin. The use of both these selectable
markers has been described in De Block et al. (1987).
[0214] The second procedure is based on the cotransformation of two
chimeric ICP genes on different plasmids in a single step. The
integration of both ICP genes can be selected by making use of the
two selectable markers conferring resistance to Km and PPT, linked
with the respective ICP genes.
[0215] For either procedure, a Ti-plasmid vector is used for
Agrobacterium-mediated transformation of each chimeric ICP gene
into plant cells.
[0216] Plasmid pGSH163, described in EP 0193259; contains the
following chimeric genes between the T-DNA border repeats: a gene
fragment encoding the toxin part of the bt2 gene under the control
of the TR2' promoter and the neo gene under control of the TR1'
promoter. The 3' ends of the T-DNA gene 7 and octopine synthase
respectively provide information for the 3' end formation of
transcripts.
[0217] A chimeric bt15 gene containing a gene fragment encoding the
toxin of the Bt15 ICP under the control of the TR2' promoter, was
constructed in the following way (FIG. 15). pOH50 consists of pUC18
with the whole bt15 gene under the control of the lac promoter. A
HindIII-BglII fragment was cloned in pMa5-8 yielding pJB3. By
site-directed mutagenesis, a NcoI site was created at the
initiation codon to yield pVE29. A fragment containing the
truncated gene fragment of the bt15 gene, with a translational stop
codon, was obtained by isolation of BclI-ClaI from pOH50 and
cloning in pLK91, yielding pHW38. The whole toxin gene fragment was
reconstructed under the control of the tac promoter, yielding
pVE35, by ligation of a ClaI-PstI fragment from pHW38, a NcoI-ClaI
fragment from pVE29 and a NcoI-PstI fragment from pOH48. A
truncated bt15 gene fragment with a NcoI site at the initiation
codon was obtained from pVE35 as a 1980 NcoI-BamHI fragment and
cloned in pGSJ141, digested with ClaI and BamHI. pGSJ141 has been
described in EPA 88402115.5. Ligation of the filled ClaI site to
the filled NcoI site yielded a chimeric TR2'--truncated bt15--3'g7
construct (pTVE47). As a selectable marker in this plasmid, the bar
gene encoding phosphinothricin acetyl transferase and conferring
resistance to PPT was used. A chimeric bar gene containing the bar
gene under the control of the 35S promoter and followed by the 3'
end of the octopine synthase was introduced in pTVE47. From pDE110,
a 35S-bar-3' ocs fragment was obtained as a StuI-HindIII fragment
and was cloned in pTVE47 digested with PstI and HindIII. This
yielded the plasmid PTHW88 (FIG. 15) which contains the truncated
bt15 gene under the control of the TR2' promoter and the bar gene
under the control of the 35S promoter between the T-DNA border
repeats. Plasmid pGSH163 is cointegration type Ti-plasmid vector,
whereas pTHW88 is a binary type Ti-plasmid vector as described in
EPA 0193259.
[0218] Both plasmids were mobilized in the A. tumefaciens strain
C58C1Rif (pGV2260) according to Deblaere et al. (1988). In the
sequential transformation procedure, tomato was transformed
according to De Block et al. (1987) with the A. tumefaciens strain
C58C1Rif carrying pGS1163 resulting from the cointegration of
pGSH163 and pGV2260. Individual transformants were selected for
kanamycin resistance, and regenerated plants were characterized for
expression of the truncated bt2 gene according to Vaeck et al.
(1987). One representative transformant was subsequently
retransformed with the A. tumefaciens strain C58C1Rif (pGV2260 and
pTHW88), and transformants were selected for PPT resistance. Using
this cotransformation procedure, the respective Agrobacteria
strains, carrying the cointegrate vector pGS1163 and the binary
vector pTHW88, were used for transformation of tomato. Individual
plants were selected for resistance to Km and PPT.
[0219] Schematically shown in FIG. 15 are:
[0220] a) construction of pVE29: bt15 N-terminal gene fragment with
NcoI site introduced at ATG initiation codon.
[0221] b) construction of pVE35: bt15 C-terminal truncated gene
fragment under control of the tac promoter.
[0222] c) construction of pTHW88: binary T-DNA vector with a
chimeric bt15 gene and a chimeric bar gene within the T-DNA border
repeats.
[0223] In both cases, co-expression of the two ICP genes in the
individual transformants was evaluated by insect toxicity tests as
described in EP 0193259 and by biochemical means. Specific RNA
probes allowed the quantitive analysis of the transcript levels;
monoclonal antibodies cross-reacting with the respective gene
products allowed the quantitative analysis of the respective gene
products in ELISA tests (EP 0193259); and specific DNA probes
allowed the characterization of the genomic integrations of the bt2
and bt15 genes in the transformants. It was found that the
transformed tomato plants simultaneously expressed both the bt2
gene (8.1 ng/mg) and the bt15 gene (7.6 ng/mg) as measured by
ELISA, which would prevent or delay development of resistance of M.
sexta to the insecticidal effects of the Bt2 and Bt15 toxins, being
expressed.
[0224] These procedures also could be applied when one or both ICP
genes are part of a hybrid gene. For example, the same strategy as
described above could be followed with the plasmid vectors pGSH152,
containing a chimeric truncated bt2-neo hybrid gene under control
of the TR2' promoter, and pTHW88 in suitable Agrobacterium
strains.
EXAMPLE 10
Separate Transfer of Two ICP Genes to the Nuclear Genome of
Separate Plants in Independent Transformation Events and Subsequent
Combination in a Single Plant Through Crossing
[0225] Tobacco plants have been transformed with either the bt18
gene or the bt15 gene by applying the same cloning strategies as
described in EP 0358557 and EP 193259, respectively. For both
genes, the plants were transformed with plant expression vectors
containing either the truncated bt18 or bt15 gene, which just
encode the Bt18 or Bt15 toxin, respectively.
[0226] The mortality rate of Spodoptera littoralis larvae feeding
on the transformed plants is significantly higher than the
mortality rate of larvae fed on untransformed plants.
[0227] The bt18-transformed plant, which is homozygous for the bt18
gene, is then crossed with the bt15-transformed plant, which is
homozygous for the bt15 gene. After selfing, a plant homozygous for
both genes is obtained.
[0228] The resulting tobacco plants, expressing both the bt18 and
bt15 genes, delay significantly development of resistance by S.
littoralis to either the Bt18 or Bt15 toxin expressed by the
plants.
EXAMPLE 11
Transfer of Two Chimeric ICP Genes Linked Within the Same DNA to
the Genome of Plant Cells
[0229] The strategy used is based on the organization of two
independent chimeric ICP genes between the T-DNA border repeats of
a single vector. Binding studies indicated that the Bt2 and Bt14
toxins are two non-competitively binding ICPs with insecticidal
activity towards Pieris brassicae. For expression in plants, both
the bt2 and bt14 genes can be co-expressed to prevent insect
resistance development. For the design of a plasmid vector with
each ICP gene under the control of a separate promoter, two
possibilities can be envisaged: 1) three chimeric constructs
carrying the truncated bt2 and bt14 genes and a selectable marker,
respectively; or 2) a hybrid of a truncated gene fragment (bt2 or
bt14) and the neo gene can be used in combination with a truncated
bt14 or bt2 gene.
[0230] This Example describes the construction of the vector pTHW94
for plant transformations carrying the following chimeric ICP genes
between the T-DNA border repeats: a truncated bt2 gene fragment
under the control of the TR2' promoter and a hybrid truncated
bt14-neo gene under the control of the TR1' promoter. The 3' end of
the T-DNA gene 7 and octopine synthase, respectively, provide
information for proper 3' end formation. pTHW94 has been deposited
at the DSM under accession no. 5514 on Aug. 28, 1989.
[0231] Schematically shown in FIG. 16 are the:
[0232] a) construction of pHW44: bt14 N-terminal gene fragment with
NcoI site introduced at ATG initiation codon.
[0233] b) construction of pHW67: reconstruction of the bt14 gene
under the control of the tac promoter.
[0234] c) construction of pHW71: construction of a hybrid truncated
bt14-neo gene under the control of the tac promoter.
[0235] d) construction of pTHW94: binary T-DNA vector with a
chimeric bt14 gene and a chimeric bt2 gene within the T-DNA border
repeats.
[0236] The pTHW94 vector is mobilized into the Agrobacterium strain
C58C1Rif (pMP90) which is used to transform Brassica napus
according to the procedure described by De Block et al. (1989).
Transformants are selected on Km, and regenerated plants are found
to express both ICP gene products in insect toxicity tests and
biochemical tests.
EXAMPLE 12
Expression of Two ICP Genes in a Hybrid Construct
[0237] In order to obtain a combined and simultaneous expression of
two ICP genes, truncated gene fragments encoding the toxic parts of
two different ICPs can be fused in a proper reading frame and
placed, as a hybrid gene, under the control of the same promoter in
a chimaeric gene construct. Toxic cores from certain ICPs can be
liberated from their protoxins by protease activation at the N-
and/or C-terminal end. Thus, hybrid genes can be designed with one
or more regions encoding protease cleavage site(s) at the fusion
point(s) of two or more ICP genes.
[0238] The simultaneous co-expression of the bt2 and bt14 genes is
obtained by constructing a hybrid gene composed of a truncated bt14
gene fragment fused to a truncated bt2 gene fragment. Schematically
shown in FIG. 17 is the construction of such a hybrid bt2-bt14 gene
with a C-terminal bt2 gene fragment (bt860) encoding the toxic core
of the Bt2 protoxin in frame with a C-terminal truncated bt14 gene
fragment encoding the toxic core of the Bt14 protoxin. The BclI
site in the bt2 gene, localized downstream of the trypsin cleavage
site, is fused in frame with the NcoI site introduced at the
N-terminal end of the truncated bt14 gene fragment. To this end,
the plasmids pLBKm860 (EP 0193259) and pHW67 are used. pLBKm860
contains a hybrid bt2-neo gene under control of the lambda P.sub.L
promoter. The bt2 gene moiety in the hybrid gene is a C-terminal
truncated bt2 gene fragment, indicated as bt860 (in FIG. 17) (see
also Vaeck et al, 1987). The construction of pHW67 is described in
FIG. 16. pHW67 contains a C-terminal truncated bt14 gene fragment
(bt14tox) with a NcoI site at the ATG initiation codon, a
translation stop codon located at the BclI site of the intact bt14
gene and a BamHI site downstream of the whole gene fragment. To
fuse both gene fragments in the proper reading frame, the BclI and
NcoI ends of the respective plasmids are treated with Klenow DNA
polymerase and S1 nuclease as indicated in FIG. 16. The resulting
plasmid pJB100 contains the hybrid bt860-bt14tox gene under control
of the lambda P.sub.L promoter and directs the expression in E.
coli of a fusion protein with the expected mobility on
SDS-PAGE.
[0239] Crude extracts of the E. coli strain show the toxicity of
the fusion protein, expressed by the strain, against P. brassicae.
It has also been confirmed by N-terminal amino acid sequence
analyses of the fusion protein produced by the E. coli strain that
the N-terminal amino acids from the Bt14 protoxin are processed
upon activation. The bt2-bt14 hybrid gene product has thus two
potential protease cleavage sites.
[0240] Subsequently, this hybrid gene is inserted into a vector for
plant transformations and placed under control of a suitable
promoter and transferred to the genome of brassica (EP 0193259)
where both the bt2 and bt14 genes are expressed in insect toxicity
tests.
5TABLE 2 predicted Disclosure amino MW (kDa) of Host acids of
encoded nucleotide Gene Bt strain range encoded aminoacids sequence
bt3 HD-1 kurstaki L 1176 133.2 Schnepf et al., 1985 bt2 berliner
1715 L 1155 131 Hofte et al., 1986 bt73 HD-73 L 1178 133.3 Adang et
al, 1985 bt14 entomocidus L 1207 138 Brizzard HD-110 and Whiteley,
1988 bt15 entomocidus L 1189 134.8 HD-110 bt4 HD-68 L 1165 132.5
aizawai bt18 darmstadiensis L 1171 133 EP HD-146 appln. 88402241.9
bt13 BtS1, DSM4288 C 644 73.1 EP 22/10/87 appln. 88402115.5 bt21
BtPGSI208, C 651 74.2 EP DSM 5131, appln. 19/1/89 89400428.2 bt22
BtPGSI245, C 1138 129 EP DSM 5132, appln. 19/1/89 8940028.2 P2
HD-263 L/D 633 70.9 Donovan et al, 1988 Cry HD-1 L 633 70.8 Widner
and B2 Whiteley, 1989
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Sequence CWU 1
1
10 1 12 DNA Bacillus thuringiensis 1 tggccagcgc ca 12 2 15 DNA
Bacillus thuringiensis 2 tgccagcgcc accat 15 3 28 DNA Bacillus
thuringiensis 3 cggaggtatt ccatggagga aaataatc 28 4 34 DNA Bacillus
thuringiensis 4 cctatttgaa gccatggtaa ctcctccttt tatg 34 5 3903 DNA
Bacillus thuringiensis CDS (264)..(3761) 5 ggatctgttt taatataagg
gatttgtgcc cttctcgtta tattctttta ttagccccaa 60 aaactagtgc
aactaaatat ttttataatt acactgatta aatactttat ttttgggagt 120
aagatttatg ctgaaatgta ataaaattcg ttccattttc tgtattttct cataaaatgt
180 ttcatatgct ttaaattgta gtaaagaaaa acagtacaaa cttaaaagga
ctttagtaat 240 ttaataaaaa aaggggatag ttt atg gaa ata aat aat caa
aac caa tgt gtg 293 Met Glu Ile Asn Asn Gln Asn Gln Cys Val 1 5 10
cct tac aat tgt tta agt aat cct aag gag ata ata tta ggc gag gaa 341
Pro Tyr Asn Cys Leu Ser Asn Pro Lys Glu Ile Ile Leu Gly Glu Glu 15
20 25 agg cta gaa aca ggg aat act gta gca gac att tca tta ggg ctt
att 389 Arg Leu Glu Thr Gly Asn Thr Val Ala Asp Ile Ser Leu Gly Leu
Ile 30 35 40 aat ttt cta tat tct aat ttt gta cca gga gga gga ttt
ata gta ggt 437 Asn Phe Leu Tyr Ser Asn Phe Val Pro Gly Gly Gly Phe
Ile Val Gly 45 50 55 tta cta gaa tta ata tgg gga ttt ata ggg cct
tcg caa tgg gat att 485 Leu Leu Glu Leu Ile Trp Gly Phe Ile Gly Pro
Ser Gln Trp Asp Ile 60 65 70 ttt tta gct caa att gag caa ttg att
agt caa aga ata gaa gaa ttt 533 Phe Leu Ala Gln Ile Glu Gln Leu Ile
Ser Gln Arg Ile Glu Glu Phe 75 80 85 90 gct agg aat cag gca att tca
aga ttg gag ggg cta agc aat ctt tat 581 Ala Arg Asn Gln Ala Ile Ser
Arg Leu Glu Gly Leu Ser Asn Leu Tyr 95 100 105 aag gtc tat gtt aga
gcg ttt agc gac tgg gag aaa gat cct act aat 629 Lys Val Tyr Val Arg
Ala Phe Ser Asp Trp Glu Lys Asp Pro Thr Asn 110 115 120 cct gct tta
agg gaa gaa atg cgt ata caa ttt aat gac atg aat agt 677 Pro Ala Leu
Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser 125 130 135 gct
ctc ata acg gct att cca ctt ttt aga gtt caa aat tat gaa gtt 725 Ala
Leu Ile Thr Ala Ile Pro Leu Phe Arg Val Gln Asn Tyr Glu Val 140 145
150 gct ctt tta tct gta tat gtt caa gcc gca aac tta cat tta tct att
773 Ala Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Ile
155 160 165 170 tta agg gat gtt tca gtt ttc gga gaa aga tgg gga tat
gat aca gcg 821 Leu Arg Asp Val Ser Val Phe Gly Glu Arg Trp Gly Tyr
Asp Thr Ala 175 180 185 act atc aat aat cgc tat agt gat ctg act agc
ctt att cat gtt tat 869 Thr Ile Asn Asn Arg Tyr Ser Asp Leu Thr Ser
Leu Ile His Val Tyr 190 195 200 act aac cat tgt gtg gat acg tat aat
cag gga tta agg cgt ttg gaa 917 Thr Asn His Cys Val Asp Thr Tyr Asn
Gln Gly Leu Arg Arg Leu Glu 205 210 215 ggt cgt ttt ctt agc gat tgg
att gta tat aat cgt ttc cgg aga caa 965 Gly Arg Phe Leu Ser Asp Trp
Ile Val Tyr Asn Arg Phe Arg Arg Gln 220 225 230 ttg aca att tca gta
tta gat att gtt gcg ttt ttt cca aat tat gat 1013 Leu Thr Ile Ser
Val Leu Asp Ile Val Ala Phe Phe Pro Asn Tyr Asp 235 240 245 250 att
aga aca tat cca att caa aca gct act cag cta acg agg gaa gtc 1061
Ile Arg Thr Tyr Pro Ile Gln Thr Ala Thr Gln Leu Thr Arg Glu Val 255
260 265 tat ctg gat tta cct ttt att aat caa aat ctt tct cct gca gca
agc 1109 Tyr Leu Asp Leu Pro Phe Ile Asn Gln Asn Leu Ser Pro Ala
Ala Ser 270 275 280 tat cca acc ttt tca gct gct gaa agt gct ata att
aga agt cct cat 1157 Tyr Pro Thr Phe Ser Ala Ala Glu Ser Ala Ile
Ile Arg Ser Pro His 285 290 295 tta gta gac ttt tta aat agc ttt acc
att tat aca gat agt ctg gca 1205 Leu Val Asp Phe Leu Asn Ser Phe
Thr Ile Tyr Thr Asp Ser Leu Ala 300 305 310 cgt tat gca tat tgg gga
ggg cac ttg gta aat tct ttc cgc aca gga 1253 Arg Tyr Ala Tyr Trp
Gly Gly His Leu Val Asn Ser Phe Arg Thr Gly 315 320 325 330 acc act
act aat ttg ata aga tcc cct tta tat gga agg gaa gga aat 1301 Thr
Thr Thr Asn Leu Ile Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn 335 340
345 aca gag cgc ccc gta act att acc gca tca cct agc gta cca ata ttt
1349 Thr Glu Arg Pro Val Thr Ile Thr Ala Ser Pro Ser Val Pro Ile
Phe 350 355 360 aga aca ctt tca tat att aca ggc ctt gac aat tca aat
cct gta gct 1397 Arg Thr Leu Ser Tyr Ile Thr Gly Leu Asp Asn Ser
Asn Pro Val Ala 365 370 375 gga atc gag gga gtg gaa ttc caa aat act
ata agt aga agt atc tat 1445 Gly Ile Glu Gly Val Glu Phe Gln Asn
Thr Ile Ser Arg Ser Ile Tyr 380 385 390 cgt aaa agc ggt cca ata gat
tct ttt agt gaa tta cca cct caa gat 1493 Arg Lys Ser Gly Pro Ile
Asp Ser Phe Ser Glu Leu Pro Pro Gln Asp 395 400 405 410 gcc agc gta
tct cct gca att ggg tat agt cac cgt tta tgc cat gca 1541 Ala Ser
Val Ser Pro Ala Ile Gly Tyr Ser His Arg Leu Cys His Ala 415 420 425
aca ttt tta gaa cgg att agt gga cca aga ata gca ggc acc gta ttt
1589 Thr Phe Leu Glu Arg Ile Ser Gly Pro Arg Ile Ala Gly Thr Val
Phe 430 435 440 tct tgg aca cac cgt agt gcc agc cct act aat gaa gta
agt cca tct 1637 Ser Trp Thr His Arg Ser Ala Ser Pro Thr Asn Glu
Val Ser Pro Ser 445 450 455 aga att aca caa att cca tgg gta aag gcg
cat act ctt gca tct ggt 1685 Arg Ile Thr Gln Ile Pro Trp Val Lys
Ala His Thr Leu Ala Ser Gly 460 465 470 gcc tcc gtc att aaa ggt cct
gga ttt aca ggt gga gat att ctg act 1733 Ala Ser Val Ile Lys Gly
Pro Gly Phe Thr Gly Gly Asp Ile Leu Thr 475 480 485 490 agg aat agt
atg ggc gag ctg ggg acc tta cga gta acc ttc aca gga 1781 Arg Asn
Ser Met Gly Glu Leu Gly Thr Leu Arg Val Thr Phe Thr Gly 495 500 505
aga tta cca caa agt tat tat ata cgt ttc cgt tat gct tcg gta gca
1829 Arg Leu Pro Gln Ser Tyr Tyr Ile Arg Phe Arg Tyr Ala Ser Val
Ala 510 515 520 aat agg agt ggt aca ttt aga tat tca cag cca cct tcg
tat gga att 1877 Asn Arg Ser Gly Thr Phe Arg Tyr Ser Gln Pro Pro
Ser Tyr Gly Ile 525 530 535 tca ttt cca aaa act atg gac gca ggt gaa
cca cta aca tct cgt tcg 1925 Ser Phe Pro Lys Thr Met Asp Ala Gly
Glu Pro Leu Thr Ser Arg Ser 540 545 550 ttc gct cat aca aca ctc ttc
act cca ata acc ttt tca cga gct caa 1973 Phe Ala His Thr Thr Leu
Phe Thr Pro Ile Thr Phe Ser Arg Ala Gln 555 560 565 570 gaa gaa ttt
gat cta tac atc caa tcg ggt gtt tat ata gat cga att 2021 Glu Glu
Phe Asp Leu Tyr Ile Gln Ser Gly Val Tyr Ile Asp Arg Ile 575 580 585
gaa ttt ata ccg gtt act gca aca ttt gag gca gaa tat gat tta gaa
2069 Glu Phe Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu
Glu 590 595 600 aga gcg caa aag gtg gtg aat gcc ctg ttt acg tct aca
aac caa cta 2117 Arg Ala Gln Lys Val Val Asn Ala Leu Phe Thr Ser
Thr Asn Gln Leu 605 610 615 ggg cta aaa aca gat gtg acg gat tat cat
att gat cag gta tcc aat 2165 Gly Leu Lys Thr Asp Val Thr Asp Tyr
His Ile Asp Gln Val Ser Asn 620 625 630 cta gtt gcg tgt tta tcg gat
gaa ttt tgt ctg gat gaa aag aga gaa 2213 Leu Val Ala Cys Leu Ser
Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu 635 640 645 650 ttg tcc gag
aaa gtt aaa cat gca aag cga ctc agt gat gag cgg aat 2261 Leu Ser
Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn 655 660 665
tta ctt caa gat cca aac ttc aga ggg atc aat agg caa cca gac cgt
2309 Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp
Arg 670 675 680 ggc tgg aga gga agt acg gat att act atc caa gga gga
gat gac gta 2357 Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly
Gly Asp Asp Val 685 690 695 ttc aaa gag aat tac gtt acg cta ccg ggt
acc ttt gat gag tgc tat 2405 Phe Lys Glu Asn Tyr Val Thr Leu Pro
Gly Thr Phe Asp Glu Cys Tyr 700 705 710 cca acg tat tta tat caa aaa
ata gat gag tcg aaa tta aaa gcc tat 2453 Pro Thr Tyr Leu Tyr Gln
Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr 715 720 725 730 acc cgt tat
caa tta aga ggg tat atc gaa gat agt caa gac tta gaa 2501 Thr Arg
Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu 735 740 745
atc tat tta att cgt tac aat gca aaa cac gaa ata gta aat gta cca
2549 Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val Asn Val
Pro 750 755 760 ggt aca gga agt tta tgg cct ctt tct gta gaa aat caa
att gga cct 2597 Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Glu Asn
Gln Ile Gly Pro 765 770 775 tgt gga gaa ccg aat cga tgc gcg cca cac
ctt gaa tgg aat cct gat 2645 Cys Gly Glu Pro Asn Arg Cys Ala Pro
His Leu Glu Trp Asn Pro Asp 780 785 790 tta cac tgt tcc tgc aga gac
ggg gaa aaa tgt gca cat cat tct cat 2693 Leu His Cys Ser Cys Arg
Asp Gly Glu Lys Cys Ala His His Ser His 795 800 805 810 cat ttc tct
ttg gac att gat gtt gga tgt aca gac tta aat gag gac 2741 His Phe
Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp 815 820 825
tta ggt gta tgg gtg ata ttc aag att aag acg caa gat ggc cac gca
2789 Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His
Ala 830 835 840 cga cta ggg aat cta gag ttt ctc gaa gag aaa cca tta
tta gga gaa 2837 Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro
Leu Leu Gly Glu 845 850 855 gca cta gct cgt gtg aaa aga gcg gag aaa
aaa tgg aga gac aaa cgc 2885 Ala Leu Ala Arg Val Lys Arg Ala Glu
Lys Lys Trp Arg Asp Lys Arg 860 865 870 gaa aca tta caa ttg gaa aca
act atc gtt tat aaa gag gca aaa gaa 2933 Glu Thr Leu Gln Leu Glu
Thr Thr Ile Val Tyr Lys Glu Ala Lys Glu 875 880 885 890 tct gta gat
gct tta ttt gta aac tct caa tat gat aga tta caa gcg 2981 Ser Val
Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala 895 900 905
gat acg aac atc gcg atg att cat gcg gca gat aaa cgc gtt cat aga
3029 Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His
Arg 910 915 920 att cga gaa gcg tat ctg ccg gag ctg tct gtg att ccg
ggt gtc aat 3077 Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile
Pro Gly Val Asn 925 930 935 gcg gct att ttt gaa gaa tta gaa gag cgt
att ttc act gca ttt tcc 3125 Ala Ala Ile Phe Glu Glu Leu Glu Glu
Arg Ile Phe Thr Ala Phe Ser 940 945 950 cta tat gat gcg aga aat att
att aaa aat ggc gat ttc aat aat ggc 3173 Leu Tyr Asp Ala Arg Asn
Ile Ile Lys Asn Gly Asp Phe Asn Asn Gly 955 960 965 970 tta tta tgc
tgg aac gtg aaa ggg cat gta gag gta gaa gaa caa aac 3221 Leu Leu
Cys Trp Asn Val Lys Gly His Val Glu Val Glu Glu Gln Asn 975 980 985
aat cac cgt tca gtc ctg gtt atc cca gaa tgg gag gca gaa gtg tca
3269 Asn His Arg Ser Val Leu Val Ile Pro Glu Trp Glu Ala Glu Val
Ser 990 995 1000 caa gag gtt cgt gtc tgt cca ggt cgt ggc tat atc
ctt cgt gtt aca 3317 Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr
Ile Leu Arg Val Thr 1005 1010 1015 gcg tac aaa gag gga tat gga gaa
ggt tgc gta acg atc cat gag atc 3365 Ala Tyr Lys Glu Gly Tyr Gly
Glu Gly Cys Val Thr Ile His Glu Ile 1020 1025 1030 gag aac aat aca
gac gaa ctg aaa ttc aac aac tgt gta gaa gag gaa 3413 Glu Asn Asn
Thr Asp Glu Leu Lys Phe Asn Asn Cys Val Glu Glu Glu 1035 1040 1045
1050 gta tat cca aac aac acg gta acg tgt att aat tat act gcg act
caa 3461 Val Tyr Pro Asn Asn Thr Val Thr Cys Ile Asn Tyr Thr Ala
Thr Gln 1055 1060 1065 gaa gaa tat gag ggt acg tac act tct cgt aat
cga gga tat gac gaa 3509 Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg
Asn Arg Gly Tyr Asp Glu 1070 1075 1080 gcc tat ggt aat aac cct tcc
gta cca gct gat tat gcg tca gtc tat 3557 Ala Tyr Gly Asn Asn Pro
Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr 1085 1090 1095 gaa gaa aaa
tcg tat aca gat aga cga aga gag aat cct tgt gaa tct 3605 Glu Glu
Lys Ser Tyr Thr Asp Arg Arg Arg Glu Asn Pro Cys Glu Ser 1100 1105
1110 aac aga gga tat gga gat tac aca cca cta cca gct ggt tat gta
aca 3653 Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr
Val Thr 1115 1120 1125 1130 aag gaa tta gag tac ttc cca gag acc gat
aag gta tgg att gag att 3701 Lys Glu Leu Glu Tyr Phe Pro Glu Thr
Asp Lys Val Trp Ile Glu Ile 1135 1140 1145 gga gaa aca gaa gga aca
ttc atc gtg gac agc gtg gaa tta ctc ctt 3749 Gly Glu Thr Glu Gly
Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu 1150 1155 1160 atg gag
gaa tag gaccatccga gtatagcagt ttaataaata ttaattaaaa 3801 Met Glu
Glu 1165 tagtagtcta acttccgttc caattaaata agtaaattac agttgtaaaa
aaaaacgaac 3861 attactcttc aaagagcgat gtccgttttt tatatggtgt gt 3903
6 1165 PRT Bacillus thuringiensis 6 Met Glu Ile Asn Asn Gln Asn Gln
Cys Val Pro Tyr Asn Cys Leu Ser 1 5 10 15 Asn Pro Lys Glu Ile Ile
Leu Gly Glu Glu Arg Leu Glu Thr Gly Asn 20 25 30 Thr Val Ala Asp
Ile Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser Asn 35 40 45 Phe Val
Pro Gly Gly Gly Phe Ile Val Gly Leu Leu Glu Leu Ile Trp 50 55 60
Gly Phe Ile Gly Pro Ser Gln Trp Asp Ile Phe Leu Ala Gln Ile Glu 65
70 75 80 Gln Leu Ile Ser Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln
Ala Ile 85 90 95 Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Lys Val
Tyr Val Arg Ala 100 105 110 Phe Ser Asp Trp Glu Lys Asp Pro Thr Asn
Pro Ala Leu Arg Glu Glu 115 120 125 Met Arg Ile Gln Phe Asn Asp Met
Asn Ser Ala Leu Ile Thr Ala Ile 130 135 140 Pro Leu Phe Arg Val Gln
Asn Tyr Glu Val Ala Leu Leu Ser Val Tyr 145 150 155 160 Val Gln Ala
Ala Asn Leu His Leu Ser Ile Leu Arg Asp Val Ser Val 165 170 175 Phe
Gly Glu Arg Trp Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg Tyr 180 185
190 Ser Asp Leu Thr Ser Leu Ile His Val Tyr Thr Asn His Cys Val Asp
195 200 205 Thr Tyr Asn Gln Gly Leu Arg Arg Leu Glu Gly Arg Phe Leu
Ser Asp 210 215 220 Trp Ile Val Tyr Asn Arg Phe Arg Arg Gln Leu Thr
Ile Ser Val Leu 225 230 235 240 Asp Ile Val Ala Phe Phe Pro Asn Tyr
Asp Ile Arg Thr Tyr Pro Ile 245 250 255 Gln Thr Ala Thr Gln Leu Thr
Arg Glu Val Tyr Leu Asp Leu Pro Phe 260 265 270 Ile Asn Gln Asn Leu
Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser Ala 275 280 285 Ala Glu Ser
Ala Ile Ile Arg Ser Pro His Leu Val Asp Phe Leu Asn 290 295 300 Ser
Phe Thr Ile Tyr Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp Gly 305 310
315 320 Gly His Leu Val Asn Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu
Ile 325 330 335 Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn Thr Glu Arg
Pro Val Thr 340 345 350 Ile Thr Ala Ser Pro Ser Val Pro Ile Phe Arg
Thr Leu Ser Tyr Ile 355 360 365 Thr Gly Leu Asp Asn Ser Asn Pro Val
Ala Gly Ile Glu Gly Val Glu 370 375 380 Phe Gln Asn Thr Ile Ser Arg
Ser Ile Tyr Arg Lys Ser Gly Pro Ile 385 390 395 400 Asp Ser Phe Ser
Glu Leu Pro Pro Gln Asp Ala Ser Val Ser Pro Ala 405 410 415 Ile Gly
Tyr Ser His Arg Leu
Cys His Ala Thr Phe Leu Glu Arg Ile 420 425 430 Ser Gly Pro Arg Ile
Ala Gly Thr Val Phe Ser Trp Thr His Arg Ser 435 440 445 Ala Ser Pro
Thr Asn Glu Val Ser Pro Ser Arg Ile Thr Gln Ile Pro 450 455 460 Trp
Val Lys Ala His Thr Leu Ala Ser Gly Ala Ser Val Ile Lys Gly 465 470
475 480 Pro Gly Phe Thr Gly Gly Asp Ile Leu Thr Arg Asn Ser Met Gly
Glu 485 490 495 Leu Gly Thr Leu Arg Val Thr Phe Thr Gly Arg Leu Pro
Gln Ser Tyr 500 505 510 Tyr Ile Arg Phe Arg Tyr Ala Ser Val Ala Asn
Arg Ser Gly Thr Phe 515 520 525 Arg Tyr Ser Gln Pro Pro Ser Tyr Gly
Ile Ser Phe Pro Lys Thr Met 530 535 540 Asp Ala Gly Glu Pro Leu Thr
Ser Arg Ser Phe Ala His Thr Thr Leu 545 550 555 560 Phe Thr Pro Ile
Thr Phe Ser Arg Ala Gln Glu Glu Phe Asp Leu Tyr 565 570 575 Ile Gln
Ser Gly Val Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val Thr 580 585 590
Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Val Val 595
600 605 Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asp
Val 610 615 620 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Ala
Cys Leu Ser 625 630 635 640 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu
Leu Ser Glu Lys Val Lys 645 650 655 His Ala Lys Arg Leu Ser Asp Glu
Arg Asn Leu Leu Gln Asp Pro Asn 660 665 670 Phe Arg Gly Ile Asn Arg
Gln Pro Asp Arg Gly Trp Arg Gly Ser Thr 675 680 685 Asp Ile Thr Ile
Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val 690 695 700 Thr Leu
Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln 705 710 715
720 Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gln Leu Arg
725 730 735 Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile
Arg Tyr 740 745 750 Asn Ala Lys His Glu Ile Val Asn Val Pro Gly Thr
Gly Ser Leu Trp 755 760 765 Pro Leu Ser Val Glu Asn Gln Ile Gly Pro
Cys Gly Glu Pro Asn Arg 770 775 780 Cys Ala Pro His Leu Glu Trp Asn
Pro Asp Leu His Cys Ser Cys Arg 785 790 795 800 Asp Gly Glu Lys Cys
Ala His His Ser His His Phe Ser Leu Asp Ile 805 810 815 Asp Val Gly
Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 820 825 830 Phe
Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 835 840
845 Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala Arg Val Lys
850 855 860 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Thr Leu Gln
Leu Glu 865 870 875 880 Thr Thr Ile Val Tyr Lys Glu Ala Lys Glu Ser
Val Asp Ala Leu Phe 885 890 895 Val Asn Ser Gln Tyr Asp Arg Leu Gln
Ala Asp Thr Asn Ile Ala Met 900 905 910 Ile His Ala Ala Asp Lys Arg
Val His Arg Ile Arg Glu Ala Tyr Leu 915 920 925 Pro Glu Leu Ser Val
Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu 930 935 940 Leu Glu Glu
Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn 945 950 955 960
Ile Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Leu Cys Trp Asn Val 965
970 975 Lys Gly His Val Glu Val Glu Glu Gln Asn Asn His Arg Ser Val
Leu 980 985 990 Val Ile Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val
Arg Val Cys 995 1000 1005 Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr
Ala Tyr Lys Glu Gly Tyr 1010 1015 1020 Gly Glu Gly Cys Val Thr Ile
His Glu Ile Glu Asn Asn Thr Asp Glu 1025 1030 1035 1040 Leu Lys Phe
Asn Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr 1045 1050 1055
Val Thr Cys Ile Asn Tyr Thr Ala Thr Gln Glu Glu Tyr Glu Gly Thr
1060 1065 1070 Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Glu Ala Tyr Gly
Asn Asn Pro 1075 1080 1085 Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr
Glu Glu Lys Ser Tyr Thr 1090 1095 1100 Asp Arg Arg Arg Glu Asn Pro
Cys Glu Ser Asn Arg Gly Tyr Gly Asp 1105 1110 1115 1120 Tyr Thr Pro
Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe 1125 1130 1135
Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr
1140 1145 1150 Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu
1155 1160 1165 7 3923 DNA Bacillus thuringiensis CDS (234)..(3803)
7 aatagaatct caaatctcga tgactgctta gtctttttaa tactgtctac ttgacagggg
60 taggaacata atcggtcaat tttaaatatg gggcatatat tgatatttta
taaaatttgt 120 tacgtttttt gtattttttc ataagatgtg tcatatgtat
taaatcgtgg taatgaaaaa 180 cagtatcaaa ctatcagaac tttggtagtt
taataaaaaa acggaggtat ttt atg 236 Met 1 gag gaa aat aat caa aat caa
tgc ata cct tac aat tgt tta agt aat 284 Glu Glu Asn Asn Gln Asn Gln
Cys Ile Pro Tyr Asn Cys Leu Ser Asn 5 10 15 cct gaa gaa gta ctt ttg
gat gga gaa cgg ata tca act ggt aat tca 332 Pro Glu Glu Val Leu Leu
Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser 20 25 30 tca att gat att
tct ctg tca ctt gtt cag ttt atg gta tct aac ttt 380 Ser Ile Asp Ile
Ser Leu Ser Leu Val Gln Phe Met Val Ser Asn Phe 35 40 45 gta cca
ggg gga gga ttt tta gtt gga tta ata gat ttt gta tgg gga 428 Val Pro
Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly 50 55 60 65
ata gtt ggc cct tct caa tgg gat gca ttt cta gta caa att gaa caa 476
Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln 70
75 80 tta att aat gaa aga ata gct gaa ttt gct agg aat gct gct att
gct 524 Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile
Ala 85 90 95 aat tta gaa gga tta gaa aac aat tta aat ata tat gtg
gaa gca ttt 572 Asn Leu Glu Gly Leu Glu Asn Asn Leu Asn Ile Tyr Val
Glu Ala Phe 100 105 110 aaa gaa tgg gaa gaa gat cct aat aat cca gaa
acc agg acc aga gta 620 Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu
Thr Arg Thr Arg Val 115 120 125 att gat cgc ttt cgt ata ctt gat ggg
cta ctt gaa agg gac att cct 668 Ile Asp Arg Phe Arg Ile Leu Asp Gly
Leu Leu Glu Arg Asp Ile Pro 130 135 140 145 tcg ttt cga att tct gga
ttt gaa gta ccc ctt tta tcc gtt tat gct 716 Ser Phe Arg Ile Ser Gly
Phe Glu Val Pro Leu Leu Ser Val Tyr Ala 150 155 160 caa gcg gcc aat
ctg cat cta gct ata tta aga gat tct gta att ttt 764 Gln Ala Ala Asn
Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe 165 170 175 gga gaa
aga tgg gga ttg aca acg ata aat gtc aat gaa aac tat aat 812 Gly Glu
Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn 180 185 190
aga cta att agg cat att gat gaa tat gct gat cac tgt gca aat acg 860
Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr 195
200 205 tat aat cgg gga tta aat aat tta ccg aaa tct acg tat caa gat
tgg 908 Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp
Trp 210 215 220 225 ata aca tat aat cga tta cgg aga gac tta aca ttg
act gta tta gat 956 Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu
Thr Val Leu Asp 230 235 240 atc gcc gct ttc ttt cca aac tat gac aat
agg aga tat cca att cag 1004 Ile Ala Ala Phe Phe Pro Asn Tyr Asp
Asn Arg Arg Tyr Pro Ile Gln 245 250 255 cca gtt ggt caa cta aca agg
gaa gtt tat acg gac cca tta att aat 1052 Pro Val Gly Gln Leu Thr
Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn 260 265 270 ttt aat cca cag
tta cag tct gta gct caa tta cct act ttt aac gtt 1100 Phe Asn Pro
Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val 275 280 285 atg
gag agc agc gca att aga aat cct cat tta ttt gat ata ttg aat 1148
Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn 290
295 300 305 aat ctt aca atc ttt acg gat tgg ttt agt gtt gga cgc aat
ttt tat 1196 Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg
Asn Phe Tyr 310 315 320 tgg gga gga cat cga gta ata tct agc ctt ata
gga ggt ggt aac ata 1244 Trp Gly Gly His Arg Val Ile Ser Ser Leu
Ile Gly Gly Gly Asn Ile 325 330 335 aca tct cct ata tat gga aga gag
gcg aac cag gag cct cca aga tcc 1292 Thr Ser Pro Ile Tyr Gly Arg
Glu Ala Asn Gln Glu Pro Pro Arg Ser 340 345 350 ttt act ttt aat gga
ccg gta ttt agg act tta tca aat cct act tta 1340 Phe Thr Phe Asn
Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu 355 360 365 cga tta
tta cag caa cct tgg cca gcg cca cca ttt aat tta cgt ggt 1388 Arg
Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly 370 375
380 385 gtt gaa gga gta gaa ttt tct aca cct aca aat agc ttt acg tat
cga 1436 Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr
Tyr Arg 390 395 400 gga aga ggt acg gtt gat tct tta act gaa tta ccg
cct gag gat aat 1484 Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu
Pro Pro Glu Asp Asn 405 410 415 agt gtg cca cct cgc gaa gga tat agt
cat cgt tta tgt cat gca act 1532 Ser Val Pro Pro Arg Glu Gly Tyr
Ser His Arg Leu Cys His Ala Thr 420 425 430 ttt gtt caa aga tct gga
aca cct ttt tta aca act ggt gta gta ttt 1580 Phe Val Gln Arg Ser
Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe 435 440 445 tct tgg acg
cat cgt agt gca act ctt aca aat aca att gat cca gag 1628 Ser Trp
Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu 450 455 460
465 aga att aat caa ata cct tta gtg aaa gga ttt aga gtt tgg ggg ggc
1676 Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly
Gly 470 475 480 acc tct gtc att aca gga cca gga ttt aca gga ggg gat
atc ctt cga 1724 Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly
Asp Ile Leu Arg 485 490 495 aga aat acc ttt ggt gat ttt gta tct cta
caa gtc aat att aat tca 1772 Arg Asn Thr Phe Gly Asp Phe Val Ser
Leu Gln Val Asn Ile Asn Ser 500 505 510 cca att acc caa aga tac cgt
tta aga ttt cgt tac gct tcc agt agg 1820 Pro Ile Thr Gln Arg Tyr
Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg 515 520 525 gat gca cga gtt
ata gta tta aca gga gcg gca tcc aca gga gtg gga 1868 Asp Ala Arg
Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly 530 535 540 545
ggc caa gtt agt gta aat atg cct ctt cag aaa act atg gaa ata ggg
1916 Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile
Gly 550 555 560 gag aac tta aca tct aga aca ttt aga tat acc gat ttt
agt aat cct 1964 Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp
Phe Ser Asn Pro 565 570 575 ttt tca ttt aga gct aat cca gat ata att
ggg ata agt gaa caa cct 2012 Phe Ser Phe Arg Ala Asn Pro Asp Ile
Ile Gly Ile Ser Glu Gln Pro 580 585 590 cta ttt ggt gca ggt tct att
agt agc ggt gaa ctt tat ata gat aaa 2060 Leu Phe Gly Ala Gly Ser
Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys 595 600 605 att gaa att att
cta gca gat gca aca ttt gaa gca gaa tct gat tta 2108 Ile Glu Ile
Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu 610 615 620 625
gaa aga gca caa aag gcg gtg aat gcc ctg ttt act tct tcc aat caa
2156 Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn
Gln 630 635 640 atc ggg tta aaa acc gat gtg acg gat tat cat att gat
caa gta tcc 2204 Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile
Asp Gln Val Ser 645 650 655 aat tta gtg gat tgt tta tca gat gaa ttt
tgt ctg gat gaa aag cga 2252 Asn Leu Val Asp Cys Leu Ser Asp Glu
Phe Cys Leu Asp Glu Lys Arg 660 665 670 gaa ttg tcc gag aaa gtc aaa
cat gcg aag cga ctc agt gat gag cgg 2300 Glu Leu Ser Glu Lys Val
Lys His Ala Lys Arg Leu Ser Asp Glu Arg 675 680 685 aat tta ctt caa
gat cca aac ttc aga ggg atc aat aga caa cca gac 2348 Asn Leu Leu
Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp 690 695 700 705
cgt ggc tgg aga gga agt aca gat att acc atc caa gga gga gat gac
2396 Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp
Asp 710 715 720 gta ttc aaa gag aat tac gtc aca cta ccg ggt acc gtt
gat gag tgc 2444 Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr
Val Asp Glu Cys 725 730 735 tat cca acg tat tta tat cag aaa ata gat
gag tcg aaa tta aaa gct 2492 Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile
Asp Glu Ser Lys Leu Lys Ala 740 745 750 tat acc cgt tat gaa tta aga
ggg tat atc gaa gat agt caa gac tta 2540 Tyr Thr Arg Tyr Glu Leu
Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu 755 760 765 gaa atc tat ttg
atc cgt tac aat gca aaa cac gaa ata gta aat gtg 2588 Glu Ile Tyr
Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val Asn Val 770 775 780 785
cca ggc acg ggt tcc tta tgg ccg ctt tca gcc caa agt cca atc gga
2636 Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile
Gly 790 795 800 aag tgt gga gaa ccg aat cga tgc gcg cca cac ctt gaa
tgg aat cct 2684 Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu
Glu Trp Asn Pro 805 810 815 gat cta gat tgt tcc tgc aga gac ggg gaa
aaa tgt gca cat cat tcc 2732 Asp Leu Asp Cys Ser Cys Arg Asp Gly
Glu Lys Cys Ala His His Ser 820 825 830 cat cat ttc acc ttg gat att
gat gtt gga tgt aca gac tta aat gag 2780 His His Phe Thr Leu Asp
Ile Asp Val Gly Cys Thr Asp Leu Asn Glu 835 840 845 gac tta ggt gta
tgg gtg ata ttc aag att aag acg caa gat ggc cat 2828 Asp Leu Gly
Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His 850 855 860 865
gca aga cta ggg aat cta gag ttt ctc gaa gag aaa cca tta tta ggg
2876 Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu
Gly 870 875 880 gaa gca cta gct cgt gtg aaa aga gcg gag aag aag tgg
aga gac aaa 2924 Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys
Trp Arg Asp Lys 885 890 895 cga gag aaa ctg cag ttg gaa aca aat att
gtt tat aaa gag gca aaa 2972 Arg Glu Lys Leu Gln Leu Glu Thr Asn
Ile Val Tyr Lys Glu Ala Lys 900 905 910 gaa tct gta gat gct tta ttt
gta aac tct caa tat gat aga tta caa 3020 Glu Ser Val Asp Ala Leu
Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln 915 920 925 gtg gat acg aac
atc gcg atg att cat gcg gca gat aaa cgc gtt cat 3068 Val Asp Thr
Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His 930 935 940 945
aga atc cgg gaa gcg tat ctg cca gag ttg tct gtg att cca ggt gtc
3116 Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly
Val 950 955 960 aat gcg gcc att ttc gaa gaa tta gag gga cgt att ttt
aca gcg tat 3164 Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile
Phe Thr Ala Tyr 965 970 975 tcc tta tat gat gcg aga aat gtc att aaa
aat ggc gat ttc aat aat 3212 Ser Leu Tyr Asp Ala Arg Asn Val Ile
Lys Asn Gly Asp Phe Asn Asn 980 985 990 ggc tta tta tgc tgg aac gtg
aaa ggt cat gta gat gta gaa gag caa 3260 Gly Leu Leu Cys Trp Asn
Val Lys Gly His Val Asp Val Glu Glu Gln 995 1000 1005 aac aac cac
cgt tcg gtc ctt gtt atc cca gaa tgg gag gca gaa gtg 3308 Asn Asn
His Arg Ser Val Leu Val Ile Pro Glu
Trp Glu Ala Glu Val 1010 1015 1020 1025 tca caa gag gtt cgt gtc tgt
cca ggt cgt ggc tat atc ctt cgt gtc 3356 Ser Gln Glu Val Arg Val
Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val 1030 1035 1040 aca gca tat
aaa gag gga tat gga gag ggc tgc gta acg atc cat gag 3404 Thr Ala
Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu 1045 1050
1055 atc gaa gac aat aca gac gaa ctg aaa ttc agc aac tgt gta gaa
gag 3452 Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val
Glu Glu 1060 1065 1070 gaa gta tat cca aac aac aca gta acg tgt aat
aat tat act ggg act 3500 Glu Val Tyr Pro Asn Asn Thr Val Thr Cys
Asn Asn Tyr Thr Gly Thr 1075 1080 1085 caa gaa gaa tat gag ggt acg
tac act tct cgt aat caa gga tat gac 3548 Gln Glu Glu Tyr Glu Gly
Thr Tyr Thr Ser Arg Asn Gln Gly Tyr Asp 1090 1095 1100 1105 gaa gcc
tat ggt aat aac cct tcc gta cca gct gat tac gct tca gtc 3596 Glu
Ala Tyr Gly Asn Asn Pro Ser Val Pro Ala Asp Tyr Ala Ser Val 1110
1115 1120 tat gaa gaa aaa tcg tat aca gat gga cga aga gag aat cct
tgt gaa 3644 Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn
Pro Cys Glu 1125 1130 1135 tct aac aga ggc tat ggg gat tac aca cca
cta ccg gct ggt tat gta 3692 Ser Asn Arg Gly Tyr Gly Asp Tyr Thr
Pro Leu Pro Ala Gly Tyr Val 1140 1145 1150 aca aag gat tta gag tac
ttc cca gag acc gat aag gta tgg att gag 3740 Thr Lys Asp Leu Glu
Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu 1155 1160 1165 atc gga
gaa aca gaa gga aca ttc atc gtg gat agc gtg gaa tta ctc 3788 Ile
Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu 1170
1175 1180 1185 ctt atg gag gaa taa gatacgttat aaaatgtaac gtatgcaaat
aaagaatgat 3843 Leu Met Glu Glu tactgaccta tattaacaga taaataagaa
aatttttata cgaataaaaa acggacatca 3903 ctcttaagag aatgatgtcc 3923 8
1189 PRT Bacillus thuringiensis 8 Met Glu Glu Asn Asn Gln Asn Gln
Cys Ile Pro Tyr Asn Cys Leu Ser 1 5 10 15 Asn Pro Glu Glu Val Leu
Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn 20 25 30 Ser Ser Ile Asp
Ile Ser Leu Ser Leu Val Gln Phe Met Val Ser Asn 35 40 45 Phe Val
Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp 50 55 60
Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu 65
70 75 80 Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala
Ala Ile 85 90 95 Ala Asn Leu Glu Gly Leu Glu Asn Asn Leu Asn Ile
Tyr Val Glu Ala 100 105 110 Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn
Pro Glu Thr Arg Thr Arg 115 120 125 Val Ile Asp Arg Phe Arg Ile Leu
Asp Gly Leu Leu Glu Arg Asp Ile 130 135 140 Pro Ser Phe Arg Ile Ser
Gly Phe Glu Val Pro Leu Leu Ser Val Tyr 145 150 155 160 Ala Gln Ala
Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile 165 170 175 Phe
Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr 180 185
190 Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn
195 200 205 Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr
Gln Asp 210 215 220 Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr
Leu Thr Val Leu 225 230 235 240 Asp Ile Ala Ala Phe Phe Pro Asn Tyr
Asp Asn Arg Arg Tyr Pro Ile 245 250 255 Gln Pro Val Gly Gln Leu Thr
Arg Glu Val Tyr Thr Asp Pro Leu Ile 260 265 270 Asn Phe Asn Pro Gln
Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn 275 280 285 Val Met Glu
Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu 290 295 300 Asn
Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe 305 310
315 320 Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly
Asn 325 330 335 Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu
Pro Pro Arg 340 345 350 Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr
Leu Ser Asn Pro Thr 355 360 365 Leu Arg Leu Leu Gln Gln Pro Trp Pro
Ala Pro Pro Phe Asn Leu Arg 370 375 380 Gly Val Glu Gly Val Glu Phe
Ser Thr Pro Thr Asn Ser Phe Thr Tyr 385 390 395 400 Arg Gly Arg Gly
Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp 405 410 415 Asn Ser
Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala 420 425 430
Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val 435
440 445 Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp
Pro 450 455 460 Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg
Val Trp Gly 465 470 475 480 Gly Thr Ser Val Ile Thr Gly Pro Gly Phe
Thr Gly Gly Asp Ile Leu 485 490 495 Arg Arg Asn Thr Phe Gly Asp Phe
Val Ser Leu Gln Val Asn Ile Asn 500 505 510 Ser Pro Ile Thr Gln Arg
Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser 515 520 525 Arg Asp Ala Arg
Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val 530 535 540 Gly Gly
Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile 545 550 555
560 Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn
565 570 575 Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser
Glu Gln 580 585 590 Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu
Leu Tyr Ile Asp 595 600 605 Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr
Phe Glu Ala Glu Ser Asp 610 615 620 Leu Glu Arg Ala Gln Lys Ala Val
Asn Ala Leu Phe Thr Ser Ser Asn 625 630 635 640 Gln Ile Gly Leu Lys
Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val 645 650 655 Ser Asn Leu
Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys 660 665 670 Arg
Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu 675 680
685 Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro
690 695 700 Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly
Gly Asp 705 710 715 720 Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro
Gly Thr Val Asp Glu 725 730 735 Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys
Ile Asp Glu Ser Lys Leu Lys 740 745 750 Ala Tyr Thr Arg Tyr Glu Leu
Arg Gly Tyr Ile Glu Asp Ser Gln Asp 755 760 765 Leu Glu Ile Tyr Leu
Ile Arg Tyr Asn Ala Lys His Glu Ile Val Asn 770 775 780 Val Pro Gly
Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile 785 790 795 800
Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn 805
810 815 Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His
His 820 825 830 Ser His His Phe Thr Leu Asp Ile Asp Val Gly Cys Thr
Asp Leu Asn 835 840 845 Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile
Lys Thr Gln Asp Gly 850 855 860 His Ala Arg Leu Gly Asn Leu Glu Phe
Leu Glu Glu Lys Pro Leu Leu 865 870 875 880 Gly Glu Ala Leu Ala Arg
Val Lys Arg Ala Glu Lys Lys Trp Arg Asp 885 890 895 Lys Arg Glu Lys
Leu Gln Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala 900 905 910 Lys Glu
Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu 915 920 925
Gln Val Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val 930
935 940 His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro
Gly 945 950 955 960 Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg
Ile Phe Thr Ala 965 970 975 Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile
Lys Asn Gly Asp Phe Asn 980 985 990 Asn Gly Leu Leu Cys Trp Asn Val
Lys Gly His Val Asp Val Glu Glu 995 1000 1005 Gln Asn Asn His Arg
Ser Val Leu Val Ile Pro Glu Trp Glu Ala Glu 1010 1015 1020 Val Ser
Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg 1025 1030
1035 1040 Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr
Ile His 1045 1050 1055 Glu Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe
Ser Asn Cys Val Glu 1060 1065 1070 Glu Glu Val Tyr Pro Asn Asn Thr
Val Thr Cys Asn Asn Tyr Thr Gly 1075 1080 1085 Thr Gln Glu Glu Tyr
Glu Gly Thr Tyr Thr Ser Arg Asn Gln Gly Tyr 1090 1095 1100 Asp Glu
Ala Tyr Gly Asn Asn Pro Ser Val Pro Ala Asp Tyr Ala Ser 1105 1110
1115 1120 Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn
Pro Cys 1125 1130 1135 Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro
Leu Pro Ala Gly Tyr 1140 1145 1150 Val Thr Lys Asp Leu Glu Tyr Phe
Pro Glu Thr Asp Lys Val Trp Ile 1155 1160 1165 Glu Ile Gly Glu Thr
Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu 1170 1175 1180 Leu Leu
Met Glu Glu 1185 9 3687 DNA Bacillus thuringiensis CDS (1)..(3687)
9 ttg act tca aat agg aaa aat gag aat gaa att ata aat gct gta tcg
48 Leu Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Val Ser
1 5 10 15 aat cat tcc gca caa atg gat cta tta cca gat gct cgt att
gag gat 96 Asn His Ser Ala Gln Met Asp Leu Leu Pro Asp Ala Arg Ile
Glu Asp 20 25 30 agc ttg tgt ata gcc gag ggg aac aat att gat cca
ttt gtt agc gca 144 Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asp Pro
Phe Val Ser Ala 35 40 45 tca aca gtc caa acg ggt att aac ata gct
ggt aga ata cta ggc gta 192 Ser Thr Val Gln Thr Gly Ile Asn Ile Ala
Gly Arg Ile Leu Gly Val 50 55 60 ttg ggc gta ccg ttt gct gga caa
cta gct agt ttt tat agt ttt ctt 240 Leu Gly Val Pro Phe Ala Gly Gln
Leu Ala Ser Phe Tyr Ser Phe Leu 65 70 75 80 gtt ggt gaa tta tgg ccc
cgc ggc aga gat cag tgg gaa att ttc cta 288 Val Gly Glu Leu Trp Pro
Arg Gly Arg Asp Gln Trp Glu Ile Phe Leu 85 90 95 gaa cat gtc gaa
caa ctt ata aat caa caa ata aca gaa aat gct agg 336 Glu His Val Glu
Gln Leu Ile Asn Gln Gln Ile Thr Glu Asn Ala Arg 100 105 110 aat acg
gct ctt gct cga tta caa ggt tta gga gat tcc ttc aga gcc 384 Asn Thr
Ala Leu Ala Arg Leu Gln Gly Leu Gly Asp Ser Phe Arg Ala 115 120 125
tat caa cag tca ctt gaa gat tgg cta gaa aac cgt gat gat gca aga 432
Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Arg 130
135 140 acg aga agt gtt ctt cat acc caa tat ata gct tta gaa ctt gat
ttt 480 Thr Arg Ser Val Leu His Thr Gln Tyr Ile Ala Leu Glu Leu Asp
Phe 145 150 155 160 ctt aat gcg atg ccg ctt ttc gca att aga aac caa
gaa gtt cca tta 528 Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn Gln
Glu Val Pro Leu 165 170 175 ttg atg gta tat gct caa gct gca aat tta
cac cta tta tta ttg aga 576 Leu Met Val Tyr Ala Gln Ala Ala Asn Leu
His Leu Leu Leu Leu Arg 180 185 190 gat gcc tct ctt ttt ggt agt gaa
ttt ggg ctt aca tcg cag gaa att 624 Asp Ala Ser Leu Phe Gly Ser Glu
Phe Gly Leu Thr Ser Gln Glu Ile 195 200 205 caa cgc tat tat gag cgc
caa gtg gaa cga acg aga gat tat tcc gac 672 Gln Arg Tyr Tyr Glu Arg
Gln Val Glu Arg Thr Arg Asp Tyr Ser Asp 210 215 220 tat tgc gta gaa
tgg tat aat aca ggt cta aat agc ttg aga ggg aca 720 Tyr Cys Val Glu
Trp Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr 225 230 235 240 aat
gcc gca agt tgg gta cgg tat aat caa ttc cgt aga gat cta acg 768 Asn
Ala Ala Ser Trp Val Arg Tyr Asn Gln Phe Arg Arg Asp Leu Thr 245 250
255 tta gga gta tta gat cta gtg gca cta ttc cca agc tat gac act cgc
816 Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg
260 265 270 act tat cca ata aat acg agt gct cag tta aca aga gaa gtt
tat aca 864 Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr Arg Glu Val
Tyr Thr 275 280 285 gac gca att gga gca aca ggg gta aat atg gca agt
atg aat tgg tat 912 Asp Ala Ile Gly Ala Thr Gly Val Asn Met Ala Ser
Met Asn Trp Tyr 290 295 300 aat aat aat gca cct tcg ttc tct gcc ata
gag gct gcg gct atc cga 960 Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile
Glu Ala Ala Ala Ile Arg 305 310 315 320 agc ccg cat cta ctt gat ttt
cta gaa caa ctt aca att ttt agc gct 1008 Ser Pro His Leu Leu Asp
Phe Leu Glu Gln Leu Thr Ile Phe Ser Ala 325 330 335 tca tca cga tgg
agt aat act agg cat atg act tat tgg cgg ggg cac 1056 Ser Ser Arg
Trp Ser Asn Thr Arg His Met Thr Tyr Trp Arg Gly His 340 345 350 acg
att caa tct cgg cca ata gga ggc gga tta aat acc tca acg cat 1104
Thr Ile Gln Ser Arg Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr His 355
360 365 ggg gct acc aat act tct att aat cct gta aca tta cgg ttc gca
tct 1152 Gly Ala Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Arg Phe
Ala Ser 370 375 380 cga gac gtt tat agg act gaa tca tat gca gga gtg
ctt cta tgg gga 1200 Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly
Val Leu Leu Trp Gly 385 390 395 400 att tac ctt gaa cct att cat ggt
gtc cct act gtt agg ttt aat ttt 1248 Ile Tyr Leu Glu Pro Ile His
Gly Val Pro Thr Val Arg Phe Asn Phe 405 410 415 acg aac cct cag aat
att tct gat aga ggt acc gct aac tat agt caa 1296 Thr Asn Pro Gln
Asn Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gln 420 425 430 cct tat
gag tca cct ggg ctt caa tta aaa gat tca gaa act gaa tta 1344 Pro
Tyr Glu Ser Pro Gly Leu Gln Leu Lys Asp Ser Glu Thr Glu Leu 435 440
445 cca cca gaa aca aca gaa cga cca aat tat gaa tct tac agt cac agg
1392 Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His
Arg 450 455 460 tta tct cat ata ggt ata att tta caa tcc agg gtg aat
gta ccg gta 1440 Leu Ser His Ile Gly Ile Ile Leu Gln Ser Arg Val
Asn Val Pro Val 465 470 475 480 tat tct tgg acg cat cgt agt gca gat
cgt acg aat acg att gga cca 1488 Tyr Ser Trp Thr His Arg Ser Ala
Asp Arg Thr Asn Thr Ile Gly Pro 485 490 495 aat aga atc acc caa atc
cca atg gta aaa gca tcc gaa ctt cct caa 1536 Asn Arg Ile Thr Gln
Ile Pro Met Val Lys Ala Ser Glu Leu Pro Gln 500 505 510 ggt acc act
gtt gtt aga gga cca gga ttt act ggt ggg gat att ctt 1584 Gly Thr
Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu 515 520 525
cga aga acg aat act ggt gga ttt gga ccg ata aga gta act gtt aac
1632 Arg Arg Thr Asn Thr Gly Gly Phe Gly Pro Ile Arg Val Thr Val
Asn 530 535 540 gga cca tta aca caa aga tat cgt ata gga ttc cgc tat
gct tca act 1680 Gly Pro Leu Thr Gln Arg Tyr Arg Ile Gly Phe Arg
Tyr Ala Ser Thr 545 550 555 560 gta gat ttt gat ttc ttt gta tca cgt
gga ggt act act gta aat aat 1728 Val Asp Phe Asp Phe Phe Val Ser
Arg Gly Gly Thr Thr Val Asn Asn 565 570 575 ttt aga ttc cta cgt aca
atg aac agt gga gac gaa cta aaa tac gga 1776 Phe Arg Phe Leu Arg
Thr Met Asn Ser Gly Asp Glu
Leu Lys Tyr Gly 580 585 590 aat ttt gtg aga cgt gct ttt act aca cct
ttt act ttt aca caa att 1824 Asn Phe Val Arg Arg Ala Phe Thr Thr
Pro Phe Thr Phe Thr Gln Ile 595 600 605 caa gat ata att cga acg tct
att caa ggc ctt agt gga aat ggg gaa 1872 Gln Asp Ile Ile Arg Thr
Ser Ile Gln Gly Leu Ser Gly Asn Gly Glu 610 615 620 gtg tat ata gat
aaa att gaa att att cca gtt act gca acc ttc gaa 1920 Val Tyr Ile
Asp Lys Ile Glu Ile Ile Pro Val Thr Ala Thr Phe Glu 625 630 635 640
gca gaa tat gat tta gaa aga gcg caa gag gcg gtg aat gct ctg ttt
1968 Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu Ala Val Asn Ala Leu
Phe 645 650 655 act aat acg aat cca aga aga ttg aaa aca gat gtg aca
gat tat cat 2016 Thr Asn Thr Asn Pro Arg Arg Leu Lys Thr Asp Val
Thr Asp Tyr His 660 665 670 att gat caa gta tcc aat tta gtg gcg tgt
tta tcg gat gaa ttc tgc 2064 Ile Asp Gln Val Ser Asn Leu Val Ala
Cys Leu Ser Asp Glu Phe Cys 675 680 685 ttg gat gaa aag aga gaa tta
ctt gag aaa gtg aaa tat gcg aaa cga 2112 Leu Asp Glu Lys Arg Glu
Leu Leu Glu Lys Val Lys Tyr Ala Lys Arg 690 695 700 ctc agt gat gaa
aga aac tta ctc caa gat cca aac ttc aca tcc atc 2160 Leu Ser Asp
Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Thr Ser Ile 705 710 715 720
aat aag caa cca gac ttc ata tct act aat gag caa tcg aat ttc aca
2208 Asn Lys Gln Pro Asp Phe Ile Ser Thr Asn Glu Gln Ser Asn Phe
Thr 725 730 735 tct atc cat gaa caa tct gaa cat gga tgg tgg gga agt
gag aac att 2256 Ser Ile His Glu Gln Ser Glu His Gly Trp Trp Gly
Ser Glu Asn Ile 740 745 750 acc atc cag gaa gga aat gac gta ttt aaa
gag aat tac gtc aca cta 2304 Thr Ile Gln Glu Gly Asn Asp Val Phe
Lys Glu Asn Tyr Val Thr Leu 755 760 765 ccg ggt act ttt aat gag tgt
tat ccg acg tat tta tat caa aaa ata 2352 Pro Gly Thr Phe Asn Glu
Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile 770 775 780 ggg gag tcg gaa
tta aaa gct tat act cgc tac caa tta aga ggt tat 2400 Gly Glu Ser
Glu Leu Lys Ala Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr 785 790 795 800
att gaa gat agt caa gat tta gag ata tat ttg att cgt tat aat gcg
2448 Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn
Ala 805 810 815 aaa cat gaa aca ttg gat gtt cca ggt acc gag tcc cta
tgg ccg ctt 2496 Lys His Glu Thr Leu Asp Val Pro Gly Thr Glu Ser
Leu Trp Pro Leu 820 825 830 tca gtt gaa agc cca atc gga agg tgc gga
gaa ccg aat cga tgc gca 2544 Ser Val Glu Ser Pro Ile Gly Arg Cys
Gly Glu Pro Asn Arg Cys Ala 835 840 845 cca cat ttt gaa tgg aat cct
gat cta gat tgt tcc tgc aga gat gga 2592 Pro His Phe Glu Trp Asn
Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly 850 855 860 gaa aaa tgt gcg
cat cat tcc cat cat ttc tct ttg gat att gat gtt 2640 Glu Lys Cys
Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val 865 870 875 880
gga tgc aca gac ttg cat gag aat cta ggc gtg tgg gtg gta ttc aag
2688 Gly Cys Thr Asp Leu His Glu Asn Leu Gly Val Trp Val Val Phe
Lys 885 890 895 att aag acg cag gaa ggt cat gca aga cta ggg aat ctg
gaa ttt att 2736 Ile Lys Thr Gln Glu Gly His Ala Arg Leu Gly Asn
Leu Glu Phe Ile 900 905 910 gaa gag aaa cca tta tta gga gaa gca ctg
tct cgt gtg aag agg gca 2784 Glu Glu Lys Pro Leu Leu Gly Glu Ala
Leu Ser Arg Val Lys Arg Ala 915 920 925 gag aaa aaa tgg aga gac aaa
cgt gaa aaa cta caa ttg gaa aca aaa 2832 Glu Lys Lys Trp Arg Asp
Lys Arg Glu Lys Leu Gln Leu Glu Thr Lys 930 935 940 cga gta tat aca
gag gca aaa gaa gct gtg gat gct tta ttc gta gat 2880 Arg Val Tyr
Thr Glu Ala Lys Glu Ala Val Asp Ala Leu Phe Val Asp 945 950 955 960
tct caa tat gat aga tta caa gcg gat aca aac atc ggc atg att cat
2928 Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile Gly Met Ile
His 965 970 975 gcg gca gat aaa ctt gtt cat cga att cga gag gcg tat
ctt tca gaa 2976 Ala Ala Asp Lys Leu Val His Arg Ile Arg Glu Ala
Tyr Leu Ser Glu 980 985 990 tta cct gtt atc cca ggt gta aat gcg gaa
att ttt gaa gaa tta gaa 3024 Leu Pro Val Ile Pro Gly Val Asn Ala
Glu Ile Phe Glu Glu Leu Glu 995 1000 1005 ggt cac att atc act gca
atc tcc tta tac gat gcg aga aat gtc 3069 Gly His Ile Ile Thr Ala
Ile Ser Leu Tyr Asp Ala Arg Asn Val 1010 1015 1020 gtt aaa aat ggt
gat ttt aat aat gga tta aca tgt tgg aat gta 3114 Val Lys Asn Gly
Asp Phe Asn Asn Gly Leu Thr Cys Trp Asn Val 1025 1030 1035 aaa ggg
cat gta gat gta caa cag agc cat cat cgt tct gac ctt 3159 Lys Gly
His Val Asp Val Gln Gln Ser His His Arg Ser Asp Leu 1040 1045 1050
gtt atc cca gaa tgg gaa gca gaa gtg tca caa gca gtt cgc gtc 3204
Val Ile Pro Glu Trp Glu Ala Glu Val Ser Gln Ala Val Arg Val 1055
1060 1065 tgt ccg ggg tgt ggc tat atc ctt cgt gtc aca gcg tac aaa
gag 3249 Cys Pro Gly Cys Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys
Glu 1070 1075 1080 gga tat gga gag ggc tgc gta acg atc cat gaa atc
gag aac aat 3294 Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile
Glu Asn Asn 1085 1090 1095 aca gac gaa cta aaa ttt aaa aac cgt gaa
gaa gag gaa gtg tat 3339 Thr Asp Glu Leu Lys Phe Lys Asn Arg Glu
Glu Glu Glu Val Tyr 1100 1105 1110 cca acg gat aca gga acg tgt aat
gat tat act gca cac caa ggt 3384 Pro Thr Asp Thr Gly Thr Cys Asn
Asp Tyr Thr Ala His Gln Gly 1115 1120 1125 aca gct gga tgc gca gat
gca tgt aat tcc cgt aat gct gga tat 3429 Thr Ala Gly Cys Ala Asp
Ala Cys Asn Ser Arg Asn Ala Gly Tyr 1130 1135 1140 gag gat gca tat
gaa gtt gat act aca gca tct gtt aat tac aaa 3474 Glu Asp Ala Tyr
Glu Val Asp Thr Thr Ala Ser Val Asn Tyr Lys 1145 1150 1155 ccg act
tat gaa gaa gaa acg tat aca gat gta aga aga gat aat 3519 Pro Thr
Tyr Glu Glu Glu Thr Tyr Thr Asp Val Arg Arg Asp Asn 1160 1165 1170
cat tgt gaa tat gac aga ggg tat gtc aat tat cca cca gta cca 3564
His Cys Glu Tyr Asp Arg Gly Tyr Val Asn Tyr Pro Pro Val Pro 1175
1180 1185 gct ggt tat gtg aca aaa gaa tta gaa tac ttc cca gaa aca
gat 3609 Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr
Asp 1190 1195 1200 aca gta tgg att gag att gga gaa acg gaa gga aag
ttt att gta 3654 Thr Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Lys
Phe Ile Val 1205 1210 1215 gat agc gtg gaa tta ctc ctc atg gaa gaa
tag 3687 Asp Ser Val Glu Leu Leu Leu Met Glu Glu 1220 1225 10 1228
PRT Bacillus thuringiensis 10 Leu Thr Ser Asn Arg Lys Asn Glu Asn
Glu Ile Ile Asn Ala Val Ser 1 5 10 15 Asn His Ser Ala Gln Met Asp
Leu Leu Pro Asp Ala Arg Ile Glu Asp 20 25 30 Ser Leu Cys Ile Ala
Glu Gly Asn Asn Ile Asp Pro Phe Val Ser Ala 35 40 45 Ser Thr Val
Gln Thr Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly Val 50 55 60 Leu
Gly Val Pro Phe Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe Leu 65 70
75 80 Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Gln Trp Glu Ile Phe
Leu 85 90 95 Glu His Val Glu Gln Leu Ile Asn Gln Gln Ile Thr Glu
Asn Ala Arg 100 105 110 Asn Thr Ala Leu Ala Arg Leu Gln Gly Leu Gly
Asp Ser Phe Arg Ala 115 120 125 Tyr Gln Gln Ser Leu Glu Asp Trp Leu
Glu Asn Arg Asp Asp Ala Arg 130 135 140 Thr Arg Ser Val Leu His Thr
Gln Tyr Ile Ala Leu Glu Leu Asp Phe 145 150 155 160 Leu Asn Ala Met
Pro Leu Phe Ala Ile Arg Asn Gln Glu Val Pro Leu 165 170 175 Leu Met
Val Tyr Ala Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg 180 185 190
Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu Thr Ser Gln Glu Ile 195
200 205 Gln Arg Tyr Tyr Glu Arg Gln Val Glu Arg Thr Arg Asp Tyr Ser
Asp 210 215 220 Tyr Cys Val Glu Trp Tyr Asn Thr Gly Leu Asn Ser Leu
Arg Gly Thr 225 230 235 240 Asn Ala Ala Ser Trp Val Arg Tyr Asn Gln
Phe Arg Arg Asp Leu Thr 245 250 255 Leu Gly Val Leu Asp Leu Val Ala
Leu Phe Pro Ser Tyr Asp Thr Arg 260 265 270 Thr Tyr Pro Ile Asn Thr
Ser Ala Gln Leu Thr Arg Glu Val Tyr Thr 275 280 285 Asp Ala Ile Gly
Ala Thr Gly Val Asn Met Ala Ser Met Asn Trp Tyr 290 295 300 Asn Asn
Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile Arg 305 310 315
320 Ser Pro His Leu Leu Asp Phe Leu Glu Gln Leu Thr Ile Phe Ser Ala
325 330 335 Ser Ser Arg Trp Ser Asn Thr Arg His Met Thr Tyr Trp Arg
Gly His 340 345 350 Thr Ile Gln Ser Arg Pro Ile Gly Gly Gly Leu Asn
Thr Ser Thr His 355 360 365 Gly Ala Thr Asn Thr Ser Ile Asn Pro Val
Thr Leu Arg Phe Ala Ser 370 375 380 Arg Asp Val Tyr Arg Thr Glu Ser
Tyr Ala Gly Val Leu Leu Trp Gly 385 390 395 400 Ile Tyr Leu Glu Pro
Ile His Gly Val Pro Thr Val Arg Phe Asn Phe 405 410 415 Thr Asn Pro
Gln Asn Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gln 420 425 430 Pro
Tyr Glu Ser Pro Gly Leu Gln Leu Lys Asp Ser Glu Thr Glu Leu 435 440
445 Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg
450 455 460 Leu Ser His Ile Gly Ile Ile Leu Gln Ser Arg Val Asn Val
Pro Val 465 470 475 480 Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr
Asn Thr Ile Gly Pro 485 490 495 Asn Arg Ile Thr Gln Ile Pro Met Val
Lys Ala Ser Glu Leu Pro Gln 500 505 510 Gly Thr Thr Val Val Arg Gly
Pro Gly Phe Thr Gly Gly Asp Ile Leu 515 520 525 Arg Arg Thr Asn Thr
Gly Gly Phe Gly Pro Ile Arg Val Thr Val Asn 530 535 540 Gly Pro Leu
Thr Gln Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser Thr 545 550 555 560
Val Asp Phe Asp Phe Phe Val Ser Arg Gly Gly Thr Thr Val Asn Asn 565
570 575 Phe Arg Phe Leu Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr
Gly 580 585 590 Asn Phe Val Arg Arg Ala Phe Thr Thr Pro Phe Thr Phe
Thr Gln Ile 595 600 605 Gln Asp Ile Ile Arg Thr Ser Ile Gln Gly Leu
Ser Gly Asn Gly Glu 610 615 620 Val Tyr Ile Asp Lys Ile Glu Ile Ile
Pro Val Thr Ala Thr Phe Glu 625 630 635 640 Ala Glu Tyr Asp Leu Glu
Arg Ala Gln Glu Ala Val Asn Ala Leu Phe 645 650 655 Thr Asn Thr Asn
Pro Arg Arg Leu Lys Thr Asp Val Thr Asp Tyr His 660 665 670 Ile Asp
Gln Val Ser Asn Leu Val Ala Cys Leu Ser Asp Glu Phe Cys 675 680 685
Leu Asp Glu Lys Arg Glu Leu Leu Glu Lys Val Lys Tyr Ala Lys Arg 690
695 700 Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Thr Ser
Ile 705 710 715 720 Asn Lys Gln Pro Asp Phe Ile Ser Thr Asn Glu Gln
Ser Asn Phe Thr 725 730 735 Ser Ile His Glu Gln Ser Glu His Gly Trp
Trp Gly Ser Glu Asn Ile 740 745 750 Thr Ile Gln Glu Gly Asn Asp Val
Phe Lys Glu Asn Tyr Val Thr Leu 755 760 765 Pro Gly Thr Phe Asn Glu
Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile 770 775 780 Gly Glu Ser Glu
Leu Lys Ala Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr 785 790 795 800 Ile
Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala 805 810
815 Lys His Glu Thr Leu Asp Val Pro Gly Thr Glu Ser Leu Trp Pro Leu
820 825 830 Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu Pro Asn Arg
Cys Ala 835 840 845 Pro His Phe Glu Trp Asn Pro Asp Leu Asp Cys Ser
Cys Arg Asp Gly 850 855 860 Glu Lys Cys Ala His His Ser His His Phe
Ser Leu Asp Ile Asp Val 865 870 875 880 Gly Cys Thr Asp Leu His Glu
Asn Leu Gly Val Trp Val Val Phe Lys 885 890 895 Ile Lys Thr Gln Glu
Gly His Ala Arg Leu Gly Asn Leu Glu Phe Ile 900 905 910 Glu Glu Lys
Pro Leu Leu Gly Glu Ala Leu Ser Arg Val Lys Arg Ala 915 920 925 Glu
Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Gln Leu Glu Thr Lys 930 935
940 Arg Val Tyr Thr Glu Ala Lys Glu Ala Val Asp Ala Leu Phe Val Asp
945 950 955 960 Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile Gly
Met Ile His 965 970 975 Ala Ala Asp Lys Leu Val His Arg Ile Arg Glu
Ala Tyr Leu Ser Glu 980 985 990 Leu Pro Val Ile Pro Gly Val Asn Ala
Glu Ile Phe Glu Glu Leu Glu 995 1000 1005 Gly His Ile Ile Thr Ala
Ile Ser Leu Tyr Asp Ala Arg Asn Val 1010 1015 1020 Val Lys Asn Gly
Asp Phe Asn Asn Gly Leu Thr Cys Trp Asn Val 1025 1030 1035 Lys Gly
His Val Asp Val Gln Gln Ser His His Arg Ser Asp Leu 1040 1045 1050
Val Ile Pro Glu Trp Glu Ala Glu Val Ser Gln Ala Val Arg Val 1055
1060 1065 Cys Pro Gly Cys Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys
Glu 1070 1075 1080 Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile
Glu Asn Asn 1085 1090 1095 Thr Asp Glu Leu Lys Phe Lys Asn Arg Glu
Glu Glu Glu Val Tyr 1100 1105 1110 Pro Thr Asp Thr Gly Thr Cys Asn
Asp Tyr Thr Ala His Gln Gly 1115 1120 1125 Thr Ala Gly Cys Ala Asp
Ala Cys Asn Ser Arg Asn Ala Gly Tyr 1130 1135 1140 Glu Asp Ala Tyr
Glu Val Asp Thr Thr Ala Ser Val Asn Tyr Lys 1145 1150 1155 Pro Thr
Tyr Glu Glu Glu Thr Tyr Thr Asp Val Arg Arg Asp Asn 1160 1165 1170
His Cys Glu Tyr Asp Arg Gly Tyr Val Asn Tyr Pro Pro Val Pro 1175
1180 1185 Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr
Asp 1190 1195 1200 Thr Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Lys
Phe Ile Val 1205 1210 1215 Asp Ser Val Glu Leu Leu Leu Met Glu Glu
1220 1225
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