U.S. patent application number 10/855535 was filed with the patent office on 2004-11-04 for antibodies immunoreactive with lepidopteran-toxic polypeptides and methods of use.
This patent application is currently assigned to Monsanto Technology LLC.. Invention is credited to Baum, James A., Gilmer, Amy Jelen, Mettus, Anne-Marie Light.
Application Number | 20040221334 10/855535 |
Document ID | / |
Family ID | 25048194 |
Filed Date | 2004-11-04 |
United States Patent
Application |
20040221334 |
Kind Code |
A1 |
Baum, James A. ; et
al. |
November 4, 2004 |
Antibodies immunoreactive with lepidopteran-toxic polypeptides and
methods of use
Abstract
Disclosed are novel synthetically-modified B. thuringiensis
nucleic acid segments encoding .delta.-endotoxins having
insecticidal activity against lepidopteran insects. Also disclosed
are synthetic crystal proteins encoded by these novel nucleic acid
sequences. Methods of making and using these genes and proteins are
disclosed as well as methods for the recombinant expression, and
transformation of suitable host cells. Transformed host cells and
transgenic plants expressing the modified endotoxin are also
aspects of the invention. Also disclosed are methods for modifying,
altering, and mutagenizing specific loop regions between the
.alpha. helices in domain 1 of these crystal proteins, including
Cry1C, to produce genetically-engineered recombinant cry* genes,
and the proteins they encode which have improved insecticidal
activity. In preferred embodiments, novel Cry1C* amino acid
segments and the modified cry1C* nucleic acid sequences which
encode them are disclosed.
Inventors: |
Baum, James A.; (Doylestown,
PA) ; Gilmer, Amy Jelen; (Langhorne, PA) ;
Mettus, Anne-Marie Light; (Feasterville, PA) |
Correspondence
Address: |
Any G. Klann
Howrey Simon Arnold & White, LLP
750 Bering Drive
Houston
TX
77057-2198
US
|
Assignee: |
Monsanto Technology LLC.
|
Family ID: |
25048194 |
Appl. No.: |
10/855535 |
Filed: |
May 27, 2004 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10855535 |
May 27, 2004 |
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09972175 |
Oct 5, 2001 |
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09972175 |
Oct 5, 2001 |
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09337635 |
Jun 21, 1999 |
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6313378 |
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09337635 |
Jun 21, 1999 |
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08980071 |
Nov 26, 1997 |
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5914318 |
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08980071 |
Nov 26, 1997 |
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08757536 |
Nov 27, 1996 |
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5942664 |
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Current U.S.
Class: |
800/279 ;
435/419; 435/468; 536/23.7 |
Current CPC
Class: |
Y10S 428/913 20130101;
C12N 1/205 20210501; Y10S 428/914 20130101; C12N 15/8286 20130101;
B41M 5/395 20130101; C07K 14/325 20130101; Y02A 40/146 20180101;
Y10T 428/31855 20150401; C12R 2001/075 20210501 |
Class at
Publication: |
800/279 ;
536/023.7; 435/419; 435/468 |
International
Class: |
A01H 001/00; C12N
015/82; C07H 021/04; C12N 005/04 |
Claims
1-40. (Cancelled).
41. A purified antibody generated by using a polypeptide comprising
SEQ ID NO: 59 or 61 as an immunogen.
42. (Cancelled)
43. (Cancelled)
44. (Cancelled)
45-58. (Cancelled).
59. A purified antibody generated by using a Bacillus thuringiensis
Cry1C .delta. endotoxin polypeptide as an immunogen, wherein: the
polypeptide comprises one or more amino acid mutations in the loop
region between .alpha. helices 4 and 5 of domain 1; and the
polypeptide comprises one or more amino acid mutations in the loop
region between .alpha. helices 6 and 7 of domain 1; and the
polypeptide exhibits improved insecticidal activity against insects
relative to a native Cry1C .delta.-endotoxin polypeptide.
60. The antibody of claim 59, wherein said polypeptide has an
alanine, arginine, asparagine, aspartic acid, cysteine, glutamic
acid, glutamine, glycine, histidine, isoleucine, leucine,
methionine, phenylalanine, proline, serine, threonine, tryptophan,
tyrosine, or valine residue for amino acid 219, and an alanine,
asparagine, aspartic acid, cysteine, glutamic acid, glutamine,
glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or
valine residue for amino acid 148.
61. The antibody of claim 60, wherein said polypeptide has an
alanine, leucine, methionine, glycine, or aspartic acid residues
for amino acids 219 and 148.
62. The antibody of claim 59, wherein said antibody is generated by
using a polypeptide comprising SEQ ID NO: 59 or 61 as an
immunogen.
63. The antibody of claim 41 or 59, operatively attached to a
detectable label.
64. An immunodetection kit comprising, in suitable container means,
the antibody according to claim 41 or 59, and an immunodetection
reagent.
65. A method for detecting an insecticidal polypeptide in a
biological sample comprising contacting a biological sample
suspected of containing said insecticidal polypeptide with an
antibody in accordance with claim 41 or 59, under conditions
effective to allow the formation of immunocomplexes, and detecting
the immunocomplexes so formed.
Description
[0001] The present invention is a continuation-in-part of U.S.
patent application Ser. No. 08/757,536, filed Nov. 27, 1996, the
entire contents of which is specifically incorporated herein by
reference.
1.0 BACKGROUND OF THE INVENTION
[0002] 1.1 Field of the Invention
[0003] The present invention relates generally to the fields of
insect control. Certain embodiments concern methods and
compositions comprising nucleic acid segments which encode Bacillus
thuringiensis-derived .delta.-endotoxins. Disclosed are methods of
altering Cry1 crystal proteins by mutagenesis of the loop regions
between the .alpha.-helices of the protein's domain 1 or of the
loop region between .alpha.-helix 7 of domain 1 and .beta.-strand 1
of domain 2 to give rise to modified Cry1 proteins (Cry1 .star.)
which have improved activity against Lepidopteran insects. Various
methods for making and using these recombinantly-engineered
proteins and nucleic acid segments, including development of
transgenic plant cells and recombinant host cells are also
disclosed.
[0004] 1.2 Description of the Related Art
[0005] The most widely used microbial pesticides are derived from
the bacterium Bacillus thuringiensis. B. thuringiensis is a
Gram-positive bacterium that produces crystal proteins which are
specifically toxic to certain orders and species of insects. Many
different strains of B. thuringiensis have been shown to produce
insecticidal crystal proteins. Compositions including B.
thuringiensis strains which produce insecticidal proteins have been
commercially-available and used as environmentally-acceptable
insecticides because they are quite toxic to the specific target
insect, but are harmless to plants and other non-targeted
organisms.
[0006] .delta.-endotoxins are used to control a wide range of
leaf-eating caterpillars and beetles, as well as mosquitoes. B.
thuringiensis produces a proteinaceous parasporal body or crystal
which is toxic upon ingestion by a susceptible insect host. For
example, B. thuringiensis subsp. kurstaki HD-1 produces a crystal
inclusion comprising .delta.-endotoxins which are toxic to the
larvae of a number of insects in the order Lepidoptera (Schnepf and
Whiteley, 1981).
1.2.1 .delta.-Endotoxins
[0007] .delta.-endotoxins are a large collection of insecticidal
proteins produced by B. thuringiensis. Over the past decade
research on the structure and function of B. thuringiensis toxins
has covered all of the major toxin categories, and while these
toxins differ in specific structure and function, general
similarities in the structure and function are assumed. Based on
the accumulated knowledge of B. thuringiensis toxins, a generalized
mode of action for B. thuringiensis toxins has been created and
includes: ingestion by the insect, solubilization in the insect
midgut (a combination stomach and small intestine), resistance to
digestive enzymes sometimes with partial digestion actually
"activating" the toxin, binding to the midgut cells, formation of a
pore in the insect cells and the disruption of cellular homeostasis
(English and Slatin, 1992).
1.2.2 Genes Encoding Crystal Proteins
[0008] Many of the .delta.-endotoxins are related to various
degrees by similarities in their amino acid sequences.
Historically, the proteins and the genes which encode them were
classified based largely upon their spectrum of insecticidal
activity. The review by Hofte and Whiteley (1989) discusses the
genes and proteins that were identified in B. thuringiensis prior
to 1990, and sets forth the nomenclature and classification scheme
which has traditionally been applied to B. thuringiensis genes and
proteins. cryI genes encode lepidopteran-toxic CryI proteins. cryII
genes encode CryII proteins that are toxic to both lepidopterans
and dipterans. cryIII genes encode coleopteran-toxic CryIII
proteins, while cryIV genes encode dipteran-toxic CryIV
proteins.
[0009] Based on the degree of sequence similarity, the proteins
were further classified into subfamilies; more highly related
proteins within each family were assigned divisional letters such
as CryIA, CryIB, CryIC, etc. Even more closely related proteins
within each division were given names such as CryIC1, CryIC2,
etc.
[0010] Recently a new nomenclature has been proposed which
systematically classifies the Cry proteins based upon amino acid
sequence homology rather than upon insect target specificities.
This classification scheme is summarized in Table 1.
1TABLE 1 REVISED B. THURINGIENSIS .delta.-ENDOTOXIN
NOMENCLATURE.sup.a New Old GenBank Accession # Cry1Aa CryIA(a)
M11250 Cry1Ab CryIA(b) M13898 Cry1Ac CryIA(c) M11068 Cry1Ad
CryIA(d) M73250 Cry1Ae CryIA(e) M65252 Cry1Ba CryIB X06711 Cry1Bb
ET5 L32020 Cry1Bc PEG5 Z46442 Cry1Bd CryE1 U70726 Cry1Ca CryIC
X07518 Cry1Cb CryIC(b) M97880 Cry1Da CryID X54160 Cry1Db PrtB
Z22511 Cry1Ea CryIE X53985 Cry1Eb CryIE(b) M73253 Cry1Fa CryIF
M63897 Cry1Fb PrtD Z22512 Cry1Ga PrtA Z22510 Cry1Gb CryH2 U70725
Cry1Ha PrtC Z22513 Cry1Hb U35780 Cry1Ia CryV X62821 Cry1Ib CryV
U07642 Cry1Ja ET4 L32019 Cry1Jb ET1 U31527 Cry1K U28801 Cry2Aa
CryIIA M31738 Cry2Ab CryIIB M23724 Cry2Ac CryIIC X57252 Cry3A
CryIIIA M22472 Cry3Ba CryIIIB X17123 Cry3Bb CryIIIB2 M89794 Cry3C
CryIIID X59797 Cry4A CryIVA Y00423 Cry4B CryIVB X07423 Cry5Aa
CryVA(a) L07025 Cry5Ab CryVA(b) L07026 Cry5B U19725 Cry6A CryVIA
L07022 Cry6B CryVIB L07024 Cry7Aa CryIIIC M64478 Cry7Ab CryIIICb
U04367 Cry8A CryIIIE U04364 Cry8B CryIIIG U04365 Cry8C CryIIIF
U04366 Cry9A CryIG X58120 Cry9B CryIX X75019 Cry9C CryIH Z37527
Cry10A CryIVC M12662 Cry11A CryIVD M31737 Cry11B Jeg80 X86902
Cry12A CryVB L07027 Cry13A CryVC L07023 Cry14A CryVD U13955 Cry15A
34 kDa M76442 Cry16A cbm71 X94146 Cry17A cbm71 X99478 Cry18A CryBP1
X99049 Cry19A Jeg65 Y08920 Cyt1Aa CytA X03182 Cyt1Ab CytM X98793
Cyt1B U37196 Cyt2A CytB Z14147 Cyt2B CytB U52043 .sup.aAdapted
from: http://epunix.biols.susx.ac.uk/Home/Neil_Cri-
ckmore/Bt/index.html
1.2.3 Crystal Proteins Find Utility as Bioinsecticides
[0011] The utility of bacterial crystal proteins as insecticides
was extended when the first isolation of a coleopteran-toxic B.
thuringiensis strain was reported (Krieg et al., 1983; 1984). This
strain (described in U.S. Pat. No. 4,766,203, specifically
incorporated herein by reference), designated B. thuringiensis var.
tenebrionis, is reported to be toxic to larvae of the coleopteran
insects Agelastica alni (blue alder leaf beetle) and Leptinotarsa
decemlineata (Colorado potato beetle).
[0012] U.S. Pat. No. 5,024, 837 also describes hybrid B.
thuringiensis var. kurstaki strains which showed activity against
lepidopteran insects. U.S. Pat. No. 4,797,279 (corresponding to EP
0221024) discloses a hybrid B. thuringiensis containing a plasmid
from B. thuringiensis var. kurstaki encoding a lepidopteran-toxic
crystal protein-encoding gene and a plasmid from B. thuringiensis
tenebrionis encoding a coleopteran-toxic crystal protein-encoding
gene. The hybrid B. thuringiensis strain produces crystal proteins
characteristic of those made by both B. thuringiensis kurstaki and
B. thuringiensis tenebrionis. U.S. Pat. No. 4,910,016
(corresponding to EP 0303379) discloses a B. thuringiensis isolate
identified as B. thuringiensis MT 104 which has insecticidal
activity against coleopterans and lepidopterans.
1.2.4 Cry1 Endotoxins
[0013] The characterization of the lepidopteran-toxic B.
thuringiensis Cry1Aa crystal protein, and the cloning, DNA
sequencing, and expression of the gene which encodes it have been
described (Schnepf and Whitely, 1981; Schnepf et al., 1985). In
related publications, U.S. Pat. No. 4,448,885 and U.S. Pat. No.
4,467,036 (specifically incorporated herein by reference), the
expression of the native B. thuringiensis Cry1Aa crystal protein in
E. coli is disclosed.
[0014] Several cry1C genes have been described in the prior art. A
cry1C gene truncated at the 3' end was isolated from B.
thuringiensis subsp. aizawai 7.29 by Sanchis et al. (1988). The
truncated protein exhibited toxicity towards Spodoptera species.
The sequence of the truncated cry1C gene and its encoded protein
was disclosed in PCT WO 88/09812 and in Sanchis et al., (1989). The
sequence of a cry1C gene isolated from B. thuringiensis subsp.
entomocidus 60.5 was described by Honee et al., (1988). This gene
is recognized as the holotype cry1C gene by Hofte and Whiteley
(1989). The sequence of a cry1C gene is also described in U.S. Pat.
No. 5,126,133.
[0015] The cry1C gene from B. thuringiensis subsp. aizawai EG6346,
contained on plasmids pEG315 and pEG916 described herein, encodes a
Cry1C protein identical to that described in the aforementioned
U.S. Pat. No. 5,126,133. The Cry1C protein described by Sanchis et
al., (1989) and in PCT WO 88/09812 differs from the EG6346 Cry1C
protein at several positions that can be described as substitutions
within the EG6346 Cry1C protein: Cry1C N366I, W376C, P377Q, A378R,
P379H, P380H, V386G, R775A.
[0016] Significantly, the amino acid positions 376-380 correspond
to amino acid residues predicted to lie within the loop region
between .beta. strand 6 and .beta. strand 7 of Cry1C, using the
nomenclature adopted by Li et al. (1991) for identifying structures
within Cry3A. Bioassay comparisons between the Cry1C protein of
strain EG6346 and the Cry1C protein of strain aizawai 7.29 revealed
no significant differences in insecticidal activity towards S.
exigua, T. ni, or P. xylostella. These results suggested that the
two Cry1C proteins exhibited the same insecticidal specificity in
spite of their different amino acid sequences within the predicted
loop region between .beta. strand 6 and .beta. strand 7.
[0017] Smith and Ellar (1994) reported the cloning of a cry1C gene
from B. thuringiensis strain HD229 and demonstrated that amino acid
substitutions within the putative loop region between .beta. strand
6 and .beta. strand 7 ("loop .beta. 6-7") altered the insecticidal
specificity of Cry1C towards Spodoptera frugiperda and Aedes
aegypti but did not improve the toxicity of Cry1C towards either
insect pest. These results appeared to conflict with the
aforementioned bioassay comparison between the EG6346 Cry1C protein
and the aizawai 7.29 Cry1C protein showing no effect of amino acid
substitutions within loop .beta. 6-7 of Cry1C on insecticidal
specificity. Accordingly, the cry1C gene from strain aizawai 7.29
was re-sequenced where variant codons for the active toxin region
were reported by Sanchis et al., (1989) and in PCT WO 88/09812. The
results of that sequence analysis revealed no differences in the
amino acid sequences of the active toxins of Cry1C from strain
EG6346 and of Cry1C from strain aizawai 7.29. Thus, the prior art
on the Cry1C protein of strain aizawai 7.29, in light of the
aforementioned bioassay comparisons with the Cry1C protein of
strain EG6346, incorrectly taught that multiple amino acid
substitutions within loop .beta. 6-7 of Cry1C had no effect on
insecticidal specificity. Recently, Smith et al., (1996) also
reported unspecified sequencing errors in the aizawai 7.29 cry1C
gene.
1.2.5 Molecular Genetic Techniques Facilitate Protein
Engineering
[0018] The revolution in molecular genetics over the past decade
has facilitated a logical and orderly approach to engineering
proteins with improved properties. Site specific and random
mutagenesis methods, the advent of polymerase chain reaction
(PCR.TM.) methodologies, and related advances in the field have
permitted an extensive collection of tools for changing both amino
acid sequence, and underlying genetic sequences for a variety of
proteins of commercial, medical, and agricultural interest.
[0019] Following the rapid increase in the number and types of
crystal proteins which have been identified in the past decade,
researchers began to theorize about using such techniques to
improve the insecticidal activity of various crystal proteins. In
theory, improvements to .delta.-endotoxins should be possible using
the methods available to protein engineers working in the art, and
it was logical to assume that it would be possible to isolate
improved variants of the wild-type crystal proteins isolated to
date. By strengthening one or more of the aforementioned steps in
the mode of action of the toxin, improved molecules should provide
enhanced activity, and therefore, represent a breakthrough in the
field. If specific amino acid residues on the protein are
identified to be responsible for a specific step in the mode of
action, then these residues can be targeted for mutagenesis to
improve performance.
1.2.6 Structural Analyses of Crystal Proteins
[0020] The combination of structural analyses of B. thuringiensis
toxins followed by an investigation of the function of such
structures, motifs, and the like has taught that specific regions
of crystal protein endotoxins are, in a general way, responsible
for particular functions.
[0021] For example, the structure of Cry3A (Li et al., 1991) and
Cry1Aa (Grochulski et al., 1995) illustrated that the Cry1 and Cry3
.delta.-endotoxins have three distinct domains. Each of these
domains has, to some degree, been experimentally determined to
assist in a particular function. Domain 1, for example, from Cry3B2
and Cry1Ac has been found to be responsible for ion channel
activity, the initial step in formation of a pore (Walters et al.,
1993; Von Tersch et al., 1994). Domains 2 and 3 have been found to
be responsible for receptor binding and insecticidal specificity
(Aronson et al., 1995; Caramori et al., 1991; Chen et al. 1993; de
Maagd et al., 1996; Ge et al., 1991; Lee et al., 1992; Lee et al.,
1995; Lu et al., 1994; Smedley and Ellar, 1996; Smith and Ellar,
1994; Rajamohan et al., 1995; Rajamohan et al., 1996; Wu and Dean,
1996). Regions in domain 3 can also impact the ion channel activity
of some toxins (Chen et al., 1993, Wolfersberger et al., 1996).
1.3 Deficiencies in the Prior Art
[0022] Unfortunately, while many laboratories have attempted to
make mutated crystal proteins, few have succeeded in making mutated
crystal proteins with improved lepidopteran toxicity. In almost all
of the examples of genetically-engineered B. thuringiensis toxins
in the literature, the biological activity of the mutated crystal
protein is no better than that of the wild-type protein, and in
many cases, the activity is decreased or destroyed altogether
(Almond and Dean, 1993; Aronson et al., 1995; Chen et al., 1993,
Chen et al., 1995; Ge et al., 1991; Kwak et al., 1995; Lu et al.,
1994; Rajamohan et al., 1995; Rajamohan et al., 1996; Smedley and
Ellar, 1996; Smith and Ellar, 1994; Wolfersberger et al., 1996; Wu
and Aronson, 1992). For a crystal protein having approximately 650
amino acids in the sequence of its active toxin, and the
possibility of 20 different amino acids at each of these sites, the
likelihood of arbitrarily creating a successful new structure is
remote, even if a general function to a stretch of 250-300 amino
acids can be assigned. Indeed, the above prior art with respect to
crystal protein gene mutagenesis has been concerned primarily with
studying the structure and function of the crystal proteins, using
mutagenesis to perturb some step in the mode of action, rather than
with engineering improved toxins.
[0023] Several examples, however, do exist in the prior art where
improvements to biological activity were achieved by preparing a
recombinant crystal protein. Angsuthanasamnbat et al. (1993)
demonstrated that a stretch of amino acids in the dipteran-toxic
Cry4B delta-endotoxin is proteolytically sensitive and, by
repairing this site, the dipteran toxicity of this protein was
increased three-fold. In contrast, the elimination of a trypsin
cleavage site on the lepidopteran-toxic Cry9C protein was reported
to have no effect on insecticidal activity (Lambert et al., 1996).
In another example, Wu and Dean (1996) demonstrated that specific
changes to amino acids at residues 481-486 (domain 2) in the
coleopteran-toxic Cry3A protein increased the biological activity
of this protein by 2.4-fold against one target insect, presumably
by altering toxin binding. Finally, chimeric Cry1 proteins
containing exchanges of domain 2 or domain 3 sequences and
exhibiting improved toxicity have been reported, but there is no
evidence that toxicity has been improved for more than one
lepidopteran insect pest or that insecticidal activity towards
other lepidopteran pests has been retained (Caramori et al., 1991;
Ge et al., 1991, de Maagd et al., 1996). Based on the prior art,
exchanges involving domain 2 or domain 3 would be expected to
change insecticidal specificity.
[0024] The prior art also provides examples of Cry1A mutants
containing mutations encoding amino acid substitutions within the
predicted a helices of domain 1 (Wu and Aronson, 1992; Aronson et
al., 1995, Chen et al., 1995). None of these mutations resulted in
improved insecticidal activity and many resulted in a reduction in
activity, particularly those encoding substitutions within the
predicted helix 5 (Wu and Aronson, 1992). Extensive mutagenesis of
loop regions within domain 2 have been shown to alter the
insecticidal specificity of Cry1C but to not improve its toxicity
towards any one insect pest (Smith and Ellar, 1994). Similarly,
extensive mutagenesis of loop regions in domain 2 and of
.beta.-strand structures in domain 3 of the Cry1A proteins have
failed to produce Cry1A mutants with improved toxicity (Aronson et
al., 1995; Chen et al., 1993; Kwak et al., 1995; Smedley and Ellar,
1996; Rajamohan et al., 1995; Rajamohan et al., 1996). These
results demonstrate the difficulty in engineering improved
insecticidal proteins and illustrate that successful engineering of
B. thuringiensis toxins does not follow simple and predictable
rules.
[0025] Collectively, the limited successes in the art to develop
synthetic toxins with improved insecticidal activity have stifled
progress in this area and confounded the search for improved
endotoxins or crystal proteins. Rather than following simple and
predictable rules, the successful engineering of an improved
crystal protein may involve different strategies, depending on the
crystal protein being improved and the insect pests being targeted.
Thus, the process is highly empirical.
[0026] Accordingly, traditional recombinant DNA technology is
clearly not routine experimentation for providing improved
insecticidal crystal proteins. What are lacking in the prior art
are rational methods for producing genetically-engineered B.
thuringiensis Cry1 crystal proteins that have improved insecticidal
activity and, in particular, improved toxicity towards a wide range
of lepidopteran insect pests.
2.0 SUMMARY OF THE INVENTION
[0027] The present invention seeks to overcome these and other
drawbacks inherent in the prior art by providing
genetically-engineered modified B. thuringiensis Cry1
.delta.-endotoxin genes, and in particular, cry1C genes, that
encode modified crystal proteins having improved insecticidal
activity against lepidopterans. Disclosed are novel methods for
constructing synthetic Cry1 proteins, synthetically-modified
nucleic acid sequences encoding such proteins, and compositions
arising therefrom. Also provided are synthetic cry1* expression
constructs and various methods of using the improved genes and
vectors. In a preferred embodiment, the invention discloses and
claims Cry1C* proteins and cry1C* genes which encode the modified
proteins.
[0028] An isolated nucleic acid segment that encodes a polypeptide
having insecticidal activity against Lepidopterans is one aspect of
the invention. Such a nucleic acid segment is isolatable from
Bacillus thuringiensis NRRL B-21590, NRRL B-21591, NRRL B-21592,
NRRL B-21638, NRRL B-21639, NRRL B-21640, NRRL B-21609, or NRRL
B-21610, and preferably encodes a polypeptide comprising the amino
acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:59 or SEQ ID NO:61. Exemplary
nucleic acid segments specifically hybridizes to, or comprise the
nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:58, or SEQ ID NO:60
or a complement thereof.
[0029] In certain embodiments, such a nucleic acid segment may be
operably linked to a promoter that expresses the nucleic acid
segment in a host cell. In those instances, the nucleic acid
segment is typically comprised within a recombinant vector such as
a plasmid, cosmid, phage, phagemid, viral, baculovirus, bacterial
artificial chromosome, or yeast artificial chromsome. As such, the
nucleic acid segment may be used in a recombinant expression method
to prepare a recombinant polypeptide, to prepare an insect
resistant transgenic plant, or to express the nucleic acid segment
in a host cell.
[0030] A further aspect of the invention is a host cell which
comprises one or more of the nucleic acid segment disclosed herein
which encode a modified Cry1* protein. Preferred host cells include
bacterial cells, such as E. coli, B. thuringiensis, B. subtilis, B.
megalerium, or Pseudomonas spp. cells, with B. thuringiensis NRRL
B-21590, NRRL B-21591, NRRL B-21592, NRRL B-21638, NRRL B-21639,
NRRL B-21640, NRRL B-21609, and NRRL B-21610 cells being highly
preferred. Another preferred host cell is an eukaryotic cell such
as a fungal, animal, or plant cell, with plant cells such as grain,
tree, vegetable, fruit, berry, nut, grass, cactus, succulent, and
ornamental plant cells being highly preferred. Transgenic plant
cells such as corn, rice, tobacco, potato, tomato, flax, canola,
sunflower, cotton, wheat, oat, barley, and rye cells are
particularly preferred.
[0031] Host cells which produce one or more of the polypeptide
having insecticidal activity against Lepidopterans, host cells
which are useful in preparation of recombinant toxin polypeptides,
and host cells used in the preparation of a transgenic plant or in
generation of pluripotent plant cells represent important aspects
of the invention. Such host cells may find particular use in the
preparation of an insecticidal polypeptide formulation, such as a
polypeptide that comprises the amino acid sequence of SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:59, or SEQ ID NO:61, and which is insecticidally active
against Lepidopterans.
[0032] A polypeptide composition such as those described herein are
particularly desirable for use in killing an insect cell, and in
the preparation of an insecticidal formulation, such as a plant
protective spray formulation. The polypeptide composition may be
prepared by culturing a B. thuringiensis NRRL B-21590, NRRL
B-21591, NRRL B-21592, NRRL B-21638, NRRL B-21639, NRRL B-21640,
NRRL B-21609, or NRRL B-21610 cell under conditions effective to
produce a B. thuringiensis crystal protein; and obtaining the B.
thuringiensis crystal protein from the cell.
[0033] The polypeptide may be used in a method of killing an insect
cell. This method generally involves providing to an insect cell an
insecticidally-effective amount of the polypeptide composition.
Typically, the insect cell is comprised within an insect, and the
insect is killed by ingesting the composition directly, or
alternatively by ingesting a plant coated with the composition, or
ingesting a transgenic plant which expresses the polypeptide
composition.
[0034] Another important embodiment of the invention is a purified
antibody that specifically binds to a polypeptide having the amino
acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:59, or SEQ ID NO:61.
Such antibody compositions may be operatively attached to a
detectable label, or comprised within an immunodetection kit. Such
antibodies find particular use in methods for detecting an
insecticidal polypeptide in a biological sample. The method
generally involves contacting a biological sample suspected of
containing such a polypeptide with an antibody under conditions
effective to allow the formation of immunecomplexes, and detecting
the immunecomplexes so formed.
[0035] A transgenic plant having incorporated into its genome a
transgene that encodes a polypeptide comprising the amino sequence
of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO: 12, SEQ ID NO:59, or SEQ ID NO:61 also represents
an important embodiment of the present invention. Such a transgenic
plant preferably comprises the nucleic acid sequence of SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:58, or SEQ ID NO:60. Progeny and seed from such a
plant and its progeny are also important aspects of the
invention.
[0036] A method of selecting a Cry1 polypeptide having increased
insecticidal activity against a Lepidopteran insect comprising
mutagenizing a population of polynucleotides to prepare a
population of polypeptides encoded by said polynucleotides and
testing said population of polypeptides and identifying a
polypeptide having one or more modified amino acids in a loop
region of domain I or in a loop region between domain 1 and domain
2, wherein said polypeptide has increased insecticidal activity
against said insects.
[0037] Another important embodiment of the invention is a method of
generating a Cry1 polypeptide having increased insecticidal
activity against a Lepidopteran insect. Such a method generally
involves identifying in such a polypeptide a loop region between
adjacent .alpha.-helices of domain 1 or between an .alpha.-helix of
domain 1 and a .beta. strand of domain 2, then mutagenizing the
polypeptide in at least one or more amino acids of one or more of
the identified loop regions; and, finally, testing the mutagenized
polypeptide to identify a polypeptide having increased insecticidal
activity against a Lepidopteran pest.
[0038] A method of mutagenizing a Cry1 polypeptide to increase the
insecticidal activity of the polypeptide against a Lepidopteran
insect is also provided by the invention. This method comprises
predicting in such a polypeptide a contiguous amino acid sequence
encoding a loop region between adjacent .alpha.-helices of domain 1
or between an .alpha.-helix of domain 1 and a .beta. strand of
domain 2; mutagenizing one or more of these amino acid residues to
produce a population of polypeptides having one or more altered
loop regions; testing the population of polypeptides for
insecticidal activity against Lepidopterans; and identifying a
polypeptide in the population which has increased insecticidal
activity against a Lepidopteran insect.
[0039] In such methods, the modified amino acid sequence preferably
comprises a loop region between .alpha. helices 1 and 2a, .alpha.
helices 2b and 3, .alpha. helices 3 and 4, .alpha. helices 4 and 5,
.alpha. helices 5 and 6, or .alpha. helices 6 and 7 of domain 1, or
between .alpha. helix 7 of domain 1 and .beta. strand 1 of domain
2. Preferably, the loop region between .alpha. helices 1 and 2a
comprises an amino acid sequence of from about amino acid 41 to
about amino acid 47 of a Cry1 protein. Likewise, the loop region
between .alpha. helices 2b and 3 comprises an amino acid sequence
of from about amino acid 83 to about amino acid 89 of a Cry1
protein, and the loop region between .alpha. helices 3 and 4
comprises an amino acid sequence of from about amino acid 118 to
about amino acid 124 of a Cry1 protein. The loop region between
.alpha. helices 4 and 5 preferably comprises an amino acid sequence
of from about amino acid 148 to about amino acid 156 of a Cry1
protein, while the loop region between a helices 5 and 6 comprises
an amino acid sequence of from about amino acid 176 to about amino
acid 85 of a Cry1 protein. The loop loop region between .alpha.
helices 6 and 7 preferably comprises an amino acid sequence of from
about amino acid 217 to about amino acid 222 of a Cry1 protein,
while the loop region between .alpha. helix 7 of domain 1 and
.beta. strand 1 of domain 2 preferably comprises an amino acid
sequence of from about amino acid 249 to about amino acid 259 of a
Cry1 protein.
[0040] Exemplary Cry1 proteins include Cry1A, Cry1B, Cry1C, Cry1D,
Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, and Cry1K crystal
proteins, with Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Ba,
Cry1Bb, Cry1Bc, Cry1Ca, Cry1Cb, Cry1Da, Cry1Db, Cry1Ea, Cry1Eb,
Cry1Fa, Cry1Fb, Cry1Hb, Cry1Ia, Cry1Ib, Cry1Ja, and Cry1Jb crystal
proteins being highly preferred.
[0041] These loop region mutations may include changing any one or
more amino acids to any other amino acid, so long as the resulting
protein has increased Lepidopteran insecticidal activity. The
inventors have shown that exemplary substitutions such as changing
one or more arginine residues to any other amino acid results in
polypeptides having increased insecticidal activity. Particularly
preferred substitutions of arginine residues include those
substituted by alanine, leucine, methionine, glycine or aspartic
acid. Likewise, the inventors have shown that substitution of
lysine residues by any other amino acid, such as an alanine
residue, also results in insecticidally-activetoxins. Indeed any
such modification is contemplated by the inventors to be useful, so
long as the substitution, addition, deletion, or modification of
one or more of the amino acid residues in the preferred loop region
results in a polypeptide which has improved insecticidal activity
when compared to an unmodified Cry1 polypeptide. The inventors
contemplate that combinatorial mutants as described herein will
find particular use in the generation of a polypeptide having one
or more mutations in multiple loop regions, or alternatively, in
the generation of a polypeptide having multiple mutations with a
single loop region. Such combinatorial mutants, as the inventors
have shown herein often result in mutagenized polypeptides which
have significantly improved insecticidal activity over the
wild-type unmodified sequence.
[0042] Of course, one of skill in the art will realize that these
amino acid modifications need not be made in the polypeptides
themselves (although chemical synthesis of such polypeptides is
well-known to those of skill in the art), but may also be made via
mutagenesis of a nucleic acid segment which encodes such a
polypeptide. Means for such DNA mutagenesis are described herein in
detail, and exemplary polypeptides constructed using such methods
are described in detail in the Examples which follow herein.
2.1 Mutagenized Cry1 Genes and Polypeptides
[0043] Accordingly, the present invention provides mutagenized
Cry1C protein genes and methods of making and using such genes. As
used herein the term "mutagenized Cry1C protein gene(s)" means one
or more genes that have been mutagenized or altered to contain one
or more nucleotide sequences which are not present in the wild type
sequences, and which encode mutant Cry1C crystal proteins (Cry1C*)
showing improved insecticidal activity. Preferably the novel
sequences comprise nucleic acid sequences in which at least one,
and preferably, more than one, and most preferably, a significant
number, of wild-type Cry1C nucleotides have been replaced with one
or more nucleotides, or where one or more nucleotides have been
added to or deleted from the native nucleotide sequence for the
purpose of altering, adding, or deleting the corresponding amino
acids encoded by the nucleic acid sequence so mutagenized. The
desired result, therefore, is alteration of the amino acid sequence
of the encoded crystal protein to provide toxins having improved or
altered activity and/or specificity compared to that of the
unmodified crystal protein. Modified cry1C gene sequences have been
termed cry1C* by the inventors, while modified Cry1C crystal
proteins encoded therein are termed Cry1C* proteins.
[0044] Contrary to the teachings of the prior art which have
focused attention on the .alpha.-helices of crystal proteins as
sites for genetic engineering to improve toxin activity, the
present invention differs markedly by providing methods for
creating modified loop regions between adjacent .alpha.-helices
within one or more of the protein's domains. In a particular
illustrative embodiment, the inventors have shown remarkable
success in generating toxins with improved insecticidal activity
using these methods. In particular, the inventors have identified
unique loop regions within domain 1 of a Cry1 crystal protein which
have been targeted for specific and random mutagenesis.
[0045] In a preferred embodiment, the inventors have identified the
predicted loop regions between .alpha.-helices 1 and 2a;
.alpha.-helices 2b and 3; .alpha.-helices 3 and 4; .alpha.-helices
4 and 5; .alpha.-helices 5 and 6, .alpha.-helices 6 and 7; and
between .alpha.-helix 7 and .beta.-strand 1in Cry1 crystal
proteins. Using Cry1C as an exemplary model, the inventors have
generated amino acid substitutions within or adjacent to these
predicted loop regions to produce synthetically-modified Cry1C
toxins which demonstrated improved insecticidal activity. In
mutating specific residues within these loop regions, the inventors
were able to produce synthetic crystal proteins which retained or
possessed enhanced insecticidal activity against certain
lepidopteran pests, including the beet armyworm, S. exigua.
[0046] Claimed is an isolated B. thuringiensis crystal protein that
has one or more modified amino acid sequences in one or more loop
regions of domain 1, or between .alpha. helix 7 of domain 1 and
.beta. strand 1 of domain 2. These synthetically-modified crystal
proteins have insecticidal activity against Lepidopteran insects.
The modified amino acid sequences may occur in one or more of the
following loop regions: between .alpha. helices 1 and 2a, .alpha.
helices 2b and 3, .alpha. helices 3 and 4, .alpha. helices 4 and 5,
.alpha. helices 5 and 6, .alpha. helices 6 and 7 of domain 1, or
between the .alpha. helix 7 of domain 1 and .beta. strand 1 of
domain 2.
[0047] In an illustrative embodiment, the invention encompasses
modifications which may be made in or immediately adjacent to the
loop region between .alpha. helices 1 and 2a of a Cry1C protein.
This loop region extends from about amino acid 42 to about amino
acid 46, with adjacent amino acids extending from about amino acid
39 to about amino acid 41 and from about amino acid 47 to about
amino acid 49.
[0048] The invention also encompasses modifications which may be
made in or immediately adjacent to the loop region between .alpha.
helices 2b and 3 of a Cry1C protein. This loop region extends from
about amino acid 84 to about amino acid 88, with adjacent amino
acids extending from about amino acid 81 to about amino acid 83,
and from about amino acid 89 to about amino acid 91.
[0049] The invention also encompasses modifications which may be
made in or immediately adjacent to the loop region between .alpha.
helices 3 and 4 of a Cry1C protein. This loop region extends from
about amino acid 119 to about amino acid 123. with the adjacent
amino acids extending from about amino acid 116 to about amino acid
118, and from about amino acid 124 to about amino acid 126.
[0050] Likewise, the invention also encompasses modifications which
may be made in or immediately adjacent to the loop region between
.alpha. helices4 and 5 of a Cry1C protein. This loop region extends
from about amino acid 149 to about amino acid 155, with the
adjacent amino acids extending from about amino acid 146 to about
amino acid 148, and from about amino acid 156 to about amino acid
158.
[0051] The invention further encompasses modifications which may be
made in or immediately adjacent to the loop region between a
helices 5 and 6 of a Cry1C protein. This loop region extends from
about amino acid 177 to about amino acid 184, with the adjacent
amino acids extending from about amino acid 174 to about amino acid
176, and from about amino acid 185 to about amino acid 187.
[0052] Another aspect of the invention encompasses modifications in
the amino acid sequence which may be made in or immediately
adjacent to the loop region between .alpha. helices 6 and 7 of a
Cry1C protein. This loop region extends from about amino acid 218
to about amino acid 221, with the adjacent amino acids extending
from about amino acid 215 to about amino acid 217, and from about
amino acid 222 to about amino acid 224.
[0053] In a similar fashion, the invention also encompasses
modifications in the amino acid sequence which may be made in or
immediately adjacent to the loop region between .alpha. helix 7 of
domain 1 and .beta. strand 1 of domain 2 of a Cry1C protein. This
loop region extends from about amino acid 250 to about amino acid
259, with the adjacent amino acids extending from about amino acid
247 to about amino acid 249, and from about amino acid 260 to about
amino acid 262.
[0054] In addition to modifications of Cry1C peptides, those having
benefit of the present teaching are now also able to make mutations
in the loop regions of proteins which are related to Cry1C
structurally. In fact, the inventors contemplate that any crystal
protein or peptide having helices which are linked together by loop
regions may be altered using the methods disclosed herein to
produce crystal proteins having altered loop regions. For example,
the inventors contemplate that the particular Cry1 crystal proteins
in which such modifications may be made include the Cry1A, Cry1B,
Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, and Cry1K
crystal proteins which are known in the art, as well as other
crystal proteins not yet described or characterized which may be
classified as a Cry1 crystal protein based upon amino acid
similarity to the known Cry1 proteins. Preferred Cry1 proteins
presently described which are contemplated by the inventors to be
modified by the methods disclosed herein for the purpose of
producing crystal proteins with altered activity or specificity
include, but are not limited to Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ad,
Cry1Ae, Cry1Ba, Cry1Bb, Cry1Bc, Cry1Ca, Cry1Cb, Cry1Da, Cry1Db,
Cry1Ea, Cry1Eb, Cry1Fa, Cry1Fb, Cry1Hb, Cry1Ia, Cry1Ib, Cry1Ja, and
Cry1Jb crystal proteins, with Cry1Ca crystal proteins being
particularly preferred.
[0055] Modifications which may be made to these loop regions which
are contemplated by the inventors to be most preferred in producing
crystal proteins with improved insecticidal activity include, but
are not limited to, substitution of one or more amino acids by one
or more amino acids not normally found at the particular site of
substitution in the wild-type protein. In particular, substitutions
of one or more arginine residues by an alanine, leucine,
methionine, glycine, or aspartic acid residues have been shown to
be particularly useful in the production of such enhanced proteins.
Likewise, the inventors have demonstrated that substitutions of one
or more lysine residues contained within or immediately adjacent to
the loop regions with an alanine residue produce mutant proteins
which have desirable insecticidal properties not found in the
parent, or wild-type protein. Particularly preferred arginine
residues in the Cry1C protein include Arg86, Arg148, Arg180,
Arg252, and Arg253, while a particularly preferred lysine residue
in Cry1C is Lys219.
[0056] Mutant proteins which have been developed by the inventors
demonstrating the efficiency and efficacy of this mutagenesis
strategy include the Cry1C-R148L, Cry1C-R148M, Cry1C-R148D,
Cry1C-R148A, Cry1C-R148G, and Cry1C-R180A strains described in
detail herein.
[0057] Disclosed and claimed herein is a method for preparing a
modified crystal protein which generally involves the steps of
identifying a crystal protein having one or more loop regions
between adjacent .alpha.-helices, introducing one or more mutations
into at least one of those loop regions, or alternatively, into the
amino acid residues immediately flanking the loop regions, and then
obtaining the modified crystal protein so produced. The modified
crystal proteins obtained by such a method are also important
aspects of this invention.
[0058] According to the invention, base substitutions may be made
in the cry1C nucleotide sequence in order to change particular
amino acids within or near the predicted loop regions of Cry1C
between the .alpha.-helices of domain 1. The resulting Cry1C*
proteins may then be assayed for bioinsecticide activity using the
techniques disclosed herein to identifying proteins having improved
toxin activity.
[0059] As an illustrative embodiment, changes in three such amino
acids within the loop region between .alpha.-helices 3 and 4 of
domain 1 produced modified crystal proteins with enhanced
insecticidal activity (Cry1C.499, Cry1C.563, Cry1C.579).
[0060] As a second illustrative embodiment, an alanine substitution
for an arginine residue within or adjacent to the loop region
between .alpha.-helices 4 and 5 produced a modified crystal protein
with enhanced insecticidal activity (Cry1C-R148A). Although this
substitution removes a potential trypsin-cleavage site within
domain 1, trypsin digestion of this modified crystal protein
revealed no difference in proteolytic stability from the native
Cry1C protein.
[0061] As a third illustrative embodiment, an alanine substitution
for an arginine residue within or adjacent to the loop region
between .alpha.-helices 5 and 6, the R180A substitution in Cry1C
(Cry1C-R180A) also removes a potential trypsin cleavage site in
domain 1, yet this substitution has no effect on insecticidal
activity. Thus, the steps in the Cry1C protein mode-of-action
impacted by these amino acid substitutions have not been determined
nor is it obvious what substitutions need to be made to improve
insecticidal activity.
[0062] Because the structures for Cry3A and Cry1Aa show a
remarkable conservation of protein tertiary structure (Grochulski
et al., 1995), and because many crystal proteins show significant
amino acid sequence identity to the Cry1C amino acid sequence
within domain 1, including proteins of the Cry1, Cry2, Cry3, Cry4,
Cry5, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, and
Cry16 classes (Table 1), now in light of the inventors' surprising
discovery, for the first time, those of skill in the art having
benefit of the teachings disclosed herein will be able to broadly
apply the methods of the invention to modifying a host of crystal
proteins with improved activity or altered specificity. Such
methods will not only be limited to the crystal proteins disclosed
in Table 1, but may also been applied to any other related crystal
protein, including those yet to be identified, which comprise one
or more loop regions between one or more pairs of adjacent
.alpha.-helices.
[0063] In particular, such methods may be now applied to
preparation of modified crystal proteins having one or more
alterations in the loop regions of domain 1. The inventors further
contemplate that similar loop regions may be identified in other
domains of crystal proteins which may be similarly modified through
site-specific or random mutagenesis to generate toxins having
improved activity, or alternatively, altered insect specificity. In
certain applications, the creation of altered toxins having
increased activity against one or more insects is desired.
Alternatively, it may be desirable to utilize the methods described
herein for creating and identifying altered crystal proteins which
are active against a wider spectrum of susceptible insects. The
inventors further contemplate that the creation of chimeric crystal
proteins comprising one or more loop regions as described herein
may be desirable for preparing "super" toxins which have the
combined advantages of increased insecticidal activity and
concomitant broad specificity.
[0064] In light of the present disclosure, the mutagenesis of
codons encoding amino acids within or adjacent to the loop regions
between the .alpha.-helices of domain 1 of these proteins may also
result in the generation of a host of related insecticidal proteins
having improved activity. As an illustrative example, alignment of
Cry1 amino acid sequences spanning the loop region between
.alpha.-helices 4 and 5 reveals that several Cry1 proteins contain
an arginine residue at the position homologous to R148 of Cry1C.
Since the Cry1C R148A mutant exhibits improved toxicity towards a
number of lepidopteran pests, it is contemplated by the inventors
that similar substitutions in these other Cry1 proteins will also
yield improved insecticidal proteins. While exemplary mutations
have been described for three of the loop regions which resulted in
crystal proteins having improved toxicity, the inventors
contemplate that mutations may also be made in other loop regions
or other portions of the active toxin which will give rise to
functional bioinsecticidal crystal proteins. All such mutations are
considered to fall within the scope of this disclosure.
[0065] In one illustrative embodiment, mutagenized cry1C* genes are
obtained which encode Cry1C* variants that are generally based upon
the wild-type Cry1C sequence, but that have one or more changes
incorporated into or adjacent to the loop regions in domain 1. A
particular example is a mutated cry1C-R148A gene (SEQ ID NO:1) that
encodes a Cry1C* with an amino acid sequence of SEQ ID NO:2 in
which Arginine at position 148 has been replaced by Alanine.
[0066] In a second illustrative embodiment, mutagenized cry1C*
genes will encode Cry1C* variants that are generally based upon the
wild-type Cry1C sequence, but that have certain changes. A
particular example is a mutated cry1C-R180A gene (SEQ ID NO:5) that
encodes a Cry1C* with an amino acid sequence of SEQ ID NO:6 in
which Arginine at position 180 has been replaced by Alanine.
[0067] In a third illustrative embodiment, mutagenized cry1C* genes
will encode Cry1C* variants that are generally based upon the
wild-type Cry1C sequence, but that have certain changes. A
particular example is a mutated cry1C.563 gene (SEQ ID NO:7) that
encodes a Cry1C with an amino acid sequence of SEQ ID NO:8 in which
mutations in nucleic acid residues 354, 361, 369, and 370, resulted
in point mutations A to T, A to C, A to C, and G to A,
respectively. These mutations modified the amino acid sequence at
positions 118 (Glu to Asp), 121 (Asn to His), and 124 (Ala to Thr).
Using the nomenclature convention described above, such a mutation
could also properly be described as a Cry1C-E118D-N121H-A124T
mutant.
[0068] In a fourth illustrative embodiment, mutagenized cry1C*
genes will encode Cry1C* variants that are generally based upon the
wild-type Cry1C sequence, but that have certain changes. A
particular example is a mutated cry1C.579 gene (SEQ ID NO:9) that
encodes a Cry1C* with an amino acid sequence of SEQ ID NO:10 in
which mutations in nucleic acid residues 353, 369, and 371,
resulted in point mutations A to T, A to T, and C to G,
respectively. These mutations modified the amino acid sequence at
positions 118 (Glu to Val) and 124 (Ala to Gly). Using the
nomenclature convention described above, such a mutation could also
properly be described as a Cry1C-E118V-A124G mutant.
[0069] In a fifth illustrative embodiment, mutagenized cry1C* genes
will encode Cry1C* variants that are generally based upon the
wild-type Cry1C sequence, but that have certain changes. A
particular example is a mutated cry1C.499 gene (SEQ ID NO:11) that
encodes a Cry1C* with an amino acid sequence of SEQ ID NO:12 in
which mutations in nucleic acid residues 360 and 361 resulted in
point mutations T to C and A to C, respectively. These mutations
modified the amino acid sequence at position 121 (Asn to His).
Using the nomenclature convention described above, such a mutation
could also properly be described as a Cry1C-N121H mutant.
[0070] In a sixth illustrative embodiment, mutagenized cry1C* genes
will encode Cry1C* variants that are generally based upon the
wild-type Cry1C sequence, but that have certain changes. A
particular example is a mutated cry1C-R148D gene (SEQ ID NO:3) that
encodes a Cry1C* with an amino acid sequence of SEQ ID NO:4 in
which Arg at position 148 has been replaced by Asp.
[0071] The mutated genes of the present invention are also
definable by genes in which at least one or more of the codon
positions contained within or adjacent to one or more loop regions
between 2 or more .alpha.-helices contain one or more substituted
codons. That is, they contain one or more codons that are not
present in the wild-type gene at the particular site(s) of
mutagenesis and that encode one or more amino acid
substitutions.
[0072] In other embodiments, the mutated genes will have at least
about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,
about 40%, about 45%, or even about 50% or more of the codon
positions within a loop region between 2 .alpha.-helices
substituted by one or more codons not present in the wild-type gene
sequence at the particular site of mutagenesis and/or amino acid
substitution. Mutated cry1C* genes wherein at least about 50%, 60%,
70%, 80%, 90% or above of the codon positions contained within a
loop region between 2 .alpha.-helices have been altered are also
contemplated to be useful in the practice of the present
invention.
[0073] Also contemplated to fall within the scope of the invention
are combinatorial mutants which contain two or more modified loop
regions, or alternatively, contain two or more mutations within a
single loop region, or alternatively, two or more modified loop
regions with each domain containing two or more modifications.
cry1C* genes wherein modifications have been made in a combination
of two or more helices, e.g., .alpha.-helices 1 and 2a,
.alpha.-helices 2b and 3, .alpha.-helices 3 and 4, .alpha.-helices
4 and 5, .alpha.-helices 5 and 6, .alpha.-helices 6 and 7, and/or
modifications between .alpha.-helix 7 and .beta.-strand 1, are also
important aspects of the present invention.
[0074] As an illustrative example, a mutated crystal protein that
the inventors designate Cry1C-R148A.563. contains an arginine to
alanine substitution at position 148, as well as incorporate the
mutations present in Cry1C.563. Such a mutated crystal protein
would, therefore, have modified both the .alpha. 3/4 loop region
and the .alpha. 4/5 loop region. For sake of clarity, an ".alpha.
3/4 loop region" is intended to mean the loop region between the
3rd and 4th .alpha. helices, while an ".alpha. 4/5 loop region" is
intended to mean the loop region between the 4th and 5th .alpha.
helices, etc. Other helices and their corresponding loop regions
have been similarly identified throughout this specification. FIG.
1 illustrates graphically the placement of loop regions between
helices for Cry1C.
[0075] Preferred mutated cry1C genes of the invention are those
genes that contain certain key changes. Examples are genes that
comprise amino acid substitutions from Arg to Ala or Asp
(particularly at amino acid residues 86, 148, 180, 252, and 253);
or Lys to Ala or Asp (particularly at amino acid residue 219).
[0076] Genes mutated in the manner of the invention may also be
operatively linked to other protein-encoding nucleic acid
sequences. This will generally result in the production of a fusion
protein following expression of such a nucleic acid construct. Both
N-terminal and C-terminal fusion proteins are contemplated.
[0077] Virtually any protein- or peptide-encoding DNA sequence, or
combinations thereof, may be fused to a mutated cry1C* sequence in
order to encode a fusion protein. This includes DNA sequences that
encode targeting peptides, proteins for recombinant expression,
proteins to which one or more targeting peptides is attached,
protein subunits, domains from one or more crystal proteins, and
the like.
[0078] In one aspect, the invention discloses and claims host cells
comprising one or more of the modified crystal proteins disclosed
herein, and in particular, cells of the novel B. thuringiensis
strains EG12111, EG 12121, EG11811, EG11815, EG11740, EG11746,
EG11822, EG11831, EG11832, and EG11747 which comprise recombinant
DNA segments encoding synthetically-modified Cry1C* crystal
proteins which demonstrates improved insecticidal activity against
members of the Order Lepidoptera.
[0079] Likewise, the invention also discloses and claims cell
cultures of B. thuringiensis EG12111, EG12121, EG11811, EG11815,
EG11740, EG11746, EG11822, EG11831, EG11832, and EG11747. Such cell
cultures may be biologically-pure cultures consisting of a single
strain, or alternatively may be cell co-cultures consisting of one
or more strains. Such cell cultures may be cultivated under
conditions in which one or more additional B. thuringiensis or
other bacterial strains are simultaneously co-cultured with one or
more of the disclosed cultures, or alternatively, one or more of
the cell cultures of the present invention may be combined with one
or more additional B. thuringiensis or other bacterial strains
following the independent culture of each. Such procedures may be
useful when suspensions of cells containing two or more different
crystal proteins are desired.
[0080] The subject cultures have been deposited under conditions
that assure that access to the cultures will be available during
the pendency of this patent application to one determined by the
Commissioner of Patents and Trademarks to be entitled thereto under
37 C.F.R. .sctn.1.14 and 35 U.S.C. .sctn.122. The deposits are
available as required by foreign patent laws in countries wherein
counterparts of the subject application, or its progeny, are filed.
However, it should be understood that the availability of a deposit
does not constitute a license to practice the subject invention in
derogation of patent rights granted by governmental action.
[0081] Further, the subject culture deposits will be stored and
made available to the public in accord with the provisions of the
Budapest Treaty for the Deposit of Microorganisms, i.e., they will
be stored with all the care necessary to keep them viable and
uncontaminated for a period of at least five years after the most
recent request for the finishing of a sample of the deposit, and in
any case, for a period of at least 30 (thirty) years after the date
of deposit or for the enforceable life of any patent which may
issue disclosing the cultures. The depositor acknowledges the duty
to replace the deposits should the depository be unable to furnish
a sample when requested, due to the condition of the deposits. All
restrictions on the availability to the public of the subject
culture deposits will be irrevocably removed upon the granting of a
patent disclosing them.
[0082] Cultures of the strains listed in Table 2 were deposited in
the permanent collection of the Agricultural Research Service
Culture Collection, Northern Regional Research Laboratory (NRRL)
under the terms of the Budapest Treaty:
2TABLE 2 STRAINS DEPOSITED UNDER THE TERMS OF THE BUDAPEST TREATY
Accession Strain Protein/Plasmid Number Deposit Date B.
thuringiensis EG11740 Cry1C.563 NRRL B-21590 Jun. 25, 1996 B.
thuringiensis EG11746 Cry1C.579 NRRL B-21591 Jun. 25, 1996 B.
thuringiensis EG11811 Cry1C-R148A NRRL B-21592 Jun. 25, 1996 B.
thuringiensis EG11747 Cry1C.499 NRRL B-21609 Aug. 2, 1996 B.
thuringiensis EG11815 Cry1C-R180A NRRL B-21610 Aug.2, 1996 B.
thuringiensis EG11822 Cry1C-R148A NRRL B-21638 Oct. 28, 1996 B.
thuringiensis EG11831 Cry1C-R148A NRRL B-21639 Oct. 28, 1996 B.
thuringiensis EG11832 Cry1C-R148D NRRL B-21640 Oct. 28, 1996 B.
thuringiensis EG12111 Cry1C-R148A-K219A NRRL B-XXXXX Nov. XX, 1997
B. thuringiensis EG12121 Cry1C-R148D-K219A NRRL B-XXXXX Nov. XX,
1997 E. coli EG1597 pEG597 NRRL B-18630 Mar. 27, 1990 E. coli
EG7529 pEG853 NRRL B-18631 Mar. 27, 1990 E. coli EG7534 pEG854 NRRL
B-18632 Mar. 27, 1990
2.2 Methods for Producing Cry1C* Protein Compositions
[0083] The modified Cry1* crystal proteins of the present invention
are preparable by a process which generally involves the steps of:
(a) identifying a Cry1 crystal protein having one or more loop
regions between two adjacent .alpha. helices or between an a helix
and a .beta. strand; (b) introducing one or more mutations into at
least one of these loop regions; and (c) obtaining the modified
Cry1 * crystal protein so produced. As described 10 above, these
loop regions occur between .alpha. helices 1 and 2, .alpha. helices
2 and 3, .alpha. helices 3 and 4, .alpha. helices 4 and 5, .alpha.
helices 5 and 6, and .alpha. helices 6 and 7 of domain 1 of the
crystal protein, and between .alpha. helix 7 of domain 1 and the
.beta. strand 1 of domain 2.
[0084] Preferred crystal proteins which are preparable by this
claimed process include the crystal proteins which have the amino
acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:59, or SEQ ID NO:61,
and most preferably, the crystal proteins which are encoded by the
nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:58, or SEQ ID NO:60,
or a nucleic acid sequence which hybridizes to the nucleic acid
sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ
ID NO:9, SEQ ID NO:11, SEQ ID NO:58, or SEQ ID NO:60 under
conditions of moderate to high stringency.
[0085] A second method for preparing a modified Cry1* crystal
protein is a further embodiment of the invention. This method
generally involves identifying a Cry1 crystal protein having one or
more loop regions, introducing one or more mutations into one or
more of the loop regions, and obtaining the resulting modified
crystal protein. Preferred Cry1* crystal proteins preparable by
either of these methods include the Cry1A*, Cry1B *, Cry1C*,
Cry1D*, Cry1E*, Cry1F*, Cry1G*, Cry1H*, Cry1I*, Cry1J*, and Cry1K*
crystal proteins, and more preferably, the Cry1Aa*, Cry1Ab*,
Cry1Ac*, Cry1Ad*, Cry1Ae*, Cry1Ba*, Cry1Bb*, Cry1Bc*, Cry1Ca*,
Cry1Cb*, Cry1Da*, Cry1Db*, Cry1Ea*, Cry1Eb*, Cry1Fa*, Cry1Fb*,
Cry1Hb*, Cry1Ia*, Cry1Ib*, Cry1Ja*, and Cry1Jb* crystal proteins.
Highly preferred proteins include Cry1Ca* crystal proteins, such as
those comprising the amino acid sequence of SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID
NO:59, or SEQ ID NO:61, and those encoded by a nucleic acid
sequence having the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:58, or SEQ
ID NO:60, or a nucleic acid sequence which hybridizes to the
nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:58, or SEQ ID NO:60
under conditions of moderate stringency.
[0086] Amino acid, peptide and protein sequences within the scope
of the present invention include, and are not limited to the
sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ
ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:59, and SEQ ID
NO:61, and alterations in the amino acid sequences including
alterations, deletions, mutations, and homologs. Compositions which
comprise from about 0.5% to about 99% by weight of the crystal
protein, or more preferably from about 5% to about 75%, or from
about 25% to about 50% by weight of the crystal protein are
provided herein. Such compositions may readily be prepared using
techniques of protein production and purification well-known to
those of skill, and the methods disclosed herein. Such a process
for preparing a Cry1C* crystal protein generally involves the steps
of culturing a host cell which expresses the Cry1C* protein (such
as a Bacillus thuringiensis NRRL B-21590, NRRL B-21591, NRRL
B-21638, NRRL B-21639, NRRL, B-21640, NRRL, B-21609, NRRL, B-21610,
or NRRL B-21592 cell) under conditions effective to produce the
crystal protein, and then obtaining the crystal protein so
produced. The protein may be present within intact cells, and as
such, no subsequent protein isolation or purification steps may be
required. Alternatively, the cells may be broken, sonicated, lysed,
disrupted, or plasmolyzed to free the crystal protein(s) from the
remaining cell debris. In such cases, one may desire to isolate,
concentrate, or further purify the resulting crystals containing
the proteins prior to use, such as, for example, in the formulation
of insecticidal compositions. The composition may ultimately be
purified to consist almost entirely of the pure protein, or
alternatively, be purified or isolated to a degree such that the
composition comprises the crystal protein(s) in an amount of from
between about 0.5% and about 99% by weight, or in an amount of from
between about 5% and about 90% by weight, or in an amount of from
between about 25% and about 75% by weight, etc.
2.3 Recombinant Vectors Expressing the Mutagenized Cry1 Genes
[0087] One important embodiment of the invention is a recombinant
vector which comprises a nucleic acid segment encoding one or more
B. thuringiensis crystal proteins having a modified amino acid
sequence in one or more loop regions of domain 1, or between
.alpha. helix 7 of domain 1 and .beta. strand 1 of domain 2. Such a
vector may be transferred to and replicated in a prokaryotic or
eukaryotic host, with bacterial cells being particularly preferred
as prokaryotic hosts, and plant cells being particularly preferred
as eukaryotic hosts.
[0088] The amino acid sequence modifications may include one or
more modified loop regions between .alpha. helices 1 and 2, .alpha.
helices 2 and 3, .alpha. helices 3 and 4, .alpha. helices 4 and 5,
.alpha. helices 5 and 6, or .alpha. helices 6 and 7 of domain 1, or
between .alpha. helix 7 of domain 1 and .beta. strand 1 of domain
2. Preferred recombinant vectors are those which contain one or
more nucleic acid segments which encode modified Cry1A, Cry1B,
Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, or Cry1K
crystal proteins. Particularly preferred recombinant vectors are
those which contain one or more nucleic acid segments which encode
modified Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Ba, Cry1Bb,
Cry1Bc, Cry1Ca, Cry1 Cb, Cry1Da, Cry1Db, Cry1Ea, Cry1Eb, Cry1Fa,
Cry1Fb, Cry1Hb, Cry1Ia, Cry1Ib, Cry1Ja, or Cry1Jb crystal proteins,
with modified Cry1Ca crystal proteins being particularly
preferred.
[0089] In preferred embodiments, the recombinant vector comprises a
nucleic acid segment encoding the amino acid sequence of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:59, or SEQ ID NO:61. Highly preferred nucleic acid
segments are those which have the sequence of SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID
NO:58, or SEQ ID NO:60.
[0090] Another important embodiment of the invention is a
transformed host cell which expresses one or more of these
recombinant vectors. The host cell may be either prokaryotic or
eukaryotic, and particularly preferred host cells are those which
express the nucleic acid segment(s) comprising the recombinant
vector which encode one or more B. thuringiensis crystal protein
comprising modified amino acid sequences in one or more loop
regions of domain 1, or between .alpha. helix 7 of domain 1 and
.beta. strand 1 of domain 2. Bacterial cells are particularly
preferred as prokaryotic hosts, and plant cells are particularly
preferred as eukaryotic hosts In an important embodiment, the
invention discloses and claims a host cell wherein the modified
amino acid sequences comprise one or more loop regions between
.alpha. helices 1 and 2, .alpha. helices 2 and 3, .alpha. helices 3
and 4, .alpha. helices 4 and 5, .alpha. helices 5 and 6 or .alpha.
helices 6 and 7 of domain 1, or between .alpha. helix 7 of domain 1
and .beta. strand 1 of domain 2. A particularly preferred host cell
is one that comprises the amino acid sequence of SEQ ID NO:2, SEQ
ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ
ID NO:59, or SEQ ID NO:61, and more preferably, one that comprises
the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:58, or SEQ ID
NO:60.
[0091] Bacterial host cells transformed with a nucleic acid segment
encoding a modified Cry1C crystal protein according to the present
invention are disclosed and claimed herein, and in particular, a
Bacillus thuringiensis cell having the NRRL accession NRRL B-21590,
NRRL B-21591, NRRL B-21592, NRRL B-21638, NRRL B-21639, NRRL
B-21640, NRRL B-21609, or NRRL B-21610.
[0092] In another embodiment, the invention encompasses a method of
using a nucleic acid segment of the present invention that encodes
a cry1C* gene. The method generally comprises the steps of: (a)
preparing a recombinant vector in which the cry1C* gene is
positioned under the control of a promoter; (b) introducing the
recombinant vector into a host cell; (c) culturing the host cell
under conditions effective to allow expression of the Cry1C*
crystal protein encoded by said cry1C* gene; and (d) obtaining the
expressed Cry1C* crystal protein or peptide.
[0093] A wide variety of ways are available for introducing a B.
thuringiensis gene expressing a toxin into the microorganism host
under conditions which allow for stable maintenance and expression
of the gene. One can provide for DNA constructs which include the
transcriptional and translational regulatory signals for expression
of the toxin gene, the toxin gene under their regulatory control
and a DNA sequence homologous with a sequence in the host organism,
whereby integration will occur, and/or a replication system which
is functional in the host, whereby integration or stable
maintenance will occur.
[0094] The transcriptional initiation signals will include a
promoter and a transcriptional initiation start site. In some
instances, it may be desirable to provide for regulative expression
of the toxin, where expression of the toxin will only occur after
release into the environment. This can be achieved with operators
or a region binding to an activator or enhancers, which are capable
of induction upon a change in the physical or chemical environment
of the microorganisms. For example, a temperature sensitive
regulatory region may be employed, where the organisms may be grown
up in the laboratory without expression of a toxin, but upon
release into the environment, expression would begin. Other
techniques may employ a specific nutrient medium in the laboratory,
which inhibits the expression of the toxin, where the nutrient
medium in the environment would allow for expression of the toxin.
For translational initiation, a ribosomal binding site and an
initiation codon will be present.
[0095] Various manipulations may be employed for enhancing the
expression of the messenger RNA, particularly by using an active
promoter, as well as by employing sequences, which enhance the
stability of the messenger RNA. The transcriptional and
translational termination region will involve stop codon(s), a
terminator region, and optionally, a polyadenylation signal. A
hydrophobic "leader" sequence may be employed at the amino terminus
of the translated polypeptide sequence in order to promote
secretion of the protein across the inner membrane.
[0096] In the direction of transcription, namely in the 5' to 3'
direction of the coding or sense sequence, the construct will
involve the transcriptional regulatory region, if any, and the
promoter, where the regulatory region may be either 5' or 3' of the
promoter, the ribosomal binding site, the initiation codon, the
structural gene having an open reading frame in phase with the
initiation codon, the stop codon(s), the polyadenylation signal
sequence, if any, and the terminator region. This sequence as a
double strand may be used by itself for transformation of a
microorganism host, but will usually be included with a DNA
sequence involving a marker, where the second DNA sequence may be
joined to the toxin expression construct during introduction of the
DNA into the host.
[0097] By a marker is intended a structural gene which provides for
selection of those hosts which have been modified or transformed.
The marker will normally provide for selective advantage, for
example, providing for biocide resistance, e.g., resistance to
antibiotics or heavy metals; complementation, so as to provide
prototropy to an auxotrophic host, or the like. Preferably,
complementation is employed, so that the modified host may not only
be selected, but may also be competitive in the field. One or more
markers may be employed in the development of the constructs, as
well as for modifying the host. The organisms may be further
modified by providing for a competitive advantage against other
wild-type microorganisms in the field. For example, genes
expressing metal chelating agents, e.g., siderophores, may be
introduced into the host along with the structural gene expressing
the toxin. In this manner, the enhanced expression of a siderophore
may provide for a competitive advantage for the toxin-producing
host, so that it may effectively compete with the wild-type
microorganisms and stably occupy a niche in the environment.
[0098] Where no functional replication system is present, the
construct will also include a sequence of at least 50 basepairs
(bp), preferably at least about 100 bp, more preferably at least
about 1000 bp, and usually not more than about 2000 bp of a
sequence homologous with a sequence in the host. In this way, the
probability of legitimate recombination is enhanced, so that the
gene will be integrated into the host and stably maintained by the
host. Desirably, the toxin gene will be in close proximity to the
gene providing for complementation as well as the gene providing
for the competitive advantage. Therefore, in the event that a toxin
gene is lost, the resulting organism will be likely to also lost
the complementing gene and/or the gene providing for the
competitive advantage, so that it will be unable to compete in the
environment with the gene retaining the intact construct.
[0099] A large number of transcriptional regulatory regions are
available from a wide variety of microorganism hosts, such as
bacteria, bacteriophage, cyanobacteria, algae, fungi, and the like.
Various transcriptional regulatory regions include the regions
associated with the trp gene, lac gene, gal gene, the
.lambda..sub.L and .lambda..sub.R promoters, the tac promoter, the
naturally-occurring promoters associated with the .delta.-endotoxin
gene, where functional in the host. See for example, U.S. Pat. No.
4,332,898; U.S. Pat. No. 4,342,832; and U.S. Pat. No. 4,356,270.
The termination region may be the termination region normally
associated with the transcriptional initiation region or a
different transcriptional initiation region, so long as the two
regions are compatible and functional in the host.
[0100] Where stable episomal maintenance or integration is desired,
a plasmid will be employed which has a replication system which is
functional in the host. The replication system may be derived from
the chromosome, an episomal element normally present in the host or
a different host, or a replication system from a virus which is
stable in the host. A large number of plasmids are available, such
as pBR322, pACYC184, RSF1010, pR01614, and the like. See for
example, Olson et al. (1982); Bagdasarian et al. (1981), Baum et
al., 1990, and U.S. Pat. Nos. 4,356,270; 4,362,817; 4,371,625, and
5,441,884, each incorporated specifically herein by reference.
[0101] The B. thuringiensis gene can be introduced between the
transcriptional and translational initiation region and the
transcriptional and translational termination region, so as to be
under the regulatory control of the initiation region. This
construct will be included in a plasmid, which will include at
least one replication system, but may include more than one, where
one replication system is employed for cloning during the
development of the plasmid and the second replication system is
necessary for functioning in the ultimate host. In addition, one or
more markers may be present, which have been described previously.
Where integration is desired, the plasmid will desirably include a
sequence homologous with the host genome.
[0102] The transformants can be isolated in accordance with
conventional ways, usually employing a selection technique, which
allows for selection of the desired organism as against unmodified
organisms or transferring organisms, when present. The
transformants then can be tested for pesticidal activity. If
desired, unwanted or ancillary DNA sequences may be selectively
removed from the recombinant bacterium by employing site-specific
recombination systems, such as those described in U.S. Pat. No.
5,441,884 (specifically incorporated herein by reference).
2.4 Synthetic Cry1C* DNA Segments
[0103] A B. thuringiensis cry1* gene encoding a crystal protein
having insecticidal activity against Lepidopteran
insects-comprising a modified amino acid sequence in one or more
loop regions of domain 1 or in a loop region between domain 1 and
domain 2 represents an important aspect of the invention.
Preferably, the cry1* gene encodes an amino acid sequence in which
one or more loop regions have been modified for the purpose of
altering the insecticidal activity of the crystal protein. As
described above, such loop domains include those between .alpha.
helices 1 and 2, .alpha. helices 2 and 3, .alpha. helices 3 and 4,
.alpha. helices 4 and 5, .alpha. helices 5 and 6, or .alpha.
helices 6 and 7 of domain 1, or between .alpha. helix 7 of domain 1
and .beta. strand 1 of domain 2 (FIG. 1). Preferred cry1* genes of
the invention include cry1A*, cry1B*, cry1C*, cry1D*, cry1E*,
cry1F*, cry1G*, cry1H*, cry1I*, cry1J*, and cry1K* genes, with
cry1Aa*, cry1Ab*, cry1Ac*, cry1Ad*, cry1Ae*, cry1Ba** cry1Bb*,
cry1Bc*, cry1Ca* cry1Cb** cry1Da*, cry1Db* cry1Ea*, cry1Eb*,
cry1Fa*, cry1Fb*, cry1Hb*, cry1Ia*, cry1Ib*, cry1Ja*, and cry1Jb*
genes being highly preferred.
[0104] In accordance with the present invention, nucleic acid
sequences include and are not limited to DNA, including and not
limited to cDNA and genomic DNA, genes; RNA, including and not
limited to mRNA and tRNA; antisense sequences, nucleosides, and
suitable nucleic acid sequences such as those set forth in SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:58, and SEQ ID NO:60 and alterations in the
nucleic acid sequences including alterations, deletions, mutations,
and homologs capable of expressing the B. thuringiensis modified
toxins of the present invention.
[0105] In an illustrative embodiment, the inventors used the
methods described herein to produce modified cry1Ca* genes which
had improved insecticidal activity against lepidopterans. In these
illustrative examples, loop regions were modified by changing one
or more arginine residues to alanine or aspartic acid residues,
such as mutations at arginine residues Arg148 and Arg180.
[0106] As such the present invention also concerns DNA segments,
that are free from total genomic DNA and that encode the novel
synthetically-modified crystal proteins disclosed herein. DNA
segments encoding these peptide species may prove to encode
proteins, polypeptides, subunits, functional domains, and the like
of crystal protein-related or other non-related gene products. In
addition these DNA segments may be synthesized entirely in vitro
using methods that are well-known to those of skill in the art.
[0107] As used herein, the term "DNA segment" refers to a DNA
molecule that has been isolated free of total genomic DNA of a
particular species. Therefore, a DNA segment encoding a crystal
protein or peptide refers to a DNA segment that contains crystal
protein coding sequences yet is isolated away from, or purified
free from, total genomic DNA of the species from which the DNA
segment is obtained, which in the instant case is the genome of the
Gram-positive bacterial genus, Bacillus, and in particular, the
species of Bacillus known as B. thuringiensis. Included within the
term "DNA segment", are DNA segments and smaller fragments of such
segments, and also recombinant vectors, including, for example,
plasmids, cosmids, phagemids, phage, viruses, and the like.
[0108] Similarly, a DNA segment comprising an isolated or purified
crystal protein-encoding gene refers to a DNA segment which may
include in addition to peptide encoding sequences, certain other
elements such as, regulatory sequences, isolated substantially away
from other naturally occurring genes or protein-encoding sequences.
In this respect, the term "gene" is used for simplicity to refer to
a functional protein-, polypeptide- or peptide-encoding unit. As
will be understood by those in the art, this functional term
includes both genomic sequences, operon sequences and smaller
engineered gene segments that express, or may be adapted to
express, proteins, polypeptides or peptides.
[0109] "Isolated substantially away from other coding sequences"
means that the gene of interest, in this case, a gene encoding a
bacterial crystal protein, forms the significant part of the coding
region of the DNA segment, and that the DNA segment does not
contain large portions of naturally-occurring coding DNA, such as
large chromosomal fragments or other functional genes or operon
coding regions. Of course, this refers to the DNA segment as
originally isolated, and does not exclude genes, recombinant genes,
synthetic linkers, or coding regions later added to the segment by
the hand of man.
[0110] Particularly preferred DNA sequences are those encoding
Cry1C-R148A, Cry1C-R148D, Cry1C-R180A, Cry1C.499, Cry1C.563 or
Cry1C.579 crystal proteins, and in particular cry1C* genes such as
cry1C-R148A, cry1C-R148D, cry1C-R180A, cry1C.499, cry1C.563 and
cry1C.579 nucleic acid sequences. In particular embodiments, the
invention concerns isolated DNA segments and recombinant vectors
incorporating DNA sequences that encode a Cry peptide species that
includes within its amino acid sequence an amino acid sequence
essentially as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:59, or SEQ ID
NO:61.
[0111] The term "a sequence essentially as set forth in SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:59, or SEQ ID NO:61 " means that the sequence
substantially corresponds to a portion of the sequence of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:59, or SEQ ID NO:61, and has relatively few amino
acids that are not identical to, or a biologically functional
equivalent of, the amino acids of any of these sequences. The term
"biologically functional equivalent" is well understood in the art
and is further defined in detail herein (e.g., see Illustrative
Embodiments). Accordingly, sequences that have between about 70%
and about 80%, or more preferably between about 81% and about 90%,
or even more preferably between about 91% and about 99% amino acid
sequence identity or functional equivalence to the amino acids of
SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:59, or SEQ ID NO:61 will be sequences that
are "essentially as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6 SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:59, or SEQ
ID NO:61."
[0112] It will also be understood that amino acid and nucleic acid
sequences may include additional residues, such as additional N- or
C-terminal amino acids or 5' or 3' sequences, and yet still be
essentially as set forth in one of the sequences disclosed herein,
so long as the sequence meets the criteria set forth above,
including the maintenance of biological protein activity where
protein expression is concerned. The addition of terminal sequences
particularly applies to nucleic acid sequences that may, for
example, include various non-coding sequences flanking either of
the 5' or 3' portions of the coding region or may include various
internal sequences, i.e., introns, which are known to occur within
genes.
[0113] The nucleic acid segments of the present invention,
regardless of the length of the coding sequence itself, may be
combined with other DNA sequences, such as promoters,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length may vary considerably. It is therefore
contemplated that a nucleic acid fragment of almost any length may
be employed, with the total length preferably being limited by the
ease of preparation and use in the intended recombinant DNA
protocol. For example, nucleic acid fragments may be prepared that
include a short contiguous stretch encoding the peptide sequence
disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:59, or SEQ ID NO:61, or that
are identical to or complementary to DNA sequences which encode the
peptide disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:59, or SEQ ID NO:61,
and particularly the DNA segments disclosed in SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID
NO:58, and SEQ ID NO:60. For example, DNA sequences such as about
14 nucleotides, and that are up to about 10,000, about 5,000, about
3,000, about 2,000, about 1,000, about 500, about 200, about 100,
about 50, and about 14 base pairs in length (including all
intermediate lengths) are also contemplated to be useful.
[0114] It will be readily understood that "intermediate lengths",
in these contexts, means any length between the quoted ranges, such
as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32,
etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,
152, 153, etc.; including all integers through the 200-500;
500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; and up to and
including sequences of about 10,000 nucleotides and the like.
[0115] It will also be understood that this invention is not
limited to the particular nucleic acid sequences which encode
peptides of the present invention, or which encode the amino acid
sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12, SEQ ID NO:59, or SEQ ID NO:61, including
the DNA sequences which are particularly disclosed in SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9. SEQ ID NO:11,
SEQ ID NO:58, or SEQ ID NO:60. Recombinant vectors and isolated DNA
segments may therefore variously include the peptide-coding regions
themselves, coding regions bearing selected alterations or
modifications in the basic coding region, or they may encode larger
polypeptides that nevertheless include these peptide-coding regions
or may encode biologically functional equivalent proteins or
peptides that have variant amino acids sequences.
[0116] The DNA segments of the present invention encompass
biologically-functional, equivalent peptides. Such sequences may
arise as a consequence of codon redundancy and functional
equivalency that are known to occur naturally within nucleic acid
sequences and the proteins thus encoded. Alternatively,
functionally-equivalent proteins or peptides may be created via the
application of recombinant DNA technology, in which changes in the
protein structure may be engineered, based on considerations of the
properties of the amino acids being exchanged. Changes designed by
man may be introduced through the application of site-directed
mutagenesis techniques, e.g., to introduce improvements to the
antigenicity of the protein or to test mutants in order to examine
activity at the molecular level.
[0117] If desired, one may also prepare fusion proteins and
peptides, e.g., where the peptide-coding regions are aligned within
the same expression unit with other proteins or peptides having
desired functions, such as for purification or immunodetection
purposes (e.g., proteins that may be purified by affinity
chromatography and enzyme label coding regions, respectively).
[0118] Recombinant vectors form further aspects of the present
invention. Particularly useful vectors are contemplated to be those
vectors in which the coding portion of the DNA segment, whether
encoding a full-length protein or smaller peptide, is positioned
under the control of a promoter. The promoter may be in the form of
the promoter that is naturally associated with a gene encoding
peptides of the present invention, as may be obtained by isolating
the 5' non-coding sequences located upstream of the coding segment
or exon, for example, using recombinant cloning and/or PCR.TM.
technology, in connection with the compositions disclosed
herein.
2.5 Recombinant Vectors and Protein Expression
[0119] In other embodiments, it is contemplated that certain
advantages will be gained by positioning the coding DNA segment
under the control of a recombinant, or heterologous, promoter. As
used herein, a recombinant or heterologous promoter is intended to
refer to a promoter that is not normally associated with a DNA
segment encoding a crystal protein or peptide in its natural
environment. Such promoters may include promoters normally
associated with other genes, and/or promoters isolated from any
bacterial, viral, eukaryotic, or plant cell. Naturally, it will be
important to employ a promoter that effectively directs the
expression of the DNA segment in the cell type, organism, or even
animal, chosen for expression. The use of promoter and cell type
combinations for protein expression is generally known to those of
skill in the art of molecular biology, for example, see Sambrook et
al., 1989. The promoters employed may be constitutive, or
inducible, and can be used under the appropriate conditions to
direct high level expression of the introduced DNA segment, such as
is advantageous in the large-scale production of recombinant
proteins or peptides. Appropriate promoter systems contemplated for
use in high-level expression include, but are not limited to, the
Pichia expression vector system (Pharmacia LKB Biotechnology).
[0120] In connection with expression embodiments to prepare
recombinant proteins and peptides, it is contemplated that longer
DNA segments will most often be used, with DNA segments encoding
the entire peptide sequence being most preferred. However, it will
be appreciated that the use of shorter DNA segments to direct the
expression of crystal peptides or epitopic core regions, such as
may be used to generate anti-crystal protein antibodies, also falls
within the scope of the invention. DNA segments that encode peptide
antigens from about 8 to about 50 amino acids in length, or more
preferably, from about 8 to about 30 amino acids in length, or even
more preferably, from about 8 to about 20 amino acids in length are
contemplated to be particularly useful. Such peptide epitopes may
be amino acid sequences which comprise contiguous amino acid
sequence from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:59, or SEQ ID NO:61.
2.6 Methods for Preparing Mutagenized Cry1* Gene Segments
[0121] The present invention encompasses both site-specific
mutagenesis methods and random mutagenesis of a nucleic acid
segment encoding one of the crystal proteins described herein. In
particular, methods are disclosed for the random mutagenesis of
nucleic acid segments encoding the amino acid sequences identified
as being in, or immediately adjacent to, a loop region of domain 1
of the crystal protein, or between the last a helix of domain one
and the first .beta. strand of domain 2. The mutagenesis of this
nucleic acid segment results in one or more modifications to one or
more loop regions of the encoded crystal protein. Using the assay
methods described herein, one may then identify mutants arising
from this procedure which have improved insecticidal properties or
altered specificity, either intraorder or interorder.
[0122] In a preferred embodiment, the randomly-mutagenized
contiguous nucleic acid segment encodes an amino acid sequence in a
loop region of domain 1 or a modified amino acid sequence in a loop
region between domain 1 and domain 2of a B. thuringiensis crystal
protein having insecticidal activity against Lepidopteran insects.
Preferably, the modified amino acid sequence comprises a loop
region between .alpha. helices 1 and 2, .alpha. helices 2 and 3,
.alpha. helices 3 and 4, .alpha. helices 4 and 5, .alpha. helices 5
and 6, or .alpha. helices 6 and 7 of domain 1, or between .alpha.
helix 7 of domain 1 and .beta. strand 1 of domain 2. Preferred
crystal proteins include Cry1A, Cry1B, Cry1C Cry1D Cry1E, Cry1F
Cry1G, Cry1H, Cry1I, Cry1J, and Cry1K crystal protein, with Cry1Aa,
Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Ba, Cry1Bb, Cry1Bc, Cry1Ca,
Cry1Cb, Cry1Da, Cry1Db, Cry1Ea, Cry1Eb, Cry1Fa, Cry1Fb, Cry1Hb,
Cry1Ia, Cry1Ib, Cry1Ja, and Cry1Ib crystal proteins being
particularly preferred.
[0123] In an illustrative embodiment, a nucleic acid segment (SEQ
ID NO:7).encoding a Cry1Ca crystal protein was mutagenized in a
region corresponding to about amino acid residue 118 to about amino
acid residue 124 of the Cry1Ca protein (SEQ ID NO:8). The modified
Cry1Ca* resulting from the mutagenesis was termed, Cry1C.563.
[0124] In a second illustrative embodiment, a nucleic acid segment
(SEQ ID NO:9).encoding a Cry1Ca crystal protein was mutagenized in
a region corresponding to about amino acid residue 118 to about
amino acid residue 124 of the Cry1Ca protein (SEQ ID NO:10). The
modified Cry1Ca* resulting from the mutagenesis was termed,
Cry1C.579.
[0125] In a third illustrative embodiment. a nucleic acid segment
(SEQ ID NO:11 ).encoding a Cry1Ca crystal protein was mutagenized
in a region corresponding to about amino acid residue 118 to about
amino acid residue 124 of the Cry1Ca protein (SEQ ID NO:12). The
modified Cry1Ca* resulting from the mutagenesis was termed,
Cry1C.499.
[0126] The means for mutagenizing a DNA segment encoding a crystal
protein having one or more loop regions in its amino acid sequence
are well-known to those of skill in the art. Modifications to such
loop regions may be made by random, or site-specific mutagenesis
procedures. The loop region may be modified by altering its
structure through the addition or deletion of one or more
nucleotides from the sequence which encodes the corresponding
un-modified loop region.
[0127] Mutagenesis may be performed in accordance with any of the
techniques known in the art such as and not limited to synthesizing
an oligonucleotide having one or more mutations within the sequence
of a particular crystal protein. A "suitable host" is any host
which will express Cry, such as and not limited to Bacillus
thuringiensis and Escherichia coli. Screening for insecticidal
activity, in the case of Cry1C includes and is not limited to
lepidopteran-toxic activity which may be screened for by techniques
known in the art.
[0128] In particular, site-specific mutagenesis is a technique
useful in the preparation of individual peptides, or biologically
functional equivalent proteins or peptides, through specific
mutagenesis of the underlying DNA. The technique further provides a
ready ability to prepare and test sequence variants, for example,
incorporating one or more of the foregoing considerations, by
introducing one or more nucleotide sequence changes into the DNA.
Site-specific mutagenesis allows the production of mutants through
the use of specific oligonucleotide sequences which encode the DNA
sequence of the desired mutation, as well as a sufficient number of
adjacent nucleotides, to provide a primer sequence of sufficient
size and sequence complexity to form a stable duplex on both sides
of the deletion junction being traversed. Typically, a primer of
about 17 to about 75 nucleotides or more in length is preferred,
with about 10 to about 25 or more residues on both sides of the
junction of the sequence being altered.
[0129] In general, the technique of site-specific mutagenesis is
well known in the art, as exemplified by various publications. As
will be appreciated, the technique typically employs a phage vector
which exists in both a single stranded and double stranded form.
Typical vectors useful in site-directed mutagenesis include vectors
such as the M13 phage. These phage are readily commercially
available and their use is generally well known to those skilled in
the art. Double stranded plasmids are also routinely employed in
site directed mutagenesis which eliminates the step of transferring
the gene of interest from a plasmid to a phage.
[0130] In general, site-directed mutagenesis in accordance herewith
is performed by first obtaining a single-stranded vector or melting
apart of two strands of a double stranded vector which includes
within its sequence a DNA sequence which encodes the desired
peptide. An oligonucleotide primer bearing the desired mutated
sequence is prepared, generally synthetically. This primer is then
annealed with the single-stranded vector, and subjected to DNA
polymerizing enzymes such as E. coli polymerase I Klenow fragment,
in order to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed wherein one strand encodes the
original non-mutated sequence and the second strand bears the
desired mutation. This heteroduplex vector is then used to
transform or transfect appropriate cells, such as E. coli cells,
and clones are selected which include recombinant vectors bearing
the mutated sequence arrangement. A genetic selection scheme was
devised by Kunkel et al. (1987) to enrich for clones incorporating
the mutagenic oligonucleotide. Alternatively, the use of PCR.TM.
with commercially available thermostable enzymes such as Taq
polymerase may be used to incorporate a mutagenic oligonucleotide
primer into an amplified DNA fragment that can then be cloned into
an appropriate cloning or expression vector. The PCR.TM.-mediated
mutagenesis procedures of Tomic et al. (1990) and Upender et al.
(1995) provide two examples of such protocols. A PCR.TM. employing
a thermostable ligase in addition to a thermostable polymerase may
also be used to incorporate a phosphorylated mutagenic
oligonucleotide into an amplified DNA fragment that may then be
cloned into an appropriate cloning or expression vector. The
mutagenesis procedure described by Michael (1994) provides an
example of one such protocol.
[0131] In a preferred embodiment of the invention,
oligonucleotide-directe- d mutagenesis may be used to insert or
delete amino acid residues within a loop region. For instance, this
mutagenic oligonucleotide could be used to delete a proline residue
(P120) within loop .alpha. 34 of the Cry1C protein from EG6346 or
aizawai strain 7.29: 5'-GCATTTAAAGAATGGGAAGAAGATAA-
TAATCCAGCAACCAGGACCAGAG-3' (SEQ ID NO:13)
[0132] Likewise, this mutagenic oligonucleotide may be used to add
an alanine residue between amino acid residues N121 and N122 within
loop .alpha. 3-4 of the Cry1C protein from EG6346 or aizawai strain
7.29: 5'-GCATTTAAAGAATGGGAAGAAGATCCTAATGCAAATCCAGCAACCAGGACCAGAG-3'
(SEQ ID NO:14)
[0133] The preparation of sequence variants of the selected
peptide-encoding DNA segments using site-directed mutagenesis is
provided as a means of producing potentially useful species and is
not meant to be limiting as there are other ways in which sequence
variants of peptides and the DNA sequences encoding them may be
obtained. For example, recombinant vectors encoding the desired
peptide sequence may be treated with mutagenic agents, such as
hydroxylamine, to obtain sequence variants.
[0134] As used herein, the term "oligonucleotide directed
mutagenesis procedure" refers to template-dependent processes and
vector-mediated propagation which result in an increase in the
concentration of a specific nucleic acid molecule relative to its
initial concentration, or in an increase in the concentration of a
detectable signal, such as amplification. As used herein, the term
"oligonucleotide directed mutagenesis procedure" is intended to
refer to a process that involves the template-dependent extension
of a primer molecule. The term template dependent process refers to
nucleic acid synthesis of an RNA or a DNA molecule wherein the
sequence of the newly synthesized strand of nucleic acid is
dictated by the well-known rules of complementary base pairing
(see, for example, Watson, 1987). Typically, vector mediated
methodologies involve the introduction of the nucleic acid fragment
into a DNA or RNA vector, the clonal amplification of the vector,
and the recovery of the amplified nucleic acid fragment. Examples
of such methodologies are provided by U.S. Pat. No. 4,237,224,
specifically incorporated herein by reference in its entirety.
[0135] A number of template dependent processes are available to
amplify the target sequences of interest present in a sample. One
of the best known amplification methods is the polymerase chain
reaction (PCR.TM.) which is described in detail in U.S. Pat. Nos.
4,683,195, 4,683,202 and 4,800,159, each of which is incorporated
herein by reference in its entirety. Briefly, in PCR.TM., two
primer sequences are prepared which are complementary to regions on
opposite complementary strands of the target sequence. An excess of
deoxynucleoside triphosphates are added to a reaction mixture along
with a DNA polymerase (e.g., Taq polymerase). If the target
sequence is present in a sample, the primers will bind to the
target and the polymerase will cause the primers to be extended
along the target sequence by adding on nucleotides. By raising and
lowering the temperature of the reaction mixture, the extended
primers will dissociate from the target to form reaction products,
excess primers will bind to the target and to the reaction products
and the process is repeated. Preferably a reverse transcriptase
PCR.TM. amplification procedure may be performed in order to
quantify the amount of mRNA amplified. Polymerase chain reaction
methodologies are well known in the art.
[0136] Another method for amplification is the ligase chain
reaction (referred to as LCR), disclosed in Eur. Pat. Appl. Publ.
No. 320,308, incorporated herein by reference in its entirety. In
LCR, two complementary probe pairs are prepared, and in the
presence of the target sequence, each pair will bind to opposite
complementary strands of the target such that they abut. In the
presence of a ligase, the two probe pairs will link to form a
single unit. By temperature cycling, as in PCR.TM., bound ligated
units dissociate from the target and then serve as "target
sequences" for ligation of excess probe pairs. U.S. Pat. No.
4,883,750, incorporated herein by reference in its entirety,
describes an alternative method of amplification similar to LCR for
binding probe pairs to a target sequence.
[0137] Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No.
PCT/US87/00880, incorporated herein by reference in its entirety,
may also be used as still another amplification method in the
present invention. In this method, a replicative sequence of RNA
which has a region complementary to that of a target is added to a
sample in the presence of an RNA polymerase. The polymerase will
copy the replicative sequence which can then be detected.
[0138] An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification of
target molecules that contain nucleotide
5'-[.alpha.-thio]triphosphates in one strand of a restriction site
(Walker et al., 1992, incorporated herein by reference in its
entirety), may also be useful in the amplification of nucleic acids
in the present invention.
[0139] Strand Displacement Amplification (SDA) is another method of
carrying out isothermal amplification of nucleic acids which
involves multiple rounds of strand displacement and synthesis, i.e.
nick translation. A similar method, called Repair Chain Reaction
(RCR) is another method of amplification which may be useful in the
present invention and is involves annealing several probes
throughout a region targeted for amplification, followed by a
repair reaction in which only two of the four bases are present.
The other two bases can be added as biotinylated derivatives for
easy detection. A similar approach is used in SDA.
[0140] Sequences can also be detected using a cyclic probe reaction
(CPR). In CPR, a probe having a 3' and 5' sequences of non-Cry1C
specific DNA and middle sequence of Cry1C protein specific RNA is
hybridized to DNA which is present in a sample. Upon hybridization,
the reaction is treated with RNaseH, and the products of the probe
identified as distinctive products generating a signal which are
released after digestion. The original template is annealed to
another cycling probe and the reaction is repeated. Thus, CPR
involves amplifying a signal generated by hybridization of a probe
to a cry1C specific expressed nucleic acid.
[0141] Still other amplification methods described in Great Britain
Pat. Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No.
PCT/US89/01025, each of which is incorporated herein by reference
in its entirety, may be used in accordance with the present
invention. In the former application, "modified" primers are used
in a PCR like, template and enzyme dependent synthesis. The primers
may be modified by labeling with a capture moiety (e.g., biotin)
and/or a detector moiety (e.g., enzyme). In the latter application,
an excess of labeled probes are added to a sample. In the presence
of the target sequence, the probe binds and is cleaved
catalytically. After cleavage, the target sequence is released
intact to be bound by excess probe. Cleavage of the labeled probe
signals the presence of the target sequence.
[0142] Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS) (Kwoh et al., 1989;
PCT Intl. Pat. Appl. Publ. No. WO 88/10315, incorporated herein by
reference in its entirety), including nucleic acid sequence based
amplification (NASBA) and 3SR. In NASBA, the nucleic acids can be
prepared for amplification by standard phenol/chloroform
extraction, heat denaturation of a sample, treatment with lysis
buffer and minispin columns for isolation of DNA and RNA or
guanidinium chloride extraction of RNA. These amplification
techniques involve annealing a primer which has crystal
protein-specific sequences. Following polymerization, DNA/RNA
hybrids are digested with RNase H while double stranded DNA
molecules are heat denatured again. In either case the single
stranded DNA is made fully double stranded by addition of second
crystal protein-specific primer, followed by polymerization. The
double stranded DNA molecules are then multiply transcribed by a
polymerase such as T7 or SP6. In an isothermal cyclic reaction, the
RNAs are reverse transcribed into double stranded DNA, and
transcribed once against with a polymerase such as T7 or SP6. The
resulting products, whether truncated or complete, indicate crystal
protein-specific sequences.
[0143] Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by
reference in its entirety, disclose a nucleic acid amplification
process involving cyclically synthesizing single-stranded RNA
("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be
used in accordance with the present invention. The ssRNA is a first
template for a first primer oligonucleotide, which is elongated by
reverse transcriptase (RNA-dependent DNA polymerase). The RNA is
then removed from resulting DNA:RNA duplex by the action of
ribonuclease H (RNase H, an RNase specific for RNA in a duplex with
either DNA or RNA). The resultant ssDNA is a second template for a
second primer, which also includes the sequences of an RNA
polymerase promoter (exemplified by T7 RNA polymerase) 5' to its
homology to its template. This primer is then extended by DNA
polymerase (exemplified by the large "Klenow" fragment of E. coli
DNA polymerase 1), resulting as a double-stranded DNA ("dsDNA")
molecule, having a sequence identical to that of the original RNA
between the primers and having additionally, at one end, a promoter
sequence. This promoter sequence can be used by the appropriate RNA
polymerase to make many RNA copies of the DNA. These copies can
then re-enter the cycle leading to very swift amplification. With
proper choice of enzymes, this amplification can be done
isothermally without addition of enzymes at each cycle. Because of
the cyclical nature of this process, the starting sequence can be
chosen to be in the form of either DNA or RNA.
[0144] PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated
herein by reference in its entirety, disclose a nucleic acid
sequence amplification scheme based on the hybridization of a
promoter/primer sequence to a target single-stranded DNA ("ssDNA")
followed by transcription of many RNA copies of the sequence. This
scheme is not cyclic; i.e. new templates are not produced from the
resultant RNA transcripts. Other amplification methods include
"RACE" (Frohman, 1990), and "one-sided PCR" (Ohara, 1989) which are
well-known to those of skill in the art.
[0145] Methods based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the
resulting "di-oligonucleotide", thereby amplifying the
di-oligonucleotide (Wu and Dean, 1996, incorporated herein by
reference in its entirety), may also be used in the amplification
of DNA sequences of the present invention.
2.7 Phage-Resistant Variants
[0146] To prepare phage resistant variants of the B. thuringiensis
mutants, an aliquot of the phage lysate is spread onto nutrient
agar and allowed to dry. An aliquot of the phage sensitive
bacterial strain is then plated directly over the dried lysate and
allowed to dry. The plates are incubated at 30.degree. C. The
plates are incubated for 2 days and, at that time, numerous
colonies could be seen growing on the agar. Some of these colonies
are picked and subcultured onto nutrient agar plates. These
apparent resistant cultures are tested for resistance by cross
streaking with the phage lysate. A line of the phage lysate is
streaked on the plate and allowed to dry. The presumptive resistant
cultures are then streaked across the phage line. Resistant
bacterial cultures show no lysis anywhere in the streak across the
phage line after overnight incubation at 30.degree. C. The
resistance to phage is then reconfirmed by plating a lawn of the
resistant culture onto a nutrient agar plate. The sensitive strain
is also plated in the same manner to serve as the positive control.
After drying, a drop of the phage lysate is plated in the center of
the plate and allowed to dry. Resistant cultures showed no lysis in
the area where the phage lysate has been placed after incubation at
30.degree. C. for 24 hours.
2.8 Transgenic Hosts/Transformed Cells Comprising Cry1C* DNA
Segments
[0147] The invention also discloses and claims host cells, both
native, and genetically engineered, which express the novel cry1C*
genes to produce Cry1C* polypeptides. Preferred examples of
bacterial host cells include Bacillus thuringiensis . NRRL B-21590,
NRRL B-21591, NRRL B-21592, NRRL B-21638, NRRL B-21639, NRRL
B-21640, NRRL B-21609. and NRRL B-21610.
[0148] Methods of using such cells to produce Cry1C* crystal
proteins are also disclosed. Such methods generally involve
culturing the host cell (such as Bacillus thuringiensis NRRL
B-21590, NRRL B-21591, NRRL B-21592, NRRL B-21638, NRRL B-21639,
NRRL B-21640, NRRL B-21609. or NRRL B-21610) under conditions
effective to produce a Cry1C* crystal protein, and obtaining the
Cry1C* crystal protein from said cell.
[0149] In yet another aspect, the present invention provides
methods for producing a transgenic plant which expresses a nucleic
acid segment encoding the novel recombinant crystal proteins of the
present invention. The process of producing transgenic plants is
well-known in the art. In general, the method comprises
transforming a suitable host cell with one or more DNA segments
which contain one or more promoters operatively linked to a coding
region that encodes one or more of the novel B. thuringiensis
Cry1C-R148A, Cry1C-R148G, Cry1C-R148M, Cry1C-R148L, Cry1C-R180A,
Cry1C-R148D, Cry1C.499, Cry1C563 and Cry1C.579 crystal proteins.
Such a coding region is generally operatively linked to a
transcription-terminating region, whereby the promoter is capable
of driving the transcription of the coding region in the cell, and
hence providing the cell the ability to produce the recombinant
protein in vivo. Alternatively, in instances where it is desirable
to control, regulate, or decrease the amount of a particular
recombinant crystal protein expressed in a particular transgenic
cell, the invention also provides for the expression of crystal
protein antisense mRNA. The use of antisense mRNA as a means of
controlling or decreasing the amount of a given protein of interest
in a cell is well-known in the art.
[0150] Another aspect of the invention comprises a transgenic plant
which express a gene or gene segment encoding one or more of the
novel polypeptide compositions disclosed herein. As used herein,
the term "transgenic plant" is intended to refer to a plant that
has incorporated DNA sequences, including but not limited to genes
which are perhaps not normally present, DNA sequences not normally
transcribed into RNA or translated into a protein ("expressed"), or
any other genes or DNA sequences which one desires to introduce
into the non-transformed plant, such as genes which may normally be
present in the non-transformed plant but which one desires to
either genetically engineer or to have altered expression.
[0151] It is contemplated that in some instances the genome of a
transgenic plant of the present invention will have been augmented
through the stable introduction of one or more Cry1C-R148A-,
Cry1C-R148D-, Cry1C-R148G, Cry1C-R148M, Cry1C-R148L,
Cry1C-R180A-Cry1C.499-, Cry1C.563-, or Cry1C.579-encoding
transgenes, either native, synthetically modified, or mutated. In
some instances, more than one transgene will be incorporated into
the genome of the transformed host plant cell. Such is the case
when more than one crystal protein-encoding DNA segment is
incorporated into the genome of such a plant. In certain
situations, it may be desirable to have one, two, three, four, or
even more B. thuringiensis crystal proteins (either native or
recombinantly-engineered) incorporated and stably expressed in the
transformed transgenic plant.
[0152] A preferred gene which may be introduced includes, for
example, a crystal protein-encoding a DNA sequence from bacterial
origin, and particularly one or more of those described herein
which are obtained from Bacillus spp. Highly preferred nucleic acid
sequences are those obtained from B. thuringiensis, or any of those
sequences which have been genetically engineered to decrease or
increase the insecticidal activity of the crystal protein in such a
transformed host cell.
[0153] Means for transforming a plant cell and the preparation of a
transgenic cell line are well-known in the art, and are discussed
herein. Vectors, plasmids, cosmids, YACs (yeast artificial
chromosomes) and DNA segments for use in transforming such cells
will, of course, generally comprise either the operons, genes, or
gene-derived sequences of the present invention, either native, or
synthetically-derived, and particularly those encoding the
disclosed crystal proteins. These DNA constructs can further
include structures such as promoters, enhancers, polylinkers, or
even gene sequences which have positively- or negatively-regulating
activity upon the particular genes of interest as desired. The DNA
segment or gene may encode either a native or modified crystal
protein, which will be expressed in the resultant recombinant
cells, and/or which will impart an improved phenotype to the
regenerated plant.
[0154] Such transgenic plants may be desirable for increasing the
insecticidal resistance of a monocotyledonous or dicotyledonous
plant, by incorporating into such a plant, a transgenic DNA segment
encoding a Cry1C-R148A, Cry1C-R148D, Cry1C-R148G, Cry1C-R148L,
Cry1C-R148M, Cry1C-R180A, Cry1C.499, Cry1C.563, and/or Cry1C.579
crystal protein which is toxic to lepidopteran insects.
Particularly preferred plants include grains such as corn, wheat,
barley, maize, and oats; legumes such as soybeans; cotton; turf and
pasture grasses; ornamental plants; shrubs; trees; vegetables,
berries, fruits, and other commercially-important crops including
garden and houseplants.
[0155] In a related aspect, the present invention also encompasses
a seed produced by the transformed plant, a progeny from such seed,
and a seed produced by the progeny of the original transgenic
plant, produced in accordance with the above process. Such progeny
and seeds will have one or more crystal protein transgene(s) stably
incorporated into its genome, and such progeny plants will inherit
the traits afforded by the introduction of a stable transgene in
Mendelian fashion. All such transgenic plants having incorporated
into their genome transgenic DNA segments encoding one or more
Cry1C-R148A, Cry1C-R148D, Cry1C-R148G, Cry1C-R148M, Cry1C-R148L,
Cry1C-R180A. Cry1C.499, Cry1 C.563 or Cry1C.579 crystal proteins or
polypeptides are aspects of this invention. Particularly preferred
transgenes for the practice of the invention include nucleic acid
segments comprising one or more cry1C-R148A, cry1C-R148D,
cry1C-R148G, cry1C-R148M, cry1C-R148L, cry1C-R180A, cry1C.499,
cry1C.563 or cry1C.579 gene(s).
2.9 Crystal Protein Compositions as Insecticides and Methods of
Use
[0156] The inventors contemplate that the crystal protein
compositions disclosed herein will find particular utility as
insecticides for topical and/or systemic application to field
crops, grasses, fruits and vegetables, and ornamental plants.
[0157] Disclosed and claimed is a composition comprising an
insecticidally-effective amount of a Cry1C* crystal protein
composition. The composition preferably comprises the amino acid
sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12, SEQ ID NO:59, or SEQ ID NO:61 or
biologically-functional equivalents thereof. The insecticide
composition may also comprise a Cry1C* crystal protein that is
encoded by a nucleic acid sequence having the sequence of SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:58, or SEQ ID NO:60, or, alternatively, a nucleic
acid sequence which hybridizes to the nucleic acid sequence of SEQ
ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:58, or SEQ ID NO:60 under conditions of moderate
stringency.
[0158] The insecticide comprises a Bacillus thuringiensis NRRL
B-21590, NRRL B-21591, NRRL B-21592, NRRL B-21638, NRRL B-21639,
NRRL B-21640, NRRL B-21609, or NRRL B-21610 cell, or a culture of
these cells, or a mixture of one or more B. thuringiensis cells
which express one or more of the novel crystal proteins of the
invention. In certain aspects it may be desirable to prepare
compositions which contain a plurality of crystal proteins, either
native or modified, for treatment of one or more types of
susceptible insects.
[0159] The inventors contemplate that any formulation methods known
to those of skill in the art may be employed using the proteins
disclosed herein to prepare such bioinsecticide compositions. It
may be desirable to formulate whole cell preparations, cell
extracts, cell suspensions, cell homogenates, cell lysates, cell
supernatants, cell filtrates, or cell pellets of a cell culture
(preferably a bacterial cell culture such as a Bacillus
thuringiensis NRRL B-21590, NRRL B-21591, NRRL B-21592, NRRL
B-21638, NRRL B-21639, NRRL B-21640, NRRL B-21609, or NRRL B-21610
culture) that expresses one or more cry1C* DNA segments to produce
the encoded Cry1C* protein(s) or peptide(s). The methods for
preparing such formulations are known to those of skill in the art,
and may include, e.g., desiccation, lyophilization, homogenization,
extraction, filtration, centrifugation, sedimentation, or
concentration of one or more cultures of bacterial cells, such as
Bacillus NRRL B-21590, NRRL B-21591, NRRL B-21592, NRRL B-21638,
NRRL B-21639, NRRL B-21640, NRRL B-21609, or NRRL B-21610 cells,
which express the Cry1C * peptide(s) of interest.
[0160] In one preferred embodiment, the bioinsecticide composition
comprises an oil flowable suspension comprising lysed or unlysed
bacterial cells, spores, or crystals which contain one or more of
the novel crystal proteins disclosed herein. Preferably the cells
are B. thuringiensis cells, however, any such bacterial host cell
expressing the novel nucleic acid segments disclosed herein and
producing a crystal protein is contemplated to be useful, such as
Bacillus spp., including B. megaterium, B. subtilis; B. cereus,
Escherichia spp., including E. coli, and/or Pseudomonas spp.,
including P. cepacia, P. aeruginosa, and P. fluorescens.
Alternatively, the oil flowable suspension may consist of a
combination of one or more of the following compositions: lysed or
unlysed bacterial cells, spores, crystals, and/or purified crystal
proteins.
[0161] In a second preferred embodiment, the bioinsecticide
composition comprises a water dispersible granule or powder. This
granule or powder may comprise lysed or unlysed bacterial cells,
spores, or crystals which contain one or more of the novel crystal
proteins disclosed herein. Preferred sources for these compositions
include bacterial cells such as B. thuringiensis cells, however,
bacteria of the genera Bacillus, Escherichia, and Pseudomonas which
have been transformed with a DNA segment disclosed herein and
expressing the crystal protein are also contemplated to be useful.
Alternatively, the granule or powder may consist of a combination
of one or more of the following compositions: lysed or unlysed
bacterial cells, spores, crystals, and/or purified crystal
proteins.
[0162] In a third important embodiment, the bioinsecticide
composition comprises a wettable powder, spray, emulsion, colloid,
aqueous or organic solution, dust, pellet, or collodial
concentrate. Such a composition may contain either unlysed or lysed
bacterial cells, spores, crystals, or cell extracts as described
above, which contain one or more of the novel crystal proteins
disclosed herein. Preferred bacterial cells are B. thuringiensis
cells, however, bacteria such as B. megaterium, B. subtilis, B.
cereus, E. coli, or Pseudomonas spp. cells transformed with a DNA
segment disclosed herein and expressing the crystal protein are
also contemplated to be useful. Such dry forms of the insecticidal
compositions may be formulated to dissolve immediately upon
wetting, or alternatively, dissolve in a controlled-release,
sustained-release, or other time-dependent manner. Alternatively,
such a composition may consist of a combination of one or more of
the following compositions: lysed or unlysed bacterial cells,
spores, crystals, and/or purified crystal proteins.
[0163] In a fourth important embodiment, the bioinsecticide
composition comprises an aqueous solution or suspension or cell
culture of lysed or unlysed bacterial cells, spores, crystals, or a
mixture of lysed or unlysed bacterial cells, spores, and/or
crystals, such as those described above which contain one or more
of the novel crystal proteins disclosed herein. Such aqueous
solutions or suspensions may be provided as a concentrated stock
solution which is diluted prior to application, or alternatively,
as a diluted solution ready-to-apply.
[0164] For these methods involving application of bacterial cells,
the cellular host containing the Crystal protein gene(s) may be
grown in any convenient nutrient medium, where the DNA construct
provides a selective advantage, providing for a selective medium so
that substantially all or all of the cells retain the B.
thuringiensis gene. These cells may then be harvested in accordance
with conventional ways. Alternatively, the cells can be treated
prior to harvesting.
[0165] When the insecticidal compositions comprise B. thuringiensis
cells, spores, and/or crystals containing the modified crystal
protein(s) of interest, such compositions may be formulated in a
variety of ways. They may be employed as wettable powders, granules
or dusts, by mixing with various inert materials, such as inorganic
minerals (phyllosilicates, carbonates, sulfates, phosphates, and
the like) or botanical materials (powdered corncobs, rice hulls,
walnut shells, and the like). The formulations may include
spreader-sticker adjuvants, stabilizing agents, other pesticidal
additives, or surfactants. Liquid formulations may be aqueous-based
or non-aqueous and employed as foams, suspensions, emulsifiable
concentrates, or the like. The ingredients may include rheological
agents, surfactants, emulsifiers, dispersants, or polymers.
[0166] Alternatively, the novel Cry1C-derived mutated crystal
proteins may be prepared by native or recombinant bacterial
expression systems in vitro and isolated for subsequent field
application. Such protein may be either in crude cell lysates,
suspensions, colloids, etc., or alternatively may be purified,
refined, buffered, and/or further processed, before formulating in
an active biocidal formulation. Likewise, under certain
circumstances, it may be desirable to isolate crystals and/or
spores from bacterial cultures expressing the crystal protein and
apply solutions, suspensions, or collodial preparations of such
crystals and/or spores as the active bioinsecticidal
composition.
[0167] Another important aspect of the invention is a method of
controlling lepidopteran insects which are susceptible to the novel
compositions disclosed herein. Such a method generally comprises
contacting the insect or insect population, colony, etc., with an
insecticidally-effective amount of a Cry1C* crystal protein
composition. The method may utilize Cry1C* crystal proteins such as
those disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:59, or SEQ ID NO:61, or
biologically functional equivalents thereof Alternatively, the
method may utilize one or more Cry1C* crystal proteins which are
encoded by the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:58,
or SEQ ID NO:60, or by one or more nucleic acid sequences which
hybridize to the sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:58, or SEQ
ID NO:60, under conditions of moderate, or higher, stringency. The
methods for identifying sequences which hybridize to those
disclosed under conditions of moderate or higher stringency are
well-known to those of skill in the art, and are discussed
herein.
[0168] Regardless of the method of application, the amount of the
active component(s) are applied at an insecticidally-effective
amount, which will vary depending on such factors as, for example,
the specific lepidopteran insects to be controlled, the specific
plant or crop to be treated, the environmental conditions, and the
method, rate, and quantity of application of the
insecticidally-active composition.
[0169] The insecticide compositions described may be made by
formulating either the bacterial cell, crystal and/or spore
suspension, or isolated protein component with the desired
agriculturally-acceptable carrier. The compositions may be
formulated prior to administration in an appropriate means such as
lyophilized, freeze-dried, dessicated, or in an aqueous carrier,
medium or suitable diluent, such as saline or other buffer. The
formulated compositions may be in the form of a dust or granular
material, or a suspension in oil (vegetable or mineral), or water
or oil/water emulsions, or as a wettable powder, or in combination
with any other carrier material suitable for agricultural
application. Suitable agricultural carriers can be solid or liquid
and are well known in the art. The term "agriculturally-acceptable
carrier" covers all adjuvants, e.g., inert components, dispersants,
surfactants, tackifiers, binders, etc. that are ordinarily used in
insecticide formulation technology; these are well known to those
skilled in insecticide formulation. The formulations may be mixed
with one or more solid or liquid adjuvants and prepared by various
means, e.g., by homogeneously mixing, blending and/or grinding the
insecticidal composition with suitable adjuvants using conventional
formulation techniques.
[0170] The insecticidal compositions of this invention are applied
to the environment of the target lepidopteran insect, typically
onto the foliage of the plant or crop to be protected, by
conventional methods, preferably by spraying. The strength and
duration of insecticidal application will be set with regard to
conditions specific to the particular pest(s), crop(s) to be
treated and particular environmental conditions. The proportional
ratio of active ingredient to carrier will naturally depend on the
chemical nature, solubility, and stability of the insecticidal
composition, as well as the particular formulation
contemplated.
[0171] Other application techniques, e.g., dusting, sprinkling,
soaking, soil injection, seed coating, seedling coating, spraying,
aerating, misting, atomizing, and the like, are also feasible and
may be required under certain circumstances such as e.g., insects
that cause root or stalk infestation, or for application to
delicate vegetation or ornamental plants. These application
procedures are also well-known to those of skill in the art.
[0172] The insecticidal composition of the invention may be
employed in the method of the invention singly or in combination
with other compounds, including and not limited to other
pesticides. The method of the invention may also be used in
conjunction with other treatments such as surfactants, detergents,
polymers or time-release formulations. The insecticidal
compositions of the present invention may be formulated for either
systemic or topical use.
[0173] The concentration of insecticidal composition which is used
for environmental, systemic, or foliar application will vary widely
depending upon the nature of the particular formulation, means of
application, environmental conditions, and degree of biocidal
activity. Typically, the bioinsecticidal composition will be
present in the applied formulation at a concentration of at least
about 1% by weight and may be up to and including about 99% by
weight. Dry formulations of the compositions may be from about 1%
to about 99% or more by weight of the composition, while liquid
formulations may generally comprise from about 1% to about 99% or
more of the active ingredient by weight. Formulations which
comprise intact bacterial cells will generally contain from about
10.sup.4 to about 10.sup.12 cells/mg.
[0174] The insecticidal formulation may be administered to a
particular plant or target area in one or more applications as
needed, with a typical field application rate per hectare ranging
on the order of from about 1 g to about 1 kg, 2 kg, 5, kg, or more
of active ingredient.
2.10 Biological Functional Equivalents
[0175] Modification and changes may be made in the structure of the
peptides of the present invention and DNA segments which encode
them and still obtain a functional molecule that encodes a protein
or peptide with desirable characteristics. The following is a
discussion based upon changing the amino acids of a protein to
create an equivalent, or even an improved, second-generation
molecule. In particular embodiments of the invention, mutated
crystal proteins are contemplated to be useful for increasing the
insecticidal activity of the protein, and consequently increasing
the insecticidal activity and/or expression of the recombinant
transgene in a plant cell. The amino acid changes may be achieved
by changing the codons of the DNA sequence, according to the codons
given in Table 3.
3TABLE 3 Amino Acid Codons Alanine Ala A GCA GCC GCG GCU Cysteine
Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA
GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K
AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG
Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine
Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S
AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val
V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
[0176] For example, certain amino acids may be substituted for
other amino acids in a protein structure without appreciable loss
of interactive binding capacity with structures such as, for
example, antigen-binding regions of antibodies or binding sites on
substrate molecules. Since it is the interactive capacity and
nature of a protein that defines that protein's biological
functional activity, certain amino acid sequence substitutions can
be made in a protein sequence, and, of course, its underlying DNA
coding sequence, and nevertheless obtain a protein with like
properties. It is thus contemplated by the inventors that various
changes may be made in the peptide sequences of the disclosed
compositions, or corresponding DNA sequences which encode said
peptides without appreciable loss of their biological utility or
activity.
[0177] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a protein is
generally understood in the art (Kyte and Doolittle, 1982,
incorporate herein by reference). It is accepted that the relative
hydropathic character of the amino acid contributes to the
secondary structure of the resultant protein, which in turn defines
the interaction of the protein with other molecules, for example,
enzymes, substrates, receptors, DNA, antibodies, antigens, and the
like.
[0178] Each amino acid has been assigned a hydropathic index on the
basis of their hydrophobicity and charge characteristics (Kyte and
Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-4); threonine (-0.7);
serine (-8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6);
histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate
(-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
[0179] It is known in the art that certain amino acids may be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, i.e., still obtain a biological functionally equivalent
protein. In making such changes, the substitution of amino acids
whose hydropathic indices are within .+-.2 is preferred, those
which are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred.
[0180] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with a biological property of the protein.
[0181] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
[0182] It is understood that an amino acid can be substituted for
another having a similar hydrophilicity value and still obtain a
biologically equivalent, and in particular, an immunologically
equivalent protein. In such changes, the substitution of amino
acids whose hydrophilicity values are within .+-.2 is preferred,
those which are within .+-.1 are particularly preferred, and those
within .+-.0.5 are even more particularly preferred.
[0183] As outlined above, amino acid substitutions are generally
therefore based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
which take various of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine.
3.0 BRIEF DESCRIPTION OF THE DRAWINGS
[0184] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0185] FIG. 1. Schematic diagram of the Cry1C crystal protein from
B. thuringiensis. .alpha. helices are depicted by the rectangles
and are labeled according to the convention adopted by Li et al.,
(1991). Adopting the convention of Li et al., the present inventors
have designated helix two as comprising two portions helix 2a and
helix 2b.
[0186] FIG. 2. Shown are the structural maps of pEG315, pEG916,
pEG359, and p154. Boxed arrows and segments indicate genes or
functional DNA elements. Designations: pTZ19u=E. coli phagemid
vector pTZ19u, cat=chloramphenicol (Cml) acetyltransferase gene,
ori43 and ori60=B. thuringiensis plasmid replication origins,
cry1C=cry1C insecticidal crystal protein gene. Restriction site
abbreviations: Ag=AgeI, Asp=Asp718, Ba=BamHI, Bb=BbuI, Bg=BglII,
Bln=BlnI, P=PstI, S=SalI, X=XhoI. The 1 kb scale refers to only the
cry1C gene segment. pEG315 gave rise to pEG 1635 and pEG1636, which
contain the Arg148Ala and Arg180Ala mutations, respectively. pEG916
gave rise to pEG370, pEG373, and pEG374, which contain the
cry1C.563, cry1C.579,. and cry1C.499 mutations, respectively. These
mutants are described in detail in Section 5.
[0187] FIG. 3. Shown is the structural map of pEG345. Boxed arrows
and segments indicate genes or functional DNA elements.
Designations: pTZ19u=E. coli phagemid vector pTZ19u, cat=Cml
acetyltransferase gene, ori44=B. thuringiensis plasmid replication
origin, cry1C=cry/C insecticidal crystal protein gene. Restriction
site abbreviations: Ag=AgeI, Asp=Asp718, Bb=BbuI, Bg=BglII,
E=EcoRI, H=HindIII, Sm=SmaI. The 1 kb scale refers to only the
cry1C gene segment.
[0188] FIG. 4. Depicted is a flow chart indicating the mutations
contained within the cry1C gene encoded by pEG359 and the mutations
contained within the cry1C.563, cry1C.579, and cry1C.499 genes
generated by random mutagenesis.
[0189] FIG. 5. Shown is the PCR.TM.-mediated mutagenesis procedure
used to generate the mutant cry1C.499, cry1C.563, and cry1C.579
genes in strains EG11747, EG11740, and EG11746, respectively. The
asterisk denotes mutations incorporated into the cry1C gene
sequence. Restriction sites abbreviations: Ag=AgeI, Bb=BbuI, and
Bg=BglII.
[0190] FIG. 6. Shown is the alignment of a loop region of 24
related Cry1 proteins.
[0191] FIG. 7. Structural maps of the cry1C-encoding plasmids
pEG348 and pEG348.DELTA.. Boxed arrows and segments indicate genes
or functional DNA elements. Designations: pTZ19u=E. coli phagemid
vector pTZ19u, tet=tetracycline resistance gene, ori60=B.
thuringiensis plasmid replication origin, cry1C=cry1C insecticidal
crystal protein gene, IRS=DNA fragment containing the internal
resolution site region of transposon Tn5401. Restriction site
abbreviations: A=Asp718, H=HindIII, Nsi=NsiI, Nsp=NspI, P=PstI,
Sp=SphI.
[0192] FIG. 8. Structural maps of the cry1C-encoding plasmids
pEG1641 and pEG1641.DELTA.. Boxed arrows and segments indicate
genes or functional DNA elements. Designations: pTZ19u=E. coli
phagemid vector pTZ19u, tet=tetracycline resistance gene, ori60=B.
thuringiensis plasmid replication origin, cry1C=cry1C insecticidal
crystal protein gene, IRS=DNA fragment containing the internal
resolution site region of transposon Tn5401. Restriction site
abbreviations: A=Asp718, H=HindIII, Nsi=NsiI, Nsp=NspI, P=PstI,
Sp=SphI.
[0193] FIG. 9. Shown is the structural map of pEG943. Boxed arrows
and segments indicate genes or functional DNA elements.
Designations: pTZ19u=E. coli phagemid vector pTZ19u cat=Cml
acetyltransferase gene, ori44=B. thuringiensis plasmid replication
origin, cry1C=cry1C insecticidal crystal protein gene. Restriction
site abbreviations: Ag=AgeI, Asp=Asp718, Bb=BbuI, Bg=BglII,
E=EcoRI, H=HindIII, Nh=NheI, Sm=SmaI. The 1 kb scale refers to only
the cry1C gene segment.
[0194] FIG. 10. Shown is the overlap extension PCR.TM. procedure
used to generate Cry1C-R148D combinatorial mutants with amino acid
substitutions in loop .alpha.6-7. The asterisk denotes mutations
incorporated into the cry1C gene sequence. The PCR.TM. with the
flanking primers H and L yielded a sub-population of fragments
encoding mutations in loop .alpha.6-7 and lacking the NheI site
derived from the pEG943 template. Restriction site abbreviations:
Ag=AgeI, Asp=Asp718, Bb=BbuI, Bg=BglII, E=EcoRI, H=HindIII,
Nh=NheI, Sm=SmaI.
[0195] FIG. 11. Shown is the overlap extension PCR.TM. procedure
used to generate Cry1C-R148D combinatorial mutants with amino acid
substitutions in loop .alpha.5-6. The asterisk denotes mutations
incorporated into the cry1C gene sequence. The PCR.TM. with the
flanking primers H and L yielded a sub-population of fragments
encoding mutations in loop .alpha.5-6 and lacking the NheI site
derived from the pEG943 template. Restriction site abbreviations:
Ag=AgeI, Asp=Asp718, Bb=BbuI, Bg=BglII, E=EcoRI, H=HindIII,
Nh=NheI, Sm=SmaI.
4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
4.1 Some Advantages of the Invention
[0196] Mutagenesis experiments with cry1 genes have failed to
identify mutant crystal proteins with improved broad-spectrum
insecticidal activity, that is, with improved toxicity towards a
range of insect pest species. Since agricultural crops are
typically threatened by more than one insect pest species at any
given time, desirable mutant crystal proteins are preferably those
that exhibit improvements in toxicity towards multiple insect pest
species. Previous failures to identify such mutants may be
attributed to the choice of sites targeted for mutagenesis. Sites
within domain 2 and domain 3 have been the principal targets of
previous Cry1 mutagenesis efforts, primarily because these domains
are believed to be important for receptor binding and in
determining insecticidal specificity (Aronson et al., 1995; Chen et
al. 1993; de Maagd et al., 1996; Lee et al., 1992; Lee et al.,
1995; Lu et al. 1994; Smedley and Ellar, 1996; Smith and Ellar,
1994; Rajamohan et al., 1995; Rajamohan et al., 1996).
[0197] In contrast, the present inventors reasoned that the
toxicity of Cry1 proteins, and specifically the toxicity of the
Cry1C protein, may be improved against a broader array of
lepidopteran pests by targeting regions involved in ion channel
function rather than regions of the molecule directly involved in
receptor interactions, namely domains 2 and 3. Accordingly, the
inventors opted to target regions within domain 1 of Cry1C for
mutagenesis in the hopes of isolating Cry1C mutants with improved
broad spectrum toxicity. Indeed, in the present invention, Cry1C
mutants are described that show improved toxicity towards several
lepidopteran pests, including Spodoptera exigua, Spodoptera
frugiperda, Trichoplusia ni, and Heliothis virescens, while
maintaining excellent activity against Plutella xylostella.
[0198] At least one, and probably more than one, .alpha. helix of
domain 1 is involved in the formation of ion channels and pores
within the insect midgut epithelium (Gazit and Shai, 1993; Gazit
and Shai, 1995). Rather than target for mutagenesis the sequences
encoding the .alpha. helices of domain 1 as others have (Wu and
Aronson, 1992; Aronson et al., 1995; Chen et al., 1995), the
present inventors opted to target exclusively sequences encoding
amino acid residues adjacent to or lying within the predicted loop
regions of Cry1C that separate these .alpha. helices. Amino acid
residues within these loop regions or amino acid residues capping
the end of an .alpha. helix and lying adjacent to these loop
regions may affect the spatial relationships among these .alpha.
helices. Consequently, the substitution of these amino acid
residues may result in subtle changes in tertiary structure, or
even quaternary structure, that positively impact the function of
the ion channel. Amino acid residues in the loop regions of domain
1 are exposed to the solvent and thus are available for various
molecular interactions. Altering these amino acids could result in
greater stability of the protein by eliminating or occluding
protease-sensitive sites. Amino acid substitutions that change the
surface charge of domain 1 could alter ion channel efficiency or
alter interactions with the brush border membrane or with other
portions of the toxin molecule, allowing binding or insertion to be
more effective.
[0199] In mutating specific residues within these loop regions, the
inventors were able to produce synthetic crystal proteins which
retained or even enhanced insecticidal activity against
lepidopteran insects.
[0200] According to this invention, base substitutions are made in
cry1C codons in order to change the particular codons with the loop
regions of the polypeptides, and particularly, in those loop
regions between .alpha.-helices. As an illustrative embodiment,
changes in three such amino acids within the loop region between
.alpha.-helices 3 and 4 of domain 1 produced modified crystal
proteins with enhanced insecticidal activity.
[0201] The insecticidal activity of a crystal protein ultimately
dictates the level of crystal protein required for effective insect
control. The potency of an insecticidal protein should be maximized
as much as possible in order to provide for its economic and
efficient utilization in the field. The increased potency of an
insecticidal protein in a bioinsecticide formulation would be
expected to improve the field performance of the bioinsecticide
product. Alternatively, increased potency of an insecticidal
protein in a bioinsecticide formulation may promote use of reduced
amounts of bioinsecticide per unit area of treated crop, thereby
allowing for more cost-effective use of the bioinsecticide product.
When expressed in planta, the production of crystal proteins with
improved insecticidal activity can be expected to improve plant
resistance to susceptible insect pests.
[0202] The most effective crystal protein against the beet
armyworm, Spodoptera exigua, is the Cry1C protein, yet the toxicity
of this toxin towards S. exigua is .about.40-fold less than the
toxicity of Cry1Ac towards the tobacco budworm, Heliothis
virescens, and .about.50-fold less than the toxicity of Cry1Ba
towards the diamondback moth, Plutella xylostella (Lambert et al.,
1996). Accordingly, there is a need to improve the toxicity of
Cry1C towards S. exigua as well as towards other lepidopteran
pests. Previously, site-directed mutagenesis was used to probe the
function of two surface-exposed loop regions found in domain 2 of
the Cry1C protein (Smith and Ellar, 1994). Although amino acid
substitutions within domain 2 were found to affect insecticidal
specificity, Cry1C mutants with improved insecticidal activity were
not obtained.
[0203] In sharp contrast to the prior art which has focused on
generating amino acid substitutions within the predicted
.alpha.-helices of domain 1 in Cry1A, the novel mutagenesis
strategies of the present invention focus on generating amino acid
substitutions at positions near or within the predicted loop
regions connecting the .alpha.-helices of domain 1. These loop
regions are shown in the schematic of crystal protein domains shown
in FIG. 1. In mutating specific residues within these loop regions,
the inventors were able to produce synthetic crystal proteins which
retained or possessed enhanced insecticidal activity against
certain lepidopteran pests, including the beet armyworm, S.
exigua.
[0204] According to this invention, base substitutions are made in
cry1C codons in order to change the particular codons encoding
amino acids within or near the predicted loop regions between the
.alpha.-helices of domain 1. As an illustrative embodiment, changes
in three such amino acids within the loop region between
.alpha.-helices 3 and 4 of domain 1 produced modified crystal
proteins with enhanced insecticidal activity (Cry1C.499, Cry1C.563,
Cry1C.579). As a second illustrative embodiment, an alanine
substitution for an arginine residue within or adjacent to the loop
region between .alpha.-helices 4 and 5 produced a modified crystal
protein with enhanced insecticidal activity (Cry1C-R148A). Although
this substitution removes a potential trypsin-cleavage site within
domain 1, trypsin digestion of this modified crystal protein
revealed no difference in proteolytic stability from the native
Cry1C protein. Furthermore, the R180A substitution in Cry1C
(Cry1C-R180A) also removes a potential trypsin cleavage site in
domain 1, yet this substitution has no effect on insecticidal
activity. Thus, the steps in the Cry1C protein mode-of-action
impacted by these amino acid substitutions have not been determined
nor is it obvious what substitutions need to be made to improve
insecticidal activity.
[0205] Many crystal proteins show significant amino acid sequence
identity to the Cry1C amino acid sequence within domain 1,
including proteins of the Cry1, Cry2, Cry3, Cry4, Cry5, Cry7, Cry8,
Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, and Cry16 classes defined
by the new cry gene nomenclature (Table 1). Furthermore, the
structures for CryIIIA (Cry3A) and CryIAa (Cry1Aa) show a
remarkable conservation of protein tertiary structure (Grochulski
et al., 1995). Thus, it is anticipated that the mutagenesis of
codons encoding amino acids within or near the loop regions between
the .alpha.-helices of domain 1 of these proteins may also result
in the generation of improved insecticidal proteins. Indeed, an
alignment of Cry1 amino acid sequences spanning the loop region
between .alpha.-helices 4 and 5 reveals that several Cry1 proteins
contain an arginine residue at the position homologous to R148 of
Cry1C. Since the Cry1C R148A mutant exhibits improved toxicity
towards a number of lepidopteran pests, the inventors contemplate
that similar substitutions in these other Cry1 proteins will also
yield improved insecticidal proteins.
4.2 Methods for Producing Cry1C* Proteins
[0206] The B. thuringiensis strains described herein may be
cultured using standard known media and fermentation techniques.
Upon completion of the fermentation cycle, the bacteria may be
harvested by first separating the B. thuringiensis spores and
crystals from the fermentation broth by means well known in the
art. The recovered B. thuringiensis spores and crystals can be
formulated into a wettable powder, a liquid concentrate, granules
or other formulations by the addition of surfactants, dispersants,
inert carriers and other components to facilitate handling and
application for particular target pests. The formulation and
application procedures are all well known in the art and are used
with commercial strains of B. thuringiensis (HD-1) active against
Lepidoptera, e.g., caterpillars.
4.3 Recombinant Host Cells for Expressing the Cry1C* genes
[0207] The nucleotide sequences of the subject invention can be
introduced into a wide variety of microbial hosts. Expression of
the toxin gene results, directly or indirectly, in the
intracellular production and maintenance of the pesticide. With
suitable hosts, e.g., Pseudomonas, the microbes can be applied to
the sites of lepidopteran insects where they will proliferate and
be ingested by the insects. The result is a control of the unwanted
insects. Alternatively, the microbe hosting the toxin gene can be
treated under conditions that prolong the activity of the toxin
produced in the cell. The treated cell then can be applied to the
environment of target pest(s). The resulting product retains the
toxicity of the B. thuringiensis toxin.
[0208] Suitable host cells, where the pesticide-containing cells
will be treated to prolong the activity of the toxin in the cell
when the then treated cell is applied to the environment of target
pest(s), may include either prokaryotes or eukaryotes, normally
being limited to those cells which do not produce substances toxic
to higher organisms, such as mammals. However, organisms which
produce substances toxic to higher organisms could be used, where
the toxin is unstable or the level of application sufficiently low
as to avoid any possibility or toxicity to a mammalian host. As
hosts, of particular interest will be the prokaryotes and the lower
eukaryotes, such as fungi. Illustrative prokaryotes, both
Gram-negative and Gram-positive, include Enterobacteriaceae, such
as Escherichia, Erwinia, Shigella, Salmonella, and Proteus,
Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as
photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,
Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such
as Pseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales,
and Nitrobacteraceae. Among eukaryotes are fungi, such as
Phycomycetes and Ascomycetes, which includes yeast, such as
Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast,
such as Rhodotorula, Aureobasidium, Sporobolomyces, and the
like.
[0209] Characteristics of particular interest in selecting a host
cell for purposes of production include ease of introducing the B.
thuringiensis gene into the host, availability of expression
systems, efficiency of expression, stability of the pesticide in
the host, and the presence of auxiliary genetic capabilities.
Characteristics of interest for use as a pesticide microcapsule
include protective qualities for the pesticide, such as thick cell
walls, pigmentation, and intracellular packaging or formation of
inclusion bodies; leaf affinity; lack of mammalian toxicity;
attractiveness to pests for ingestion; ease of killing and fixing
without damage to the toxin; and the like. Other considerations
include ease of formulation and handling, economics, storage
stability, and the like.
[0210] Host organisms of particular interest include yeast, such as
Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and
Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp.,
Erwinia sp. and Flavobacterium sp.; or such other organisms as
Escherichia, Lactobacillus sp., Bacillus sp., Streptomyces sp., and
the like. Specific organisms include Pseudomonas aeruginosa,
Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus
thuringiensis , Escherichia coli, Bacillus subtilis, Bacillus
megaterium, Bacillus cereus, Streptomyces lividans and the
like.
[0211] Treatment of the microbial cell, e.g., a microbe containing
the B. thuringiensis toxin gene, can be by chemical or physical
means, or by a combination of chemical and/or physical means, so
long as the technique does not deleteriously affect the properties
of the toxin, nor diminish the cellular capability in protecting
the toxin. Examples of chemical reagents are halogenating agents,
particularly halogens of atomic no. 17-80. More particularly,
iodine can be used under mild conditions and for sufficient time to
achieve the desired results. Other suitable techniques include
treatment with aldehydes, such as formaldehyde and glutaraldehye;
anti-infectives, such as zephiran chloride and cetylpyridinium
chloride; alcohols, such as isopropyl and ethanol; various
histologic fixatives, such as Lugol's iodine, Bouin's fixative, and
Helly's fixatives, (see e.g., Humason, 1967); or a combination of
physical (heat) and chemical agents that preserve and prolong the
activity of the toxin produced in the cell when the cell is
administered to the host animal. Examples of physical means are
short wavelength radiation such as .gamma.-radiation and
X-radiation, freezing, UV irradiation, lyophilization, and the
like. The cells employed will usually be intact and be
substantially in the proliferative form when treated, rather than
in a spore form, although in some instances spores may be
employed.
[0212] Where the B. thuringiensis toxin gene is introduced via a
suitable vector into a microbial host, and said host is applied to
the environment in a living state, it is essential that certain
host microbes be used. Microorganism hosts are selected which are
known to occupy the "phytosphere" (phylloplane, phyllosphere,
rhizosphere, and/or rhizoplane) of one or more crops of interest.
These microorganisms are selected so as to be capable of
successfully competing in the particular environment (crop and
other insect habitats) with the wild-type microorganisms, provide
for stable maintenance and expression of the gene expressing the
polypeptide pesticide, and, desirably, provide for improved
protection of the pesticide from environmental degradation and
inactivation.
[0213] A large number of microorganisms are known to inhabit the
phylloplane (the surface of the plant leaves) and/or the
rhizosphere (the soil surrounding plant roots) of a wide variety of
important crops. These microorganisms include bacteria, algae, and
fungi. Of particular interest are microorganisms, such as bacteria,
e.g., genera Bacillus, Pseudomonas, Erwinia, Serratia, Klebsiella,
Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas,
Methylophilius, Agrobacterium, Acetobacter, Lactobacillus,
Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi,
particularly yeast, e.g., genera Saccharomyces, Cryptococcus,
Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of
particular interest are such phytosphere bacterial species as
Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,
Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobacter
sphaeroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes
eutrophus, and Azotobacter vinlandii; and phytosphere yeast species
such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,
Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces
rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S.
odorus, Kluyveromyces veronae, and Aureobasidium pollulans.
4.4 Definitions
[0214] As used herein, the designations "Cry1" and "Cry1" are
synonymous, as are the designations "Cry1C" and "Cry1C." Likewise,
the inventors have utilized the generic term Cry1C* to denote any
and all Cry1C variants which comprise amino acid sequences modified
in the loop region of domain 1. Similarly, cry1C* is meant to
denote any and all nucleic acid segments and/or genes which encode
such modified Cry1C* proteins. In similar regard, the inventors
have used the terms Cry1* to denote any and all Cry1 variants which
comprise amino acid sequences modified in the loop region of domain
1. Similarly, cry1* is meant to denote any and all nucleic acid
segments and/or genes which encode such modified Cry1* proteins. A
similar convention is used to described modified loop domain
variants in any of the related crystal proteins and genes which
encode them.
[0215] In accordance with the present invention, nucleic acid
sequences include and are not limited to DNA (including and not
limited to genomic or extragenomic DNA), genes, RNA (including and
not limited to mRNA and tRNA), nucleosides, and suitable nucleic
acid segments either obtained from native sources, chemically
synthesized, modified, or otherwise prepared by the hand of man.
The following words and phrases have the meanings set forth
below.
[0216] Broad spectrum: refers to a wide range of insect
species.
[0217] Broad spectrum insecticidal activity: toxicity towards a
wide range of insect species.
[0218] Expression: The combination of intracellular processes,
including transcription and translation undergone by a coding DNA
molecule such as a structural gene to produce a polypeptide.
[0219] Insecticidal activity: toxicity towards insects.
[0220] Insecticidal specificity: the toxicity exhibited by a
crystal protein towards multiple insect species.
[0221] Intraorder specificity: the toxicity of a particular crystal
protein towards insect species within an Order of insects (e.g.,
Order Lepidoptera).
[0222] Interorder specificity: the toxicity of a particular crystal
protein towards insect species of different Orders (e.g., Orders
Lepidoptera and Diptera).
[0223] LC.sub.50: the lethal concentration of crystal protein that
causes 50% mortality of the insects treated.
[0224] LC.sub.95: the lethal concentration of crystal protein that
causes 95% mortality of the insects treated.
[0225] Promoter: A recognition site on a DNA sequence or group of
DNA sequences that provide an expression control element for a
structural gene and to which RNA polymerase specifically binds and
initiates RNA synthesis (transcription) of that gene.
[0226] Regeneration: The process of growing a plant from a plant
cell (e.g., plant protoplast or explant).
[0227] Structural gene: A gene that is expressed to produce a
polypeptide.
[0228] Transformation: A process of introducing an exogenous DNA
sequence (e.g., a vector, a recombinant DNA molecule) into a cell
or protoplast in which that exogenous DNA is incorporated into a
chromosome or is capable of autonomous replication.
[0229] Transformed cell: A cell whose DNA has been altered by the
introduction of an exogenous DNA molecule into that cell.
[0230] Transgenic cell: Any cell derived or regenerated from a
transformed cell or derived from a transgenic cell. Exemplary
transgenic cells include plant calli derived from a transformed
plant cell and particular cells such as leaf, root, stem, e.g.,
somatic cells, or reproductive (germ) cells obtained from a
transgenic plant.
[0231] Transgenic plant: A plant or progeny thereof derived from a
transformed plant cell or protoplast, wherein the plant DNA
contains an introduced exogenous DNA molecule not originally
present in a native, non-transgenic plant of the same strain. The
terms "transgenic plant" and "transformed plant" have sometimes
been used in the art as synonymous terms to define a plant whose
DNA contains an exogenous DNA molecule. However, it is thought more
scientifically correct to refer to a regenerated plant or callus
obtained from a transformed plant cell or protoplast as being a
transgenic plant, and that usage will be followed herein.
[0232] Vector: A DNA molecule capable of replication in a host cell
and/or to which another DNA segment can be operatively linked so as
to bring about replication of the attached segment. Plasmids,
phagemids, cosmids, phage, virus, YACs, and BACs are all exemplary
vectors.
4.5 Probes and Primers
[0233] In another aspect, DNA sequence information provided by the
invention allows for the preparation of relatively short DNA (or
RNA) sequences having the ability to specifically hybridize to gene
sequences of the selected polynucleotides disclosed herein. In
these aspects, nucleic acid probes of an appropriate length are
prepared based on a consideration of a selected crystal protein
gene sequence, e.g., a sequence such as that shown in SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,
SEQ ID NO:58, or SEQ ID NO:60. The ability of such nucleic acid
probes to specifically hybridize to a crystal protein-encoding gene
sequence lends them particular utility in a variety of embodiments.
Most importantly, the probes may be used in a variety of assays for
detecting the presence of complementary sequences in a given
sample.
[0234] In certain embodiments, it is advantageous to use
oligonucleotide primers. The sequence of such primers is designed
using a polynucleotide of the present invention for use in
detecting, amplifying or mutating a defined segment of a crystal
protein gene from B. thuringiensis using PCR.TM. technology.
Segments of related crystal protein genes from other species may
also be amplified by PCR.TM. using such primers.
[0235] To provide certain of the advantages in accordance with the
present invention, a preferred nucleic acid sequence employed for
hybridization studies or assays includes sequences that are
complementary to at least a 14 to 30 or so long nucleotide stretch
of a crystal protein-encoding sequence, such as that shown in SEQ
ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:58, or SEQ ID NO:60. A size of at least 14
nucleotides in length helps to ensure that the fragment will be of
sufficient length to form a duplex molecule that is both stable and
selective. Molecules having complementary sequences over stretches
greater than 14 bases in length are generally preferred, though, in
order to increase stability and selectivity of the hybrid, and
thereby improve the quality and degree of specific hybrid molecules
obtained. One will generally prefer to design nucleic acid
molecules having gene-complementary stretches of 14 to 20
nucleotides, or even longer where desired. Such fragments may be
readily prepared by, for example, directly synthesizing the
fragment by chemical means, by application of nucleic acid
reproduction technology, such as the PCR.TM. technology of U.S.
Pat. Nos. 4,683,195, and 4,683,202, herein incorporated by
reference, or by excising selected DNA fragments from recombinant
plasmids containing appropriate inserts and suitable restriction
sites.
[0236] A particularly preferred oligonucleotide is the 63-mer
identified in SEQ ID NO:18. The oligonucleotide is particularly
preferred for preparation of mutagenized nucleic acid sequences to
produce toxins with improved properties. Mutagenic oligonucleotides
may be prepared with known or random substitutions, by methods
well-known to those of skill in the art. Such oligonucleotides may
be provided by commercial firms that perform custom syntheses.
[0237] Accordingly, a nucleotide sequence of the invention can be
used for its ability to selectively form duplex molecules with
complementary stretches of the gene. Depending on the application
envisioned, one will desire to employ varying conditions of
hybridization to achieve varying degree of selectivity of the probe
toward the target sequence. For applications requiring a high
degree of selectivity, one will typically desire to employ
relatively stringent conditions to form the hybrids, for example,
one will select relatively low salt and/or high temperature
conditions, such as provided by about 0.02 M to about 0.15 M NaCl
at temperatures of about 50.degree. C. to about 70.degree. C. These
conditions are particularly selective, and tolerate little, if any,
mismatch between the probe and the template or target strand.
[0238] Of course, for some applications, for example, where one
desires to prepare mutants employing a mutant primer strand
hybridized to an underlying template or where one seeks to isolate
a crystal protein-coding sequences for related species, functional
equivalents, or the like, less stringent hybridization conditions
will typically be needed in order to allow formation of the
heteroduplex. In these circumstances, one may desire to employ
conditions such as about 0.15 M to about 0.9 M salt, at
temperatures ranging from about 20.degree. C. to about 55.degree.
C. Cross-hybridizing species can thereby be readily identified as
positively hybridizing signals with respect to control
hybridizations. In any case, it is generally appreciated that
conditions can be rendered more stringent by the addition of
increasing amounts of formamide, which serves to destabilize the
hybrid duplex in the same manner as increased temperature. Thus,
hybridization conditions can be readily manipulated, and thus will
generally be a method of choice depending on the desired
results.
4.6 Expression Vectors
[0239] The present invention contemplates an expression vector
comprising a polynucleotide of the present invention. Thus, in one
embodiment an expression vector is an isolated and purified DNA
molecule comprising a promoter operatively linked to an coding
region that encodes a polypeptide of the present invention, which
coding region is operatively linked to a transcription-terminating
region, whereby the promoter drives the transcription of the coding
region.
[0240] As used herein, the term "operatively linked" means that a
promoter is connected to an coding region in such a way that the
transcription of that coding region is controlled and regulated by
that promoter. Means for operatively linking a promoter to a coding
region are well known in the art.
[0241] In a preferred embodiment, the recombinant expression of
DNAs encoding the crystal proteins of the present invention is
preferable in a Bacillus host cell. Preferred host cells include B.
thuringiensis, B. megaterium, B. cereus, B. subtilis, and related
bacilli, with B. thuringiensis host cells being highly preferred.
Promoters that function in bacteria are well-known in the art. An
exemplary and preferred promoter for the Bacillus crystal proteins
include any of the known crystal protein gene promoters, including
native crystal protein encoding gene promoters. Alternatively,
mutagenized or recombinant crystal protein-encoding gene promoters
may be engineered by the hand of man and used to promote expression
of the novel gene segments disclosed herein.
[0242] In an alternate embodiment, the recombinant expression of
DNAs encoding the crystal proteins of the present invention is
performed using a transformed Gram-negative bacterium such as an E.
coli or Pseudomonas spp. host cell. Promoters which function in
high-level expression of target polypeptides in E. coli and other
Gram-negative host cells are also well-known in the art.
[0243] Where an expression vector of the present invention is to be
used to transform a plant, a promoter is selected that has the
ability to drive expression in plants. Promoters that function in
plants are also well known in the art. Useful in expressing the
polypeptide in plants are promoters that are inducible, viral,
synthetic, constitutive as described (Poszkowski et al., 1989;
Odell et al., 1985), and temporally regulated, spatially regulated,
and spatio-temporally regulated (Chau et al., 1989).
[0244] A promoter is also selected for its ability to direct the
transformed plant cell's or transgenic plant's transcriptional
activity to the coding region. Structural genes can be driven by a
variety of promoters in plant tissues. Promoters can be
near-constitutive, such as the CaMV 35S promoter, or
tissue-specific or developmentally specific promoters affecting
dicots or monocots.
[0245] Where the promoter is a near-constitutive promoter such as
CaMV 35S, increases in polypeptide expression are found in a
variety of transformed plant tissues (e.g., callus, leaf, seed and
root). Alternatively, the effects of transformation can be directed
to specific plant tissues by using plant integrating vectors
containing a tissue-specific promoter.
[0246] An exemplary tissue-specific promoter is the lectin
promoter, which is specific for seed tissue. The Lectin protein in
soybean seeds is encoded by a single gene (Le1) that is only
expressed during seed maturation and accounts for about 2 to about
5% of total seed mRNA. The lectin gene and seed-specific promoter
have been fully characterized and used to direct seed specific
expression in transgenic tobacco plants (Vodkin et al., 1983;
Lindstrom et al., 1990.) An expression vector containing a coding
region that encodes a polypeptide of interest is engineered to be
under control of the lectin promoter and that vector is introduced
into plants using, for example, a protoplast transformation method
(Dhir et al., 1991). The expression of the polypeptide is directed
specifically to the seeds of the transgenic plant.
[0247] A transgenic plant of the present invention produced from a
plant cell transformed with a tissue specific promoter can be
crossed with a second transgenic plant developed from a plant cell
transformed with a different tissue specific promoter to produce a
hybrid transgenic plant that shows the effects of transformation in
more than one specific tissue.
[0248] Exemplary tissue-specific promoters are corn sucrose
synthetase 1 (Yang et al., 1990), corn alcohol dehydrogenase 1
(Vogel et al., 1989), corn light harvesting complex (Simpson,
1986), corn heat shock protein (Odell et al., 1985), pea small
subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al.,
1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti
plasmid nopaline synthase (Langridge et al., 1989), petunia
chalcone isomerase (Van Tunen et al., 1988), bean glycine rich
protein 1 (Keller et al., 1989), CaMV 35s transcript (Odell et al.,
1985) and Potato patatin (Wenzler et al., 1989). Preferred
promoters are the cauliflower mosaic virus (CaMV 35S) promoter and
the S-E9 small subunit RuBP carboxylase promoter.
[0249] The choice of which expression vector and ultimately to
which promoter a polypeptide coding region is operatively linked
depends directly on the functional properties desired, e.g., the
location and timing of protein expression, and the host cell to be
transformed. These are well known limitations inherent in the art
of constructing recombinant DNA molecules. However, a vector useful
in practicing the present invention is capable of directing the
expression of the polypeptide coding region to which it is
operatively linked.
[0250] Typical vectors useful for expression of genes in higher
plants are well known in the art and include vectors derived from
the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens
described (Rogers et al., 1987). However, several other plant
integrating vector systems are known to function in plants
including pCaMVCN transfer control vector described (Fromm et al.,
1985). Plasmid pCaMVCN (available from Pharmacia, Piscataway, N.J.)
includes the cauliflower mosaic virus CaMV 35S promoter.
[0251] In preferred embodiments, the vector used to express the
polypeptide includes a selection marker that is effective in a
plant cell, preferably a drug resistance selection marker. One
preferred drug resistance marker is the gene whose expression
results in kanamycin resistance; i.e., the chimeric gene containing
the nopaline synthase promoter, Tn5 neomycin phosphotransferase II
(nptII) and nopaline synthase 3' non-translated region described
(Rogers et al., 1988).
[0252] RNA polymerase transcribes a coding DNA sequence through a
site where polyadenylation occurs. Typically, DNA sequences located
a few hundred base pairs downstream of the polyadenylation site
serve to terminate transcription. Those DNA sequences are referred
to herein as transcription-termination regions. Those regions are
required for efficient polyadenylation of transcribed messenger RNA
(mRNA).
[0253] Means for preparing expression vectors are well known in the
art. Expression (transformation vectors) used to transform plants
and methods of making those vectors are described in U.S. Pat. Nos.
4,971,908, 4,940,835, 4,769,061 and 4,757,011, the disclosures of
which are incorporated herein by reference. Those vectors can be
modified to include a coding sequence in accordance with the
present invention.
[0254] A variety of methods has been developed to operatively link
DNA to vectors via complementary cohesive termini or blunt ends.
For instance, complementary homopolymer tracts can be added to the
DNA segment to be inserted and to the vector DNA. The vector and
DNA segment are then joined by hydrogen bonding between the
complementary homopolymeric tails to form recombinant DNA
molecules.
[0255] A coding region that encodes a polypeptide having the
ability to confer insecticidal activity to a cell is preferably a
Cry1C-R148A, Cry1C-R180A, Cry1C.563, Cry1C.579 or Cry1C.499 B.
thuringiensis crystal protein-encoding gene. In preferred
embodiments, such a polypeptide has the amino acid residue sequence
of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID
NO:10, or SEQ ID NO::12 respectively, or a functional equivalent of
those sequences. In accordance with such embodiments, a coding
region comprising the DNA sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ
ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:58, or
SEQ ID NO:60 is also preferred.
4.7 DNA Segments as Hybridization Probes and Primers
[0256] In addition to their use in directing the expression of
crystal proteins or peptides of the present invention, the nucleic
acid sequences contemplated herein also have a variety of other
uses. For example, they also have utility as probes or primers in
nucleic acid hybridization embodiments. As such, it is contemplated
that nucleic acid segments that comprise a sequence region that
consists of at least a 14 nucleotide long contiguous sequence that
has the same sequence as, or is complementary to, a 14 nucleotide
long contiguous DNA segment of SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:58, or SEQ
ID NO:60 will find particular utility. Longer contiguous identical
or complementary sequences, e.g., those of about 20, 30, 40, 50,
100, 200, 500, 1000, 2000, 5000, 10000 etc. (including all
intermediate lengths and up to and including full-length sequences
will also be of use in certain embodiments.
[0257] The ability of such nucleic acid probes to specifically
hybridize to crystal protein-encoding sequences will enable them to
be of use in detecting the presence of complementary sequences in a
given sample. However, other uses are envisioned, including the use
of the sequence information for the preparation of mutant species
primers, or primers for use in preparing other genetic
constructions.
[0258] Nucleic acid molecules having sequence regions consisting of
contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of
100-200 nucleotides or so, identical or complementary to DNA
sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:58, or SEQ ID NO:60 are
particularly contemplated as hybridization probes for use in, e.g.,
Southern and Northern blotting. Smaller fragments will generally
find use in hybridization embodiments, wherein the length of the
contiguous complementary region may be varied, such as between
about 10-14 and about 100 or 200 nucleotides, but larger contiguous
complementarity stretches may be used, according to the length
complementary sequences one wishes to detect.
[0259] The use of a hybridization probe of about 14 nucleotides in
length allows the formation of a duplex molecule that is both
stable and selective. Molecules having contiguous complementary
sequences over stretches greater than 14 bases in length are
generally preferred, though, in order to increase stability and
selectivity of the hybrid, and thereby improve the quality and
degree of specific hybrid molecules obtained. One will generally
prefer to design nucleic acid molecules having gene-complementary
stretches of 15 to 20 contiguous nucleotides, or even longer where
desired.
[0260] Of course, fragments may also be obtained by other
techniques such as, e.g., by mechanical shearing or by restriction
enzyme digestion. Small nucleic acid segments or fragments may be
readily prepared by, for example, directly synthesizing the
fragment by chemical means, as is commonly practiced using an
automated oligonucleotide synthesizer. Also, fragments may be
obtained by application of nucleic acid reproduction technology,
such as the PCR.TM. technology of U.S. Pat. Nos. 4,683,195 and
4,683,202 (each incorporated herein by reference), by introducing
selected sequences into recombinant vectors for recombinant
production, and by other recombinant DNA techniques generally known
to those of skill in the art of molecular biology.
[0261] Accordingly, the nucleotide sequences of the invention may
be used for their ability to selectively form duplex molecules with
complementary stretches of DNA fragments. Depending on the
application envisioned, one will desire to employ varying
conditions of hybridization to achieve varying degrees of
selectivity of probe towards target sequence. For applications
requiring high selectivity, one will typically desire to employ
relatively stringent conditions to form the hybrids, e.g., one will
select relatively low salt and/or high temperature conditions, such
as provided by about 0.02 M to about 0.15 M NaCl at temperatures of
about 50.degree. C. Go about 70.degree. C. Such selective
conditions tolerate little, if any, mismatch between the probe and
the template or target strand, and would be particularly suitable
for isolating crystal protein-encoding DNA segments. Detection of
DNA segments via hybridization is well-known to those of skill in
the art, and the teachings of U.S. Pat. Nos. 4,965,188 and
5,176,995 (each incorporated herein by reference) are exemplary of
the methods of hybridization analyses. Teachings such as those
found in the texts of Maloy et al., 1994; Segal 1976; Prokop, 1991;
and Kuby, 1994, are particularly relevant.
[0262] Of course, for some applications, for example, where one
desires to prepare mutants employing a mutant primer strand
hybridized to an underlying template or where one seeks to isolate
crystal protein-encoding sequences from related species, functional
equivalents, or the like, less stringent hybridization conditions
will typically be needed in order to allow formation of the
heteroduplex. In these circumstances, one may desire to employ
conditions such as about 0.15 M to about 0.9 M salt, at
temperatures ranging from about 20.degree. C. Go about 55.degree.
C. Cross-hybridizing species can thereby be readily identified as
positively hybridizing signals with respect to control
hybridizations. In any case, it is generally appreciated that
conditions can be rendered more stringent by the addition of
increasing amounts of formamide, which serves to destabilize the
hybrid duplex in the same manner as increased temperature. Thus,
hybridization conditions can be readily manipulated, and thus will
generally be a method of choice depending on the desired
results.
[0263] In certain embodiments, it will be advantageous to employ
nucleic acid sequences of the present invention in combination with
an appropriate means, such as a label, for determining
hybridization. A wide variety of appropriate indicator means are
known in the art, including fluorescent, radioactive, enzymatic or
other ligands, such as avidin/biotin, which are capable of giving a
detectable signal. In preferred embodiments, one will likely desire
to employ a fluorescent label or an enzyme tag, such as urease,
alkaline phosphatase or peroxidase, instead of radioactive or other
environmental undesirable reagents. In the case of enzyme tags,
colorimetric indicator substrates are known that can be employed to
provide a means visible to the human eye or spectrophotometrically,
to identify specific hybridization with complementary nucleic
acid-containing samples.
[0264] In general, it is envisioned that the hybridization probes
described herein will be useful both as reagents in solution
hybridization as well as in embodiments employing a solid phase. In
embodiments involving a solid phase, the test DNA (or RNA) is
adsorbed or otherwise affixed to a selected matrix or surface. This
fixed, single-stranded nucleic acid is then subjected to specific
hybridization with selected probes under desired conditions. The
selected conditions will depend on the particular circumstances
based on the particular criteria required (depending, for example,
on the G+C content, type of target nucleic acid, source of nucleic
acid, size of hybridization probe, etc.). Following washing of the
hybridized surface so as to remove nonspecifically bound probe
molecules, specific hybridization is detected, or even quantitated,
by means of the label.
4.8 Characteristics of Cry1C* Proteins
[0265] The present invention provides novel polypeptides that
define a whole or a portion of a B. thuringiensis Cry1C-R180A,
Cry1C-R148A, Cry1C-R148D, Cry1C-R148L, Cry1C-R148M, Cry1C-R148G,
Cry1C.563, Cry1C.499, or Cry1C.579 crystal protein.
[0266] In a preferred embodiment, the invention discloses and
claims a purified Cry1C-R148A protein. The Cry1C-R148A protein
comprises an 1189-amino acid sequence, which is given in SEQ ID
NO:2.
[0267] In a second embodiment, the invention discloses and claims a
purified Cry1C-R148D protein. The Cry1C-R148D protein comprises an
1189-amino acid sequence, which is given in SEQ ID NO:4.
[0268] In a third embodiment, the invention discloses and claims a
purified Cry1C-R180A protein. The Cry1C-R180A protein comprises an
1189-amino acid sequence, which is given in SEQ ID NO:6.
[0269] In a fourth embodiment, the invention discloses and claims a
purified Cry1C.563 protein. The Cry1C.563 protein comprises an
1189-amino acid sequence which is given in SEQ ID NO:8.
[0270] In a fifth embodiment, the invention discloses and claims a
purified Cry1C.579 protein. The Cry1C.579 protein comprises an
1189-amino acid sequence, which is given in SEQ ID NO:10.
[0271] In a sixth embodiment, the invention discloses and claims a
purified Cry1C.499 protein. The Cry1C.499 protein comprises an I
189-amino acid sequence, which is given in SEQ ID NO:12.
4.9 Nomenclature of Cry* Proteins
[0272] The inventors have arbitrarily assigned the designations
Cry1C-R148A, Cry1C-R148D, Cry1C-R148L, Cry1C-R148M, Cry1C-R148G,
Cry1C-R180A, Cry1C.563, Cry1C.579 and Cry1C.499 to the novel
proteins of the invention. Likewise, the arbitrary designations of
cry1C-R148A, cry1C-R148D, cry1C-R148L, cry1C-R148M, cry1C-R148G,
cry1C-R180A, cry1C.563, cry1C579 and cry1C.499 have been assigned
to the novel nucleic acid sequences which encode these
polypeptides, respectively. While formal assignment of gene and
protein designations based on the revised nomenclature of crystal
protein endotoxins (Table 1) may be made by the committee on the
nomenclature of B. thuringiensis, any re-designations of the
compositions of the present invention are also contemplated to be
fully within the scope of the present disclosure.
4.10 Transformed Host Cells and Transgenic Plants
[0273] A bacterium, a yeast cell, or a plant cell or a plant
transformed with an expression vector of the present invention is
also contemplated. A transgenic bacterium, yeast cell, plant cell
or plant derived from such a transformed or transgenic cell is also
contemplated. Means for transforming bacteria and yeast cells are
well known in the art. Typically, means of transformation are
similar to those well known means used to transform other bacteria
or yeast such as E. coli or Saccharomyces cerevisiae.
[0274] Methods for DNA transformation of plant cells include
Agrobacterium-mediated plant transformation, protoplast
transformation, gene transfer into pollen, injection into
reproductive organs, injection into immature embryos and particle
bombardment. Each of these methods has distinct advantages and
disadvantages. Thus, one particular method of introducing genes
into a particular plant strain may not necessarily be the most
effective for another plant strain, but it is well known which
methods are useful for a particular plant strain.
[0275] There are many methods for introducing transforming DNA
segments into cells, but not all are suitable for delivering DNA to
plant cells. Suitable methods are believed to include virtually any
method by which DNA can be introduced into a cell, such as by
Agrobacterium infection, direct delivery of DNA such as, for
example, by PEG-mediated transformation of protoplasts (Omirulleh
et al, 1993), by desiccation/inhibition-mediated DNA uptake, by
electroporation, by agitation with silicon carbide fibers, by
acceleration of DNA coated particles, etc. In certain embodiments,
acceleration methods are preferred and include, for example,
microprojectile bombardment and the like.
[0276] Technology for introduction of DNA into cells is well-known
to those of skill in the art. Four general methods for delivering a
gene into cells have been described: (1) chemical methods (Graham
and van der Eb, 1973; Zatloukal et al., 1992); (2) physical methods
such as microinjection (Capecchi, 1980), electroporation (Wong and
Neumann, 1982; Fromm et al., 1985) and the gene gun (Johnston and
Tang, 1994; Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu
et al., 1993; Eglitis and Anderson, 1988a; 1988b); and (4)
receptor-mediated mechanisms (Curiel et al., 199 1; 1992; Wagner et
al., 1992).
4.10.1 Electroporation
[0277] The application of brief, high-voltage electric pulses to a
variety of animal and plant cells leads to the formation of
nanometer-sized pores in the plasma membrane. DNA is taken directly
into the cell cytoplasm either through these pores or as a
consequence of the redistribution of membrane components that
accompanies closure of the pores. Electroporation can be extremely
efficient and can be used both for transient expression of clones
genes and for establishment of cell lines that carry integrated
copies of the gene of interest. Electroporation, in contrast to
calcium phosphate-mediated transfection and protoplast fusion,
frequently gives rise to cell lines that carry one, or at most a
few, integrated copies of the foreign DNA.
[0278] The introduction of DNA by means of electroporation, is
well-known to those of skill in the art. In this method, certain
cell wall-degrading enzymes, such as pectin-degrading enzymes, are
employed to render the target recipient cells more susceptible to
transformation by electroporation than untreated cells.
Alternatively, recipient cells are made more susceptible to
transformation, by mechanical wounding. To effect transformation by
electroporation one may employ either friable tissues such as a
suspension culture of cells, or embryogenic callus, or
alternatively, one may transform immature embryos or other
organized tissues directly. One would partially degrade the cell
walls of the chosen cells by exposing them to pectin-degrading
enzymes (pectolyases) or mechanically wounding in a controlled
manner. Such cells would then be recipient to DNA transfer by
electroporation, which may be carried out at this stage, and
transformed cells then identified by a suitable selection or
screening protocol dependent on the nature of the newly
incorporated DNA.
4.10.2 Microprojectile Bombardment
[0279] A further advantageous method for delivering transforming
DNA segments to plant cells is microprojectile bombardment. In this
method, particles may be coated with nucleic acids and delivered
into cells by a propelling force. Exemplary particles include those
comprised of tungsten, gold, platinum, and the like.
[0280] An advantage of microprojectile bombardment, in addition to
it being an effective means of reproducibly stably transforming
monocots, is that neither the isolation of protoplasts (Cristou et
al., 1988) nor the susceptibility to Agrobacterium infection is
required. An illustrative embodiment of a method for delivering DNA
into maize cells by acceleration is a Biolistics Particle Delivery
System, which can be used to propel particles coated with DNA or
cells through a screen, such as a stainless steel or Nytex screen,
onto a filter surface covered with corn cells cultured in
suspension. The screen disperses the particles so that they are not
delivered to the recipient cells in large aggregates. It is
believed that a screen intervening between the projectile apparatus
and the cells to be bombarded reduces the size of projectiles
aggregate and may contribute to a higher frequency of
transformation by reducing damage inflicted on the recipient cells
by projectiles that are too large.
[0281] For the bombardment, cells in suspension are preferably
concentrated on filters or solid culture medium. Alternatively,
immature embryos or other target cells may be arranged on solid
culture medium. The cells to be bombarded are positioned at an
appropriate distance below the macroprojectile stopping plate. If
desired, one or more screens are also positioned between the
acceleration device and the cells to be bombarded. Through the use
of techniques set forth herein one may obtain up to 1000 or more
foci of cells transiently expressing a marker gene. The number of
cells in a focus which express the exogenous gene product 48 hours
post-bombardment often range from 1 to 10 and average 1 to 3.
[0282] In bombardment transformation, one may optimize the
prebombardment culturing conditions and the bombardment parameters
to yield the maximum numbers of stable transformants. Both the
physical and biological parameters for bombardment are important in
this technology. Physical factors are those that involve
manipulating the DNA/microprojectile precipitate or those that
affect the flight and velocity of either the macro- or
microprojectiles. Biological factors include all steps involved in
manipulation of cells before and immediately after bombardment, the
osmotic adjustment of target cells to help alleviate the trauma
associated with bombardment, and also the nature of the
transforming DNA, such as linearized DNA or intact supercoiled
plasmids. It is believed that pre-bombardment manipulations are
especially important for successful transformation of immature
embryos.
[0283] Accordingly, it is contemplated that one may wish to adjust
various of the bombardment parameters in small scale studies to
fully optimize the conditions. One may particularly wish to adjust
physical parameters such as gap distance, flight distance, tissue
distance, and helium pressure. One may also minimize the trauma
reduction factors (TRFs) by modifying conditions which influence
the physiological state of the recipient cells and which may
therefore influence transformation and integration efficiencies.
For example, the osmotic state, tissue hydration and the subculture
stage or cell cycle of the recipient cells may be adjusted for
optimum transformation. The execution of other routine adjustments
will be known to those of skill in the art in light of the present
disclosure.
4.10.3 Agrobacterium-Mediated transfer
[0284] Agrobacterium-mediated transfer is a widely applicable
system for introducing genes into plant cells because the DNA can
be introduced into whole plant tissues, thereby bypassing the need
for regeneration of an intact plant from a protoplast. The use of
Agrobacterium-mediated plant integrating vectors to introduce DNA
into plant cells is well known in the art. See, for example, the
methods described (Fraley et al., 1985; Rogers et al., 1987).
Further, the integration of the Ti-DNA is a relatively precise
process resulting in few rearrangements. The region of DNA to be
transferred is defined by the border sequences, and intervening DNA
is usually inserted into the plant genome as described (Spielmann
et a5., 1986; Jorgensen et al., 1987).
[0285] Modem Agrobacterium transformation vectors are capable of
replication in E. coli as well as Agrobacterium, allowing for
convenient manipulations as described (Klee et al., 1985).
Moreover, recent technological advances in vectors for
Agrobacterium-mediated gene transfer have improved the arrangement
of genes and restriction sites in the vectors to facilitate
construction of vectors capable of expressing various polypeptide
coding genes. The vectors described (Rogers et al., 1987), have
convenient multi-linker regions flanked by a promoter and a
polyadenylation site for direct expression of inserted polypeptide
coding genes and are suitable for present purposes. In addition,
Agrobacterium containing both armed and disarmed Ti genes can be
used for the transformations. In those plant strains where
Agrobacterium-mediated transformation is efficient, it is the
method of choice because of the facile and defined nature of the
gene transfer.
[0286] Agrobacterium-mediated transformation of leaf disks and
other tissues such as cotyledons and hypocotyls appears to be
limited to plants that Agrobacterium naturally infects.
Agrobacterium-mediated transformation is most efficient in
dicotyledonous plants. Few monocots appear to be natural hosts for
Agrobacterium, although transgenic plants have been produced in
asparagus using Agrobacterium vectors as described (Bytebier et
al., 1987). Therefore, commercially important cereal grains such as
rice, corn, and wheat must usually be transformed using alternative
methods. However, as mentioned above, the transformation of
asparagus using Agrobacterium can also be achieved (see, for
example, Bytebier et al., 1987).
[0287] A transgenic plant formed using Agrobacterium transformation
methods typically contains a single gene on one chromosome. Such
transgenic plants can be referred to as being heterozygous for the
added gene. However, inasmuch as use of the word "heterozygous"
usually implies the presence of a complementary gene at the same
locus of the second chromosome of a pair of chromosomes, and there
is no such gene in a plant containing one added gene as here, it is
believed that a more accurate name for such a plant is an
independent segregant, because the added, exogenous gene segregates
independently during mitosis and meiosis.
[0288] More preferred is a transgenic plant that is homozygous for
the added structural gene; i.e., a transgenic plant that contains
two added genes, one gene at the same locus on each chromosome of a
chromosome pair. A homozygous transgenic plant can be obtained by
sexually mating (selfing) an independent segregant transgenic plant
that contains a single added gene, germinating some of the seed
produced and analyzing the resulting plants produced for enhanced
carboxylase activity relative to a control (native, non-transgenic)
or an independent segregant transgenic plant.
[0289] It is to be understood that two different transgenic plants
can also be mated to produce offspring that contain two
independently segregating added, exogenous genes. Selfing of
appropriate progeny can produce plants that are homozygous for both
added, exogenous genes that encode a polypeptide of interest.
Back-crossing to a parental plant and out-crossing with a
non-transgenic plant are also contemplated.
[0290] Transformation of plant protoplasts can be achieved using
methods based on calcium phosphate precipitation, polyethylene
glycol treatment, electroporation, and combinations of these
treatments (see, e.g., Potrykus et al., 1985; Lorz et al., 1985;
Fromm et al., 1985; Uchimiya et al., 1986; Callis et al., 1987;
Marcotte et al., 1988).
[0291] Application of these systems to different plant strains
depends upon the ability to regenerate that particular plant strain
from protoplasts. Illustrative methods for the regeneration of
cereals from protoplasts are described (Fujimura et al., 1985;
Toriyama et al., 1986; Yamada et al., 1986; Abdullah et al.,
1986).
[0292] To transform plant strains that cannot be successfully
regenerated from protoplasts, other ways to introduce DNA into
intact cells or tissues can be utilized. For example, regeneration
of cereals from immature embryos or explants can be effected as
described (Vasil, 1988). In addition, "particle gun" or
high-velocity microprojectile technology can be utilized (Vasil,
1992).
[0293] Using that latter technology, DNA is carried through the
cell wall and into the cytoplasm on the surface of small metal
particles as described (Klein et al., 1987; Klein et al., 1988;
McCabe et al., 1988). The metal particles penetrate through several
layers of cells and thus allow the transformation of cells within
tissue explants.
4.10.4 Gene Expression in Plants
[0294] The fact that plant codon usage more closely resembles that
of humans and other higher organisms than unicellular organisms,
such as bacteria, unmodified bacterial genes are often poorly
expressed in transgenic plant cells. The apparent overall
preference for GC content in codon position three has been
described in detail by Murray et al. (1990). The 207 plant genes
described in this work permitted the compilation of codon
preferences for amino acids in plants. These authors describe the
difference between codon usage in monocots and dicots, as well as
differences between chloroplast encoded genes and those which are
nuclear encoded. Utilizing the codon frequency tables provided,
those of skill in the art can engineer such a bacterial sequence
for expression in plants by modifying the DNA sequences to provide
a codon bias for G or C in the third position. The reference
provides an exhaustive list of tables to guide molecular
geneticists in preparing synthetic gene sequences which encode the
polypeptides of the invention, and which are expressed in
transformed plant cells in a suitable fashion to permit synthesis
of the polypeptide of interest in planta.
[0295] A similar work by Diehn et al. (1996) details the
modification of prokaryotic-derived gene sequences necessary to
permit expression in plants.
[0296] Iannacone et al. (1997) describe the transformation of egg
plant with a genetically engineered B. thuringiensis gene encoding
a cry3 class endotoxin. Utilizing sequences which avoid
polyadenylation sequences, ATRIA sequences, and splicing sites a
synthetic gene was constructed which permitted expression of the
encoded toxin in planta.
[0297] Expression of heterologous proteins in transgenic tobacco
has been described by Rouwendal et al. (1997). Using a synthetic
gene, the third position codon bias for C+G was created to permit
expression of the jellyfish green fluorescent protein-encoding gene
in planta. Futterer and Hohn (1996) describe the effects of mRNA
sequence, leader sequences, polycistronic messages, and internal
ribosome binding site motis, on expression in plants. Modification
of such sequences by construction of synthetic genes permitted
expression of viral mRNAs in transgenic plant cells.
[0298] Preparation of transgenic plants which express genes
encoding non-native proteins (such as B. thuringiensis crystal
proteins) is becoming a critical step in the formulation of plant
varieties which express insect resistance genes. In recent years
considerable research has yielded tools for the manipulation of
endotoxin-encoding genes to permit expression of their encoded
proteins in planta. Scientists have shown that maintaining a
significant level of an mRNA species in a plant is often a critical
factor. Unfortunately, the causes for low steady state levels of
mRNA encoding foreign proteins are many. First, full-length RNA
synthesis may not occur at a high frequency. This could, for
example, be caused by the premature termination of RNA during
transcription or due to unexpected mRNA processing during
transcription. Second, full-length RNA may be produced in the plant
cell, but then processed (splicing, polyA addition) in the nucleus
in a fashion that creates a nonfunctional mRNA. If the RNA is not
properly synthesized, terminated and polyadenylated, it cannot move
to the cytoplasm for translation. Similarly, in the cytoplasm, if
mRNAs have reduced half lives (which are determined by their
primary or secondary sequence) inisufficient protein product will
be produced. In addition, there is an effect, whose magnitude is
uncertain, of translational efficiency on mRNA half-life. In
addition, every RNA molecule folds into a particular structure, or
perhaps family of structures, which is determined by its sequence.
The particular structure of any RNA might lead to greater or lesser
stability in the cytoplasm. Structure per se is probably also a
determinant of mRNA processing in the nucleus. Unfortunately, it is
impossible to predict, and nearly impossible to determine, the
structure of any RNA (except for tRNA) in vitro or in vivo.
However, it is likely that dramatically changing the sequence of an
RNA will have a large effect on its folded structure It is likely
that structure per se or particular structural features also have a
role in determining RNA stability.
[0299] To overcome these limitations in foreign gene expression,
researchers have identified particular sequences and signals in
RNAs that have the potential for having a specific effect on RNA
stability. In certain embodiments of the invention, therefore,
there is a desire to optimize expression of the disclosed nucleic
acid segments in planta. One particular method of doing so, is by
alteration of the bacterial gene to remove sequences or motifs
which decrease expression in a transformed plant cell. The process
of engineering a coding sequence for optimal expression in planta
is often referred to as "plantizing" a DNA sequence.
[0300] Particularly problematic sequences are those which are A+T
rich. Unfortunately, since B. thuringiensis has an A+T rich genome,
native crystal protein gene sequences must often be modified for
optimal expression in a plant. The sequence motif ATTTA (or AUUUA
as it appears in RNA) has been implicated as a destabilizing
sequence in mammalian cell mRNA (Shaw and Kamen, 1986). Many short
lived mRNAs have A+T rich 3' untranslated regions, and these
regions often have the ATTTA sequence, sometimes present in
multiple copies or as multimers (e.g., ATTTATTTA . . . ). Shaw and
Kamen showed that the transfer of the 3' end of an unstable mRNA to
a stable RNA (globin or VA1) decreased the stable RNA's half life
dramatically. They further showed that a pentamer of ATTTA had a
profound destabilizing effect on a stable message, and that this
signal could exert its effect whether it was located at the 3' end
or within the coding sequence. However, the number of ATTTA
sequences and/or the sequence context in which they occur also
appear to be important in determining whether they function as
destabilizing sequences. Shaw and Kamen showed that a trimer of
ATTTA had much less effect than a pentamer on mRNA stability and a
dimer or a monomer had no effect on stability (Shaw and Kamen,
1987). Note that multimers of ATTTA such as a pentamer
automatically create an A+T rich region. This was shown to be a
cytoplasmic effect, not nuclear. In other unstable mRNAs, the ATTTA
sequence may be present in only a single copy, but it is often
contained in an A+T rich region. From the animal cell data
collected to date, it appears that ATTTA at least in some contexts
is important in stability, but it is not yet possible to predict
which occurrences of ATTTA are destabiling elements or whether any
of these effects are likely to be seen in plants.
[0301] Some studies on mRNA degradation in animal cells also
indicate that RNA degradation may begin in some cases with
nucleolytic attack in A+T rich regions. It is not clear if these
cleavages occur at ATTTA sequences. There are also examples of
mRNAs that have differential stability depending on the cell type
in which they are expressed or on the stage within the cell cycle
at which they are expressed. For example, histone mRNAs are stable
during DNA synthesis but unstable if DNA synthesis is disrupted.
The 3' end of some histone mRNAs seems to be responsible for this
effect (Pandey and Marzluff, 1987). It does not appear to be
mediated by ATTTA, nor is it clear what controls the differential
stability of this mRNA. Another example is the differential
stability of IgG mRNA in B lymphocytes during B cell maturation
(Genovese and Milcarek, 1988). A final example is the instability
of a mutant .beta.-thallesemic globin mRNA. In bone marrow cells,
where this gene is normally expressed, the mutant mRNA is unstable,
while the wild-type mRNA is stable. When the mutant gene is
expressed in HeLa or L cells in vitro, the mutant mRNA shows no
instability (Lim et al., 1992). These examples all provide evidence
that mRNA stability can be mediated by cell type or cell cycle
specific factors. Furthermore this type of instability is not yet
associated with specific sequences. Given these uncertainties, it
is not possible to predict which RNAs are likely to be unstable in
a given cell. In addition, even the ATTTA motif may act
differentially depending on the nature of the cell in which the RNA
is present. Shaw and Kamen (1987) have reported that activation of
protein kinase C can block degradation mediated by ATTTA.
[0302] The addition of a polyadenylate string to the 3' end is
common to most eukaryotic mRNAs, both plant and animal. The
currently accepted view of polyA addition is that the nascent
transcript extends beyond the mature 3' terminus. Contained within
this transcript are signals for polyadenylation and proper 3' end
formation. This processing at the 3' end involves cleavage of the
mRNA and addition of polyA to the mature 3' end. By searching for
consensus sequences near the polyA tract in both plant and animal
mRNAs, it has been possible to identify consensus sequences that
apparently are involved in polyA addition and 3' end cleavage. The
same consensus sequences seem to be important to both of these
processes. These signals are typically a variation on the sequence
AATAAA. In animal cells, some variants of this sequence that are
functional have been identified; in plant cells there seems to be
an extended range of functional sequences (Wickens and Stephenson,
1984; Dean et al., 1986). Because all of these consensus sequences
are variations on AATAAA, they all are A+T rich sequences. This
sequence is typically found 15 to 20 bp before the polyA tract in a
mature mRNA. Studies in animal cells indicate that this sequence is
involved in both polyA addition and 3' maturation. Site directed
mutations in this sequence can disrupt these functions (Conway and
Wickens, 1988; Wickens et al., 1987). However, it has also been
observed that sequences up to 50 to 100 bp 3' to the putative polyA
signal are also required; i.e., a gene that has a normal AATAAA but
has been replaced or disrupted downstream does not get properly
polyadenylated (Gil and Proudfoot, 1984; Sadofsky and Alwine, 1984;
McDevitt et al., 1984). That is, the polyA signal itself is not
sufficient for complete and proper processing. It is not yet known
what specific downstream sequences are required in addition to the
polyA signal, or if there is a specific sequence that has this
function. Therefore, sequence analysis can only identify potential
polyA signals.
[0303] In naturally occurring mRNAs that are normally
polyadenylated, it has been observed that disruption of this
process either by altering the polyA signal or other sequences in
the mRNA, profound effects can be obtained in the level of
functional mRNA. This has been observed in several naturally
occurring mRNAs, with results that are gene-specific so far.
[0304] It has been shown that in natural mRNAs proper
polyadenylation is important in mRNA accumulation, and that
disruption of this process can effect mRNA levels significantly.
However, insufficient knowledge exists to predict the effect of
changes in a normal gene. In a heterologous gene, it is even harder
to predict the consequences. However, it is possible that the
putative sites identified are dysfunctional. That is, these sites
may not act as proper polyA sites, but instead function as aberrant
sites that give rise to unstable mRNAs.
[0305] In animal cell systems, AATAAA is by far the most common
signal identified in mRNAs upstream of the polyA, but at least four
variants have also been found (Wickens and Stephenson, 1984). In
plants, not nearly so much analysis has been done, but it is clear
that multiple sequences similar to AATAAA can be used. The plant
sites in Table 4 called major or minor refer only to the study of
Dean et al. (1986) which analyzed only three types of plant gene.
The designation of polyadenylation sites as major or minor refers
only to the frequency of their occurrence as functional sites in
naturally occurring genes that have been analyzed. In the case of
plants this is a very limited database. It is hard to predict with
any certainty that a site designated major or minor is more or less
likely to function partially or completely when found in a
heterologous gene such as those encoding the crystal proteins of
the present invention.
4TABLE 4 POLYADENYLATION SITES IN PLANT GENES PA AATAAA Major
consensus site P1A AATAAT Major plant site P2A AACCAA Minor plant
site P3A ATATAA " P4A AATCAA " P5A ATACTA " P6A ATAAAA " P7A ATGAAA
" P8A AAGCAT " P9A ATTAAT " P10A ATACAT " P11A AAAATA " P12A ATTAAA
Minor animal site P13A AATTAA " P14A AATACA " P15A CATAAA "
[0306] The present invention provides a method for preparing
synthetic plant genes which genes express their protein product at
levels significantly higher than the wild-type genes which were
commonly employed in plant transformation heretofore. In another
aspect, the present invention also provides novel synthetic plant
genes which encode non-plant proteins.
[0307] As described above, the expression of native B.
thuringiensis genes in plants is often problematic. The nature of
the coding sequences of B. thuringiensis genes distinguishes them
from plant genes as well as many other heterologous genes expressed
in plants. In particular, B. thuringiensis genes are very rich
(.about.62%) in adenine (A) and thymine (T) while plant genes and
most other bacterial genes which have been expressed in plants are
on the order of 45-55% A+T.
[0308] Due to the degeneracy of the genetic code and the limited
number of codon choices for any amino acid, most of the "excess"
A+T of the structural coding sequences of some Bacillus species are
found in the third position of the codons. That is, genes of some
Bacillus species have A or T as the third nucleotide in many
codons. Thus A+T content in part can determine codon usage bias. In
addition, it is clear that genes evolve for maximum function in the
organism in which they evolve. This means that particular
nucleotide sequences found in a gene from one organism, where they
may play no role except to code for a particular stretch of amino
acids, have the potential to be recognized as gene control elements
in another organism (such as transcriptional promoters or
terminators, polyA addition sites, intron splice sites, or specific
mRNA degradation signals). It is perhaps surprising that such
misread signals are not a more common feature of heterologous gene
expression but this can be explained in part by the relatively
homogeneous A+T content (.about.50%) of many organisms. This A+T
content plus the nature of the genetic code put clear constraints
on the likelihood of occurrence of any particular oligonucleotide
sequence. Thus, a gene from E. coli with a 50% A+T content is much
less likely to contain any particular A+T rich segment than a gene
from B. thuringiensis.
[0309] Typically, to obtain high-level expression of the
S-endotoxin genes in plants, existing structural coding sequence
("structural gene") which codes for the S-endotoxin are modified by
removal of ATTTA sequences and putative polyadenylation signals by
site directed mutagenesis of the DNA comprising the structural
gene. It is most preferred that substantially all the
polyadenylation signals and ATTTA sequences are removed although
enhanced expression levels are observed with only partial removal
of either of the above identified sequences. Alternately if a
synthetic gene is prepared which codes for the expression of the
subject protein, codons are selected to avoid the ATTTA sequence
and putative polyadenylation signals. For purposes of the present
invention putative polyadenylation signals include, but are not
necessarily limited to, AATAAA, AATAAT, AACCAA, ATATAA, AATCAA,
ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA,
AATTAA, AATACA and CATAAA. In replacing the ATTTA sequences and
polyadenylation signals, codons are preferably utilized which avoid
the codons which are rarely found in plant genomes.
[0310] The selected DNA sequence is scanned to identify regions
with greater than four consecutive adenine (A) or thymine (T)
nucleotides. The A+T regions are scanned for potential plant
polyadenylation signals. Although the absence of five or more
consecutive A or T nucleotides eliminates most plant
polyadenylation signals, if there are more than one of the minor
polyadenylation signals identified within ten nucleotides of each
other, then the nucleotide sequence of this region is preferably
altered to remove these signals while maintaining the original
encoded amino acid sequence.
[0311] The second step is to consider the about 15 to about 30 or
so nucleotide residues surrounding the A+T rich region identified
in step one. If the A+T content of the surrounding region is less
than 80%, the region should be examined for polyadenylation
signals. Alteration of the region based on polyadenylation signals
is dependent upon (1) the number of polyadenylation signals present
and (2) presence of a major plant polyadenylation signal.
[0312] The extended region is examined for the presence of plant
polyadenylation signals. The polyadenylation signals are removed by
site-directed mutagenesis of the DNA sequence. The extended region
is also examined for multiple copies of the ATTTA sequence which
are also removed by mutagenesis.
[0313] It is also preferred that regions comprising many
consecutive A+T bases or G+C bases are disrupted since these
regions are predicted to have a higher likelihood to form hairpin
structure due to self-complementarity. Therefore, insertion of
heterogeneous base pairs would reduce the likelihood of
self-complementary secondary structure formation which are known to
inhibit transcription and/or translation in some organisms. In most
cases, the adverse effects may be minimized by using sequences
which do not contain more than five consecutive A+T or G+C.
4.11 Methods for Producing Insect-Resistant Transgenic Plants
[0314] By transforming a suitable host cell, such as a plant cell,
with a recombinant cry1C* gene-containing segment, the expression
of the encoded crystal protein (i.e., a bacterial crystal protein
or polypeptide having insecticidal activity against lepidopterans)
can result in the formation of insect-resistant plants.
[0315] By way of example, one may utilize an expression vector
containing a coding region for a B. thuringiensis crystal protein
and an appropriate selectable marker to transform a suspension of
embryonic plant cells, such as wheat or corn cells using a method
such as particle bombardment (Maddock et al., 1991; Vasil et al.,
1992) to deliver the DNA coated on microprojectiles into the
recipient cells. Transgenic plants are then regenerated from
transformed embryonic calli that express the insecticidal
proteins.
[0316] The formation of transgenic plants may also be accomplished
using other methods of cell transformation which are known in the
art such as Agrobacterium-mediated DNA transfer (Fraley et al.,
1983). Alternatively, DNA can be introduced into plants by direct
DNA transfer into pollen (Zhou et al., 1983; Hess, 1987; Luo et
al., 1988), by injection of the DNA into reproductive organs of a
plant (Pena et al., 1987), or by direct injection of DNA into the
cells of immature embryos followed by the rehydration of desiccated
embryos (Neuhaus et al., 1987; Benbrook et al., 1986).
[0317] The regeneration, development, and cultivation of plants
from single plant protoplast transformants or from various
transformed explants is well known in the art (Weissbach and
Weissbach, 1988). This regeneration and growth process typically
includes the steps of selection of transformed cells, culturing
those individualized cells through the usual stages of embryonic
development through the rooted plantlet stage. Transgenic embryos
and seeds are similarly regenerated. The resulting transgenic
rooted shoots are thereafter planted in an appropriate plant growth
medium such as soil.
[0318] The development or regeneration of plants containing the
foreign, exogenous gene that encodes a polypeptide of interest
introduced by Agrobacterium from leaf explants can be achieved by
methods well known in the art such as described (Horsch et al.,
1985). In this procedure, transformants are cultured in the
presence of a selection agent and in a medium that induces the
regeneration of shoots in the plant strain being transformed as
described (Fraley et al., 1983).
[0319] This procedure typically produces shoots within two to four
months and those shoots are then transferred to an appropriate
root-inducing medium containing the selective agent and an
antibiotic to prevent bacterial growth. Shoots that rooted in the
presence of the selective agent to form plantlets are then
transplanted to soil or other media to allow the production of
roots. These procedures vary depending upon the particular plant
strain employed, such variations being well known in the art.
[0320] Preferably, the regenerated plants are self-pollinated to
provide homozygous transgenic plants, as discussed before.
Otherwise, pollen obtained from the regenerated plants is crossed
to seed-grown plants of agronomically important, preferably inbred
lines. Conversely, pollen from plants of those important lines is
used to pollinate regenerated plants. A transgenic plant of the
present invention containing a desired polypeptide is cultivated
using methods well known to one skilled in the art.
[0321] A transgenic plant of this invention thus has an increased
amount of a coding region (e.g., a cry1C* gene) that encodes the
Cry1C* polypeptide of interest. A preferred transgenic plant is an
independent segregant and can transmit that gene and its activity
to its progeny. A more preferred transgenic plant is homozygous for
that gene, and transmits that gene to all of its offspring on
sexual mating. Seed from a transgenic plant may be grown in the
field or greenhouse, and resulting sexually mature transgenic
plants are self-pollinated to generate true breeding plants. The
progeny from these plants become true breeding lines that are
evaluated for, by way of example, increased insecticidal capacity
against lepidopteran insects, preferably in the field, under a
range of environmental conditions. The inventors contemplate that
the present invention will find particular utility in the creation
of transgenic plants of commercial interest including various turf
grasses, wheat, corn, rice, barley, oats, a variety of ornamental
plants and vegetables, as well as a number of nut- and
fruit-bearing trees and plants.
4.12 Methods for Producing Cry1C* Proteins Having Multiple
Mutations
[0322] Cry1C mutants containing substitutions in multiple loop
regions may be constructed via a number of techniques. For
instance, sequences of highly related genes can be readily shuffled
using the PCR-based technique described by Stemmer (1994).
Alternatively, if suitable restriction sites are available, the
mutations of one cry1C gene may be combined with the mutations of a
second cry1C gene by routine subcloning methodologies. If a
suitable restriction site is not available, one may be generated by
oligonucleotide directed mutagenesis using any number of procedures
known to those skilled in the art. Alternatively, splice-overlap
extension PCR (Horton et al., 1989) may be used to combine
mutations in different loop regions of Cry1C. In this procedure,
overlapping DNA fragments generated by the PCR and containing
different mutations within their unique sequences may be annealed
and used as a template for amplification using flanking primers to
generate a hybrid gene sequence. Finally, cry1C mutants may be
combined by simply using one cry1C mutant as a template for
oligonucleotide-directed mutagenesis using any number of protocols
such as those described herein.
4.13 Ribozymes
[0323] Ribozymes are enzymatic RNA molecules which cleave
particular mRNA species. In certain embodiments, the inventors
contemplate the selection and utilization of ribozymes capable of
cleaving the RNA segments of the present invention, and their use
to reduce activity of target mRNAs in particular cell types or
tissues.
[0324] Six basic varieties of naturally-occurring enzymatic RNAs
are known presently. Each can catalyze the hydrolysis of RNA
phosphodiester bonds in trans (and thus can cleave other RNA
molecules) under physiological conditions. In general, enzymatic
nucleic acids act by first binding to a target RNA. Such binding
occurs through the target binding portion of a enzymatic nucleic
acid which is held in close proximity to an enzymatic portion of
the molecule that acts to cleave the target RNA. Thus, the
enzymatic nucleic acid first recognizes and then binds a target RNA
through complementary base-pairing, and once bound to the correct
site, acts enzymatically to cut the target RNA. Strategic cleavage
of such a target RNA will destroy its ability to direct synthesis
of an encoded protein. After an enzymatic nucleic acid has bound
and cleaved its RNA target, it is released from that RNA to search
for another target and can repeatedly bind and cleave new
targets.
[0325] The enzymatic nature of a ribozyme is advantageous over many
technologies, such as antisense technology (where a nucleic acid
molecule simply binds to a nucleic acid target to block its
translation) since the concentration of ribozyme necessary to
affect a therapeutic treatment is lower than that of an antisense
oligonucleotide. This advantage reflects the ability of the
ribozyme to act enzymatically. Thus, a single ribozyme molecule is
able to cleave many molecules of target RNA. In addition, the
ribozyme is a highly specific inhibitor, with the specificity of
inhibition depending not only on the base pairing mechanism of
binding to the target RNA, but also on the mechanism of target RNA
cleavage. Single mismatches, or base-substitutions, near the site
of cleavage can completely eliminate catalytic activity of a
ribozyme. Similar mismatches in antisense molecules do not prevent
their action (Woolf et al., 1992). Thus, the specificity of action
of a ribozyme is greater than that of an antisense oligonucleotide
binding the same RNA site.
[0326] The enzymatic nucleic acid molecule may be formed in a
hammerhead, hairpin, a hepatitis .delta. virus, group I intron or
RNaseP RNA (in association with an RNA guide sequence) or
Neurospora VS RNA motif. Examples of hammerhead motifs are
described by Rossi et al. (1992); examples of hairpin motifs are
described by Hampel et al. (Eur. Pat. EP 0360257), Hampel and Tritz
(1989), Hampel et al. (1990) and Cech et al. (U.S. Pat. No.
5,631,359; an example of the hepatitis .delta. virus motif is
described by Perrotta and Been (1992); an example of the RNaseP
motif is described by Guerrier-Takada et al. (1983); Neurospora VS
RNA ribozyme motif is described by Collins (Saville and Collins,
1990; Saville and Collins, 1991; Collins and Olive, 1993); and an
example of the Group I intron is described by Cech et al. (U.S.
Pat. No. 4,987,071). All that is important in an enzymatic nucleic
acid molecule of this invention is that it has a specific substrate
binding site which is complementary to one or more of the target
gene RNA regions, and that it have nucleotide sequences within or
surrounding that substrate binding site which impart an RNA
cleaving activity to the molecule. Thus the ribozyme constructs
need not be limited to specific motifs mentioned herein.
[0327] The invention provides a method for producing a class of
enzymatic cleaving agents which exhibit a high degree of
specificity for the RNA of a desired target. The enzymatic nucleic
acid molecule is preferably targeted to a highly conserved sequence
region of a target mRNA such that specific treatment of a disease
or condition can be provided with either one or several enzymatic
nucleic acids. Such enzymatic nucleic acid molecules can be
delivered exogenously to specific cells as required. Alternatively,
the ribozymes can be expressed from DNA or RNA vectors that are
delivered to specific cells.
[0328] Small enzymatic nucleic acid motifs (e.g., of the hammerhead
or the hairpin structure) may be used for exogenous delivery. The
simple structure of these molecules increases the ability of the
enzymatic nucleic acid to invade targeted regions of the mRNA
structure. Alternatively, catalytic RNA molecules can be expressed
within cells from eukaryotic promoters (e.g., Scanlon et al., 1991;
Kashani-Sabet et al., 1992; Dropulic et al., 1992; Weerasinghe et
al., 1991; Ojwang et al., 1992; Chen et al., 1992; Sarver et al.,
1990). Those skilled in the art realize that any ribozyme can be
expressed in eukaryotic cells from the appropriate DNA vector. The
activity of such ribozymes can be augmented by their release from
the primary transcript by a second ribozyme (Draper et al., Int.
Pat. Appl. Publ. No. WO 93/23569, and Sullivan et al., Int. Pat.
Appl. Publ. No. WO 94/02595, both hereby incorporated in their
totality by reference herein; Ohkawa et al., 1992; Taira et al.,
1991; Ventura et al., 1993).
[0329] Ribozymes may be added directly, or can be complexed with
cationic lipids, lipid complexes, packaged within liposomes, or
otherwise delivered to target cells. The RNA or RNA complexes can
be locally administered to relevant tissues ex vivo, or in vivo
through injection, aerosol inhalation, infusion pump or stent, with
or without their incorporation in biopolymers.
[0330] Ribozymes may be designed as described in Draper et al.
(Int. Pat. Appl. Publ. No. WO 93/23569), or Sullivan et al., (Int.
Pat. Appl. Publ. No. WO 94/02595) and synthesized to be tested in
vitro and in vivo, as described. Such ribozymes can also be
optimized for delivery. While specific examples are provided, those
in the art will recognize that equivalent RNA targets in other
species can be utilized when necessary.
[0331] Hammerhead or hairpin ribozymes may be individually analyzed
by computer folding (Jaeger et al., 1989) to assess whether the
ribozyme sequences fold into the appropriate secondary structure.
Those ribozymes with unfavorable intramolecular interactions
between the binding arms and the catalytic core are eliminated from
consideration. Varying binding arm lengths can be chosen to
optimize activity. Generally, at least 5 bases on each arm are able
to bind to, or otherwise interact with, the target RNA.
[0332] Ribozymes of the hammerhead or hairpin motif may be designed
to anneal to various sites in the mRNA message, and can be
chemically synthesized. The method of synthesis used follows the
procedure for normal RNA synthesis as described in Usman et al.
(1987) and in Scaringe et al. (1990) and makes use of common
nucleic acid protecting and coupling groups, such as
dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
Average stepwise coupling yields are typically >98%. Hairpin
ribozymes may be synthesized in two parts and annealed to
reconstruct an active ribozyme (Chowrira and Burke, 1992).
Ribozymes may be modified extensively to enhance stability by
modification with nuclease resistant groups, for example, 2'-amino,
2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see Usman
and Cedergren, 1992). Ribozymes may be purified by gel
electrophoresis using general methods or by high pressure liquid
chromatography and resuspended in water.
[0333] Ribozyme activity can be optimized by altering the length of
the ribozyme binding arms, or chemically synthesizing ribozymes
with modifications that prevent their degradation by serum
ribonucleases (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065;
Perrault et al, 1990; Pieken et al., 1991; Usman and Cedergren,
1992; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ.
No. WO 91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U.S. Pat.
No. 5,334,711; and Int. Pat. Appl. Publ. No. WO 94/13688, which
describe various chemical modifications that can be made to the
sugar moieties of enzymatic RNA molecules), modifications which
enhance their efficacy in cells, and removal of stem II bases to
shorten RNA synthesis times and reduce chemical requirements.
[0334] Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595)
describes the general methods for delivery of enzymatic RNA
molecules. Ribozymes may be administered to cells by a variety of
methods known to those familiar to the art, including, but not
restricted to, encapsulation in liposomes, by iontophoresis, or by
incorporation into other vehicles, such as hydrogels,
cyclodextrins, biodegradable nanocapsules, and bioadhesive
microspheres. For some indications, ribozymes may be directly
delivered ex vivo to cells or tissues with or without the
aforementioned vehicles. Alternatively, the RNA/vehicle combination
may be locally delivered by direct inhalation, by direct injection
or by use of a catheter, infusion pump or stent. Other routes of
delivery include, but are not limited to, intravascular,
intramuscular, subcutaneous or joint injection, aerosol inhalation,
oral (tablet or pill form), topical, systemic, ocular,
intraperitoneal and/or intrathecal delivery. More detailed
descriptions of ribozyme delivery and administration are provided
in Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) and
Draper et al. (Int. Pat. Appl. Publ. No. WO 93/23569) which have
been incorporated by reference herein.
[0335] Another means of accumulating high concentrations of a
ribozyme(s) within cells is to incorporate the ribozyme-encoding
sequences into a DNA expression vector. Transcription of the
ribozyme sequences are driven from a promoter for eukaryotic RNA
polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase
III (pol III). Transcripts from pol II or pol III promoters will be
expressed at high levels in all cells; the levels of a given pol II
promoter in a given cell type will depend on the nature of the gene
regulatory sequences (enhancers, silencers, etc.) present nearby.
Prokaryotic RNA polymerase promoters may also be used, providing
that the prokaryotic RNA polymerase enzyme is expressed in the
appropriate cells (Elroy-Stein and Moss, 1990; Gao and Huang, 1993;
Lieber et al., 1993; Zhou et al., 1990). Ribozymes expressed from
such promoters can function in mammalian cells (e.g. Kashani-Saber
et al., 1992; Ojwang et al., 1992; Chen et al., 1992; Yu et al.,
1993; L'Huillier et al., 1992; Lisziewicz et al., 1993). Such
transcription units can be incorporated into a variety of vectors
for introduction into mammalian cells, including but not restricted
to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or
adeno-associated vectors), or viral RNA vectors (such as
retroviral, semliki forest virus, sindbis virus vectors).
[0336] Ribozymes of this invention may be used as diagnostic tools
to examine genetic drift and mutations within cell lines or cell
types. They can also be used to assess levels of the target RNA
molecule. The close relationship between ribozyme activity and the
structure of the target RNA allows the detection of mutations in
any region of the molecule which alters the base-pairing and
three-dimensional structure of the target RNA. By using multiple
ribozymes described in this invention, one may map nucleotide
changes which are important to RNA structure and function in vitro,
as well as in cells and tissues. Cleavage of target RNAs with
ribozymes may be used to inhibit gene expression and define the
role (essentially) of specified gene products in particular cells
or cell types.
4.14 Isolating Homologous Gene and Gene Fragments
[0337] The genes and .delta.-endotoxins according to the subject
invention include not only the full-length sequences disclosed
herein but also fragments of these sequences, or fusion proteins,
which retain the characteristic insecticidal activity of the
sequences specifically exemplified herein.
[0338] It should be apparent to a person skill in this art that
insecticidal .delta.-endotoxins can be identified and obtained
through several means. The specific genes, or portions thereof, may
be obtained from a culture depository, or constructed
synthetically, for example, by use of a gene machine. Variations of
these genes may be readily constructed using standard techniques
for making point mutations. Also, fragments of these genes can be
made using commercially available exonucleases or endonucleases
according to standard procedures. For example, enzymes such as
Bal31 or site-directed mutagenesis can be used to systematically
cut off nucleotides from the ends of these genes. Also, genes which
code for active fragments may be obtained using a variety of other
restriction enzymes. Proteases may be used to directly obtain
active fragments of these .delta.-endotoxins.
[0339] Equivalent .delta.-endotoxins and/or genes encoding these
equivalent .delta.-endotoxins can also be isolated from Bacillus
strains and/or DNA libraries using the teachings provided herein.
For example, antibodies to the .delta.-endotoxins disclosed and
claimed herein can be used to identify and isolate other
.delta.-endotoxins from a mixture of proteins. Specifically,
antibodies may be raised to the portions of the .delta.-endotoxins
which are most constant and most distinct from other B.
thuringiensis .delta.-endotoxins. These antibodies can then be used
to specifically identify equivalent .delta.-endotoxins with the
characteristic insecticidal activity by immunoprecipitation, enzyme
linked immunoassay (ELISA), or Western blotting.
[0340] A further method for identifying the .delta.-endotoxins and
genes of the subject invention is through the use of
oligonucleotide probes. These probes are nucleotide sequences
having a detectable label. As is well known in the art, if the
probe molecule and nucleic acid sample hybridize by forming a
strong bond between the two molecules, it can be reasonably assumed
that the probe and sample are essentially identical. The probe's
detectable label provides a means for determining in a known manner
whether hybridization has occurred. Such a probe analysis provides
a rapid method for identifying formicidal .delta.-endotoxin genes
of the subject invention.
[0341] The nucleotide segments which are used as probes according
to the invention can be synthesized by use of DNA synthesizers
using standard procedures. In the use of the nucleotide segments as
probes, the particular probe is labeled with any suitable label
known to those skilled in the art, including radioactive and
non-radioactive labels. Typical radioactive labels include
.sup.32P, .sup.125I, .sup.35S, or the like. A probe labeled with a
radioactive isotope can be constructed from a nucleotide sequence
complementary to the DNA sample by a conventional nick translation
reaction, using a DNase and DNA polymerase. The probe and sample
can then be combined in a hybridization buffer solution and held at
an appropriate temperature until annealing occurs. Thereafter, the
membrane is washed free of extraneous materials, leaving the sample
and bound probe molecules typically detected and quantified by
autoradiography and/or liquid scintillation counting.
[0342] Non-radioactive labels include, for example, ligands such as
biotin or thyroxine, as well as enzymes such as hydrolases or
peroxidases, or the various chemiluminescers such as luciferin, or
fluorescent compounds like fluorescein and its derivatives. The
probe may also be labeled at both ends with different types of
labels for ease of separation, as, for example, by using an
isotopic label at the end mentioned above and a biotin label at the
other end.
[0343] Duplex formation and stability depend on substantial
complementarity between the two strands of a hybrid, and, as noted
above, a certain degree of mismatch can be tolerated. Therefore,
the probes of the subject invention include mutations (both single
and multiple), deletions, insertions of the described sequences,
and combinations thereof, wherein said mutations, insertions and
deletions permit formation of stable hybrids with the target
polynucleotide of interest. Mutations, insertions, and deletions
can be produced in a given polynucleotide sequence in many ways, by
methods currently known to an ordinarily skilled artisan, and
perhaps by other methods which may become known in the future.
[0344] The potential variations in the probes listed is due, in
part, to the redundancy of the genetic code. Because of the
redundancy of the genetic code, i.e., more than one coding
nucleotide triplet (codon) can be used for most of the amino acids
used to make proteins. Therefore different nucleotide sequences can
code for a particular amino acid. Thus, the amino acid sequences of
the B. thuringiensis .delta.-endotoxins and peptides can be
prepared by equivalent nucleotide sequences encoding the same amino
acid sequence of the protein or peptide. Accordingly, the subject
invention includes such equivalent nucleotide sequences. Also,
inverse or complement sequences are an aspect of the subject
invention and can be readily used by a person skilled in this art.
In addition it has been shown that proteins of identified structure
and function may be constructed by changing the amino acid sequence
if such changes do not alter the protein secondary structure
(Kaiser and Kezdy, 1984). Thus, the subject invention includes
mutants of the amino acid sequence depicted herein which do not
alter the protein secondary structure, or if the structure is
altered, the biological activity is substantially retained.
Further, the invention also includes mutants of organisms hosting
all or part of a .delta.-endotoxin encoding a gene of the
invention. Such mutants can be made by techniques well known to
persons skilled in the art. For example, UV irradiation can be used
to prepare mutants of host organisms. Likewise, such mutants may
include asporogenous host cells which also can be prepared by
procedures well known in the art.
5.0 EXAMPLES
[0345] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
5.1 Example 1
Preparation of Templates for Random Mutagenesis
[0346] Structural maps for the cry1C plasmids pEG315 and pEG916 are
shown in FIG. 2. The cry1C gene contained on these plasmids was
isolated from the B. thuringiensis strain EG6346 subsp. aizawai,
first described by Chambers et al. (1991). An .about.4 kb
Sa/I-BamHI fragment containing the intact cry1C gene from EG6346
was cloned into the unique XhoI and BamHI sites of the shuttle
vector pEG854, described by Baum et al. (1990) to yield pEG315.
pEG916 is a pEG853 derivative (also described by Baum et al., 1990)
containing the same cry1C gene fragment and a 3' transcription
terminator region derived from the cry1F gene described by Chambers
et al. (1991).
[0347] pEG345 (FIG. 3) is a pEG597 derivative (also described by
Baum et al., 1990) that contains the cry1C gene from B.
thuringiensis subsp. aizawai strain 7.29, described by Sanchis et
al. (1989) and disclosed in the European Pat. Appl. No. EP 295156A1
and Intl. Pat. Appl. Publ. No. WO 88/09812. Both genes are nearly
identical to the holotype cry1C gene described by Honee et al.
(1988).
[0348] The recombinant DNA techniques employed are familiar to
those skilled in the art of manipulating and cloning DNA fragments
and employed pursuant to the teachings of Maniatis et al. (1982)
and Sambrook et al. (1989).
[0349] A frame-shift mutation was introduced into the cry1C gene of
pEG916 at codon 118. By analogy to the published crystal structures
for Cry1Aa and Cry3A, the glutamic acid residue (E) at this
position is predicted to lie within or immediately adjacent to the
loop region between a helices 3 and 4 of Cry1C domain 1, the target
site for random mutagenesis. This mutated gene can be used as a
template for oligonucleotide-directed mutagenesis using a mutagenic
primer that corrects the frame-shift mutation, thus ensuring that
the majority of clones recovered encoding full-length protoxin
molecules will have incorporated the mutagenic oligonucleotide.
[0350] The frame-shift mutation was introduced by a
PCR.TM.-mediated mutagenesis protocol using the oligonucleotide
primers A, B, and C and pEG916 (FIG. 2) as the DNA template. The
mutagenesis protocol, described by (Michael, 1994) relies on the
use of a thermostable ligase to incorporate a phosphorylated
mutagenic oligonucleotide into an amplified DNA fragment. The DNA
sequence of these primers is shown below:
5 Primer A: (SEQ ID NO:15) 5'-CCCGATCGGCCGCATGC-3' Primer B: (SEQ
ID NO:16) 5'-GCATTTAAAGAATGGGAAGGGATCCTAGGAATCCAGCAACCAGGA-
CCAGAG-3' Primer C: (SEQ ID NO:17) 5'-GAGCTCTTGTTAAAAAAGGT-
GTTCCAGATC-3'
[0351] The mutagenic oligonucleotide, primer B, was designed to
incorporate a BamHI and BlnI restriction site in addition to the
frame-shift mutation at codon 118 (FIG. 4). The product obtained
from the PCR.TM. was resolved by electrophoresis of an agarose-TAE
gel and purified using the Geneclean II.RTM. Kit (Bio 101, Inc., La
Jolla, Calif.) following the manufacturer's suggested protocol. The
purified DNA fragment was digested with the restriction enzymes
AgeI and BbuI. pEG916 was also digested with the restriction
enzymes AgeI and BbuI and the restricted DNA fragments resolved by
agarose gel electrophoresis and the vector fragment purified as
described above. The amplified DNA fragment and the pEG916 vector
fragment were ligated together with T4 ligase, and the ligation
reaction used to transform the acrystalliferous B. thuringiensis
strain EG10368 (described in U.S. Pat. No. 5,322,687) to Cml
resistance, using the electroporation procedure described by Mettus
and Macaluso (1990). Individual transformants were selected and
many were determined to be acrystalliferous by phase-contrast
microscopy of the sporulated cultures. Recombinant plasmids were
isolated from B. thuringiensis transformants using the alkaline
lysis procedure described by Maniatis et al. (1982). Incorporation
of the frame-shift mutation into cry1C was also indicated by the
presence of the BamHI and BlnI sites, determined by restriction
enzyme analysis of the recombinant plasmids isolated from the EG
10368 transformants. The recombinant plasmid incorporating the
frame-shift mutation and the BamHI and BlnI sites was designated
pEG359 (FIG. 2 and FIG. 4).
[0352] pEG359 was introduced into the E. coli host strain
DH5.alpha. by transformation using frozen competent cells and
procedures obtained from GIBCO BRL (Gaithersburg, Md.). pEG359,
purified from E. coli using the alkaline lysis procedure (Maniatis
et al, 1982), was further modified by digestion with the
restriction enzyme BglII and religation of the vector fragment with
T4 ligase. The ligation reaction was used to transform the E. coli
host strain DH5.alpha. as before. The resulting plasmid, designated
p154 (FIG. 2), contains a deletion of the cry1C gene sequences
downstream of the unique BglII site in cry1C.
5.2 Example 2
Random Mutagnesis of Nucleotides 352-372 in Cry1C
[0353] Mutagenesis of nucleotides 352-372, encoding the putative
loop region between .alpha. helices 3 and 4 of Cry1C domain 1, was
performed according to the PCR.TM.-mediated "Megaprimer" method as
described (Upender et al., 1995), using the oligonucleotide primers
A (SEQ ID NO:15), C (SEQ ID NO:17), and D (SEQ ID NO:18).
[0354] Primer D: (SEQ ID NO:18)
[0355]
5'-GCATTTAAAGAATGGGAANNNNNNNNNNNNNNNNNNNNNACCAGGACCAGAGTAATTGATCGC--
3'
[0356] N (20, 21, 23, 28, 29, 31, 32, and 39)=82% A; 6% G, C,
T,
[0357] N (25, 26, 34, 35, and 38)=82% C; 6% G, T, A
[0358] N (19, 22, and 37)=82% G; 6% C, T, A
[0359] N (24, 27, 30, 33, and 36)=82% T; 6% G, C, A. Numbers in
parentheses correspond to the positions above in SEQ ID NO:18,
wherein the first G is position number 1.
[0360] The mutagenic primer D corrects the frame-shift mutation and
eliminates the BamHI and BlnI sites introduced into pEG359. To
accomplish this mutagenesis, the Megaprimer was first synthesized
by PCR.TM. amplification of pEG315 DNA (FIG. 2) using the mutagenic
primer D and the opposing primer C (FIG. 5). The resulting
amplified DNA fragment was purified by gel electrophoresis as
described above and used in a second PCR.TM. using primers A and C
and p154 as the template. Because the p154 template contains a
deletion of the region complementary to primer C (FIG. 5),
initiation of the PCR.TM. first requires extension of the
Megaprimer to allow annealing of primer A to the mutagenic strand,
thus ensuring that most of the amplified product obtain from the
PCR.TM. incorporates the mutagenic DNA. The resulting PCR.TM.
product was isolated and purified following gel electrophoresis in
agarose and 1.times.TAE as described above.
[0361] The amplified DNA fragment was digested with the restriction
enzymes AgeI and BbuI, to provide sticky ends suitable for cloning,
and with the enzymes BamHI and BlnI to eliminate any residual p154
template DNA. pEG359 was digested with AgeI and BbuI and the vector
fragment ligated to the restricted amplified DNA preparation. The
ligation reaction was used to transform the E. coli Sure.TM.
(Stratagene Cloning Systems, La Jolla, Calif.) strain to ampicillin
(Amp) resistance (Amp.sup.R) using a standard transformation
procedure. Amp.sup.R colonies were scraped from plates and growth
for 1-2 hr at 37.degree. C. in Luria Broth with 50 .mu.g/ml of Amp.
Plasmid DNA was isolated from this culture using the alkaline lysis
procedure described above and used to transform B. thuringiensis
EG10368 to Cml resistance (Cml.sup.R) by electroporation.
Transformants were plated on starch agar plates containing 5
.mu.g/ml Cml and incubated at 25-30.degree. C. Restriction enzyme
analysis of plasmid DNAs isolated from crystal-forming
transformants indicated that .about.75% of the transformants had
incorporated the mutagenic oligonucleotide at the target site (nt
352-372). That is, .about.75% of the crystal-forming transformants
had lost the BamHI and BlnI sites at the target site on cry1C.
5.3 Example 3
Mutagenesis of Arg Residues in Cry1C Domain 1
[0362] Arginine residues within potential loop regions of Cry1C
domain 1 were replaced by alanine residues using
oligonucleotide-directed mutagenesis. The elimination of these
arginine residues may reduce the proteolysis of toxin protein by
trypsin-like proteases in the lepidopteran midgut since trypsin is
known to cleave peptide bonds immediately C-terminal to arginine
and lysine. The arginine residues at amino acid positions 148 and
180 in the Cry1C amino acid sequence were replaced with alanine
residues. The PCR.TM.-mediated mutagenesis protocol used, described
by Michael (1994) relies on the use of a thermostable ligase to
incorporate a phosphorylated mutagenic oligonucleotide into an
amplified DNA fragment. The mutagenesis of R148 employed the
mutagenic primer E (SEQ ID NO:19) and the flanking primers A (SEQ
ID NO:15) and primer F (SEQ ID NO:20). The mutagenesis of R180
employed the mutagenic primer G (SEQ ID NO:21) and the flanking
primers A (SEQ ID NO:15) and F (SEQ ID NO:20). Both PCR.TM. studies
employed pEG315 (FIG. 2) DNA as the cry1C template. Primer E was
designed to eliminate an AsuII site within the wild-type cry1C
nucleotide sequence. Primer G was designed to introduce a HincII
site within the cry1C nucleotide sequence.
6 Primer E: (SEQ ID NO:19)
5'-GGGCTACTTGAAAGGGACATTCCTTCGTTTGCAATTT- CTGGATTTGAAGTACCCC-3'
Primer F: (SEQ ID NO:20)
5'-CCAAGAAAATACTAGAGCTCTTGTTAAAAAAGGTGTTCC-3' Primer G: (SEQ ID
NO:21) 5'-GAGATTCTGTAATTTTTGGAGAAGCATGGGGGTTGACAACGATAAATGTC-3'
[0363] The products obtained from the PCR.TM. were purified
following agarose gel electrophoresis using the Geneclean II.RTM.
procedure and reamplified using the opposing primers A and F and
standard PCR.TM. procedures. The resultant PCR.TM. products were
digested with the restriction enzymes BbuI and AgeI. pEG315,
containing the intact cry1C gene of EG6346, was digested with the
restriction enzymes BbuI and AgeI. The restricted fragments were
resolved by agarose gel electrophoresis in 1.times.TAE, the pEG315
vector fragment purified using the Geneclean II.RTM. procedure and,
subsequently ligated to the amplified DNA fragments obtained from
the mutagenesis using T4 ligase. The ligation reactions were used
to transform the E. coli DH5.alpha..TM. to Amp resistance using
standard transformation methods. Transformants were selected on
Luria plates containing 50 .mu.g/ml Amp. Plasmid DNAs isolated from
the E. coli transformants generated by the R148 mutagenesis were
used to transform B. thuringiensis EG10368 to Cml.sup.R, using the
electroporation procedure described by Mettus and Macaluso (1990).
Transformants were selected on Luria plates containing 3 .mu.g/ml
Cml: Approximately 75% of the EG10368 transformants generated by
the R148 mutagenesis had lost the AsuII site, indicating that the
mutagenic oligonucleotide primer E had been incorporated into the
cry1C gene. One transformant, designated EG11811, was chosen for
further study. Approximately 25% of the E. coli transformants
generated by the R180 mutagenesis contained the new HincII site
introduced by the mutagenic oligonucleotide primer G, indicating
that the mutagenic oligonucleotide had been incorporated into the
cry1C gene. Plasmid DNA from one such transformant was used to
transform the B. thuringiensis host strain EG10368 to Cml.sup.R by
electroporation as before. One of the resulting transformants was
designated EG 11815.
[0364] The mutagenesis of R148 was repeated using the cry1C gene
contained in plasmid pEG345. Plasmid pEG345 (FIG. 2) contains the
cry1C gene from B. thuringiensis subsp. aizawai strain 7.29
(Sanchis et al., 1989; Eur. Pat. Application EP 295156A1; Intl.
Pat. Appl. Publ. No. WO 88/09812). The mutagenesis of R148 employed
the mutagenic primer E (SEQ ID No: 19), the flanking primers H (SEQ
ID NO:52) and F (SEQ ID NO:20), and plasmid pEG345 as the source of
the cry1C DNA template. Primer E was designed to eliminate an AsuII
site within the wild-type cry1C sequence.
[0365] Primer H: 5'-GGATCCCTCGAGCTGCAGGAGC-3' (SEQ ID NO:52)
[0366] cry1C template DNA was obtained from a PCR.TM. using the
opposing primers H and F and plasmid pEG345 as a template. This DNA
was then used as the template for a PCR.TM.-mediated mutagenesis
reaction that employed the flanking primers H and F and the
mutagenic oligonucleotide E, using the procedure described by
Michael (1994). The resultant PCR.TM. products were digested with
the restriction enzymes BbuI and AgeI. The restricted DNA fragments
were resolved by agarose gel electrophoresis in 1.times.TAE and the
amplified cry1C fragment was purified using the Geneclean II.RTM.
procedure. Similarly, plasmid pEG345 was digested with the
restriction enzymes BbuI and AgeI, resolved by agarose gel
electrophoresis in 1.times.TAE and the pEG345 vector fragment
purified using the Geneclean II.RTM. procedure. The purified DNA
fragments were ligated together using T4 ligase and used to
transform E. coli DH5.alpha. using a standard transformation
procedure. Transformants were selected on Luria plates containing
50 .mu.g/ml Amp. Approximately 50% of the DH5.alpha. transformants
generated by the R148 mutagenesis had lost the AsuII site,
indicating that the mutagenic oligonucleotide primer E had been
incorporated into the cry1C gene. Plasmid DNA from one transformant
was used to transform B. thuringiensis EG10368 to Cml.sup.R, using
the electroporation procedure described by Mettus and Macaluso
(1990). Transformants were selected on Luria plates containing 3
ug/ml chloramphenicol. One of the transformants was designated EG
11822.
[0367] The arginine residue at amino acid position 148 was also
replaced with random amino acids. This mutagenesis of R148 employed
the mutagenic primer I (SEQ ID No: 53), the flanking primers H (SEQ
ID NO:52) and F (SEQ ID NO:20), and plasmid pEG345 as the source of
the cry1C DNA template. Primer I was also designed to eliminate an
AsuII site within the wild-type cry1C sequence:
[0368] Primer I: (SEQ ID NO:53)
[0369]
5'-GGGCTACTTGAAAGGGACATTCCTTCGTTTNNNATTTCTGGATTTGAAGTACCCC-3'
[0370] N (31,32,33)=25% A, 25% C, 25% G, 25% T
[0371] cry1C template DNA was obtained from a PCR.TM. using the
opposing primers H and F and plasmid pEG345 as a template. This DNA
was then used as the template for a PCR.TM.-mediated mutagenesis
reaction that employed the flanking primers H and F and the
mutagenic oligonucleotide I, using the procedure described by
Michael (1994). The resultant PCR.TM. products were digested with
the restriction enzymes BbuI and AgeI. The restricted DNA fragments
were resolved by agarose gel electrophoresis in 1.times.TAE and the
amplified cry1C fragment was purified using the Geneclean II.RTM.
procedure. Similarly, plasmid pEG345 was digested with the
restriction enzymes BbuI and AgeI, resolved by agarose gel
electrophoresis in 1.times.TAE and the pEG345 vector fragment
purified using the Geneclean II.RTM. procedure. The purified DNA
fragments were ligated together using T4 ligase and used to
transform E. coli DH5.alpha. to ampicillin resistance using a
standard transformation procedure. Transformants were selected on
Luria plates containing 50 ug/ml ampicillin. The DH5.alpha.
transformants were pooled together and plasmid DNA was prepared
using the alkaline lysis procedure. Plasmid DNA from the DH5.alpha.
transformants was used to transform B. thuringiensis EG10368 to
Cml.sup.R, using the electroporation procedure described by Mettus
and Macaluso (1990). Transformants were selected that exhibited an
opaque phenotype on starch agar plates containing 3 ug/ml
chloramphenicol, indicating crystal protein production.
Approximately 90% of the opaque EG10368 transformants generated by
the R148 mutagenesis had lost the AsuII site, indicating that the
mutagenic oligonucleotide primer I had been incorporated into the
cry1C gene.
5.4 Example 4
Bioassay Evaluation of Cry1C* Toxins
[0372] EG10368 transformants containing mutant cry1C genes were
grown in C2 medium, described by Donovan et al. (1988), for 3 days
at 25.degree. C. or until fully sporulated and lysed. The
spore-Cry1C crystal suspensions recovered from the spent C2
cultures were used for bioassay evaluation against neonate larvae
of Spodoptera exigua and 3rd instar larvae of Plutella
xylostella.
[0373] EG10368 transformants harboring Cry1C mutants generated by
random mutagenesis were grown in 2 ml of C2 medium and evaluated in
one-dose bioassay screens. Each culture was diluted with 10 ml of
0.005% Triton X-100.RTM. and 25 .mu.l of these dilutions were
seeded into an additional 4 ml of 0.005% Triton X-100.RTM. to
achieve the appropriate dilution for the bioassay screens. Fifty
.mu.l of this dilution were topically applied to 32 wells
containing 1.0 ml artificial diet per well (surface area of 175
mm.sup.2). A single neonate larvae (S. exigua) or 3rd instar larvae
(P. xylostella) was placed in each of the treated wells and the
tray was covered by a clear perforated mylar strand. Larval
mortality was scored after 7 days of feeding at 28-30.degree. C.
and percent mortality expressed as ratio of the number of dead
larvae to the total number of larvae treated.
[0374] Three EG10368 transformants, designated EG11740, EG11746,
and EG11747, were identified as showing increased insecticidal
activity against Spodoptera exigua in replicated bioassay screens.
The putative Cry1C variants in strains EG11740, EG11746, and
EG11747 were designated Cry1C.563, Cry1C.579, and Cry1C.499,
respectively. These three variants contain amino acid substitutions
within the loop region between .alpha. helices 3 and 4 of Cry1C.
EG11740, EG11746, and EG1 1747, as well as EG11726 (which contains
the wild-type cry1C gene from strain EG6346) were grown in C2
medium for 3 days at 25.degree. C. The cultures were centrifuged
and the spore/crystal pellets were washed three times in 2.times.
volumes of distilled-deionized water. The final pellet was
suspended in an original volume of 0.005% TritonX-100 and crystal
protein quantified by SDS-PAGE as described by Brussock and Currier
(1990). The procedure was modified to eliminate the neutralization
step with 3M HEPES. Eight .delta.-endotoxin concentrations of the
spore/crystal preparations were prepared by serial dilution in
0.005% Triton X-100 and each concentration was topically applied to
wells containing 1.0 ml of artificial diet. Larval mortality was
scored after 7 days of feeding at 23-30.degree. C. (32 larvae for
each .delta.-endotoxin concentration). Mortality data was expressed
as LC.sub.50 and LC.sub.95 values, in accordance with the technique
of Daum (1970), the concentration of Cry1C protein (ng/well)
causing 50% and 95% mortality, respectively (Table 5, Table 6, and
Table 7). Strains EG11740 (Cry1C.563) and EG11746 (Cry1C.579)
exhibited 3-fold lower LC.sub.95 values than the control strain
EG11726 (Cry1C) against S. exigua, while retaining a comparable
level of activity against P. xylostella. EG11740 and EG11746 also
exhibited significantly lower LC.sub.50 values against S.
exigua.
7TABLE 5 BIOASSAY OF CRY1C LOOP .alpha. 3-4 MUTANTS USING
SPODOPTERA EXIGUA LARVAE Strain Toxin LC.sub.50.sup.1 (95% C.
I.).sup.3 LC.sub.95.sup.2 (95% C. I.) EG11726 Cry1C 116 (104-131)
1601 (1253-2131) EG11740 Cry1C.563 50 (42-59) 583 (433-844) EG11747
Cry1C.499 67 (58-78) 596 (455-834) EG11746 Cry1C.579 68 (58-79) 554
(427-766) .sup.1Concentration of Cry1C protein that causes 50%
mortality expressed in ng crystal protein per 175 mm.sup.2 well.
Results of 3-7 sets of replicated bioassays. .sup.2Concentration of
Cry1C protein that causes 95% mortality expressed in ng crystal
protein per 175 mm.sup.2 well. Results of 3-7 sets of replicated
bioassays. .sup.395% confidence intervals.
[0375]
8TABLE 6 BIOASSAYS USING PLUTELLA XYLOSTELLA LARVAE Strain Toxin
LC.sub.50.sup.1 (95% C. I.).sup.3 LC.sub.95.sup.2 (95% C. I.)
EG11726 Cry1C 92 (83-102) 444 (371-549) EG11740 Cry1C.563 106
(95-119) 579 (478-728) EG11811 Cry1C R148A 61 (45-85) 400 (241-908)
.sup.1Concentration of Cry1C protein that causes 50% mortality
expressed in ng crystal protein per 175 mm.sup.2 well. Results of
two sets of replicated bioassays. .sup.2Concentration of Cry1C
protein that causes 95% mortality expressed in ng crystal protein
per 175 mm.sup.2 well. Results of two sets of replicated bioassays.
.sup.395% confidence intervals.
[0376] The Cry1C mutant strains EG11811 (Cry1C R148A) and EG11815
(Cry1C R180A) were grown in C2 medium and evaluated using the same
quantitative eight-dose bioassay procedure. The insecticidal
activities of Cry1C and Cry1C R180A against S. exigua and P.
xylostella were not significantly different, however, Cry1C R148A
exhibited a 3.6-fold lower LC.sub.50 and a 3.7-fold lower LC.sub.95
against S. exigua when compared to the original Cry1C-endotoxin
(Table 7). Cry1C R148A and Cry1C exhibited comparable insecticidal
activity against P. xylostella (Table 6).
9TABLE 7 BIOASSAYS OF CRY1C R148A USING SPODOPTERA EXIGUA LARVAE
Strain Toxin LC.sub.50.sup.1 (95% C. I.).sup.3 LC.sub.95.sup.2 (95%
C. I.) EG11726 Cry1C 141 (122-164) 1747 (1279-2563) EG11811 Cry1C
R148A 41 (33-52) 481 (314-864) .sup.1Concentration of Cry1C protein
that causes 50% mortality expressed in ng crystal protein per 175
mm.sup.2 well. Results of two sets of replicated bioassays.
.sup.2Concentration of Cry1C protein that causes 95% mortality
expressed in ng crystal protein per 175 mm.sup.2 well. Results of
two sets of replicated bioassays. .sup.395% confidence
intervals.
[0377] The Cry1C mutant strains EG11811 (Cry1C R148A), EG11740
(Cry1C.563), and EG11726 (producing wildtype Cry1C) were similarly
cultured and evaluated in bioassays using neonate larvae of
Trichoplusia ni. The insecticidal activities of Cry1C R148A and
Cry1C.563 against T. ni exhibited a lower LC.sub.50 and LC.sub.95
against T. ni when compared to EG11726 (Table 8).
10TABLE 8 BIOASSAYS USING TRICHOPLUSIA NI LARVAE Strain Toxin
LC.sub.50.sup.1 LC.sub.95.sup.2 EG11726 Cry1C 40 (31-56).sup.3 330
EG11740 Cry1C.563 20 (17-24) 104 EG11811 Cry1C-R148A 19 (16-23) 115
.sup.1Concentration of Cry1C protein that causes 50% mortality
expressed in ng crystal protein per 175 mm.sup.2 well. Results of
one set of replicated bioassays. .sup.2Concentration of Cry1C
protein that causes 95% mortality expressed in ng crystal protein
per 175 mm.sup.2 well. Results of one set of replicated bioassays.
.sup.395% confidence intervals.
[0378] Bioassay comparisons with other lepidopteran insects
revealed additional improvements in the properties of Cry1C.563 and
Cry1C-R148A, particularly in toxicity towards the fall armyworm
Spodoptera frugiperda (Table 9) The doses reported in Table 8 are
as follows: 10,000 ng/well A. epsilon, H. virescens, H. zea, O.
nubilalis, and S. frugiperda.
11TABLE 9 BIOASSAY COMPARISONS WITH OTHER LEPIDOPTERAN INSECTS
Mortality Insect Control Cry1C.563 Cry1C-R148A Native Cry1C A.
ipsilon -- -- -- -- H. virescens -- + +++ + H. zea -- -- -- -- O.
nubilalis -- +++ +++ ++ S. frugiperda -- +++ +++ + + = 20-49%
mortality ++ = 50-74% mortality +++ = 75-100% mortality
[0379] EG10368 transformants harboring random mutants at position
R148 of Cry1C were evaluated in bioassay in a one-dose screen
against S. exigua as described above. Five Cry1C mutants were
identified with improved activity over wild-type Cry1C. The mutants
were then evaluated in eight-dose bioassay against S. exigua as
described above. All five Cry1C mutants gave a significantly lower
LC.sub.50 than wild-type Cry1C (Table 10), comparable to EG11822
(R148A). One mutant, designated EG11832 (Cry1C-R148D) gave a
significantly lower LC.sub.50 and LC.sub.95 than EG11822,
indicating further improved toxicity towards S. exigua.
12TABLE 10 BIOASSAYS USING SPODOPTERA EXIGUA LARVAE Strain Mutation
LC.sub.50.sup.1 (95% C. I.).sup.3 LC.sub.95.sup.2 (95% C. I.)
EG11822 R148A 37 (32-43).sup.4 493 (375-686).sup.4 EG11832 R148D 22
(19-25).sup.4 211 (167-282).sup.4 Wild-type None 145 (117-182) 1685
(1072-3152) Mutant #1 R148L 47 (39-57) 523 (367-831) Mutant #12
R148G 65 (46-93) 549 (316-1367) Mutant #43 R148L 31 (16-54) 311
(144-1680) Mutant #45 R148M 36 (29-45) 469 (324-762)
.sup.1Concentration of Cry1C protein that causes 50% mortality
expressed in ng crystal protein per 175 mm.sup.2 well. Results of
one set of replicated bioassays. .sup.2Concentration of Cry1C
protein that causes 95% mortality expressed in ng crystal protein
per 175 mm.sup.2 well. Results of one set of replicated bioassays.
.sup.395% confidence intervals. .sup.4Results of two sets of
replicated bioassays.
5.5 Example 5
Sequence Analysis of Cry1C Mutations
[0380] Recombinant plasmids from the EG10368 transformants were
isolated using the alkaline lysis method (Maniatis et al., 1982).
Plasmids obtained from the transformants were introduced into the
E. coli host strain DH5.alpha..TM. by competent cell transformation
and used as templates for DNA sequencing using the Sequenase.RTM.
v2.0 DNA sequencing kit (U. S. Biochemical Corp., Cleveland,
Ohio).
[0381] Sequence analysis of plasmid pEG359 (FIG. 4; SEQ ID NO:24)
revealed the expected frameshift mutation at codon 118 and the
BamHI and BlnI restriction sites introduced by the mutagenic
oligonucleotide primer B (SEQ ID NO:16).
[0382] Sequence analysis of the cry1C.563 gene on plasmid pEG370
(FIG. 4; SEQ ID NO:25) revealed nucleotide substitutions at
positions 354, 361, 369, and 370, resulting in point mutations A to
T, A to C, A to C, and G to A, respectively. These mutations
resulted in amino acid substitutions in Cry1C.563 (FIG. 4; SEQ ID
NO:26) at positions 118 (E to D), 121 (N to H), and 124 (A to
T).
[0383] Sequence analysis of the cry1C.579 gene on plasmid pEG373
(FIG. 4; SEQ ID NO:54) revealed nucleotide substitutions at
positions 353, 369, and 371, resulting in point mutations A to T, A
to T, and C to G, respectively. These mutations resulted in amino
acid substitutions in Cry1C.579 (FIG. 4; SEQ ID NO:55) at positions
118 (E to V) and 124 (A to G).
[0384] Sequence analysis of the cry1C.499 gene on plasmid pEG374
(FIG. 4; SEQ ID NO:56) revealed nucleotide substitutions at
positions 360 and 361, resulting in point mutations T to C and A to
C, respectively. These mutations resulted in an amino acid
substitution in Cry1C.499 (FIG. 4; SEQ ID NO:57) at position 121 (N
to H).
[0385] Sequence analysis of the cry1C genes in EG11811 and EG11822
confirmed the substitution of alanine for arginine at position 148
(SEQ ID NO:1, SEQ ID NO:2). Nucleotide substitutions C442G and
G443C yield the codon GCA, encoding alanine.
[0386] Sequence analysis of the random R148 mutants indicate
changes of R148 to aspartic acid, methionine, leucine, and glycine.
Thus, a variety of amino acid substitutions for the
positively-charged arginine residue at position 148 in Cry1C result
in improved toxicity. None of these substitutions can be regarded
as conservative changes. Alanine, leucine, and methionine are
non-polar amino acids, aspartic acid is a negatively-charged amino
acid, and glycine is an uncharged amino acid, all possessing side
chains smaller than that of arginine. All of these amino acids,
with the exception of aspartic acid, differ significantly (.+-.2
units) from arginine using the hydropathic and hydrophilicity
indices described above.
[0387] The strain harboring the cry1C-R148D gene was designated
EG11832. The nucleotide sequence of the cry1C-R148D gene is shown
in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID
NO:4. The nucleotide substitutions C442G, G443A, and A444C yield
the codon GAC, encoding aspartic acid. The Cry1C-R148D mutant
EG11832 exhibits a 6.5-fold lower LC.sub.50 and a .about.8-fold
lower LC.sub.95 in bioassay against S. exigua when compared to the
wild-type Cry1C strain.
5.6 Example 6
Summary of Cry1C* Mutants
[0388] The cry1C mutants of the present invention are summarized in
Table 11.
13TABLE 11 SUMMARY OF CRY1C* STRAINS Cry1C Designation Strain
Plasmid Name Parental Plasmid Cry1C.563 EG11740 pEG370 pEG916
Cry1C.579 EG11746 pEG373 pEG916 Cry1C.499 EG11747 pEG374 pEG916
Cry1C R148A EG11811 pEG1635 pEG315 Cry1C R180A EG11815 pEG1636
pEG315 Cry1C R148A EG11822 pEG1639 pEG345 Cry1C R148D EG11832
pEG1642 pEG345 Cry1C R148G EG11833 pEG1643 pEG345 Cry1C R148L
EG11834 pEG1644 pEG345 Cry1C-R148A-K219A EG12111 pEG1639 pEG1639
Cry1C-R148D-K219A EG12121 pEG943 pEG1642 Cry1C R148M EG11835
pEG1645 pEG345
5.7 Example 7
Construction of B. Thuringiensis Strains Containing Multiple Cry
Genes in Addition to Cry1C and Cry1C R148A
[0389] The B. thuringiensis host strain EG4923-4 may be used as a
host strain for the native and mutant cry1C genes of the present
invention. Strain EG4923-4 contains three cry1Ac genes and one
cry2A gene on native plasmids and exhibits excellent insecticidal
activity against a variety of lepidopteran pests. Recombinant
plasmids containing the cry1C and cry1C-R148A crystal protein
genes, originally derived from aizawai strain 7.29, were introduced
into the strain EG4923-4 background using the electroporation 15
procedure described by Mettus and Macaluso (1990). The recombinant
plasmids containing cry1C and cry1C-R148A were designated pEG348
(FIG. 7) and pEG1641 (FIG. 8), respectively, and were similar in
structure to the cry1 plasmids described in U.S. Pat. No. 5,441,884
(specifically incorporated herein by reference).
[0390] Strain EG4923-4 transformants containing plasmids pEG348 and
pEG1641 were isolated on Luria plates containing 10 .mu.g/ml
tetracycline. Recombinant plasmid DNAs from the transformants were
isolated by the alkaline lysis procedure described by Baum (1995)
and confirmed by restriction enzyme analysis. The plasmid arrays of
the transformants were further confirmed by the Eckhardt agarose
gel analysis procedure described by Gonzalez Jr. et al., (1982).
The EG4923-4 recombinant derivatives were designated
EG4923-4/pEG348 and EG4923-4/pEG1641.
5.8 Example 8
Modification of EG4923-4/pEG348 and EG4923-4/pEG1641 to Remove
Foreign DNA Elements
[0391] pEG348 and pEG1641 contain duplicate copies of a
site-specific recombination site or internal resolution site (IRS)
that serves as a substrate for an in vivo site-specific
recombination reaction mediated by the TnpI recombinase of
transposon Tn5401 (described in Baum, 1995). This site-specific
recombination reaction, described in U.S. Pat. No. 5,441,884,
results in the deletion of non-B. thuringiensis DNA or foreign DNA
elements from the crystal protein-encoding recombinant plasmids.
The resulting recombinant B. thuringiensis strains are free of
foreign DNA elements, a desirable feature for genetically
engineered strains destined for use as bioinsecticides for spray-on
application. Strains EG4923-4/pEG348 and EG4923-4/pEG1641 were
modified using this in vivo site-specific recombination (SSR)
system to generate two new strains (Table 12), designated EG7841-1
(alias EG11730) and EG7841-2 (alias EG11831). The recombinant
plasmids in strains EG7841-1 and EG7841-2 were designated
pEG348.DELTA. and pEG1641.DELTA., respectively.
14TABLE 12 RECOMBINANT B. THURINGIENSIS STRAINS Strain Alias
Recombinant plasmid Progenitor strain EG7841-1 EG11730
pEG348.DELTA. EG4923-4/pEG348 EG7841-2 EG11831 pEG1641.DELTA.
EG4923-4/pEG1641
Example 9
Cry1C Combinational Mutants at AA Positions 148 and 219
[0392] The cry1C-R148A gene on pEG1639 and the cry1C-R148D gene on
pEG1642 were used as templates for additional mutagenesis studies
aimed at achieving further improvements in insecticidal
activity.
[0393] In one example, the lysine residue at position 219 (K219)
was replaced with an alanine residue, using the PCR.TM.-based
mutagenesis protocol described by Michael (1994) and the mutagenic
oligonucleotide primer J:
[0394] Primer J: (SEQ ID NO:62)
[0395] 5'-CGGGGATTAAATAATTTACCGGCTAGCACGTATCAAGATTGGATAAC-3'
[0396] Primer J also incorporates a unique NheI site (underlined
above) that can be used to distinguish the original gene from the
mutant gene by restriction enzyme analysis. The PCR.TM.-mediated
mutagenesis reactions employed the flanking primers H (SEQ ID
NO:52) and F (SEQ ID NO:20), the mutagenic oligonucleotide primer J
(SEQ ID NO:62), and pEG1639 (cry1C-R148A) as a template. In these
reactions, 5 units of Taq Extender.TM. (Stratagene) were included
to improve the efficiency of amplification with Taq polymerase. The
amplified products from the mutagenesis reaction were resolved by
agarose gel electrophoresis and the amplified DNA fragment
incorporating the mutagenic oligonucleotide primer J was excised
from the gel and purified using the Geneclean II.RTM. procedure.
This DNA fragment was cleaved with the restriction endonucleases
BbuI and AgeI.
[0397] In order to subclone the BbuI-AgeI cry1C restriction
fragment and express the mutant cry1C gene in B. thuringiensis, the
cry1C plasmid pEG345 (FIG. 3) was cleaved with BbuI and AgeI,
treated with calf intestinal alkaline phosphatase (Boehringer
Mannheim Corp.), and the resulting DNA fragments resolved by
agarose gel electrophoresis. The larger vector fragment was excised
from the gel and purified using the Geneclean II.RTM. procedure.
The pEG345 vector fragment was subsequently ligated to the
amplified cry1C fragment recovered from the mutagenesis reaction
and the ligation products used to transform E. coli Sure.TM. cells
(Stratagene) to ampicillin resistance using electroporation.
Individual colonies recovered from Luria plates containing 50
.mu.g/ml ampicillin were isolated and inoculated into 3 ml cultures
containing 1.times. brain heart infusion, 0.5% glycerol (BHIG), and
50 .mu.g/ml ampicillin.
[0398] Plasmid DNAs were prepared from the broth cultures using the
alkaline lysis method, digested with the restriction enzyme NheI,
and resolved by agarose gel electrophoresis to distinguish clones
incorporating the mutagenic sequence of primer J and therefore
encoding the alanine substitution at position 219. Incorporation of
the mutant sequence into cry1C-R148A was confirmed by DNA sequence
analysis. Plasmid DNAs from four recombinant E. coli clones were
used to transform the acrystalliferous B. thuringiensis strain
EG10368 to chloramphenicol resistance using electroporation.
Transfer of the recombinant plasmid to EG10368 was confirmed by
restriction enzyme analysis of plasmid DNAs recovered from the
EG10368 transformants. One chloramphenicol resistant colony was
selected and designated EG 12111. The cry1C gene in EG12111 was
designated cry1C-R148A K219A (SEQ ID NO:58) and the encoded crystal
protein designated Cry1C-R148A K219A (SEQ ID NO:59).
[0399] The same substitution was made in Cry1C-R148D using the same
procedures but using pEG1642 (cry1C-R148D) as the template for the
PCR.TM.-mediated mutagenesis reaction. The ligation products were
used to transform E. coli DH5.alpha. cells to ampicillin resistance
using standard transformation procedures. Plasmid DNAs were
prepared from broth cultures of selected ampicillin resistant
clones using the alkaline lysis method, digested with the
restriction enzyme NheI, and resolved by agarose gel
electrophoresis to distinguish clones incorporating the mutagenic
sequence of primer J and therefore encoding the alanine
substitution at position 219. Incorporation of the mutant sequence
into cry1C-R148D was confirmed by DNA sequence analysis.
Recombinant plasmids from three mutant clones were used to
transform the acrystalliferous B. thuringiensis strain EG10368 to
chloramphenicol resistance using electroporation. Transfer of the
recombinant plasmid to EG10368 was confirmed by restriction enzyme
analysis of plasmid DNAs recovered from the EG10368 transformants.
One chloramphenicol resistant colony was selected and designated
EG12121. The cry1C gene in EG12121 was designated cry1C-R148D K219A
(SEQ ID NO:60) and the encoded crystal protein designated
Cry1C-R148D K219A (SEQ ID NO:61). The recombinant cry1C plasmid in
EG12121 was designated pEG943 (FIG. 9).
[0400] Strains EG12115 (Cry1C wild-type), EG11822 (Cry1C-R148A),
EG12111 (Cry1C-R148A K219A), EG11832 (Cry1C-R148D), and EG12121
(Cry1C-R148D K219A) were grown in C2 medium as described in Example
4. The spore-Cry1C crystal suspensions recovered from the spent C2
cultures were used for bioassay evaluation against neonate larvae
of Spodoptera exigua and Trichoplusia ni as described in Example 4.
In two sets of replicated eight-dose bioassays against S. exigua,
the EG12111 and EG12121 Cry1C proteins were indistinguishable from
the EG11822 and EG11832 Cry1C proteins, respectively. In bioassays
against T. ni, however, further improvements in toxicity were
observed for the combinatorial mutants (Tables 12 and 13).
15TABLE 13 BIOASSAY EVALUATION OF THE COMBINATORIAL MUTANT
CRY1C-R148A K219A AGAINST NEONATE LARVAE OF TRICHOPLUSIA NI Strain
Toxin LC.sub.50.sup.1 (95% C. I).sup.2 EG12115 Cry1C 52 (32-97)
EG11822 Cry1C-R148A 24 (21-29) EG12111 Cry1C-R148A K219A 18 (16-21)
.sup.1Concentration of Cry1C protein that causes 50% mortality
expressed in ng crystal protein per 175 mm.sup.2 well. .sup.295%
confidence intervals.
[0401]
16TABLE 14 BIOASSAY EVALUATION OF THE COMBINATORIAL MUTANT
CRY1C-R148D K219A AGAINST NEONATE LARVAE OF TRICHOPLUSIA NI Strain
Toxin LC.sub.50.sup.1 (95% C. I).sup.2 EG12115 Cry1C 40 (34-48)
EG11832 Cry1C-R148D 35 (29-43) EG12121 Cry1C-R148D K219A 23 (19-28)
.sup.1Concentration of Cry1C protein that causes 50% mortality
expressed in ng crystal protein per 175 mm.sup.2 well. .sup.295%
confidence intervals.
Example 10
Cry1C-R148D Combinational Mutants Containing Other Substitutions in
Loop .alpha.6-7
[0402] Additional combinatorial mutants were constructed using
cry1C-R148D K219A, contained on pEG943, as a template for
PCR.TM.-mediated mutagenesis. A modification of the overlap
extension PCR.TM. procedure (Horton et al., 1989) was used to
generate these combinatorial mutants (FIG. 10). Briefly, a PCR.TM.
was performed using pEG943 as a template and the opposing primers H
(SEQ ID NO:52) and F (SEQ ID NO:20). The amplified DNA fragment
contained the R148D mutation as well as the unique NheI restriction
site marking the nucleotide substitutions encoding the K219A
mutation in loop .alpha.6-7. This PCR was performed using Taq
polymerase and Taq Extender.TM. and following the protocol
recommended by Stratagene. A second DNA fragment was amplified by
the PCR.TM. using pEG943 as a template and the mutagenic
oligonucleotide primer K (SEQ ID NO:63) and the opposing primer L
(SEQ ID NO:64). In this instance, the PCR.TM. was performed using
the thermostable polymerase Deep Vent.TM. and following the
protocol recommended by New England Biolabs, Inc.
[0403] Primer K: (SEQ ID NO:63)
[0404] 5'-CGGGGATTAAATAATTTACCGAAANNAACGTATCAAGATTGGATAAC-3'
[0405] N (25)=50% C; 50% G
[0406] N (26)=33.3% C; 33.3% G, 33.3% A
[0407] Primer L: (SEQ ID NO:64)
[0408] 5'-GGATAGCACTCATCAAAGGTACC-3'
[0409] The mutagenic primer K incorporated mutations in the codon
for serine (S) at position 220 of Cry1C. Six different amino acid
substitutions are predicted from the mutagenesis procedure:
arginine (R), alanine (A), glutamic acid (E), glutamine (Q),
glycine (G), and proline (P). The mutagenic primer K also
eliminates the unique NheI site in pEG943 and restores the lysine
residue at position 219. Thus, cry1C clones incorporating this
primer and containing substitutions at S220 can be distinguished
from the template cry1C-R148A K219A gene by the loss of the NheI
site.
[0410] The amplified DNA fragments were purified following agarose
gel electrophoresis using the Geneclean II.RTM. procedure. To
perform the overlap extension PCR.TM., approximately equimolar
amounts of the two DNA fragments were mixed together and amplified
using the flanking primers H (SEQ ID NO:52) and L (SEQ ID NO:64).
Annealing of complementary strands from the two DNA fragments
allows for extension from their 3' ends (FIG. 10). Fully extended
strands can then serve as templates for amplification using the
flanking primers. The resulting amplified DNA fragment was purified
following agarose gel electrophoresis using the Geneclean II.RTM.
procedure and digested with the restriction endonucleases BbuI and
AgeI. The BbuI-AgeI restriction fragment containing the 5' portion
of the cry1C gene was purified following agarose gel
electrophoresis using the Geneclean II.RTM. procedure. In order to
subclone this restriction fragment and express the mutant cry1C
genes in B. thuringiensis, the cry1C plasmid, pEG943, (FIG. 9) was
cleaved with BbuI, NheI, and AgeI, treated with calf intestinal
alkaline phosphatase, and the resulting DNA fragments resolved by
agarose gel electrophoresis. The vector fragment was excised from
the gel and purified using the Geneclean II.RTM. procedure. The
pEG943 vector fragment was subsequently ligated to the amplified
cry1C fragments recovered from the overlap extension PCR.TM. and
the ligation products used to transform E. coli Sure.TM. cells
(Stratagene) to ampicillin resistance using electroporation.
Several hundred ampicillin resistant colonies were harvested from
Luria plates containing 50 .mu.g/ml ampicillin, suspended in 10 ml
of Luria broth containing 50 .mu.g/ml ampicillin, and allowed to
grow at 37.degree. C. for 1 hour with agitation. Recombinant
plasmids from the culture were isolated using the alkaline lysis
procedure.
[0411] Approximately 0.1-1.0 microgram of the cry1C plasmid
preparation was digested with NheI to linearize plasmid molecules
harboring the NheI site of pEG943. The plasmid preparation was then
used to transform the acrystalliferous B. thuringiensis strain
EG10650 to chloramphenicol resistance using electroporation.
Because linear DNAs do not transform B. thuringiensis efficiently,
this NheI cleavage step ensures that virtually all of the clones
recovered from the transformation encode substitutions at position
220 and lysine at position 219. Individual chloramphenicol
resistant colonies were transferred to starch agar or Luria plates
containing 3 .mu.g/ml chloramphenicol. To confirm transfer of the
cry1C plasmids to EG10650, individual clones were inoculated into 3
ml of BHIG containing 3 .mu.g/ml chloramphenicol and grown at
30.degree. C. until the cultures were turbid. Plasmid DNAs were
isolated from the broth cultures using the alkaline lysis method
and the plasmid identities confirmed by restriction enzyme
analysis. Cry1C-R148D mutants containing substitutions at S220 were
designated Cry1C pr66-1, -2, -3, etc.
[0412] Amino acid substitutions were also generated at amino acid
positions 217, 218, 219, 221, and 222 in Cry1C using this procedure
and the following mutagenic oligonucleotide primers:
[0413] Position 217: Primer M (SEQ ID NO:65)
[0414] 5'-CGGGGATTAAATAATNNACCGAAAAGCACGTATCAAGATTGGATAAC-3'
[0415] N (16)=50% C; 50% G
[0416] N (17)=33.3% C; 33.3% G; 33.3% A
[0417] Position 218: Primer N (SEQ ID NO:66)
[0418] 5'-CGGGGATTAAATAATTTANNAAAAAGCACGTATCAAGATTGGATAAC-3'
[0419] N(19)=50% C; 50% G
[0420] N (20)=33.3% C; 33.3% G; 33.3% A
[0421] Position 219: Primer O (SEQ ID NO:67)
[0422] 5'-CGGGGATTAAATAATTTACCGNNAAGCACGTATCAAGATTGGATAAC-3'
[0423] N(22)=50% C; 50% G
[0424] N (23)=33.3% C; 33.3% G; 33.3% A
[0425] Position 221: Primer P (SEQ ID NO:68)
[0426]
5'-GGATTAAATAATTTACCGAAAAGCNNATATCAAGATTGGATAACATATAATCG-3'
[0427] N(25)=50% C;50% G
[0428] N (26)=33.3% C; 33.3% G; 33.3% A
[0429] Position 222: Primer Q (SEQ ID NO:69)
[0430]
5'-GGATTAAATAATTTACCGAAAAGCACGNNACAAGATTGGATAACATATAATCG-3'
[0431] N (28)=50% C; 50% G
[0432] N (29)=33.3% C; 33.3% G.33.3% A
[0433] Table 15 lists the Cry1C mutants expected from the
mutagenesis procedure.
17TABLE 15 SUMMARY OF CRY1C-R148D LOOP .alpha.6-7 MUTANTS Amino
acid Wild-type Predicted amino acid Position amino acid Primer
substitutions Mutant designation 217 leucine M R, E, Q, A, G, P
Cry1C pr67 -1, -2, -3, etc. 218 proline N R, E, Q, A, G, P Cry1C
pr65 -1, -2, -3, etc. 219 lysine O R, E, Q, A, G, P Cry1C pr70 -1,
-2, -3, etc. 221 threonine P R, E, Q, A, G, P Cry1C pr68 -1, -2,
-3, etc. 222 tyrosine Q R, E, Q, A, G, P Cry1C pr69 -1, -2, -3,
etc.
Example 11
Cry1C-R148D Loop .alpha.5-6 Combinational Mutants
[0434] A similar overlap extension PCR.TM. procedure was used to
generate Cry1C R148D mutants containing amino acid substitutions in
loop .alpha.5-6, including amino acid positions 178-184. The
mutagenic oligonucleotide primers used to generate mutations
encoding substitutions in loop .alpha.5-6 are listed below.
[0435] Position 178: Primer R (SEQ ID NO:70)
[0436]
5'-GATTCTGTAATTTTTNNAGAAAGATGGGGATTGACAACGATAAATGTCAATG-3'
[0437] N(16)=50% C;50% G
[0438] N (17)=33.3% C; 33.3% G; 33.3% A
[0439] Position 179: Primer S (SEQ ID. NO:71)
[0440]
5'-GATTCTGTAATTTTTGGANNAAGATGGGGATTGACAACGATAAATGTCAATG-3'
[0441] N (19)=50% C; 50% G
[0442] N (20)=33.3% C; 33.3% G0 33.3% A
[0443] Position 180: Primer T (SEQ ID NO: 72)
[0444]
5'-GATTCTGTAATTTTTGGAGAANNATGGGGATTGACAACGATAAATGTCAATG-3'
[0445] N(22)=50% C;50% G
[0446] N (23)=33.3% C; 33.3% G; 33.3% A
[0447] Position 181: Primer U (SEQ ID NO:73)
[0448]
5'-TCTGTAATTTTTGGAGAAAGANNAGGATTGACAACGATAAATGTCAATGAAAAC-3'
[0449] N (22)=50% C; 50% G
[0450] N (23)=33.3% C; 33.3% G; 33.3% A
[0451] Position 182: Primer V (SEQ ID NO:74)
[0452] 5'-TAATTTTTGGAGAAAGATGGNNATTGACAACGATAAATGTCAATGAAAAC-3'
[0453] N (22)=50% C; 50% G
[0454] N (23)=25% C; 25% G; 25% A; 25% T
[0455] Position 183: Primer W (SEQ ID NO:75)
[0456]
5'-GTAATTTTTGGAGAAAGATGGGGANNAACAACGATAAATGTCAATGAAAAC-3'
[0457] N (25)=50% C; 50% G
[0458] N (26)=25% C; 25% G; 25% A; 25% T
[0459] Position 184: Primer X (SEQ ID NO:76)
[0460]
5'-GTAATTTTTGGAGAAAGATGGGGATTGNNAACGATAAATGTCAATGAAAAC-3'
[0461] N (28)=50% C; 50% G
[0462] N (29)=25% C; 25% G; 25% A; 25% T
[0463] A PCR.TM. using the opposing primers H (SEQ ID NO:52) and F
(SEQ ID NO:20) and plasmid pEG943 as a template was first performed
to generate a DNA fragment containing the R148D and K219A mutations
as well as the unique NheI restriction site marking the K219A
mutation (FIG. 10). In order to generate cry1C fragments harboring
loop .alpha.5-6 mutations, PCRs were run using a mutagenic primer
(e.g., primer R) and the opposing primer L (SEQ ID NO:64) (FIG.
11). The amplified DNA fragments were purified following agarose
gel electrophoresis using the Geneclean II.RTM. procedure. For the
overlap extension PCR.TM., approximately equimolar amounts of the
two DNA fragments were mixed and amplified using the flanking
primers H (SEQ ID NO:52) and L (SEQ ID NO:64). The amplification
products were digested with the restriction enzymes BbuI and AgeI,
the resulting BbuI-AgeI cry1C fragments subcloned into a cry1C
expression vector, and the B. thuringiensis EG10650 transformants
constructed as described in Example 10. Table 16 summarizes the
Cry1C mutants predicted from the mutagenesis procedure.
18TABLE 16 SUMMARY OF CRY1C-R148D LOOP .alpha.5-6 MUTANTS Amino
Acid Wild-type Predicted Amino Acid Position Amino Acid Primer
Substitutions Mutant Designation 178 glycine R R, E, Q, A, G, P
Cry1C 1 -1, -2, -3, etc. 179 glutamic acid S R, E, Q, A, G, P Cry1C
2 -1, -2, -3, etc. 180 arginine T R, E, Q, A, G, P Cry1C 3 -1, -2,
-3, etc. 181 tryptophan U R, E, Q, A, G, P Cry1C 4 -1, -2, -3, etc.
182 glycine V R, E, Q, A, G, P, L, V Cry1C 5 -1, -2, -3, etc. 183
leucine W R, E, Q, A, G, P, L, V Cry1C 6 -1, -2, -3, etc. 184
threonine X R, E, Q, A, G, P, L, V Cry1C 7 -1, -2, -3, etc.
Example 12
Bioassay Evaluation of Cry1C-R148D Combinational Mutants
[0464] EG10650 transformants containing mutant cry1C genes were
grown in C2 medium, the spore-crystal protein suspensions
recovered, and one-dose bioassays performed against neonate larvae
of S. exigua and T. ni as described in Example 4. Strain EG11832
(Cry1C-R148D) was used as the control strain in these bioassays.
Dilutions of the spore-crystal suspensions were typically adjusted
to obtain 20-40% mortality with strain EG11832. Replicated one-dose
screens of the Cry1C-R148D combinatorial mutants identified several
mutants with increased mortality. Sixteen of these mutants were
grown again in C2 medium and their Cry1C crystal proteins
quantified as described in Example 4. One-dose bioassays were
performed against S. exigua using 50 ng Cry1C protein per diet
well. One dose bioassays were performed against T. ni using 25 ng
Cry1C protein per diet well. The results of those bioassays are
shown in Table 17. Triplicate samples of the control strain EG11832
(Cry1C-R148D) were also tested. Several Cry1C-R148D combinatorial
mutants show increased (approximately two-fold) toxicity towards S.
exigua when compared to EG11832 (Cry1C-R148D). Several of these
mutants, including Cry1C 7-3, Cry1C 66-19, and Cry1C 69-24 also
showed excellent toxicity towards T. ni.
19TABLE 17 TOXICITY OF CRY1C R148D COMBINATORIAL MUTANTS TOWARDS
TRICHOPLUSIA NI AND SPODOPTERA EXIGUA T. ni S. exigua Mutant %
mortality.sup.1 % mortality.sup.2 1C 2-7 53.1 11.29 1C 2-17 12.5
4.84 1C 3-13 51.6 29.03 1C 5-1 28.1 17.74 1C 5-3 57.8 17.74 1C 5-5
54.7 25.81 1C 6-21 14.1 19.35 1C 7-3 81.2 32.26 1C 7-16 48.44 14.52
1C 7-21 50 12.9 1C 66-14 37.5 16.13 1C 66-19 60.9 35.48 1C 66-21
78.1 29.03 1C 69-9 68.7 20.97 1C 69-15 62.5 24.19 1C 69-24 71.88
40.32 11832 #1 (Cry1C-R148D) 53 16.13 11832 #2 (Cry1C-R148D) 50
20.97 11832 #3 (Cry1C-R148D) 51.6 17.74 .sup.1Percent mortality
obtained using 25 ng Cry1C protein per 175 mm.sup.2 diet well, 64
larvae per assay. .sup.2Percent mortality obtained using 50 ng
Cry1C protein per 175 mm.sup.2 diet well, 64 larvae per assay.
5.13 Example 13
Amino Acid Sequences of the Modified Crystal Proteins
[0465]
20 5.13.1 AMINO ACID SEQUENCE OF CRY1C-R148A (SEQ ID NO:2) Met Glu
Glu Asn Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu
Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp
Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn Phe Val Pro Gly Gly
Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro Ser
Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Glu Arg Ile
Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly Leu Gly Asn
Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn
Asn Pro Ala Thr Arg Thr Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly
Leu Leu Glu Arg Asp Ile Pro Ser Phe Ala Ile Ser Gly Phe Glu Val Pro
Leu Leu Ser Val Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg
Asp Ser Val Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn
Glu Asn Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys
Ala Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln
Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu
Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln
Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe
Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met Glu
Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn Leu Thr
Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His
Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile Tyr
Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro
Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gln Gln Pro Trp
Pro Ala Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr
Pro Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr
Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His
Arg Leu Cys His Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr
Thr Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr
Ile Asp Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val
Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile
Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn
Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg
Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly
Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu Asn
Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe
Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu Phe Gly Ala
Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu
Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala
Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val
Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp
Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala
Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly
Ile Asn Arg Gln Pro Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile
Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr
Val Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys
Leu Lys Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln
Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val Asn
Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly
Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp
Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His
Phe Thr Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly
Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly
Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala Arg
Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Gln Leu
Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe
Val Asn Ser Gln Tyr Asp Arg Leu Gln Val Asp Thr Asn Ile Ala Met Ile
His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu
Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly
Arg Ile Phe Thr Ala Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn
Gly Asp Phe Asn Asn Gly Leu Leu Cys Trp Asn Val Lys Gly His Val Asp
Val Glu Glu Gln Asn Asn His Arg Ser Val Leu Val Ile Pro Glu Trp Glu
Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu
Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His
Glu Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu
Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asn Tyr Thr Gly Thr Gln
Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Gln Gly Tyr Asp Glu Ala
Tyr Gly Asn Asn Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu
Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly
Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu
Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly
Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu 5.13.2
AMINO ACID SEQUENCE OF CRY1C-R148D (SEQ ID NO:4) Met Glu Glu Asn
Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val
Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile Ser
Leu Ser Leu Val Gln Phe Leu Val Ser Asn Phe Val Pro Gly Gly Gly Phe
Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro Ser Gln Trp
Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu
Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe
Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro
Ala Thr Arg Thr Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu
Glu Arg Asp Ile Pro Ser Phe Asp Ile Ser Gly Phe Glu Val Pro Leu Leu
Ser Val Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser
Val Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn
Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn
Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp Trp
Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile
Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln Pro Val
Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro
Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met Glu Ser Ser
Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile Phe
Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His Arg Val
Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile Tyr Gly Arg
Glu Ala Asn Gln Glu Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe
Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala
Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr
Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu
Pro Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu
Cys His Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly
Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp
Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly
Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg
Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser Pro
Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala
Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gln Val
Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu Asn Leu Thr
Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala
Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly Ser
Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp
Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val Asn
Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val Thr Asp
Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe
Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg
Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn
Arg Gln Pro Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly
Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Val Asp
Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys
Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu
Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val Asn Val Pro
Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys
Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp
Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Thr
Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp
Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu
Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala Arg Val Lys
Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Gln Leu Glu Thr
Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn
Ser Gln Tyr Asp Arg Leu Gln Val Asp Thr Asn Ile Ala Met Ile His Ala
Ala Asp Lys Arg Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser
Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile
Phe Thr Ala Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp
Phe Asn Asn Gly Leu Leu Cys Trp Asn Val Lys Gly His Val Asp Val Glu
Glu Gln Asn Asn His Arg Ser Val Leu Val Ile Pro Glu Trp Glu Ala Glu
Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val
Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile
Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val
Tyr Pro Asn Asn Thr Val Thr Cys Asn Asn Tyr Thr Gly Thr Gln Glu Glu
Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr Gly
Asn Asn Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser
Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly
Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu Tyr Phe
Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe
Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu 5.13.3 AMINO ACID
SEQUENCE OF CRY1C-R180A (SEQ ID NO:6) Met Glu Glu Asn Asn Gln Asn
Gln Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val Leu Leu Asp
Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu
Val Gln Phe Leu Val Ser Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly
Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe
Leu Val Gln Ile Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg
Asn Ala Ala Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr
Val Glu Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Ala Thr Arg
Thr Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp
Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr
Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe
Gly Glu Ala Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg
Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn
Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp Trp Ile Thr Tyr
Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile Ala Ala Phe
Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln Pro Val Gly Gln Leu
Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro Gln Leu Gln
Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met Glu Ser Ser Ala Ile Arg
Asn Pro His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp
Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser
Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn
Gln Glu Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu
Ser Asn Pro Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe
Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe
Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu
Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala
Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe
Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg
Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr Ser
Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr
Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser Pro Ile Thr Gln
Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile
Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gln Val Ser Val Asn
Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr
Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp
Ile Ile Gly Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser
Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe
Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe
Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile
Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp
Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp
Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro
Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp Asp
Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Val Asp Glu Cys Tyr
Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr
Arg Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr
Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val Asn Val Pro Gly Thr Gly
Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro
Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys
Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Thr Leu Asp Ile
Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe
Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu
Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu
Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Gln Leu Glu Thr Asn Ile Val
Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr
Asp Arg Leu Gln Val Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys
Arg Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro
Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala
Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn
Gly Leu Leu Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn
Asn His Arg Ser Val Leu Val Ile Pro Glu Trp Glu Ala Glu Val Ser Gln
Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr
Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp Asn
Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr Pro Asn
Asn Thr Val Thr Cys Asn Asn Tyr Thr Gly Thr Gln Glu Glu Tyr Glu Gly
Thr Tyr Thr Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr Gly Asn Asn Pro
Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp
Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr
Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu Tyr Phe Pro Glu Thr
Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp
Ser Val Glu Leu Leu Leu Met Glu Glu 5.13.4 AMINO ACID SEQUENCE OF
CRY1C.563 (SEQ ID NO:8) Met Glu Glu Asn Asn Gln Asn Gln Cys Ile Pro
Tyr Asn Cys Leu Ser
Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser
Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn Phe Val
Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val
Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn
Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly
Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu Asp
Asp Pro His Asn Pro Thr Thr Arg Thr Arg Val Ile Asp Arg Phe Arg Ile
Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile Ser Gly Phe
Glu Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Ala Asn Leu His Leu Ala
Ile Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile
Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala
Asp His Cys Ala Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser
Thr Tyr Gln Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu
Thr Val Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr
Pro Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu
Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn
Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn
Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp
Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr Ser
Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg Ser Phe Thr Phe
Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gln
Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val Glu
Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp
Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly
Tyr Ser His Arg Leu Cys His Ala Thr Phe Val Gln Arg Ser Gly Thr Pro
Phe Leu Thr Thr Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu
Thr Asn Thr Ile Asp Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly
Phe Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly
Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val
Asn Ile Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala
Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly
Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile
Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro
Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu
Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu
Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala
Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys
Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys
Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val
Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn
Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp Arg Gly Ser Thr Asp
Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu
Pro Gly Thr Val Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp
Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu
Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu
Ile Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser
Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp
Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His
Ser His His Phe Thr Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu
Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala
Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala
Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys
Leu Gln Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp
Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Val Asp Thr Asn Ile
Ala Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu Ala Tyr
Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu
Leu Glu Gly Arg Ile Phe Thr Ala Tyr Ser Leu Tyr Asp Ala Arg Asn Val
Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Leu Cys Trp Asn Val Lys Gly
His Val Asp Val Glu Glu Gln Asn Asn His Arg Ser Val Leu Val Ile Pro
Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly
Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val
Thr Ile His Glu Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys
Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asn Tyr Thr
Gly Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Gln Gly Tyr
Asp Glu Ala Tyr Gly Asn Asn Pro Ser Val Pro Ala Asp Tyr Ala Ser Val
Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser
Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys
Asp Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu
Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu
5.13.5 AMINO ACID SEQUENCE OF CRY1C.579 (SEQ ID NO:10) Met Glu Glu
Asn Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu
Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile
Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn Phe Val Pro Gly Gly Gly
Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro Ser Gln
Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Glu Arg Ile Ala
Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn
Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu Val Asp Pro Asn Asn
Pro Gly Thr Arg Thr Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu
Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu
Leu Ser Val Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp
Ser Val Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu
Asn Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala
Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp
Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp
Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln Pro
Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe Asn
Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met Glu Ser
Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile
Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His Arg
Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile Tyr Gly
Arg Glu Ala Asn Gln Glu Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val
Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro
Ala Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro
Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu
Leu Pro Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg
Leu Cys His Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr
Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile
Asp Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp
Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu
Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser
Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp
Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gln
Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu Asn Leu
Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg
Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly
Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala
Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val
Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val Thr
Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu
Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys
Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile
Asn Arg Gln Pro Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln
Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Val
Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu
Lys Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp
Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val Asn Val
Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys
Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu
Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe
Thr Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val
Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn
Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala Arg Val
Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Gln Leu Glu
Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val
Asn Ser Gln Tyr Asp Arg Leu Gln Val Asp Thr Asn Ile Ala Met Ile His
Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu
Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg
Ile Phe Thr Ala Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly
Asp Phe Asn Asn Gly Leu Leu Cys Trp Asn Val Lys Gly His Val Asp Val
Glu Glu Gln Asn Asn His Arg Ser Val Leu Val Ile Pro Glu Trp Glu Ala
Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg
Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu
Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu
Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asn Tyr Thr Gly Thr Gln Glu
Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr
Gly Asn Asn Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys
Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr
Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu Tyr
Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr
Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu 5.13.6 AMINO
ACID SEQUENCE OF CRY1C.499 (SEQ ID NO:12) Met Glu Glu Asn Asn Gln
Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val Leu Leu
Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile Ser Leu Ser
Leu Val Gln Phe Leu Val Ser Asn Phe Val Pro Gly Gly Gly Phe Leu Val
Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala
Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala
Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile
Tyr Val Glu Ala Phe Lys Glu Trp Glu Glu Asp Pro His Asn Pro Ala Thr
Arg Thr Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg
Asp Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val
Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile
Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn
Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr
Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp Trp Ile Thr
Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile Ala Ala
Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln Pro Val Gly Gln
Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro Gln Leu
Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met Glu Ser Ser Ala Ile
Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile Phe Thr Asp
Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His Arg Val Ile Ser
Ser Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala
Asn Gln Glu Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr
Leu Ser Asn Pro Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro
Phe Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser
Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro
Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His
Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val
Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu
Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr
Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Asn
Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser Pro Ile Thr
Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val
Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gln Val Ser Val
Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg
Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro
Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser
Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr
Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu
Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His
Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu
Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser
Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln
Pro Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp
Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Val Asp Glu Cys
Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr
Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile
Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val Asn Val Pro Gly Thr
Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu
Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser
Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Thr Leu Asp
Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile
Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe
Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala Arg Val Lys Arg Ala
Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Gln Leu Glu Thr Asn Ile
Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln
Tyr Asp Arg Leu Gln Val Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp
Lys Arg Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile
Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr
Ala Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn
Asn Gly Leu Leu Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln
Asn Asn His Arg Ser Val Leu Val Ile Pro Glu Trp Glu Ala Glu Val Ser
Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala
Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp
Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr Pro
Asn Asn Thr Val Thr Cys Asn Asn Tyr Thr Gly Thr Gln Glu Glu Tyr Glu
Gly Thr Tyr Thr Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr Gly Asn Asn
Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr
Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr
Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu Tyr Phe Pro Glu
Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val
Asp Ser Val Glu Leu Leu Leu Met Glu Glu
5.14 Example 14
Nucleic Acid Sequences of the Genes Encoding Modified Cry1C*
Crystal Proteins
[0466]
21 5.14.1 NUCLEIC ACID SEQUENCE OF CRY1C-R148A (SEQ ID NO:1)
ATGGAGGAAAATAATCAAAATCAATGCATACCTTACAATTGTTTAAGTAATCCTGAAGAAGTA-
CTTTTGGAT GGAGAACGGATATCAACTGGTAATTCATCAATTGATATTTCTCTGTC-
ACTTGTTCAGTTTCTGGTATCTAAC TTTGTACCAGGGGGAGGATTTTTAGTTGGATT-
AATAGATTTTGTATGGGGAATAGTTGGCCCTTCTCAATGG
GATGCATTTCTAGTACAAATTGAACAATTAATTAATGAAAGAATAGCTGAATTTGCTAGGAATGCTGCTATT
GCTAATTTAGAAGGATTAGGAAACAATTTCAATATATATGTGGAAGCATTTAAAGAA-
TGGGAAGAAGATCCT AATAATCCAGCAACCAGGACCAGAGTAATTGATCGCTTTCGT-
ATACTTGATGGGCTACTTGAAAGGGACATT CCTTCGTTTGCAATTTCTGGATTTGAA-
GTACCCCTTTTATCCGTTTATGCTCAAGCGGCCAATCTGCATCTA
GCTATATTAAGAGATTCTGTAATTTTTGGAGAAAGATGGGGATTGACAACGATAAATGTCAATGAAAACTAT
AATAGACTAATTAGGCATATTGATGAATATGCTGATCACTGTGCAAATACGTATAAT-
CGGGGATTAAATAAT TTACCGAAATCTACGTATCAAGATTGGATAACATATAATCGA-
TTACGGAGAGACTTAACATTGACTGTATTA GATATCGCCGCTTTCTTTCCAAACTAT-
GACAATAGGAGATATCCAATTCAGCCAGTTGGTCAACTAACAAGG
GAAGTTTATACGGACCCATTAATTAATTTTAATCCACAGTTACAGTCTGTAGCTCAATTACCTACTTTTAAC
GTTATGGAGAGCAGCGCAATTAGAAATCCTCATTTATTTGATATATTGAATAATCTT-
ACAATCTTTACGGAT TGGTTTAGTGTTGGACGCAATTTTTATTGGGGAGGACATCGA-
GTAATATCTAGCCTTATAGGAGGTGGTAAC ATAACATCTCCTATATATGGAAGAGAG-
GCGAACCAGGAGCCTCCAAGATCCTTTACTTTTAATGGACCGGTA
TTTAGGACTTTATCAAATCCTACTTTACGATTATTACAGCAACCTTGGCCAGCGCCACCATTTAATTTACGT
GGTGTTGAAGGAGTAGAATTTTCTACACCTACAAATAGCTTTACGTATCGAGGAAGA-
GGTACGGTTGATTCT TTAACTGAATTACCGCCTGAGGATAATAGTGTGCCACCTCGC-
GAAGGATATAGTCATCGTTTATGTCATGCA ACTTTTGTTCAAAGATCTGGAACACCT-
TTTTTAACAACTGGTGTAGTATTTTCTTGGACGCATCGTAGTGCA
ACTCTTACAAATACAATTGATCCAGAGAGAATTAATCAAATACCTTTAGTGAAAGGATTTAGAGTTTGGGGG
GGCACCTCTGTCATTACAGGACCAGGATTTACAGGAGGGGATATCCTTCGAAGAAAT-
ACCTTTGGTGATTTT GTATCTCTACAAGTCAATATTAATTCACCAATTACCCAAAGA-
TACCGTTTAAGATTTCGTTACGCTTCCAGT AGGGATGCACGAGTTATAGTATTAACA-
GGAGCGGCATCCACAGGAGTGGGAGGCCAAGTTAGTGTAAATATG
CCTCTTCAGAAAACTATGGAAATAGGGGAGAACTTAACATCTAGAACATTTAGATATACCGATTTTAGTAAT
CCTTTTTCATTTAGAGCTAATCCAGATATAATTGGGATAAGTGAACAACCTCTATTT-
GGTGCAGGTTCTATT AGTAGCGGTGAACTTTATATAGATAAAATTGAAATTATTCTA-
GCAGATGCAACATTTGAAGCAGAATCTGAT TTAGAAAGAGCACAAAAGGCGGTGAAT-
GCCCTGTTTACTTCTTCCAATCAAATCGGGTTAAAAACCGATGTG
ACGGATTATCATATTGATCAAGTATCCAATTTAGTGGATTGTTTATCAGATGAATTTTGTCTGGATGAAAAG
CGAGAATTGTCCGAGAAAGTCAAACATGCGAAGCGACTCAGTGATGAGCGGAATTTA-
CTTCAAGATCCAAAC TTCAGAGGGATCAATAGACAACCAGACCGTGGCTGGAGAGGA-
AGTACAGATATTACCATCCAAGGAGGAGAT GACGTATTCAAAGAGAATTACGTCACA-
CTACCGGGTACCGTTGATGAGTGCTATCCAACGTATTTATATCAG
AAAATAGATGAGTCGAAATTAAAAGCTTATACCCGTTATGAATTAAGAGGGTATATCGAAGATAGTCAAGAC
TTAGAAATCTATTTGATCCGTTACAATGCAAAACACGAAATAGTAAATGTGCCAGGC-
ACGGGTTCCTTATGG CCGCTTTCAGCCCAAAGTCCAATCGGAAAGTGTGGAGAACCG-
AATCGATGCGCGCCACACCTTGAATGGAAT CCTGATCTAGATTGTTCCTGCAGAGAC-
GGGGAAAAATGTGCACATCATTCCCATCATTTCACCTTGGATATT
GATGTTGGATGTACAGACTTAAATGAGGACTTAGGTGTATGGGTGATATTCAAGATTAAGACGCAAGATGGC
CATGCAAGACTAGGGAATCTAGAGTTTCTCGAAGAGAAACCATTATTAGGGGAAGCA-
CTAGCTCGTGTGAAA AGAGCGGAGAAGAAGTGGAGAGACAAACGAGAGAAACTGCAG-
TTGGAAACAAATATTGTTTATAAAGAGGCA AAAGAATCTGTAGATGCTTTATTTGTA-
AACTCTCAATATGATAGATTACAAGTGGATACGAACATCGCAATG
ATTCATGCGGCAGATAAACGCGTTCATAGAATCCGGGAAGCGTATCTGCCAGAGTTGTCTGTGATTCCAGGT
GTCAATGCGGCCATTTTCGAAGAATTAGAGGGACGTATTTTTACAGCGTATTCCTTA-
TATGATGCGAGAAAT GTCATGAAAAATGGCGATTTCAATAATGGCTTATTATGCTGG-
AACGTGAAAGGTCATGTAGATGTAGAAGAG CAAAACAACCACCGTTCGGTCCTTGTT-
ATCCCAGAATGGGAGGCAGAAGTGTCACAAGAGGTTCGTGTCTGT
CCAGGTCGTGGCTATATCCTTCGTGTCACAGCATATAAAGAGGGATATGGAGAGGGCTGCGTAACGATCCAT
GAGATCGAAGACAATACAGACGAACTGAAATTCAGCAACTGTGTAGAAGAGGAAGTA-
TATCCAAACAACACA GTAACGTGTAATAATTATACTGGGACTCAAGAAGAATATGAG-
GGTACGTACACTTCTCGTAATCAAGGATAT GACGAAGCCTATGGTAATAACCCTTCC-
GTACCAGCTGATTACGCTTCAGTCTATGAAGAAAAATCGTATACA
GATGGACGAAGAGAGAATCCTTGTGAATCTAACAGAGGCTATGGGGATTACACACCACTACCGGCTGGTTAT
GTAACAAAGGATTTAGAGTACTTCCCAGAGACCGATAAGGTATGGATTGAGATCGGA-
GAAACAGAAGGAACA TTCATCGTGGATAGCGTGGAATTACTCCTTATGGAGGAA 5.14.2
NUCLEIC ACID SEQUENCE OF CRY1C-R148D (SEQ ID NO:3)
ATGGAGGAAAATAATCAAAATCAATGCATACCTTACAATTGTTTAAGTAATCCTGAAGA-
AGTACTTTTGGAT GGAGAACGGATATCAACTGGTAATTCATCAATTGATATTTCTC-
TGTCACTTGTTCAGTTTCTGGTATCTAAC TTTGTACCAGGGGGAGGATTTTTAGTTG-
GATTAATAGATTTTGTATGGGGAATAGTTGGCCCTTCTCAATGG
GATGCATTTCTAGTACAAATTGAACAATTAATTAATTGAAAGAATAGCTGATTTGCTAGGAATGCTGCTATT
GCTAATTTAGAAGGATTAGGAAACAATTTCAATATATATGTGGAAGCATTTAAAGAA-
TGGGAAGAAGATCCT AATAATCCAGCAACCAGGACCAGAGTTAATTCATCGTTTCGT-
ATACTTGATGGGCTACTTGAAAGGGACATT CCTTCGTTTCGAATTTCTGGATTTGAA-
GTACCCCTTTTATCCGTTTATGCTCAAGCGGCCAATCTGCATCTA
GCTATATTAAGAGATTCTGTAATTTTTGGAGAAGCATGGGGGTTGACAACGATAAATGTCAATGAAAACTAT
AATAGACTAATTAGGCATATTGATGAATATGCTGATCACTGTGCAAATACGTATAAT-
CGGGGATTAAATAAT TTACCGAAATCTACGTATCAAGATTGGATAACATATAATCGA-
TTACGGAGAGACTTAACATTGACTGTATTA GATATCGCCGCTTTCTTTCCAAACTAT-
GACAATAGGAGATATCCAATTCAGCCAGTTGGTCAACTAACAAGG
GAAGTTTATACGGACCCATTAATTAATTTTAATCCACAGTTACAGTCTGTAGCTCAATTACCTACTTTTAAC
GTTATGGAGAGCAGCGCAATTAGAAATCCTCATTTATTTGATATATTGAATAATCTT-
ACAATCTTTACGGAT TGGTTTAGTGTTGGACGCAATTTTTATTGGGGAGGACATCGA-
GTAATATCTAGCCTTATAGGAGGTGGTAAC ATAACATCTCCTATATATGGAAGAGAG-
GCGAACCAGGAGCCTCCAAGATCCTTTACTTTTAATGGACCGGTA
TTTAGGACTTTATCAAATCCTACTTTACGATTATTACAGCAACCTTGGCCAGCGCCACCATTTAATTTACGT
GGTGTTGAAGGAGTAGAATTTTCTACACCTACAAATAGCTTTACGTATCGAGGAAGA-
GGTACGGTTGATTCT TTAACTGAATTACCGCCTGAGGATAATAGTGTGCCACCTCGC-
GAAGGATATAGTCATCGTTTATGTCATGCA ACTTTTGTTCAAAGATCTGGAACACCT-
TTTTTAACAACTGGTGTAGTATTTTCTTGGACGCATCGTAGTGCA
ACTCTTACAAATACAATTGATCCAGAGAGAATTAATCAAATACCTTTAGTGAAAGGATTTAGAGTTTGGGGG
GGCACCTCTGTCATTACAGGACCAGGATTTACAGGAGGGGATATCCTTCGAAGAAAT-
ACCTTTGGTGATTTT GTATCTCTACAAGTCAATATTAATTCACCAATTACCCAAAGA-
TACCGTTTAAGATTTCGTTACGCTTCCAGT AGGGATGCACGAGTTATAGTATTAACA-
GGAGCGGCATCCACAGGAGTGGGAGGCCAAGTTAGTGTAAATATG
CCTCTTCAGAAAACTATGGAAATAGGGGAGAACTTAACATCTAGAACATTTAGATATACCGATTTTAGTAAT
CCTTTTTCATTTAGAGCTAATCCAGATATAATTGGGATAAGTGAACAACCTCTATTT-
GGTGCAGGTTCTATT AGTAGCGGTGAACTTTATATAGATAAATTGATAATTATTCTA-
GCAGATGCAACATTTGAAGCAGAATCTGAT TTAGAAAGAGCACAAAAGGCGGTGAAT-
GCCCTGTTTACTTCTTCCAATCAAATCGGGTTAAAAACCGATGTG
ACGGATTATCATATTGATCAAGTATCCAATTTAGTGGATTGTTTATCAGATGAATTTTGTCTGGATGAAAAG
CGAGAATTGTCCGAGAAAGTCAAACATGCGAAGCGACTCAGTGATGAGCGGAATTTA-
CTTCAAGATCCAAAC TTCAGAGGGATCAATAGACAACCAGACCGTGGCTGGAGAGGA-
AGTACAGATATTACCATCCAAGGAGGAGAT GACGTATTCAAAGAGAATTACGTCACA-
CTACCGGGTACCGTTGATGAGTGCTATCCAACGTATTTATATCAG
AAAATAGATGAGTCGAAATTAAAAGCTTATACCCGTTATGAATTAAGAGGGTATATCGAAGATAGTCAAGAC
TTAGAAATCTATTTGATCCGTTACAATGCAAAACACGAAATAGTAAATGTGCCAGGC-
ACGGGTTCCTTATGG CCGCTTTCAGCCCAAAGTCCAATCGGAAAGTGTGGAGAACCG-
AATCGATGCGCGCCACACCTTGAATGGAAT CCTGATCTAGATTGTTCCTGCAGAGAC-
GGGGAAAAATGTGCACATCATTCCCATCATTTCACCTTGGATATT
GATGTTGGATGTACAGACTTAAATGAGGACTTAGGTGTATGGGTGATATTCAAGATTAAGACGCAAGATGGC
CATGCAAGACTAGGGAATCTAGAGTTTCTCGAAGAGAAACCATTATTAGGGGAAGCA-
CTAGCTCGTGTGAAA AGAGCGGAGAAGAAGTGGAGAGACAAACGAGAGAAACTGCAG-
TTGGAAACAAATATTGTTTATAAAGAGGCA AAAGAATCTGTAGATGCTTTATTTGTA-
AACTCTCAATATGATAGATTACAAGTGGATACGAACATCGCAATG
ATTCATGCGGCAGATAAACGCGTTCATAGAATCCGGGAAGCGTATCTGCCAGAGTTGTCTGTGATTCCAGGT
GTCAATGCGGCCATTTTCGAAGAATTAGAGGGACGTATTTTTACAGCGTATTCCTTA-
TATGATGCGAGAAAT GTCATTAAAAATGGCGATTTCAATAATGGCTTATTATGCTGG-
AACGTGAAAGGTCATGTAGATGTAGAAGAG CAAAACAACCACCGTTCGGTCCTTGTT-
ATCCCAGAATGGGAGGCAGAAGTGTCACAAGAGGTTCGTGTCTGT
CCAGGTCGTGGCTATATCCTTCGTGTCACAGCATATAAAGAGGGATATGGAGAGGGCTGCGTAACGATCCAT
GAGATCGAAGACAATACAGACGAACTGAAATTCAGCAACTGTGTAGAAGAGGAAGTA-
TATCCAAACAACACA GTAACGTGTAATAATTATACTGGGACTCAAGAAGAATATGAG-
GGTACGTACACTTCTCGTAATCAAGGATAT GACGAAGCCTATGGTAATAACCCTTCC-
GTACCAGCTGATTACGCTTCAGTCTATGAAGAAAAATCGTATACA
GATGGACGAAGAGAGAATCCTTGTGAATCTAACAGAGGCTATGGGGATTACACACCACTACCGGCTGGTTAT
GTAACAAAGGATTTAGAGTACTTCCCAGAGACCGATAAGGTATGGATTGAGATCGGA-
GAAACAGAAGGAACA TTCATCGTGGATAGCGTGGAATTACTCCTTATGGAGGAA 5.14.3
NUCLEIC ACID SEQUENCE OF CRY1C-R180A (SEQ ID NO:5)
ATGGAGGAAAATAATCAAAATCAATGCATACCTTACAATTGTTTAAGTAATCCTGAAGA-
AGTACTTTTGGAT GGAGAACGGATATCAACTGGTAATTCATCAATTGATATTTCTC-
TGTCACTTGTTCAGTTTCTGGTATCTAAC TTTGTACCAGGGGGAGGATTTTTAGTTG-
GATTAATAGATTTTGTATGGGGAATAGTTGGCCCTTCTCAATGG
GATGCATTTCTAGTACAAATTGAACAATTAATTAATGAAAGAATAGCTGAATTTGCTAGGAATGCTGCTATT
GCTAATTTAGAAGGATTAGGAAACAATTTCAATATATATGTGGAAGCATTTAAAGAA-
TGGGAAGAAGATCCT AATAATCCAGCAACCAGGACCAGAGTAATTGATCGCTTTCGT-
ATACTTGATGGGCTACTTGAAAGGGACATT CCTTCGTTTCGAATTTCTGGATTTGAA-
GTACCCCTTTTATCCGTTTATGCTCAAGCGGCCAATCTGCATCTA
GCTATATTAAGAGATTCTGTAATTTTTGGAGAAGCATGGGGGTTGACAACGATAAATGTCAATGAAAACTAT
AATAGACTAATTAGGCATATTGATGAATATGCTGATCACTGTGCAAATACGTATAAT-
CGGGGATTAAATAAT TTACCGAAATCTACGTATCAAGATTGGATAACATATAATCGA-
TTACGGAGAGACTTAACATTGACTGTATTA GATATCGCCGCTTTCTTTCCAAACTAT-
GACAATAGGAGATATCCAATTCAGCCAGTTGGTCAACTAACAAGG
GAAGTTTATACGGACCCATTAATTAATTTTAATCCACAGTTACAGTCTGTAGCTCAATTACCTACTTTTAAC
GTTATGGAGAGCAGCGCAATTAGAAATCCTCATTTATTTGATATATTGAATAATCTT-
ACAATCTTTACGGAT TGGTTTAGTGTTGGACGCAATTTTTATTGGGGAGGACATCGA-
GTAATATCTAGCCTTATAGGAGGTGGTAAC ATAACATCTCCTATATATGGAAGAGAG-
GCGAACCAGGAGCCTCCAAGATCCTTTACTTTTAATGGACCGGTA
TTTAGGACTTTATCAAATCCTACTTTACGATTATTACAGCAACCTTGGCCAGCGCCACCATTTAATTTACGT
GGTGTTGAAGGAGTAGAATTTTCTACACCTACAAATAGCTTTACGTATCGAGGAAGA-
GGTACGGTTGATTCT TTAACTGAATTACCGCCTGAGGATAATAGTGTGCCACCTCGC-
GAAGGATATAGTCATCGTTTATGTCATGCA ACTTTTGTTCAAAGATCTGGAACACCT-
TTTTTAACAACTGGTGTAGTATTTTCTTGGACGCATCGTAGTGCA
ACTCTTACAAATACAATTGATCCAGAGAGAATTAATCAAATACCTTTAGTGAAAGGATTTAGAGTTTGGGGG
GGCACCTCTGTCATTACAGGACCAGGATTTACAGGAGGGGATATCCTTCGAAGAAAT-
ACCTTTGGTGATTTT GTATCTCTACAAGTCAATATTAATTCACCAATTACCCAAAGA-
TACCGTTTAAGATTTCGTTACGCTTCCAGT AGGGATGCACGAGTTATAGTATTAACA-
GGAGCGGCATCCACAGGAGTGGGAGGCCAAGTTAGTGTAAATATG
CCTCTTCAGAAAACTATGGAAATAGGGGAGAACTTAACATCTAGAACATTTAGATATACCGATTTTAGTAAT
CCTTTTTCATTTAGAGCTAATCCAGATATAATTGGGATAAGTGAACAACCTCTATTT-
GGTGCAGGTTCTATT AGTAGCGGTGAACTTTATATAGATAAAATTGAAATTATTCTA-
GCAGATGCAACATTTGAAGCAGAATCTGAT TTAGAAAGAGCACAAAAGGCGGTGAAT-
GCCCTGTTTACTTCTTCCAATCAAATCGGGTTAAAAACCGATGTG
ACGGATTATCATATTGATCAAGTATCCAATTTAGTGGATTGTTTATCAGATGAATTTTGTCTGGATGAAAAG
CGAGAATTGTCCGAGAAAGTCAAACATGCGAAGCGACTCAGTGATGAGCGGAATTTA-
CTTCAAGATCCAAAC TTCAGAGGGATCAATAGACAACCAGACCGTGGCTGGAGAGGA-
AGTACAGATATTACCATCCAAGGAGGAGAT GACGTATTCAAAGAGAATTACGTCACA-
CTACCGGGTACCGTTGATGAGTGCTATCCAACGTATTTATATCAG
AAAATAGATGAGTCGAAATTAAAAGCTTATACCCGTTATGAATTAAGAGGGTATATCGAAGATAGTCAAGAC
TTAGAAATCTATTTGATCCGTTACAATGCAAAACACGAAATAGTAAATGTGCCAGGC-
ACGGGTTCCTTATGG CCGCTTTCAGCCCAAAGTCCAATCGGAAAGTGTGGAGAACCG-
AATCGATGCGCGCCACACCTTGAATGGAAT CCTGATCTAGATTGTTCCTGCAGAGAC-
GGGGAAAAATGTGCACATCATTCCCATCATTTCACCTTGGATATT
GATGTTGGATGTACAGACTTAAATGAGGACTTAGGTGTATGGGTGATATTCAAGATTAAGACGCAAGATGGC
CATGCAAGACTAGGGAATCTAGAGTTTCTCGAAGAGAAACCATTATTAGGGGAAGCA-
CTAGCTCGTGTGAAA AGAGCGGAGAAGAAGTGGAGAGACAAACGAGAGAAACTGCAG-
TTGGAAACAAATATTGTTTATAAAGAGGCA AAAGAATCTGTAGATGCTTTATTTGTA-
AACTCTCAATATGATAGATTACAAGTGGATACGAACATCGCAATG
ATTCATGCGGCAGATAAACGCGTTCATAGAATCCGGGAAGCGTATCTGCCAGAGTTGTCTGTGATTCCAGGT
GTCAATGCGGCCATTTTCGAAGAATTAGAGGGACGTATTTTTACAGCGTATTCCTTA-
TATGATGCGAGAAAT GTCATTAAAAATGGCGATTTCAATAATGGCTTATTATGCTGG-
AACGTGAAAGGTCATGTAGATGTAGAAGAG CAAAACAACCACCGTTCGGTCCTTGTT-
ATCCCAGAATGGGAGGCAGAAGTGTCACAAGAGGTTCGTGTCTGT
CCAGGTCGTGGCTATATCCTTCGTGTCACAGCATATAAAGAGGGATATGGAGAGGGCTGCGTAACGATCCAT
GAGATCGAAGACAATACAGACGAACTGAAATTCAGCAACTGTGTAGAAGAGGAAGTA-
TATCCAAACAACACA GTAACGTGTAATAATTATACTGGGACTCAAGAAGAATATGAG-
GGTACGTACACTTCTCGTAATCAAGGATAT GACGAAGCCTATGGTAATAACCCTTCC-
GTACCAGCTGATTACGCTTCAGTCTATGAAGAAAAATCGTATACA
GATGGACGAAGAGAGAATCCTTGTGAATCTAACAGAGGCTATGGGGATTACACACCACTACCGGCTGGTTAT
GTAACAAAGGATTTAGAGTACTTCCCAGAGACCGATAAGGTATGGATTGAGATCGGA-
GAAACAGAAGGAACA TTCATCGTGGATAGCGTGGAATTACTCCTTATGGAGGAA 5.14.4
NUCLEIC ACID SEQUENCE OF CRY1C.563 (SEQ ID NO:7)
ATGGAGGAAAATAATCAAAATCAATGCATACCTTACAATTGTTTAAGTAATCCTGAAGAAG-
TACTTTTGGAT GGAGAACGGATATCAACTGGTAATTCATCAATTGATATTTCTCTG-
TCACTTGTTCAGTTTCTGGTATCTAAC TTTGTACCAGGGGGAGGATTTTTAGTTGGA-
TTAATAGATTTTGTATGGGGAATAGTTGGCCCTTCTCAATGG
GATGCATTTCTAGTACAAATTGAACAATTAATTAATGAAAGAATAGCTGAATTTGCTAGGAATGCTGCTATT
GCTAATTTAGAAGGATTAGGAAACAATTTCAATATATATGTGGAAGCATTTAAAGAA-
TGGGAAGTAGATCCT AATAATCCTGGAACCAGGACCAGAGTAATTGATCGCTTTCGT-
ATACTTGATGGGCTACTTGAAAGGGACATT CCTTCGTTTCGAATTTCTGGATTTGAA-
GTACCCCTTTTATCCGTTTATGCTCAAGCGGCCAATCTGCATCTA
GCTATATTAAGAGATTCTGTAATTTTTGGAGAAAGATGGGGATTGACAACGATAAATGTCAATGAAAACTAT
AATAGACTAATTAGGCATATTGATGAATATGCTGATCACTGTGCAAATACGTATAAT-
CGGGGATTAAATAAT TTACCGAAATCTACGTATCAAGATTGGATAACATATAATCGA-
TTACGGAGAGACTTAACATTGACTGTATTA GATATCGCCGCTTTCTTTCCAAACTAT-
GACAATAGGAGATATCCAATTCAGCCAGTTGGTCAACTAACAAGG
GAAGTTTATACGGACCCATTAATTAATTTTAATCCACAGTTACAGTCTGTAGCTCAATTACCTACTTTTAAC
GTTATGGAGAGCAGCGCAATTAGAAATCCTCATTTATTTGATATATTGAATAATCTT-
ACAATCTTTACGGAT TGGTTTAGTGTTGGACGCAATTTTTATTGGGGAGGACATCGA-
GTAATATCTAGCCTTATAGGAGGTGGTAAC ATAACATCTCCTATATATGGAAGAGAG-
GCGAACCAGGAGCCTCCAAGATCCTTTACTTTTAATGGACCGGTA
TTTAGGACTTTATCAAATCCTACTTTACGATTATTACGCAACCTTGGCCAGCGGCCACCATTTAATTTACGT
GGTGTTGAAGGAGTAGAATTTTCTACACCTACAAATAGCTTTACGTATCGAGGAAGA-
GGTACGGTTGATTCT TTAACTGAATTACCGCCTGAGGATAATAGTGTGCCACCTCGC-
GAAGGATATAGTCATCGTTTATGTCATGCA ACTTTTGTTCAAAGATCTGGAACACCT-
TTTTTAACAACTGGTGTAGTATTTTCTTGGACGCATCGTAGTGCA
ACTCTTACAAATACAATTGATCCAGAGAGAATTAATCAAATACCTTTAGTGAAAGGATTTAGAGTTTGGGGG
GGCACCTCTGTCATTACAGGACCAGGATTTACAGGAGGGGATATCCTTCGAAGAAAT-
ACCTTTGGTGATTTT GTATCTCTACAAGTCAATATTAATTCACCAATTACCCAAAGA-
TACCGTTTAAGATTTCGTTACGCTTCCAGT AGGGATGCACGAGTTATAGTATTAACA-
GGAGCGGCATCCACAGGAGTGGGAGGCCAAGTTAGTGTAAATATG
CCTCTTCAGAAAACTATGGAAATAGGGGAGAACTTAACATCTAGAACATTTAGATATACCGATTTTAGTAAT
CCTTTTTCATTTAGAGCTAATCCAGATATAATTGGGATAAGTGAACAACCTCTATTT-
GGTGCAGGTTCTATT AGTAGCGGTGAACTTTATATAGATAAAATTGAAATTATTCTA-
GCAGATGCAACATTTGAAGCAGAATCTGAT TTAGAAAGAGCACAAAAGGCGGTGAAT-
GCCCTGTTTACTTCTTCCAATCAAATCGGGTTAAAAACCGATGTG
ACGGATTATCATATTGATCAAGTATCCAATTTAGTGGATTGTTTATCAGATGAATTTTGTCTGGATGAAAAG
CGAGAATTGTCCGAGAAAGTCAAACATGCGAAGCGACTCAGTGATGAGCGGAATTTA-
CTTCAAGATCCAAAC TTCAGAGGGATCAATAGACAACCAGACCGTGGCTGGAGAGGA-
AGTACAGATATTACCATCCAAGGAGGAGAT GACGTATTCAAAGAGAATTACGTCACA-
CTACCGGGTACCGTTGATGAGTGCTATCCAACGTATTTATATCAG
AAAATAGATGAGTCGAAATTAAAAGCTTATACCCGTTATGAATTAAGAGGGTATATCGAAGATAGTCAAGAC
TTAGAAATCTATTTGATCCGTTACAATGCAAAACACGAAATAGTAAATGTGCCAGGC-
ACGGGTTCCTTATGG CCGCTTTCAGCCCAAAGTCCAATCGGAAAGTGTGGAGAACCG-
AATCGATGCGCGCCACACCTTGAATGGAAT CCTGATCTAGATTGTTCCTGCAGAGAC-
GGGGAAAAATGTGCACATCATTCCCATCATTTCACCTTGGATATT
GATGTTGGATGTACAGACTTAAATGAGGACTTAGGTGTATGGGTGATATTCAAGATTAAGACGCAAGATGGC
CATGCAAGACTAGGGAATCTAGAGTTTCTCGAAGAGAAACCATTATTAGGGGAAGCA-
CTAGCTCGTGTGAAA AGAGCGGAGAAGAAGTGGAGAGACAAACGAGAGAAACTGCAG-
TTGGAAACAAATATTGTTTATAAAGAGGCA AAAGAATCTGTAGATGCTTTATTTGTA-
AACTCTCAATATGATAGATTACAAGTGGATACGAACATCGCAATG
ATTCATGCGGCAGATAAACGCGTTCATAGAATCCGGGAAGCGTATCTGCCAGAGTTGTCTGTGATTCCAGGT
GTCAATGCGGCCATTTTCGAAGAATTAGAGGGACGTATTTTTACAGCGTATTCCTTA-
TATGATGCGAGAAAT GTCATTAAAATGGCGATTTCAATAATGGCTTATTATGCTGGA-
ACGTGAAATGGTCATGTAGATGTAGAAGAG CAAAACAACCACCGTTCGGTCCTTGTT-
ATCCCAGAATGGGAGGCAGAAGTGTCACAAGAGGTTCGTGTCTGT
CCAGGTCGTGGCTATATCCTTCGTGTCACAGCATATAAAGAGGGATATGGAGAGGGCTGCGTAACGATCCAT
GAGATCGAAGACAATACAGACGAACTGAAATTCAGCAACTGTGTAGAAGAGGAAGTA-
TATCCAAACAACACA GTAACGTGTAATAATTATACTGGGACTCAAGAAGAATATGAG-
GGTACGTACACTTCTCGTAATCAAGGATAT GACGAAGCCTATGGTAATAACCCTTCC-
GTACCAGCTGATTACGCTTCAGTCTATGAAGAAAAATCGTATACA
GATGGACGAAGAGAGAATCCTTGTGAATCTAACAGAGGCTATGGGGATTACACACCACTACCGGCTGGTTAT
GTAACAAAGGATTTAGAGTACTTCCCAGAGACCGATAAGGTATGGATTGAGATCGGA-
GAAACAGAAGGAACA TTCATCGTGGATAGCGTGGAATTACTCCTTATGGAGGAA 5.14.5
NUCLEIC ACID SEQUENCE OF CRY1C.579 (SEQ ID NO:9)
ATGGAGGAAAATAATCAAAATCAATGCATACCTTACAATTGTTTAAGTAATCCTGAAGAAG-
TACTTTTGGAT GGAGAACGGATATCAACTGGTAATTCATCAATTGATATTTCTCTG-
TCACTTGTTCAGTTTCTGGTATCTAAC TTTGTACCAGGGGGAGGATTTTTAGTTGGA-
TTAATAGATTTTGTATGGGGAATAGTTGGCCCTTCTCAATGG
GATGCATTTCTAGTACAAATTGAACAATTAATTAATGAAAGAATAGCTGAATTTGCTAGGAATGCTGCTATT
GCTAATTTAGAAGGATTAGGAAACAATTTCAATATATATGTGGAAGCATTTAAAGAA-
TGGGAAGTAGATCCT AATAATCCTGGAACCAGGACCAGAGTAATTGATCGCTTTCGT-
ATACTTGATGGGCTACTTGAAAGGGACATT CCTTCGTTTCGAATTTCTGGATTTGAA-
GTACCCCTTTTATCCGTTTATGCTCAAGCGGCCAATCTGCATCTA
GCTATATTAAGAGATTCTGTAATTTTTGGAGAAAGATGGGGATTGACAACGATAAATGTCAATGAAAACTAT
AATAGACTAATTAGGCATATTGATGAATATGCTGATCACTGTGCAAATACGTATAAT-
CGGGGATTAAATAAT TTACCGAAATCTACGTATCAAGATTGGATAACATATAATCGA-
TTACGGAGAGACTTAACATTGACTGTATTA GATATCGCCGCTTTCTTTCCAAACTAT-
GACAATAGGAGATATCCAATTCAGCCAGTTGGTCAACTAACAAGG
GAAGTTTATACGGACCCATTAATTAATTTTAATCCACAGTTACAGTCTGTAGCTCAATTACCTACTTTTAAC
GTTATGGAGAGCAGCGCAATTAGAAATCCTCATTTATTTGATATATTGAATAATCTT-
ACAATCTTTACGGAT TGGTTTAGTGTTGGACGCAATTTTTATTGGGGAGGACATCGA-
GTAATATCTAGCCTTATAGGAGGTGGTAAC ATAACATCTCCTATATATGGAAGAGAG-
GCGAACCAGGAGCCTCCAAGATCCTTTACTTTTAATGGACCGGTA
TTTAGGACTTTATCAAATCCTACTTTACGATTATTACGCAACCTTGGCCAGCGGCCACCATTTAATTTACGT
GGTGTTGAAGGAGTAGAATTTTCTACACCTACAAATAGCTTTACGTATCGAGGAAGA-
GGTACGGTTGATTCT TTAACTGAATTACCGCCTGAGGATAATAGTGTGCCACCTCGC-
GAAGGATATAGTCATCGTTTATGTCATGCA ACTTTTGTTCAAAGATCTGGAACACCT-
TTTTTAACAACTGGTGTAGTATTTTCTTGGACGCATCGTAGTGCA
ACTCTTACAAATACAATTGATCCAGAGAGAATTAATCAAATACCTTTAGTGAAAGGATTTAGAGTTTGGGGG
GGCACCTCTGTCATTACAGGACCAGGATTTACAGGAGGGGATATCCTTCGAAGAAAT-
ACCTTTGGTGATTTT GTATCTCTACAAGTCAATATTAATTCACCAATTACCCAAAGA-
TACCGTTTAAGATTTCGTTACGCTTCCAGT AGGGATGCACGAGTTATAGTATTAACA-
GGAGCGGCATCCACAGGAGTGGGAGGCCAAGTTAGTGTAAATATG
CCTCTTCAGAAAACTATGGAAATAGGGGAGAACTTAACATCTAGAACATTTAGATATACCGATTTTAGTAAT
CCTTTTTCATTTAGAGCTAATCCAGATATAATTGGGATAAGTGAACAACCTCTATTT-
GGTGCAGGTTCTATT AGTAGCGGTGAACTTTATATAGATAAAATTGAAATTATTCTA-
GCAGATGCAACATTTGAAGCAGAATCTGAT TTAGAAAGAGCACAAAAGGCGGTGAAT-
GCCCTGTTTACTTCTTCCAATCAAATCGGGTTAAAAACCGATGTG
ACGGATTATCATATTGATCAAGTATCCAATTTAGTGGATTGTTTATCAGATGAATTTTGTCTGGATGAAAAG
CGAGAATTGTCCGAGAAAGTCAAACATGCGAAGCGACTCAGTGATGAGCGGAATTTA-
CTTCAAGATCCAAAC TTCAGAGGGATCAATAGACAACCAGACCGTGGCTGGAGAGGA-
AGTACAGATATTACCATCCAAGGAGGAGAT GACGTATTCAAAGAGAATTACGTCACA-
CTACCGGGTACCGTTGATGAGTGCTATCCAACGTATTTATATCAG
AAAATAGATGAGTCGAAATTAAAAGCTTATACCCGTTATGAATTAAGAGGGTATATCGAAGATAGTCAAGAC
TTAGAAATCTATTTGATCCGTTACAATGCAAAACACGAAATAGTAAATGTGCCAGGC-
ACGGGTTCCTTATGG CCGCTTTCAGCCCAAAGTCCAATCGGAAAGTGTGGAGAACCG-
AATCGATGCGCGCCACACCTTGAATGGAAT CCTGATCTAGATTGTTCCTGCAGAGAC-
GGGGAAAAATGTGCACATCATTCCCATCATTTCACCTTGGATATT
GATGTTGGATGTACAGACTTAAATGAGGACTTAGGTGTATGGGTGATATTCAAGATTAAGACGCAAGATGGC
CATGCAAGACTAGGGAATCTAGAGTTTCTCGAAGAGAAACCATTATTAGGGGAAGCA-
CTAGCTCGTGTGAAA AGAGCGGAGAAGAAGTGGAGAGACAAACGAGAGAAACTGCAG-
TTGGAAACAAATATTGTTTATAAAGAGGCA AAAGAATCTGTAGATGCTTTATTTGTA-
AACTCTCAATATGATAGATTACAAGTGGATACGAACATCGCAATG
ATTCATGCGGCAGATAAACGCGTTCATAGAATCCGGGAAGCGTATCTGCCAGAGTTGTCTGTGATTCCAGGT
GTCAATGCGGCCATTTTCGAAGAATTAGAGGGACGTATTTTTACAGCGTATTCCTTA-
TATGATGCGAGAAAT GTCATTAAAATGGCGATTTCAATAATGGCTTATTATGCTGGA-
ACGTGAAATGGTCATGTAGATGTAGAAGAG CAAAACAACCACCGTTCGGTCCTTGTT-
ATCCCAGAATGGGAGGCAGAAGTGTCACAAGAGGTTCGTGTCTGT
CCAGGTCGTGGCTATATCCTTCGTGTCACAGCATATAAAGAGGGATATGGAGAGGGCTGCGTAACGATCCAT
GAGATCGAAGACAATACAGACGAACTGAAATTCAGCAACTGTGTAGAAGAGGAAGTA-
TATCCAAACAACACA GTAACGTGTAATAATTATACTGGGACTCAAGAAGAATATGAG-
GGTACGTACACTTCTCGTAATCAAGGATAT GACGAAGCCTATGGTAATAACCCTTCC-
GTACCAGCTGATTACGCTTCAGTCTATGAAGAAAAATCGTATACA
GATGGACGAAGAGAGAATCCTTGTGAATCTAACAGAGGCTATGGGGATTACACACCACTACCGGCTGGTTAT
GTAACAAAGGATTTAGAGTACTTCCCAGAGACCGATAAGGTATGGATTGAGATCGGA-
GAAACAGAAGGAACA TTCATCGTGGATAGCGTGGAATTACTCCTTATGGAGGAA 5.14.6
NUCLEIC ACID SEQUENCE OF CRY1C.499 (SEQ ID NO:11)
ATGGAGGAAAATAATCAAAATCAATGCATACCTTACAATTGTTTAAGTAATCCTGAAGAA- GTACT
TTTGGATGGAGAACGGATATCAACTGGTAATTCATCAATTGATATTTCTCT- GTCACTTGTTCAGT
TTCTGGTATCTAACTTTGTACCAGGGGGAGGATTTTTAGTTGG- ATTAATAGATTTTGTATGGGGA
ATAGTTGGCCCTTCTCAATGGGATGCATTTCTAGT- ACAAATTGAACAATTAATTAATGAAAGAAT
AGCTGAATTTGCTAGGAATGCTGCTAT- TGCTAATTTAGAAGGATTAGGAAACAATTTCAATATAT
ATGTGGAAGCATTTAAAGAATGGGAAGAAGATCCCCATAATCCAGCAACCAGGACCAGAGTAATT
GATCGCTTTCGTATACTTGATGGGCTACTTGAAAGGGACATTCCTTCGTTTCGAATTTCTGGATT
TGAAGTACCCCTTTTATCCGTTTATCATTTGGCGGCCAATCTGCATCTAGCTATATT- AAGAGATT
CTGTAATTTTTGGAGAAAGATGGGGATTGACAACGATAAATGTCAATGA- AAACTATAATAGACTA
ATTAGGCATATTGATGAATATGCTGATCACTGTGCAAATAC- GTATAATCGGGGATTAAATAATTT
ACCGAAATCTACGTATCAAGATTGGATAACATA- TAATCGATTACGGAGAGACTTAACATTGACTG
TATTAGATATCGCCGCTTTCTTTCC- AAACTATGACAATAGGAGATATCCAATTCAGCCAGTTGGT
CAACTAACAAGGGAAGTTTATACGGACCCATTAATTAATTTTAATCCACAGTTACAGTCTGTAGC
TCAATTACCTACTTTTAACGTTATGGAGAGCAGCGCAATTAGAAATCCTCATTTATTTGATATAT
TGAATAATCTTACAATCTTTACGGATTGGTTTAGTGTTGGACGCAATTTTTATTGGG- GAGGACAT
CGAGTAATATCTAGCCTTATAGGAGGTGGTAACATAACATCTCCTATAT- ATGGAAGAGAGGCGAA
CCAGGAGCCTCCAAGATCCTTTACTTTTAATGGACCGGTAT- TTAGGACTTTATCAAATCCTACTT
TACGATTATTACAGCAACCTTGGCCAGCGCCAC- CATTTAATTTACGTGGTGTTGAAGGAGTAGAA
TTTTCTACACCTACAAATAGCTTTA- CGTATCGAGGAAGAGGTACGGTTGATTCTTTAACTGAATT
ACCGCCTGAGGATAATAGTGTGCCACCTCGCGAAGGATATAGTCATCGTTTATGTCATGCAACTT
TTGTTCAAAGATCTGGAACACCTTTTTTAACAACTGGTGTAGTATTTTCTTGGACGCATCGTAGT
GCAACTCTTACAAATACAATTGATCCAGAGAGAATTAATCAAATACCTTTAGTGAAA- GGATTTAG
AGTTTGGGGGGGCACCTCTGTCATTACAGGACCAGGATTTACAGGAGGG- GATATCCTTCGAAGAA
ATACCTTTGGTGATTTTGTATCTCTACAAGTCAATATTAAT- TCACCAATTACCCAAAGATACCGT
TTAAGATTTCGTTACGCTTCCAGTAGGGATGCA- CGAGTTATAGTATTAACAGGAGCGGCATCCAC
AGGAGTGGGAGGCCAAGTTAGTGTA- AATATGCCTCTTCAGAAAACTATGGAAATAGGGGAGAACT
TAACATCTAGAACATTTAGATATACCGATTTTAGTAATCCTTTTTCATTTAGAGCTAATCCAGAT
ATAATTGGGATAAGTGAACAACCTCTATTTGGTGCAGGTTCTATTAGTAGCGGTGAACTTTATAT
AGATAAAATTGAAATTATTCTAGCAGATGCAACATTTGAAGCAGAATCTGATTTAGA- AAGAGCAC
AAAAGGCGGTGAATGCCCTGTTTACTTCTTCCAATCAAATCGGGTTAAA- AACCGATGTGACGGAT
TATCATATTGATCAAGTATCCAATTTAGTGGATTGTTTATC- AGATGAATTTTGTCTGGATGAAAA
GCGAGAATTGTCCGAGAAAGTCAAACATGCGAA- GCGACTCAGTGATGAGCGGAATTTACTTCAAG
ATCCAAACTTCAGAGGGATCAATAG- ACAACCAGACCGTGGCTGGAGAGGAAGTACAGATATTACC
ATCCAAGGAGGAGATGACGTATTCAAAGAGAATTACGTCACACTACCGGGTACCGTTGATGAGTG
CTATCCAACGTATTTATATCAGAAAATAGATGAGTCGAAATTAAAAGCTTATACCCGTTATGAAT
TAAGAGGGTATATCGAAGATAGTCAAGACTTAGAAATCTATTTGATCCGTTACAATG- CAAAACAC
GAAATAGTAAATGTGCCAGGCACGGGTTCCTTATGGCCGCTTTCAGCCC- AAAGTCCAATCGGAAA
GTGTGGAGAACCGAATCGATGCGCGCCACACCTTGAATGGA- ATCCTGATCTAGATTGTTCCTGCA
GAGACGGGGAAAAATGTGCACATCATTCCCATC- ATTTCACCTTGGATATTGATGTTGGATGTACA
GACTTAAATGAGGACTTAGGTGTAT- GGGTGATATTCAAGATTAAGACGCAAGATGGCCATGCAAG
ACTAGGGAATCTAGAGTTTCTCGAAGAGAAACCATTATTAGGGGAAGCACTAGCTCGTGTGAAAA
GAGCGGAGAAGAAGTGGAGAGACAAACGAGAGAAACTGCAGTTGGAAACAAATATTGTTTATAAA
GAGGCAAAAGAATCTGTAGATGCTTTATTTGTAAACTCTCAATATGATAGATTACAA- GTGGATAC
GAACATCGCAATGATTCATGCGGCAGATAAACGCGTTCATAGAATCCGG- GAAGCGTATCTGCCAG
AGTTGTCTGTGATTCCAGGTGTCAATGCGGCCATTTTCGAA- GAATTAGAGGGACGTATTTTTACA
GCGTATTCCTTATATGATGCGAGAAATGTCATT- AAAAATGGCGATTTCAATAATGGCTTATTATG
CTGGAACGTGAAAGGTCATGTAGAT- GTAGAAGAGCAAAACAACCACCGTTCGGTCCTTGTTATCC
CAGAATGGGAGGCAGAAGTGTCACAAGAGGTTCGTGTCTGTCCAGGTCGTGGCTATATCCTTCGT
GTCACAGCATATAAAGAGGGATATGGAGAGGGCTGCGTAACGATCCATGAGATCGAAGACAATAC
AGACGAACTGAAATTCAGCAACTGTGTAGAAGAGGAAGTATATCCAAACAACACAGT- AACGTGTA
ATAATTATACTGGGACTCAAGAAGAATATGAGGGTACGTACACTTCTCG- TAATCAAGGATATGAC
GAAGCCTATGGTAATAACCCTTCCGTACCAGCTGATTACGC- TTCAGTCTATGAAGAAAAATCGTA
TACAGATGGACGAAGAGAGAATCCTTGTGAATC- TAACAGAGGCTATGGGGATTACACACCACTAC
CGGCTGGTTATGTAACAAAGGATTT- AGAGTACTTCCCAGAGACCGATAAGGTATGGATTGAGATC
GGAGAAACAGAAGGAACATTCATCGTGGATAGCGTGGAATTACTCCTTATGGAGGAA
5.15 Example 15
Isolation of Transgenic Plants Resistant to Cry* Variants
5.15.1 Plant Gene Construction
[0467] The expression of a plant gene which exists in
double-stranded DNA form involves transcription of messenger RNA
(mRNA) from one strand of the DNA by RNA polymerase enzyme, and the
subsequent processing of the mRNA primary transcript inside the
nucleus. This processing involves a 3' non-translated region which
adds polyadenylate nucleotides to the 3' end of the RNA.
Transcription of DNA into mRNA is regulated by a region of DNA
usually referred to as the "promoter". The promoter region contains
a sequence of bases that signals RNA polymerase to associate with
the DNA and to initiate the transcription of mRNA using one of the
DNA strands as a template to make a corresponding strand of
RNA.
[0468] A number of promoters which are active in plant cells have
been described in the literature. Such promoters may be obtained
from plants or plant viruses and include, but are not limited to,
the nopaline synthase (NOS) and octopine synthase (OCS) promoters
(which are carried on tumor-inducing plasmids of Agrobacterium
tumefaciens), the cauliflower mosaic virus (CaMV) 19S and 35S
promoters, the light-inducible promoter from the small subunit of
ribulose 1,5-bisphosphate carboxylase (ssRUBISCO, a very abundant
plant polypeptide), and the Figwort Mosaic Virus (FMV) 35S
promoter. All of these promoters have been used to create various
types of DNA constructs which have been expressed in plants (see
e.g., U.S. Pat. No. 5,463,175, specifically incorporated herein by
reference).
[0469] The particular promoter selected should be capable of
causing sufficient expression of the enzyme coding sequence to
result in the production of an effective amount of protein. One set
of preferred promoters are constitutive promoters such as the
CaMV35S or FMV35S promoters that yield high levels of expression in
most plant organs (U.S. Pat. No. 5,378,619, specifically
incorporated herein by reference). Another set of preferred
promoters are root enhanced or specific promoters such as the CaMV
derived 4 as-1 promoter or the wheat POX1 promoter (U.S. Pat. No.
5,023,179, specifically incorporated herein by reference; Hertig et
al., 1991). The root enhanced or specific promoters would be
particularly preferred for the control of corn rootworm
(Diabroticus spp.) in transgenic corn plants.
[0470] The promoters used in the DNA constructs (i.e. chimeric
plant genes) of the present invention may be modified, if desired,
to affect their control characteristics. For example, the CaMV35S
promoter may be ligated to the portion of the ssRUBISCO gene that
represses the expression of ssRUBISCO in the absence of light, to
create a promoter which is active in leaves but not in roots. The
resulting chimeric promoter may be used as described herein. For
purposes of this description, the phrase "CaMV35S" promoter thus
includes variations of CaMV35S promoter, e.g., promoters derived by
means of ligation with operator regions, random or controlled
mutagenesis, etc. Furthermore, the promoters may be altered to
contain multiple "enhancer sequences" to assist in elevating gene
expression.
[0471] The RNA produced by a DNA construct of the present invention
also contains a 5' non-translated leader sequence. This sequence
can be derived from the promoter selected to express the gene, and
can be specifically modified so as to increase translation of the
mRNA. The 5' non-translated regions can also be obtained from viral
RNA's, from suitable eucaryotic genes, or from a synthetic gene
sequence. The present invention is not limited to constructs
wherein the non-translated region is derived from the 5'
non-translated sequence that accompanies the promoter sequence.
[0472] For optimized expression in monocotyledenous plants such as
maize, an intron should also be included in the DNA expression
construct. This intron would typically be placed near the 5' end of
the mRNA in untranslated sequence. This intron could be obtained
from, but not limited to, a set of introns consisting of the maize
hsp70 intron (U.S. Pat. No. 5,424,412; specifically incorporated
herein by reference) or the rice Act1 intron (McElroy et al.,
1990). As shown below, the maize hsp70 intron is useful in the
present invention.
[0473] As noted above, the 3' non-translated region of the chimeric
plant genes of the present invention contains a polyadenylation
signal which functions in plants to cause the addition of adenylate
nucleotides to the 3' end of the RNA. Examples of preferred 3'
regions are (1) the 3' transcribed, non-translated regions
containing the polyadenylate signal of Agrobacterium tumor-inducing
(Ti) plasmid genes, such as the nopaline synthase (NOS) gene and
(2) plant genes such as the pea ssRUBISCO E9 gene (Fischhoff et
al., 1987).
5.15.2 Plant Transformation and Expression
[0474] A chimeric transgene containing a structural coding sequence
of the present invention can be inserted into the genome of a plant
by any suitable method such as those detailed herein. Suitable
plant transformation vectors include those derived from a Ti
plasmid of Agrobacterium tumefaciens, as well as those disclosed,
e.g., by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and
Eur. Pat. Appl. Publ. No. EP0120516. In addition to plant
transformation vectors derived from the Ti or root-inducing (Ri)
plasmids of Agrobacterium, alternative methods can be used to
insert the DNA constructs of this invention into plant cells. Such
methods may involve, for example, the use of liposomes,
electroporation, chemicals that increase free DNA uptake, free DNA
delivery via microprojectile bombardment, and transformation using
viruses or pollen (Fromm et al., 1986; Armstrong et al., 1990;
Fromm et al., 1990).
5.15.3 Construction of Plant Expression Vectors for Cry*
Transgenes
[0475] For efficient expression of the cry* variants disclosed
herein in transgenic plants, the gene encoding the variants must
have a suitable sequence composition (Diehn et al., 1996).
[0476] To place a cry* gene in a vector suitable for expression in
monocotyledonous plants (i.e. under control of the enhanced
Cauliflower Mosaic Virus 35S promoter and link to the hsp70 intron
followed by a nopaline synthase polyadenylation site as in U.S.
Pat. No. 5,424,412, specifically incorporated herein by reference),
the vector is digested with appropriate enzymes such as NcoI and
EcoRI. The larger vector band of approximately 4.6 kb is then
electrophoresed, purified, and ligated with T4 DNA ligase to the
appropriate restriction fragment containing the plantized cry*
gene. The ligation mix is then transformed into E. coli,
carbenicillin resistant colonies recovered and plasmid DNA
recovered by DNA miniprep procedures. The DNA may then be subjected
to restriction endonuclease analysis with enzymes such as NcoI and
EcoRI (together), NotI, and PstI to identify clones containing the
cry* gene coding sequence fused to the hsp70 intron under control
of the enhanced CaMV35S promoter).
[0477] To place the gene in a vector suitable for recovery of
stably transformed and insect resistant plants, the restriction
fragment from pMON33708 containing the lysine oxidase coding
sequence fused to the hsp70 intron under control of the enhanced
CaMV35S promoter may be isolated by gel electrophoresis and
purification. This fragment can then be ligated with a vector such
as pMON30460 treated with NotI and calf intestinal alkaline
phosphatase (pMON30460 contains the neomycin phosphotransferase
coding sequence under control of the CaMV35S promoter). Kanamycin
resistant colonies may then be obtained by transformation of this
ligation mix into E. coli and colonies containing the resulting
plasmid can be identified by restriction endonuclease digestion of
plasmid miniprep DNAs. Restriction enzymes such as NotI, EcoRV,
HindIII, NcoI, EcoRI, and BglII can be used to identify the
appropriate clones containing the restriction fragment properly
inserted in the corresponding site of pMON30460, in the orientation
such that both genes are in tandem (i.e. the 3' end of the cry*
gene expression cassette is linked to the 5' end of the nptII
expression cassette). Expression of the Cry* protein by the
resulting vector is then confirmed in plant protoplasts by
electroporation of the vector into protoplasts followed by protein
blot and ELISA analysis. This vector can be introduced into the
genomic DNA of plant embryos such as maize by particle gun
bombardment followed by paromomycin selection to obtain corn plants
expressing the cry* gene essentially as described in U.S. Pat. No.
5,424,412, specifically incorporated herein by reference. In this
example, the vector was introduced via cobombardment with a
hygromycin resistance conferring plasmid into immature embryo
scutella (IES) of maize, followed by hygromycin selection, and
regeneration. Transgenic plant lines expressing the Cry* protein
are then identified by ELISA analysis. Progeny seed from these
events are then subsequently tested for protection from susceptible
insect feeding.
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forth herein, are specifically incorporated herein by
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[0751] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
Sequence CWU 1
1
76 1 3567 DNA Artificial Sequence Recombinant Delta Endotoxin 1 atg
gag gaa aat aat caa aat caa tgc ata cct tac aat tgt tta agt 48 Met
Glu Glu Asn Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser 1 5 10
15 aat cct gaa gaa gta ctt ttg gat gga gaa cgg ata tca act ggt aat
96 Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn
20 25 30 tca tca att gat att tct ctg tca ctt gtt cag ttt ctg gta
tct aac 144 Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val
Ser Asn 35 40 45 ttt gta cca ggg gga gga ttt tta gtt gga tta ata
gat ttt gta tgg 192 Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile
Asp Phe Val Trp 50 55 60 gga ata gtt ggc cct tct caa tgg gat gca
ttt cta gta caa att gaa 240 Gly Ile Val Gly Pro Ser Gln Trp Asp Ala
Phe Leu Val Gln Ile Glu 65 70 75 80 caa tta att aat gaa aga ata gct
gaa ttt gct agg aat gct gct att 288 Gln Leu Ile Asn Glu Arg Ile Ala
Glu Phe Ala Arg Asn Ala Ala Ile 85 90 95 gct aat tta gaa gga tta
gga aac aat ttc aat ata tat gtg gaa gca 336 Ala Asn Leu Glu Gly Leu
Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala 100 105 110 ttt aaa gaa tgg
gaa gaa gat cct aat aat cca gca acc agg acc aga 384 Phe Lys Glu Trp
Glu Glu Asp Pro Asn Asn Pro Ala Thr Arg Thr Arg 115 120 125 gta att
gat cgc ttt cgt ata ctt gat ggg cta ctt gaa agg gac att 432 Val Ile
Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile 130 135 140
cct tcg ttt gca att tct gga ttt gaa gta ccc ctt tta tcc gtt tat 480
Pro Ser Phe Ala Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr 145
150 155 160 gct caa gcg gcc aat ctg cat cta gct ata tta aga gat tct
gta att 528 Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser
Val Ile 165 170 175 ttt gga gaa aga tgg gga ttg aca acg ata aat gtc
aat gaa aac tat 576 Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val
Asn Glu Asn Tyr 180 185 190 aat aga cta att agg cat att gat gaa tat
gct gat cac tgt gca aat 624 Asn Arg Leu Ile Arg His Ile Asp Glu Tyr
Ala Asp His Cys Ala Asn 195 200 205 acg tat aat cgg gga tta aat aat
tta ccg aaa tct acg tat caa gat 672 Thr Tyr Asn Arg Gly Leu Asn Asn
Leu Pro Lys Ser Thr Tyr Gln Asp 210 215 220 tgg ata aca tat aat cga
tta cgg aga gac tta aca ttg act gta tta 720 Trp Ile Thr Tyr Asn Arg
Leu Arg Arg Asp Leu Thr Leu Thr Val Leu 225 230 235 240 gat atc gcc
gct ttc ttt cca aac tat gac aat agg aga tat cca att 768 Asp Ile Ala
Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile 245 250 255 cag
cca gtt ggt caa cta aca agg gaa gtt tat acg gac cca tta att 816 Gln
Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile 260 265
270 aat ttt aat cca cag tta cag tct gta gct caa tta cct act ttt aac
864 Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn
275 280 285 gtt atg gag agc agc gca att aga aat cct cat tta ttt gat
ata ttg 912 Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp
Ile Leu 290 295 300 aat aat ctt aca atc ttt acg gat tgg ttt agt gtt
gga cgc aat ttt 960 Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val
Gly Arg Asn Phe 305 310 315 320 tat tgg gga gga cat cga gta ata tct
agc ctt ata gga ggt ggt aac 1008 Tyr Trp Gly Gly His Arg Val Ile
Ser Ser Leu Ile Gly Gly Gly Asn 325 330 335 ata aca tct cct ata tat
gga aga gag gcg aac cag gag cct cca aga 1056 Ile Thr Ser Pro Ile
Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg 340 345 350 tcc ttt act
ttt aat gga ccg gta ttt agg act tta tca aat cct act 1104 Ser Phe
Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr 355 360 365
tta cga tta tta cag caa cct tgg cca gcg cca cca ttt aat tta cgt
1152 Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu
Arg 370 375 380 ggt gtt gaa gga gta gaa ttt tct aca cct aca aat agc
ttt acg tat 1200 Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn
Ser Phe Thr Tyr 385 390 395 400 cga gga aga ggt acg gtt gat tct tta
act gaa tta ccg cct gag gat 1248 Arg Gly Arg Gly Thr Val Asp Ser
Leu Thr Glu Leu Pro Pro Glu Asp 405 410 415 aat agt gtg cca cct cgc
gaa gga tat agt cat cgt tta tgt cat gca 1296 Asn Ser Val Pro Pro
Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala 420 425 430 act ttt gtt
caa aga tct gga aca cct ttt tta aca act ggt gta gta 1344 Thr Phe
Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val 435 440 445
ttt tct tgg acg cat cgt agt gca act ctt aca aat aca att gat cca
1392 Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp
Pro 450 455 460 gag aga att aat caa ata cct tta gtg aaa gga ttt aga
gtt tgg ggg 1440 Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe
Arg Val Trp Gly 465 470 475 480 ggc acc tct gtc att aca gga cca gga
ttt aca gga ggg gat atc ctt 1488 Gly Thr Ser Val Ile Thr Gly Pro
Gly Phe Thr Gly Gly Asp Ile Leu 485 490 495 cga aga aat acc ttt ggt
gat ttt gta tct cta caa gtc aat att aat 1536 Arg Arg Asn Thr Phe
Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn 500 505 510 tca cca att
acc caa aga tac cgt tta aga ttt cgt tac gct tcc agt 1584 Ser Pro
Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser 515 520 525
agg gat gca cga gtt ata gta tta aca gga gcg gca tcc aca gga gtg
1632 Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly
Val 530 535 540 gga ggc caa gtt agt gta aat atg cct ctt cag aaa act
atg gaa ata 1680 Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys
Thr Met Glu Ile 545 550 555 560 ggg gag aac tta aca tct aga aca ttt
aga tat acc gat ttt agt aat 1728 Gly Glu Asn Leu Thr Ser Arg Thr
Phe Arg Tyr Thr Asp Phe Ser Asn 565 570 575 cct ttt tca ttt aga gct
aat cca gat ata att ggg ata agt gaa caa 1776 Pro Phe Ser Phe Arg
Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln 580 585 590 cct cta ttt
ggt gca ggt tct att agt agc ggt gaa ctt tat ata gat 1824 Pro Leu
Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp 595 600 605
aaa att gaa att att cta gca gat gca aca ttt gaa gca gaa tct gat
1872 Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser
Asp 610 615 620 tta gaa aga gca caa aag gcg gtg aat gcc ctg ttt act
tct tcc aat 1920 Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe
Thr Ser Ser Asn 625 630 635 640 caa atc ggg tta aaa acc gat gtg acg
gat tat cat att gat caa gta 1968 Gln Ile Gly Leu Lys Thr Asp Val
Thr Asp Tyr His Ile Asp Gln Val 645 650 655 tcc aat tta gtg gat tgt
tta tca gat gaa ttt tgt ctg gat gaa aag 2016 Ser Asn Leu Val Asp
Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys 660 665 670 cga gaa ttg
tcc gag aaa gtc aaa cat gcg aag cga ctc agt gat gag 2064 Arg Glu
Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu 675 680 685
cgg aat tta ctt caa gat cca aac ttc aga ggg atc aat aga caa cca
2112 Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln
Pro 690 695 700 gac cgt ggc tgg aga gga agt aca gat att acc atc caa
gga gga gat 2160 Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile
Gln Gly Gly Asp 705 710 715 720 gac gta ttc aaa gag aat tac gtc aca
cta ccg ggt acc gtt gat gag 2208 Asp Val Phe Lys Glu Asn Tyr Val
Thr Leu Pro Gly Thr Val Asp Glu 725 730 735 tgc tat cca acg tat tta
tat cag aaa ata gat gag tcg aaa tta aaa 2256 Cys Tyr Pro Thr Tyr
Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys 740 745 750 gct tat acc
cgt tat gaa tta aga ggg tat atc gaa gat agt caa gac 2304 Ala Tyr
Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp 755 760 765
tta gaa atc tat ttg atc cgt tac aat gca aaa cac gaa ata gta aat
2352 Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val
Asn 770 775 780 gtg cca ggc acg ggt tcc tta tgg ccg ctt tca gcc caa
agt cca atc 2400 Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala
Gln Ser Pro Ile 785 790 795 800 gga aag tgt gga gaa ccg aat cga tgc
gcg cca cac ctt gaa tgg aat 2448 Gly Lys Cys Gly Glu Pro Asn Arg
Cys Ala Pro His Leu Glu Trp Asn 805 810 815 cct gat cta gat tgt tcc
tgc aga gac ggg gaa aaa tgt gca cat cat 2496 Pro Asp Leu Asp Cys
Ser Cys Arg Asp Gly Glu Lys Cys Ala His His 820 825 830 tcc cat cat
ttc acc ttg gat att gat gtt gga tgt aca gac tta aat 2544 Ser His
His Phe Thr Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn 835 840 845
gag gac tta ggt gta tgg gtg ata ttc aag att aag acg caa gat ggc
2592 Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp
Gly 850 855 860 cat gca aga cta ggg aat cta gag ttt ctc gaa gag aaa
cca tta tta 2640 His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu
Lys Pro Leu Leu 865 870 875 880 ggg gaa gca cta gct cgt gtg aaa aga
gcg gag aag aag tgg aga gac 2688 Gly Glu Ala Leu Ala Arg Val Lys
Arg Ala Glu Lys Lys Trp Arg Asp 885 890 895 aaa cga gag aaa ctg cag
ttg gaa aca aat att gtt tat aaa gag gca 2736 Lys Arg Glu Lys Leu
Gln Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala 900 905 910 aaa gaa tct
gta gat gct tta ttt gta aac tct caa tat gat aga tta 2784 Lys Glu
Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu 915 920 925
caa gtg gat acg aac atc gca atg att cat gcg gca gat aaa cgc gtt
2832 Gln Val Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg
Val 930 935 940 cat aga atc cgg gaa gcg tat ctg cca gag ttg tct gtg
att cca ggt 2880 His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser
Val Ile Pro Gly 945 950 955 960 gtc aat gcg gcc att ttc gaa gaa tta
gag gga cgt att ttt aca gcg 2928 Val Asn Ala Ala Ile Phe Glu Glu
Leu Glu Gly Arg Ile Phe Thr Ala 965 970 975 tat tcc tta tat gat gcg
aga aat gtc att aaa aat ggc gat ttc aat 2976 Tyr Ser Leu Tyr Asp
Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn 980 985 990 aat ggc tta
tta tgc tgg aac gtg aaa ggt cat gta gat gta gaa gag 3024 Asn Gly
Leu Leu Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu 995 1000
1005 caa aac aac cac cgt tcg gtc ctt gtt atc cca gaa tgg gag gca
3069 Gln Asn Asn His Arg Ser Val Leu Val Ile Pro Glu Trp Glu Ala
1010 1015 1020 gaa gtg tca caa gag gtt cgt gtc tgt cca ggt cgt ggc
tat atc 3114 Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly
Tyr Ile 1025 1030 1035 ctt cgt gtc aca gca tat aaa gag gga tat gga
gag ggc tgc gta 3159 Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly
Glu Gly Cys Val 1040 1045 1050 acg atc cat gag atc gaa gac aat aca
gac gaa ctg aaa ttc agc 3204 Thr Ile His Glu Ile Glu Asp Asn Thr
Asp Glu Leu Lys Phe Ser 1055 1060 1065 aac tgt gta gaa gag gaa gta
tat cca aac aac aca gta acg tgt 3249 Asn Cys Val Glu Glu Glu Val
Tyr Pro Asn Asn Thr Val Thr Cys 1070 1075 1080 aat aat tat act ggg
act caa gaa gaa tat gag ggt acg tac act 3294 Asn Asn Tyr Thr Gly
Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr 1085 1090 1095 tct cgt aat
caa gga tat gac gaa gcc tat ggt aat aac cct tcc 3339 Ser Arg Asn
Gln Gly Tyr Asp Glu Ala Tyr Gly Asn Asn Pro Ser 1100 1105 1110 gta
cca gct gat tac gct tca gtc tat gaa gaa aaa tcg tat aca 3384 Val
Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr 1115 1120
1125 gat gga cga aga gag aat cct tgt gaa tct aac aga ggc tat ggg
3429 Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly
1130 1135 1140 gat tac aca cca cta ccg gct ggt tat gta aca aag gat
tta gag 3474 Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp
Leu Glu 1145 1150 1155 tac ttc cca gag acc gat aag gta tgg att gag
atc gga gaa aca 3519 Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu
Ile Gly Glu Thr 1160 1165 1170 gaa gga aca ttc atc gtg gat agc gtg
gaa tta ctc ctt atg gag 3564 Glu Gly Thr Phe Ile Val Asp Ser Val
Glu Leu Leu Leu Met Glu 1175 1180 1185 gaa 3567 Glu 2 1189 PRT
Artificial Sequence Recombinant Delta Endotoxin 2 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 Leu 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 Gly Asn Asn
Phe Asn Ile Tyr Val Glu Ala 100 105 110 Phe Lys Glu Trp Glu Glu Asp
Pro Asn Asn Pro Ala 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
Ala 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 1010 1015 1020 Glu Val
Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile 1025 1030 1035
Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val 1040
1045 1050 Thr Ile His Glu Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe
Ser 1055 1060 1065 Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr
Val Thr Cys 1070 1075 1080 Asn Asn Tyr Thr Gly Thr Gln Glu Glu Tyr
Glu Gly Thr Tyr Thr 1085 1090 1095 Ser Arg Asn Gln Gly Tyr Asp Glu
Ala Tyr Gly Asn Asn Pro Ser 1100 1105 1110 Val Pro Ala Asp Tyr Ala
Ser Val Tyr Glu Glu Lys Ser Tyr Thr 1115 1120 1125 Asp Gly Arg Arg
Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly 1130 1135 1140 Asp Tyr
Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu 1145 1150 1155
Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr 1160
1165 1170 Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met
Glu 1175 1180 1185 Glu 3 3567 DNA Artificial Sequence Recombinant
Delta Endotoxin 3 atg gag gaa aat aat caa aat caa tgc ata cct tac
aat tgt tta agt 48 Met Glu Glu Asn Asn Gln Asn Gln Cys Ile Pro Tyr
Asn Cys Leu Ser 1 5 10 15 aat cct gaa gaa gta ctt ttg gat gga gaa
cgg ata tca act ggt aat 96 Asn Pro Glu Glu Val Leu Leu Asp Gly Glu
Arg Ile Ser Thr Gly Asn 20 25 30 tca tca att gat att tct ctg tca
ctt gtt cag ttt ctg gta tct aac 144 Ser Ser Ile Asp Ile Ser Leu Ser
Leu Val Gln Phe Leu Val Ser Asn 35 40 45 ttt gta cca ggg gga gga
ttt tta gtt gga tta ata gat ttt gta tgg 192 Phe Val Pro Gly Gly Gly
Phe Leu Val Gly Leu Ile Asp Phe Val Trp 50 55 60 gga ata gtt ggc
cct tct caa tgg gat gca ttt cta gta caa att gaa 240 Gly Ile Val Gly
Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu 65 70 75 80 caa tta
att aat gaa aga ata gct gaa ttt gct agg aat gct gct att 288 Gln Leu
Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile 85 90 95
gct aat tta gaa gga tta gga aac aat ttc aat ata tat gtg gaa gca 336
Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala 100
105 110 ttt aaa gaa tgg gaa gaa gat cct aat aat cca gca acc agg acc
aga 384 Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Ala Thr Arg Thr
Arg 115 120 125 gta att gat cgc ttt cgt ata ctt gat ggg cta ctt gaa
agg gac att 432 Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu
Arg Asp Ile 130 135 140 cct tcg ttt gac att tct gga ttt gaa gta ccc
ctt tta tcc gtt tat 480 Pro Ser Phe Asp Ile Ser Gly Phe Glu Val Pro
Leu Leu Ser Val Tyr 145 150 155 160 gct caa gcg gcc aat ctg cat cta
gct ata tta aga gat tct gta att 528 Ala Gln Ala Ala Asn Leu His Leu
Ala Ile Leu Arg Asp Ser Val Ile 165 170 175 ttt gga gaa aga tgg gga
ttg aca acg ata aat gtc aat gaa aac tat 576 Phe Gly Glu Arg Trp Gly
Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr 180 185 190 aat aga cta att
agg cat att gat gaa tat gct gat cac tgt gca aat 624 Asn Arg Leu Ile
Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn 195 200 205 acg tat
aat cgg gga tta aat aat tta ccg aaa tct acg tat caa gat 672 Thr Tyr
Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp 210 215 220
tgg ata aca tat aat cga tta cgg aga gac tta aca ttg act gta tta 720
Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu 225
230 235 240 gat atc gcc gct ttc ttt cca aac tat gac aat agg aga tat
cca att 768 Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr
Pro Ile 245 250 255 cag cca gtt ggt caa cta aca agg gaa gtt tat acg
gac cca tta att 816 Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr
Asp Pro Leu Ile 260 265 270 aat ttt aat cca cag tta cag tct gta gct
caa tta cct act ttt aac 864 Asn Phe Asn Pro Gln Leu Gln Ser Val Ala
Gln Leu Pro Thr Phe Asn 275 280 285 gtt atg gag agc agc gca att aga
aat cct cat tta ttt gat ata ttg 912 Val Met Glu Ser Ser Ala Ile Arg
Asn Pro His Leu Phe Asp Ile Leu 290 295 300 aat aat ctt aca atc ttt
acg gat tgg ttt agt gtt gga cgc aat ttt 960 Asn Asn Leu Thr Ile Phe
Thr Asp Trp Phe Ser Val Gly Arg Asn Phe 305 310 315 320 tat tgg gga
gga cat cga gta ata tct agc ctt ata gga ggt ggt aac 1008 Tyr Trp
Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn 325 330 335
ata aca tct cct ata tat gga aga gag gcg aac cag gag cct cca aga
1056 Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro
Arg 340 345 350 tcc ttt act ttt aat gga ccg gta ttt agg act tta tca
aat cct act 1104 Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu
Ser Asn Pro Thr 355 360 365 tta cga tta tta cag caa cct tgg cca gcg
cca cca ttt aat tta cgt 1152 Leu Arg Leu Leu Gln Gln Pro Trp Pro
Ala Pro Pro Phe Asn Leu Arg 370 375 380 ggt gtt gaa gga gta gaa ttt
tct aca cct aca aat agc ttt acg tat 1200 Gly Val Glu Gly Val Glu
Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr 385 390 395 400 cga gga aga
ggt acg gtt gat tct tta act gaa tta ccg cct gag gat 1248 Arg Gly
Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp 405 410 415
aat agt gtg cca cct cgc gaa gga tat agt cat cgt tta tgt cat gca
1296 Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His
Ala 420 425 430 act ttt gtt caa aga tct gga aca cct ttt tta aca act
ggt gta gta 1344 Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr
Thr Gly Val Val 435 440 445 ttt tct tgg acg cat cgt agt gca act ctt
aca aat aca att gat cca 1392 Phe Ser Trp Thr His Arg Ser Ala Thr
Leu Thr Asn Thr Ile Asp Pro 450 455 460 gag aga att aat caa ata cct
tta gtg aaa gga ttt aga gtt tgg ggg 1440 Glu Arg Ile Asn Gln Ile
Pro Leu Val Lys Gly Phe Arg Val Trp Gly 465 470 475 480 ggc acc tct
gtc att aca gga cca gga ttt aca gga ggg gat atc ctt 1488 Gly Thr
Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu 485 490 495
cga aga aat acc ttt ggt gat ttt gta tct cta caa gtc aat att aat
1536 Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile
Asn 500 505 510 tca cca att acc caa aga tac cgt tta aga ttt cgt tac
gct tcc agt 1584 Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg
Tyr Ala Ser Ser 515 520 525 agg gat gca cga gtt ata gta tta aca gga
gcg gca tcc aca gga gtg 1632 Arg Asp Ala Arg Val Ile Val Leu Thr
Gly Ala Ala Ser Thr Gly Val 530 535 540 gga ggc caa gtt agt gta aat
atg cct ctt cag aaa act atg gaa ata 1680 Gly Gly Gln Val Ser Val
Asn Met Pro Leu Gln Lys Thr Met Glu Ile 545 550 555 560 ggg gag aac
tta aca tct aga aca ttt aga tat acc gat ttt agt aat 1728 Gly Glu
Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn 565 570 575
cct ttt tca ttt aga gct aat cca gat ata att ggg ata agt gaa caa
1776 Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu
Gln 580 585 590 cct cta ttt ggt gca ggt tct att agt agc ggt gaa ctt
tat ata gat 1824 Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu
Leu Tyr Ile Asp 595 600 605 aaa att gaa att att cta gca gat gca aca
ttt gaa gca gaa tct gat 1872 Lys Ile Glu Ile Ile Leu Ala Asp Ala
Thr Phe Glu Ala Glu Ser Asp 610 615 620 tta gaa aga gca caa aag gcg
gtg aat gcc ctg ttt act tct tcc aat 1920 Leu Glu Arg Ala Gln Lys
Ala Val Asn Ala Leu Phe Thr Ser Ser Asn 625 630 635 640 caa atc ggg
tta aaa acc gat gtg acg gat tat cat att gat caa gta 1968 Gln Ile
Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val 645 650 655
tcc aat tta gtg gat tgt tta tca gat gaa ttt tgt ctg gat gaa aag
2016 Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu
Lys 660 665 670 cga gaa ttg tcc gag aaa gtc aaa cat gcg aag cga ctc
agt gat gag 2064 Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg
Leu Ser Asp Glu 675 680 685 cgg aat tta ctt caa gat cca aac ttc aga
ggg atc aat aga caa cca 2112 Arg Asn Leu Leu Gln Asp Pro Asn Phe
Arg Gly Ile Asn Arg Gln Pro 690 695 700 gac cgt ggc tgg aga gga agt
aca gat att acc atc caa gga gga gat 2160 Asp Arg Gly Trp Arg Gly
Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp 705 710 715 720 gac gta ttc
aaa gag aat tac gtc aca cta ccg ggt acc gtt gat gag 2208 Asp Val
Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Val Asp Glu 725 730 735
tgc tat cca acg tat tta tat cag aaa ata gat gag tcg aaa tta aaa
2256 Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu
Lys 740 745 750 gct tat acc cgt tat gaa tta aga ggg tat atc gaa gat
agt caa gac 2304 Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu
Asp Ser Gln Asp 755 760 765 tta gaa atc tat ttg atc cgt tac aat gca
aaa cac gaa ata gta aat 2352 Leu Glu Ile Tyr Leu Ile Arg Tyr Asn
Ala Lys His Glu Ile Val Asn 770 775 780 gtg cca ggc acg ggt tcc tta
tgg ccg ctt tca gcc caa agt cca atc 2400 Val Pro Gly Thr Gly Ser
Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile 785 790 795 800 gga aag tgt
gga gaa ccg aat cga tgc gcg cca cac ctt gaa tgg aat 2448 Gly Lys
Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn 805 810 815
cct gat cta gat tgt tcc tgc aga gac ggg gaa aaa tgt gca cat cat
2496 Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His
His 820 825 830 tcc cat cat ttc acc ttg gat att gat gtt gga tgt aca
gac tta aat 2544 Ser His His Phe Thr Leu Asp Ile Asp Val Gly Cys
Thr Asp Leu Asn 835 840 845 gag gac tta ggt gta tgg gtg ata ttc aag
att aag acg caa gat ggc 2592 Glu Asp Leu Gly Val Trp Val Ile Phe
Lys Ile Lys Thr Gln Asp Gly 850 855 860 cat gca aga cta ggg aat cta
gag ttt ctc gaa gag aaa cca tta tta 2640 His Ala Arg Leu Gly Asn
Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu 865 870 875 880 ggg gaa gca
cta gct cgt gtg aaa aga gcg gag aag aag tgg aga gac 2688 Gly Glu
Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp 885 890 895
aaa cga gag aaa ctg cag ttg gaa aca aat att gtt tat aaa gag gca
2736 Lys Arg Glu Lys Leu Gln Leu Glu Thr Asn Ile Val Tyr Lys Glu
Ala 900 905 910 aaa gaa tct gta gat gct tta ttt gta aac tct caa tat
gat aga tta 2784 Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln
Tyr Asp Arg Leu 915 920 925 caa gtg gat acg aac atc gca atg att cat
gcg gca gat aaa cgc gtt 2832 Gln Val Asp Thr Asn Ile Ala Met Ile
His Ala Ala Asp Lys Arg Val 930 935 940 cat aga atc cgg gaa gcg tat
ctg cca gag ttg tct gtg att cca ggt 2880 His Arg Ile Arg Glu Ala
Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly 945 950 955 960 gtc aat gcg
gcc att ttc gaa gaa tta gag gga cgt att ttt aca gcg 2928 Val Asn
Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala 965 970 975
tat tcc tta tat gat gcg aga aat gtc att aaa aat ggc gat ttc aat
2976 Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe
Asn 980 985 990 aat ggc tta tta tgc tgg aac gtg aaa ggt cat gta gat
gta gaa gag 3024 Asn Gly Leu Leu Cys Trp Asn Val Lys Gly His Val
Asp Val Glu Glu 995 1000 1005 caa aac aac cac cgt tcg gtc ctt gtt
atc cca gaa tgg gag gca 3069 Gln Asn Asn His Arg Ser Val Leu Val
Ile Pro Glu Trp Glu Ala 1010 1015 1020 gaa gtg tca caa gag gtt cgt
gtc tgt cca ggt cgt ggc tat atc 3114 Glu Val Ser Gln Glu Val Arg
Val Cys Pro Gly Arg Gly Tyr Ile 1025 1030 1035 ctt cgt gtc aca gca
tat aaa gag gga tat gga gag ggc tgc gta 3159 Leu Arg Val Thr Ala
Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val 1040 1045 1050 acg atc cat
gag atc gaa gac aat aca gac gaa ctg aaa ttc agc 3204 Thr Ile His
Glu Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser 1055 1060 1065 aac
tgt gta gaa gag gaa gta tat cca aac aac aca gta acg tgt 3249 Asn
Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys 1070 1075
1080 aat
aat tat act ggg act caa gaa gaa tat gag ggt acg tac act 3294 Asn
Asn Tyr Thr Gly Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr 1085 1090
1095 tct cgt aat caa gga tat gac gaa gcc tat ggt aat aac cct tcc
3339 Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr Gly Asn Asn Pro Ser
1100 1105 1110 gta cca gct gat tac gct tca gtc tat gaa gaa aaa tcg
tat aca 3384 Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser
Tyr Thr 1115 1120 1125 gat gga cga aga gag aat cct tgt gaa tct aac
aga ggc tat ggg 3429 Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn
Arg Gly Tyr Gly 1130 1135 1140 gat tac aca cca cta ccg gct ggt tat
gta aca aag gat tta gag 3474 Asp Tyr Thr Pro Leu Pro Ala Gly Tyr
Val Thr Lys Asp Leu Glu 1145 1150 1155 tac ttc cca gag acc gat aag
gta tgg att gag atc gga gaa aca 3519 Tyr Phe Pro Glu Thr Asp Lys
Val Trp Ile Glu Ile Gly Glu Thr 1160 1165 1170 gaa gga aca ttc atc
gtg gat agc gtg gaa tta ctc ctt atg gag 3564 Glu Gly Thr Phe Ile
Val Asp Ser Val Glu Leu Leu Leu Met Glu 1175 1180 1185 gaa 3567 Glu
4 1189 PRT Artificial Sequence Recombinant Delta Endotoxin 4 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 Leu 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
Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala 100 105 110 Phe Lys Glu Trp
Glu Glu Asp Pro Asn Asn Pro Ala 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 Asp 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
1010 1015 1020 Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly
Tyr Ile 1025 1030 1035 Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly
Glu Gly Cys Val 1040 1045 1050 Thr Ile His Glu Ile Glu Asp Asn Thr
Asp Glu Leu Lys Phe Ser 1055 1060 1065 Asn Cys Val Glu Glu Glu Val
Tyr Pro Asn Asn Thr Val Thr Cys 1070 1075 1080 Asn Asn Tyr Thr Gly
Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr 1085 1090 1095 Ser Arg Asn
Gln Gly Tyr Asp Glu Ala Tyr Gly Asn Asn Pro Ser 1100 1105 1110 Val
Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr 1115 1120
1125 Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly
1130 1135 1140 Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp
Leu Glu 1145 1150 1155 Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu
Ile Gly Glu Thr 1160 1165 1170 Glu Gly Thr Phe Ile Val Asp Ser Val
Glu Leu Leu Leu Met Glu 1175 1180 1185 Glu 5 3567 DNA Artificial
Sequence Recombinant Delta Endotoxin 5 atg gag gaa aat aat caa aat
caa tgc ata cct tac aat tgt tta agt 48 Met Glu Glu Asn Asn Gln Asn
Gln Cys Ile Pro Tyr Asn Cys Leu Ser 1 5 10 15 aat cct gaa gaa gta
ctt ttg gat gga gaa cgg ata tca act ggt aat 96 Asn Pro Glu Glu Val
Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn 20 25 30 tca tca att
gat att tct ctg tca ctt gtt cag ttt ctg gta tct aac 144 Ser Ser Ile
Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn 35 40 45 ttt
gta cca ggg gga gga ttt tta gtt gga tta ata gat ttt gta tgg 192 Phe
Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp 50 55
60 gga ata gtt ggc cct tct caa tgg gat gca ttt cta gta caa att gaa
240 Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu
65 70 75 80 caa tta att aat gaa aga ata gct gaa ttt gct agg aat gct
gct att 288 Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala
Ala Ile 85 90 95 gct aat tta gaa gga tta gga aac aat ttc aat ata
tat gtg gaa gca 336 Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile
Tyr Val Glu Ala 100 105 110 ttt aaa gaa tgg gaa gaa gat cct aat aat
cca gca acc agg acc aga 384 Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn
Pro Ala Thr Arg Thr Arg 115 120 125 gta att gat cgc ttt cgt ata ctt
gat ggg cta ctt gaa agg gac att 432 Val Ile Asp Arg Phe Arg Ile Leu
Asp Gly Leu Leu Glu Arg Asp Ile 130 135 140 cct tcg ttt cga att tct
gga ttt gaa gta ccc ctt tta tcc gtt tat 480 Pro Ser Phe Arg Ile Ser
Gly Phe Glu Val Pro Leu Leu Ser Val Tyr 145 150 155 160 gct caa gcg
gcc aat ctg cat cta gct ata tta aga gat tct gta att 528 Ala Gln Ala
Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile 165 170 175 ttt
gga gaa gca tgg ggg ttg aca acg ata aat gtc aat gaa aac tat 576 Phe
Gly Glu Ala Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr 180 185
190 aat aga cta att agg cat att gat gaa tat gct gat cac tgt gca aat
624 Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn
195 200 205 acg tat aat cgg gga tta aat aat tta ccg aaa tct acg tat
caa gat 672 Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr
Gln Asp 210 215 220 tgg ata aca tat aat cga tta cgg aga gac tta aca
ttg act gta tta 720 Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr
Leu Thr Val Leu 225 230 235 240 gat atc gcc gct ttc ttt cca aac tat
gac aat agg aga tat cca att 768 Asp Ile Ala Ala Phe Phe Pro Asn Tyr
Asp Asn Arg Arg Tyr Pro Ile 245 250 255 cag cca gtt ggt caa cta aca
agg gaa gtt tat acg gac cca tta att 816 Gln Pro Val Gly Gln Leu Thr
Arg Glu Val Tyr Thr Asp Pro Leu Ile 260 265 270 aat ttt aat cca cag
tta cag tct gta gct caa tta cct act ttt aac 864 Asn Phe Asn Pro Gln
Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn 275 280 285 gtt atg gag
agc agc gca att aga aat cct cat tta ttt gat ata ttg 912 Val Met Glu
Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu 290 295 300 aat
aat ctt aca atc ttt acg gat tgg ttt agt gtt gga cgc aat ttt 960 Asn
Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe 305 310
315 320 tat tgg gga gga cat cga gta ata tct agc ctt ata gga ggt ggt
aac 1008 Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly
Gly Asn 325 330 335 ata aca tct cct ata tat gga aga gag gcg aac cag
gag cct cca aga 1056 Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn
Gln Glu Pro Pro Arg 340 345 350 tcc ttt act ttt aat gga ccg gta ttt
agg act tta tca aat cct act 1104 Ser Phe Thr Phe Asn Gly Pro Val
Phe Arg Thr Leu Ser Asn Pro Thr 355 360 365 tta cga tta tta cag caa
cct tgg cca gcg cca cca ttt aat tta cgt 1152 Leu Arg Leu Leu Gln
Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg 370 375 380 ggt gtt gaa
gga gta gaa ttt tct aca cct aca aat agc ttt acg tat 1200 Gly Val
Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr 385 390 395
400 cga gga aga ggt acg gtt gat tct tta act gaa tta ccg cct gag gat
1248 Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu
Asp 405 410 415 aat agt gtg cca cct cgc gaa gga tat agt cat cgt tta
tgt cat gca 1296 Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg
Leu Cys His Ala 420 425 430 act ttt gtt caa aga tct gga aca cct ttt
tta aca act ggt gta gta 1344 Thr Phe Val Gln Arg Ser Gly Thr Pro
Phe Leu Thr Thr Gly Val Val 435 440 445 ttt tct tgg acg cat cgt agt
gca act ctt aca aat aca att gat cca 1392 Phe Ser Trp Thr His Arg
Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro 450 455 460 gag aga att aat
caa ata cct tta gtg aaa gga ttt aga gtt tgg ggg 1440 Glu Arg Ile
Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly 465 470 475 480
ggc acc tct gtc att aca gga cca gga ttt aca gga ggg gat atc ctt
1488 Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile
Leu 485 490 495 cga aga aat acc ttt ggt gat ttt gta tct cta caa gtc
aat att aat 1536 Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln
Val Asn Ile Asn 500 505 510 tca cca att acc caa aga tac cgt tta aga
ttt cgt tac gct tcc agt 1584 Ser Pro Ile Thr Gln Arg Tyr Arg Leu
Arg Phe Arg Tyr Ala Ser Ser 515 520 525 agg gat gca cga gtt ata gta
tta aca gga gcg gca tcc aca gga gtg 1632 Arg Asp Ala Arg Val Ile
Val Leu Thr Gly Ala Ala Ser Thr Gly Val 530 535 540 gga ggc caa gtt
agt gta aat atg cct ctt cag aaa act atg gaa ata 1680 Gly Gly Gln
Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile 545 550 555 560
ggg gag aac tta aca tct aga aca ttt aga tat acc gat ttt agt aat
1728 Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser
Asn 565 570 575 cct ttt tca ttt aga gct aat cca gat ata att ggg ata
agt gaa caa 1776 Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly
Ile Ser Glu Gln 580 585 590 cct cta ttt ggt gca ggt tct att agt agc
ggt gaa ctt tat ata gat 1824 Pro Leu Phe Gly Ala Gly Ser Ile Ser
Ser Gly Glu Leu Tyr Ile Asp 595 600 605 aaa att gaa att att cta gca
gat gca aca ttt gaa gca gaa tct gat 1872 Lys Ile Glu Ile Ile Leu
Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp 610 615 620 tta gaa aga gca
caa aag gcg gtg aat gcc ctg ttt act tct tcc aat 1920 Leu Glu Arg
Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn 625 630 635 640
caa atc ggg tta aaa acc gat gtg acg gat tat cat att gat caa gta
1968 Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln
Val 645 650 655 tcc
aat tta gtg gat tgt tta tca gat gaa ttt tgt ctg gat gaa aag 2016
Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys 660
665 670 cga gaa ttg tcc gag aaa gtc aaa cat gcg aag cga ctc agt gat
gag 2064 Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser
Asp Glu 675 680 685 cgg aat tta ctt caa gat cca aac ttc aga ggg atc
aat aga caa cca 2112 Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly
Ile Asn Arg Gln Pro 690 695 700 gac cgt ggc tgg aga gga agt aca gat
att acc atc caa gga gga gat 2160 Asp Arg Gly Trp Arg Gly Ser Thr
Asp Ile Thr Ile Gln Gly Gly Asp 705 710 715 720 gac gta ttc aaa gag
aat tac gtc aca cta ccg ggt acc gtt gat gag 2208 Asp Val Phe Lys
Glu Asn Tyr Val Thr Leu Pro Gly Thr Val Asp Glu 725 730 735 tgc tat
cca acg tat tta tat cag aaa ata gat gag tcg aaa tta aaa 2256 Cys
Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys 740 745
750 gct tat acc cgt tat gaa tta aga ggg tat atc gaa gat agt caa gac
2304 Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln
Asp 755 760 765 tta gaa atc tat ttg atc cgt tac aat gca aaa cac gaa
ata gta aat 2352 Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His
Glu Ile Val Asn 770 775 780 gtg cca ggc acg ggt tcc tta tgg ccg ctt
tca gcc caa agt cca atc 2400 Val Pro Gly Thr Gly Ser Leu Trp Pro
Leu Ser Ala Gln Ser Pro Ile 785 790 795 800 gga aag tgt gga gaa ccg
aat cga tgc gcg cca cac ctt gaa tgg aat 2448 Gly Lys Cys Gly Glu
Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn 805 810 815 cct gat cta
gat tgt tcc tgc aga gac ggg gaa aaa tgt gca cat cat 2496 Pro Asp
Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His 820 825 830
tcc cat cat ttc acc ttg gat att gat gtt gga tgt aca gac tta aat
2544 Ser His His Phe Thr Leu Asp Ile Asp Val Gly Cys Thr Asp Leu
Asn 835 840 845 gag gac tta ggt gta tgg gtg ata ttc aag att aag acg
caa gat ggc 2592 Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys
Thr Gln Asp Gly 850 855 860 cat gca aga cta ggg aat cta gag ttt ctc
gaa gag aaa cca tta tta 2640 His Ala Arg Leu Gly Asn Leu Glu Phe
Leu Glu Glu Lys Pro Leu Leu 865 870 875 880 ggg gaa gca cta gct cgt
gtg aaa aga gcg gag aag aag tgg aga gac 2688 Gly Glu Ala Leu Ala
Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp 885 890 895 aaa cga gag
aaa ctg cag ttg gaa aca aat att gtt tat aaa gag gca 2736 Lys Arg
Glu Lys Leu Gln Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala 900 905 910
aaa gaa tct gta gat gct tta ttt gta aac tct caa tat gat aga tta
2784 Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg
Leu 915 920 925 caa gtg gat acg aac atc gca atg att cat gcg gca gat
aaa cgc gtt 2832 Gln Val Asp Thr Asn Ile Ala Met Ile His Ala Ala
Asp Lys Arg Val 930 935 940 cat aga atc cgg gaa gcg tat ctg cca gag
ttg tct gtg att cca ggt 2880 His Arg Ile Arg Glu Ala Tyr Leu Pro
Glu Leu Ser Val Ile Pro Gly 945 950 955 960 gtc aat gcg gcc att ttc
gaa gaa tta gag gga cgt att ttt aca gcg 2928 Val Asn Ala Ala Ile
Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala 965 970 975 tat tcc tta
tat gat gcg aga aat gtc att aaa aat ggc gat ttc aat 2976 Tyr Ser
Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn 980 985 990
aat ggc tta tta tgc tgg aac gtg aaa ggt cat gta gat gta gaa gag
3024 Asn Gly Leu Leu Cys Trp Asn Val Lys Gly His Val Asp Val Glu
Glu 995 1000 1005 caa aac aac cac cgt tcg gtc ctt gtt atc cca gaa
tgg gag gca 3069 Gln Asn Asn His Arg Ser Val Leu Val Ile Pro Glu
Trp Glu Ala 1010 1015 1020 gaa gtg tca caa gag gtt cgt gtc tgt cca
ggt cgt ggc tat atc 3114 Glu Val Ser Gln Glu Val Arg Val Cys Pro
Gly Arg Gly Tyr Ile 1025 1030 1035 ctt cgt gtc aca gca tat aaa gag
gga tat gga gag ggc tgc gta 3159 Leu Arg Val Thr Ala Tyr Lys Glu
Gly Tyr Gly Glu Gly Cys Val 1040 1045 1050 acg atc cat gag atc gaa
gac aat aca gac gaa ctg aaa ttc agc 3204 Thr Ile His Glu Ile Glu
Asp Asn Thr Asp Glu Leu Lys Phe Ser 1055 1060 1065 aac tgt gta gaa
gag gaa gta tat cca aac aac aca gta acg tgt 3249 Asn Cys Val Glu
Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys 1070 1075 1080 aat aat
tat act ggg act caa gaa gaa tat gag ggt acg tac act 3294 Asn Asn
Tyr Thr Gly Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr 1085 1090 1095
tct cgt aat caa gga tat gac gaa gcc tat ggt aat aac cct tcc 3339
Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr Gly Asn Asn Pro Ser 1100
1105 1110 gta cca gct gat tac gct tca gtc tat gaa gaa aaa tcg tat
aca 3384 Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr
Thr 1115 1120 1125 gat gga cga aga gag aat cct tgt gaa tct aac aga
ggc tat ggg 3429 Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg
Gly Tyr Gly 1130 1135 1140 gat tac aca cca cta ccg gct ggt tat gta
aca aag gat tta gag 3474 Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val
Thr Lys Asp Leu Glu 1145 1150 1155 tac ttc cca gag acc gat aag gta
tgg att gag atc gga gaa aca 3519 Tyr Phe Pro Glu Thr Asp Lys Val
Trp Ile Glu Ile Gly Glu Thr 1160 1165 1170 gaa gga aca ttc atc gtg
gat agc gtg gaa tta ctc ctt atg gag 3564 Glu Gly Thr Phe Ile Val
Asp Ser Val Glu Leu Leu Leu Met Glu 1175 1180 1185 gaa 3567 Glu 6
1189 PRT Artificial Sequence Recombinant Delta Endotoxin 6 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 Leu 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 Gly
Asn Asn Phe Asn Ile Tyr Val Glu Ala 100 105 110 Phe Lys Glu Trp Glu
Glu Asp Pro Asn Asn Pro Ala 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 Ala 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 1010
1015 1020 Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr
Ile 1025 1030 1035 Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu
Gly Cys Val 1040 1045 1050 Thr Ile His Glu Ile Glu Asp Asn Thr Asp
Glu Leu Lys Phe Ser 1055 1060 1065 Asn Cys Val Glu Glu Glu Val Tyr
Pro Asn Asn Thr Val Thr Cys 1070 1075 1080 Asn Asn Tyr Thr Gly Thr
Gln Glu Glu Tyr Glu Gly Thr Tyr Thr 1085 1090 1095 Ser Arg Asn Gln
Gly Tyr Asp Glu Ala Tyr Gly Asn Asn Pro Ser 1100 1105 1110 Val Pro
Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr 1115 1120 1125
Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly 1130
1135 1140 Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu
Glu 1145 1150 1155 Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile
Gly Glu Thr 1160 1165 1170 Glu Gly Thr Phe Ile Val Asp Ser Val Glu
Leu Leu Leu Met Glu 1175 1180 1185 Glu 7 3567 DNA Artificial
Sequence Recombinant Delta Endotoxin 7 atg gag gaa aat aat caa aat
caa tgc ata cct tac aat tgt tta agt 48 Met Glu Glu Asn Asn Gln Asn
Gln Cys Ile Pro Tyr Asn Cys Leu Ser 1 5 10 15 aat cct gaa gaa gta
ctt ttg gat gga gaa cgg ata tca act ggt aat 96 Asn Pro Glu Glu Val
Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn 20 25 30 tca tca att
gat att tct ctg tca ctt gtt cag ttt ctg gta tct aac 144 Ser Ser Ile
Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn 35 40 45 ttt
gta cca ggg gga gga ttt tta gtt gga tta ata gat ttt gta tgg 192 Phe
Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp 50 55
60 gga ata gtt ggc cct tct caa tgg gat gca ttt cta gta caa att gaa
240 Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu
65 70 75 80 caa tta att aat gaa aga ata gct gaa ttt gct agg aat gct
gct att 288 Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala
Ala Ile 85 90 95 gct aat tta gaa gga tta gga aac aat ttc aat ata
tat gtg gaa gca 336 Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile
Tyr Val Glu Ala 100 105 110 ttt aaa gaa tgg gaa gat gat cct cat aat
ccc aca acc agg acc aga 384 Phe Lys Glu Trp Glu Asp Asp Pro His Asn
Pro Thr Thr Arg Thr Arg 115 120 125 gta att gat cgc ttt cgt ata ctt
gat ggg cta ctt gaa agg gac att 432 Val Ile Asp Arg Phe Arg Ile Leu
Asp Gly Leu Leu Glu Arg Asp Ile 130 135 140 cct tcg ttt cga att tct
gga ttt gaa gta ccc ctt tta tcc gtt tat 480 Pro Ser Phe Arg Ile Ser
Gly Phe Glu Val Pro Leu Leu Ser Val Tyr 145 150 155 160 gct caa gcg
gcc aat ctg cat cta gct ata tta aga gat tct gta att 528 Ala Gln Ala
Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile 165 170 175 ttt
gga gaa aga tgg gga ttg aca acg ata aat gtc aat gaa aac tat 576 Phe
Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr 180 185
190 aat aga cta att agg cat att gat gaa tat gct gat cac tgt gca aat
624 Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn
195 200 205 acg tat aat cgg gga tta aat aat tta ccg aaa tct acg tat
caa gat 672 Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr
Gln Asp 210 215 220 tgg ata aca tat aat cga tta cgg aga
gac tta aca ttg act gta tta 720 Trp Ile Thr Tyr Asn Arg Leu Arg Arg
Asp Leu Thr Leu Thr Val Leu 225 230 235 240 gat atc gcc gct ttc ttt
cca aac tat gac aat agg aga tat cca att 768 Asp Ile Ala Ala Phe Phe
Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile 245 250 255 cag cca gtt ggt
caa cta aca agg gaa gtt tat acg gac cca tta att 816 Gln Pro Val Gly
Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile 260 265 270 aat ttt
aat cca cag tta cag tct gta gct caa tta cct act ttt aac 864 Asn Phe
Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn 275 280 285
gtt atg gag agc agc gca att aga aat cct cat tta ttt gat ata ttg 912
Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu 290
295 300 aat aat ctt aca atc ttt acg gat tgg ttt agt gtt gga cgc aat
ttt 960 Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn
Phe 305 310 315 320 tat tgg gga gga cat cga gta ata tct agc ctt ata
gga ggt ggt aac 1008 Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu
Ile Gly Gly Gly Asn 325 330 335 ata aca tct cct ata tat gga aga gag
gcg aac cag gag cct cca aga 1056 Ile Thr Ser Pro Ile Tyr Gly Arg
Glu Ala Asn Gln Glu Pro Pro Arg 340 345 350 tcc ttt act ttt aat gga
ccg gta ttt agg act tta tca aat cct act 1104 Ser Phe Thr Phe Asn
Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr 355 360 365 tta cga tta
tta cag caa cct tgg cca gcg cca cca ttt aat tta cgt 1152 Leu Arg
Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg 370 375 380
ggt gtt gaa gga gta gaa ttt tct aca cct aca aat agc ttt acg tat
1200 Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr
Tyr 385 390 395 400 cga gga aga ggt acg gtt gat tct tta act gaa tta
ccg cct gag gat 1248 Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu
Leu Pro Pro Glu Asp 405 410 415 aat agt gtg cca cct cgc gaa gga tat
agt cat cgt tta tgt cat gca 1296 Asn Ser Val Pro Pro Arg Glu Gly
Tyr Ser His Arg Leu Cys His Ala 420 425 430 act ttt gtt caa aga tct
gga aca cct ttt tta aca act ggt gta gta 1344 Thr Phe Val Gln Arg
Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val 435 440 445 ttt tct tgg
acg cat cgt agt gca act ctt aca aat aca att gat cca 1392 Phe Ser
Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro 450 455 460
gag aga att aat caa ata cct tta gtg aaa gga ttt aga gtt tgg ggg
1440 Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp
Gly 465 470 475 480 ggc acc tct gtc att aca gga cca gga ttt aca gga
ggg gat atc ctt 1488 Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr
Gly Gly Asp Ile Leu 485 490 495 cga aga aat acc ttt ggt gat ttt gta
tct cta caa gtc aat att aat 1536 Arg Arg Asn Thr Phe Gly Asp Phe
Val Ser Leu Gln Val Asn Ile Asn 500 505 510 tca cca att acc caa aga
tac cgt tta aga ttt cgt tac gct tcc agt 1584 Ser Pro Ile Thr Gln
Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser 515 520 525 agg gat gca
cga gtt ata gta tta aca gga gcg gca tcc aca gga gtg 1632 Arg Asp
Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val 530 535 540
gga ggc caa gtt agt gta aat atg cct ctt cag aaa act atg gaa ata
1680 Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu
Ile 545 550 555 560 ggg gag aac tta aca tct aga aca ttt aga tat acc
gat ttt agt aat 1728 Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr
Thr Asp Phe Ser Asn 565 570 575 cct ttt tca ttt aga gct aat cca gat
ata att ggg ata agt gaa caa 1776 Pro Phe Ser Phe Arg Ala Asn Pro
Asp Ile Ile Gly Ile Ser Glu Gln 580 585 590 cct cta ttt ggt gca ggt
tct att agt agc ggt gaa ctt tat ata gat 1824 Pro Leu Phe Gly Ala
Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp 595 600 605 aaa att gaa
att att cta gca gat gca aca ttt gaa gca gaa tct gat 1872 Lys Ile
Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp 610 615 620
tta gaa aga gca caa aag gcg gtg aat gcc ctg ttt act tct tcc aat
1920 Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser
Asn 625 630 635 640 caa atc ggg tta aaa acc gat gtg acg gat tat cat
att gat caa gta 1968 Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr
His Ile Asp Gln Val 645 650 655 tcc aat tta gtg gat tgt tta tca gat
gaa ttt tgt ctg gat gaa aag 2016 Ser Asn Leu Val Asp Cys Leu Ser
Asp Glu Phe Cys Leu Asp Glu Lys 660 665 670 cga gaa ttg tcc gag aaa
gtc aaa cat gcg aag cga ctc agt gat gag 2064 Arg Glu Leu Ser Glu
Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu 675 680 685 cgg aat tta
ctt caa gat cca aac ttc aga ggg atc aat aga caa cca 2112 Arg Asn
Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro 690 695 700
gac cgt ggc tgg aga gga agt aca gat att acc atc caa gga gga gat
2160 Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly
Asp 705 710 715 720 gac gta ttc aaa gag aat tac gtc aca cta ccg ggt
acc gtt gat gag 2208 Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro
Gly Thr Val Asp Glu 725 730 735 tgc tat cca acg tat tta tat cag aaa
ata gat gag tcg aaa tta aaa 2256 Cys Tyr Pro Thr Tyr Leu Tyr Gln
Lys Ile Asp Glu Ser Lys Leu Lys 740 745 750 gct tat acc cgt tat gaa
tta aga ggg tat atc gaa gat agt caa gac 2304 Ala Tyr Thr Arg Tyr
Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp 755 760 765 tta gaa atc
tat ttg atc cgt tac aat gca aaa cac gaa ata gta aat 2352 Leu Glu
Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val Asn 770 775 780
gtg cca ggc acg ggt tcc tta tgg ccg ctt tca gcc caa agt cca atc
2400 Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro
Ile 785 790 795 800 gga aag tgt gga gaa ccg aat cga tgc gcg cca cac
ctt gaa tgg aat 2448 Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro
His Leu Glu Trp Asn 805 810 815 cct gat cta gat tgt tcc tgc aga gac
ggg gaa aaa tgt gca cat cat 2496 Pro Asp Leu Asp Cys Ser Cys Arg
Asp Gly Glu Lys Cys Ala His His 820 825 830 tcc cat cat ttc acc ttg
gat att gat gtt gga tgt aca gac tta aat 2544 Ser His His Phe Thr
Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn 835 840 845 gag gac tta
ggt gta tgg gtg ata ttc aag att aag acg caa gat ggc 2592 Glu Asp
Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly 850 855 860
cat gca aga cta ggg aat cta gag ttt ctc gaa gag aaa cca tta tta
2640 His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu
Leu 865 870 875 880 ggg gaa gca cta gct cgt gtg aaa aga gcg gag aag
aag tgg aga gac 2688 Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu
Lys Lys Trp Arg Asp 885 890 895 aaa cga gag aaa ctg cag ttg gaa aca
aat att gtt tat aaa gag gca 2736 Lys Arg Glu Lys Leu Gln Leu Glu
Thr Asn Ile Val Tyr Lys Glu Ala 900 905 910 aaa gaa tct gta gat gct
tta ttt gta aac tct caa tat gat aga tta 2784 Lys Glu Ser Val Asp
Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu 915 920 925 caa gtg gat
acg aac atc gca atg att cat gcg gca gat aaa cgc gtt 2832 Gln Val
Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val 930 935 940
cat aga atc cgg gaa gcg tat ctg cca gag ttg tct gtg att cca ggt
2880 His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro
Gly 945 950 955 960 gtc aat gcg gcc att ttc gaa gaa tta gag gga cgt
att ttt aca gcg 2928 Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly
Arg Ile Phe Thr Ala 965 970 975 tat tcc tta tat gat gcg aga aat gtc
att aaa aat ggc gat ttc aat 2976 Tyr Ser Leu Tyr Asp Ala Arg Asn
Val Ile Lys Asn Gly Asp Phe Asn 980 985 990 aat ggc tta tta tgc tgg
aac gtg aaa ggt cat gta gat gta gaa gag 3024 Asn Gly Leu Leu Cys
Trp Asn Val Lys Gly His Val Asp Val Glu Glu 995 1000 1005 caa aac
aac cac cgt tcg gtc ctt gtt atc cca gaa tgg gag gca 3069 Gln Asn
Asn His Arg Ser Val Leu Val Ile Pro Glu Trp Glu Ala 1010 1015 1020
gaa gtg tca caa gag gtt cgt gtc tgt cca ggt cgt ggc tat atc 3114
Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile 1025
1030 1035 ctt cgt gtc aca gca tat aaa gag gga tat gga gag ggc tgc
gta 3159 Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys
Val 1040 1045 1050 acg atc cat gag atc gaa gac aat aca gac gaa ctg
aaa ttc agc 3204 Thr Ile His Glu Ile Glu Asp Asn Thr Asp Glu Leu
Lys Phe Ser 1055 1060 1065 aac tgt gta gaa gag gaa gta tat cca aac
aac aca gta acg tgt 3249 Asn Cys Val Glu Glu Glu Val Tyr Pro Asn
Asn Thr Val Thr Cys 1070 1075 1080 aat aat tat act ggg act caa gaa
gaa tat gag ggt acg tac act 3294 Asn Asn Tyr Thr Gly Thr Gln Glu
Glu Tyr Glu Gly Thr Tyr Thr 1085 1090 1095 tct cgt aat caa gga tat
gac gaa gcc tat ggt aat aac cct tcc 3339 Ser Arg Asn Gln Gly Tyr
Asp Glu Ala Tyr Gly Asn Asn Pro Ser 1100 1105 1110 gta cca gct gat
tac gct tca gtc tat gaa gaa aaa tcg tat aca 3384 Val Pro Ala Asp
Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr 1115 1120 1125 gat gga
cga aga gag aat cct tgt gaa tct aac aga ggc tat ggg 3429 Asp Gly
Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly 1130 1135 1140
gat tac aca cca cta ccg gct ggt tat gta aca aag gat tta gag 3474
Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu 1145
1150 1155 tac ttc cca gag acc gat aag gta tgg att gag atc gga gaa
aca 3519 Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu
Thr 1160 1165 1170 gaa gga aca ttc atc gtg gat agc gtg gaa tta ctc
ctt atg gag 3564 Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu
Leu Met Glu 1175 1180 1185 gaa 3567 Glu 8 1189 PRT Artificial
Sequence Recombinant Delta Endotoxin 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 Leu 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 Gly Asn Asn Phe Asn Ile
Tyr Val Glu Ala 100 105 110 Phe Lys Glu Trp Glu Asp Asp Pro His Asn
Pro Thr 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 1010 1015 1020 Glu Val Ser Gln Glu Val Arg Val Cys Pro
Gly Arg Gly Tyr Ile 1025 1030 1035 Leu Arg Val Thr Ala Tyr Lys Glu
Gly Tyr Gly Glu Gly Cys Val 1040 1045 1050 Thr Ile His Glu Ile Glu
Asp Asn Thr Asp Glu Leu Lys Phe Ser 1055 1060 1065 Asn Cys Val Glu
Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys 1070 1075 1080 Asn Asn
Tyr Thr Gly Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr 1085 1090 1095
Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr Gly Asn Asn Pro Ser 1100
1105 1110 Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr
Thr 1115 1120 1125 Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg
Gly Tyr Gly 1130 1135 1140 Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val
Thr Lys Asp Leu Glu 1145 1150 1155 Tyr Phe Pro Glu Thr Asp Lys Val
Trp Ile Glu Ile Gly Glu Thr 1160 1165 1170 Glu Gly Thr Phe Ile Val
Asp Ser Val Glu Leu Leu Leu Met Glu 1175 1180 1185 Glu 9 3567 DNA
Artificial Sequence Recombinant Delta Endotoxin 9 atg gag gaa aat
aat caa aat caa tgc ata cct tac aat tgt tta agt 48 Met Glu Glu Asn
Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser 1 5 10 15 aat cct
gaa gaa gta ctt ttg gat gga gaa cgg ata tca act ggt aat 96 Asn Pro
Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn 20 25 30
tca tca att gat att tct ctg tca ctt gtt cag ttt ctg gta tct aac 144
Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn 35
40 45 ttt gta cca ggg gga gga ttt tta gtt gga tta ata gat ttt gta
tgg 192 Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val
Trp 50 55 60 gga ata gtt ggc cct tct caa tgg gat gca ttt cta gta
caa att gaa 240 Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val
Gln Ile Glu 65 70 75 80 caa tta att aat gaa aga ata gct gaa ttt gct
agg aat gct gct att 288 Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala
Arg Asn Ala Ala Ile 85 90 95 gct aat tta gaa gga tta gga aac aat
ttc aat ata tat gtg gaa gca 336 Ala Asn Leu Glu Gly Leu Gly Asn Asn
Phe Asn Ile Tyr Val Glu Ala 100 105 110 ttt aaa gaa tgg gaa gta gat
cct aat aat cct gga acc agg acc aga 384 Phe Lys Glu Trp Glu Val Asp
Pro Asn Asn Pro Gly Thr Arg Thr Arg 115 120 125 gta att gat cgc ttt
cgt ata ctt gat ggg cta ctt gaa agg gac att 432 Val Ile Asp Arg Phe
Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile 130 135 140 cct tcg ttt
cga att tct gga ttt gaa gta ccc ctt tta tcc gtt tat 480 Pro Ser Phe
Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr 145 150 155 160
gct caa gcg gcc aat ctg cat cta gct ata tta aga gat tct gta att 528
Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile 165
170 175 ttt gga gaa aga tgg gga ttg aca acg ata aat gtc aat gaa aac
tat 576 Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn
Tyr 180 185 190 aat aga cta att agg cat att gat gaa tat gct gat cac
tgt gca aat 624 Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His
Cys Ala Asn 195 200 205 acg tat aat cgg gga tta aat aat tta ccg aaa
tct acg tat caa gat 672 Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys
Ser Thr Tyr Gln Asp 210 215 220 tgg ata aca tat aat cga tta cgg aga
gac tta aca ttg act gta tta 720 Trp Ile Thr Tyr Asn Arg Leu Arg Arg
Asp Leu Thr Leu Thr Val Leu 225 230 235 240 gat atc gcc gct ttc ttt
cca aac tat gac aat agg aga tat cca att 768 Asp Ile Ala Ala Phe Phe
Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile 245 250 255 cag cca gtt ggt
caa cta aca agg gaa gtt tat acg gac cca tta att 816 Gln Pro Val Gly
Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile 260 265 270 aat ttt
aat cca cag tta cag tct gta gct caa tta cct act ttt aac 864 Asn Phe
Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn 275 280 285
gtt atg gag agc agc gca att aga aat cct cat tta ttt gat ata ttg 912
Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu 290
295 300 aat aat ctt aca atc ttt acg gat tgg ttt agt gtt gga cgc aat
ttt 960 Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn
Phe 305 310 315 320 tat tgg gga gga cat cga gta ata tct agc ctt ata
gga ggt ggt aac 1008 Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu
Ile Gly Gly Gly Asn 325 330 335 ata aca tct cct ata tat gga aga gag
gcg aac cag gag cct cca aga 1056 Ile Thr Ser Pro Ile Tyr Gly Arg
Glu Ala Asn Gln Glu Pro Pro Arg 340 345 350 tcc ttt act ttt aat gga
ccg gta ttt agg act tta tca aat cct act 1104 Ser Phe Thr Phe Asn
Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr 355 360 365 tta cga tta
tta cag caa cct tgg cca gcg cca cca ttt aat tta cgt 1152 Leu Arg
Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg 370 375 380
ggt gtt gaa gga gta gaa ttt tct aca cct aca aat agc ttt acg tat
1200 Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr
Tyr 385 390 395 400 cga gga aga ggt acg gtt gat tct tta act gaa tta
ccg cct gag gat 1248 Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu
Leu Pro Pro Glu Asp 405 410 415 aat agt gtg cca cct cgc gaa gga tat
agt cat cgt tta tgt cat gca 1296 Asn Ser Val Pro Pro Arg Glu Gly
Tyr Ser His Arg Leu Cys His Ala 420 425 430 act ttt gtt caa aga tct
gga aca cct ttt tta aca act ggt gta gta 1344 Thr Phe Val Gln Arg
Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val 435 440 445 ttt tct tgg
acg cat cgt agt gca act ctt aca aat aca att gat cca 1392 Phe Ser
Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro 450 455 460
gag aga att aat caa ata cct tta gtg aaa gga ttt aga gtt tgg ggg
1440 Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp
Gly 465 470 475 480 ggc acc tct gtc att aca gga cca gga ttt aca gga
ggg gat atc ctt 1488 Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr
Gly Gly Asp Ile Leu 485 490 495 cga aga aat acc ttt ggt gat ttt gta
tct cta caa gtc aat att aat 1536 Arg Arg Asn Thr Phe Gly Asp Phe
Val Ser Leu Gln Val Asn Ile Asn 500 505 510 tca cca att acc caa aga
tac cgt tta aga ttt cgt tac gct tcc agt 1584 Ser Pro Ile Thr Gln
Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser 515 520 525 agg gat gca
cga gtt ata gta tta aca gga gcg gca tcc aca gga gtg 1632 Arg Asp
Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val 530 535 540
gga ggc caa gtt agt gta aat atg cct ctt cag aaa act atg gaa ata
1680 Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu
Ile 545 550 555 560 ggg gag aac tta aca tct aga aca ttt aga tat acc
gat ttt agt aat 1728 Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr
Thr Asp Phe Ser Asn 565 570 575 cct ttt tca ttt aga gct aat cca gat
ata att ggg ata agt gaa caa 1776 Pro Phe Ser Phe Arg Ala Asn Pro
Asp Ile Ile Gly Ile Ser Glu Gln 580 585 590 cct cta ttt ggt gca ggt
tct att agt agc ggt gaa ctt tat ata gat 1824 Pro Leu Phe Gly Ala
Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp 595 600 605 aaa att gaa
att att cta gca gat gca aca ttt gaa gca gaa tct gat 1872 Lys Ile
Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp 610 615 620
tta gaa aga gca caa aag gcg gtg aat gcc ctg ttt act tct tcc aat
1920 Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser
Asn 625 630 635 640 caa atc ggg tta aaa acc gat gtg acg gat tat cat
att gat caa gta 1968 Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr
His Ile Asp Gln Val 645 650 655 tcc aat tta gtg gat tgt tta tca gat
gaa ttt tgt ctg gat gaa aag 2016 Ser Asn Leu Val Asp Cys Leu Ser
Asp Glu Phe Cys Leu Asp Glu Lys 660 665 670 cga gaa ttg tcc gag aaa
gtc aaa cat gcg aag cga ctc agt gat gag 2064 Arg Glu Leu Ser Glu
Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu 675 680 685 cgg aat tta
ctt caa gat cca aac ttc aga ggg atc aat aga caa cca 2112 Arg Asn
Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro 690 695 700
gac cgt ggc tgg aga gga agt aca gat att acc atc caa gga gga gat
2160 Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly
Asp 705 710 715 720 gac gta ttc aaa gag aat tac gtc aca cta ccg ggt
acc gtt gat gag 2208 Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro
Gly Thr Val Asp Glu 725 730 735 tgc tat cca acg tat tta tat cag aaa
ata gat gag tcg aaa tta aaa 2256 Cys Tyr Pro Thr Tyr Leu Tyr Gln
Lys Ile Asp Glu Ser Lys Leu Lys 740 745 750 gct tat acc cgt tat gaa
tta aga ggg tat atc gaa gat agt caa gac 2304 Ala Tyr Thr Arg Tyr
Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp 755 760 765 tta gaa atc
tat ttg atc cgt tac aat gca aaa cac gaa ata gta aat 2352 Leu Glu
Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val Asn 770 775 780
gtg cca ggc acg ggt tcc tta tgg ccg ctt tca gcc caa agt cca atc
2400 Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro
Ile 785 790 795 800 gga aag tgt gga gaa ccg aat cga tgc gcg cca cac
ctt gaa tgg aat 2448 Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro
His Leu Glu Trp Asn 805 810 815 cct gat cta gat tgt tcc tgc aga gac
ggg gaa aaa tgt gca cat cat 2496 Pro Asp Leu Asp Cys Ser Cys Arg
Asp Gly Glu Lys Cys Ala His His 820 825 830 tcc cat cat ttc acc ttg
gat att gat gtt gga tgt aca gac tta aat 2544 Ser His His Phe Thr
Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn 835 840 845 gag gac tta
ggt gta tgg gtg ata ttc aag att aag acg caa gat ggc 2592 Glu Asp
Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly 850 855 860
cat gca aga cta ggg aat cta gag ttt ctc gaa gag aaa cca tta tta
2640 His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu
Leu 865 870 875 880 ggg gaa gca cta gct cgt gtg aaa aga gcg gag aag
aag tgg aga gac 2688 Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu
Lys Lys Trp Arg Asp 885 890 895 aaa cga gag aaa ctg cag ttg gaa aca
aat att gtt tat aaa gag gca 2736 Lys Arg Glu Lys Leu Gln Leu Glu
Thr Asn Ile Val Tyr Lys Glu Ala 900 905 910 aaa gaa tct gta gat gct
tta ttt gta aac tct caa tat gat aga tta 2784 Lys Glu Ser Val Asp
Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu 915 920 925 caa gtg gat
acg aac atc gca atg att cat gcg gca gat aaa cgc gtt 2832 Gln Val
Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val 930 935 940
cat aga atc cgg gaa gcg tat ctg cca gag ttg tct gtg att cca ggt
2880 His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro
Gly 945 950 955 960 gtc aat gcg gcc att ttc gaa gaa tta gag gga cgt
att ttt aca gcg 2928 Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly
Arg Ile Phe Thr Ala 965 970 975 tat tcc tta tat gat gcg aga aat gtc
att aaa aat ggc gat ttc aat 2976 Tyr Ser Leu Tyr Asp Ala Arg Asn
Val Ile Lys Asn Gly Asp Phe Asn 980 985 990 aat ggc tta tta tgc tgg
aac gtg aaa ggt cat gta gat gta gaa gag 3024 Asn Gly Leu Leu Cys
Trp Asn Val Lys Gly His Val Asp Val Glu Glu 995 1000 1005 caa aac
aac cac cgt tcg gtc ctt gtt atc cca gaa tgg gag gca 3069 Gln Asn
Asn His Arg Ser Val Leu Val Ile Pro Glu Trp Glu Ala 1010 1015 1020
gaa gtg tca caa gag gtt cgt gtc tgt cca ggt cgt ggc tat atc 3114
Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile 1025
1030 1035 ctt cgt gtc aca gca tat aaa gag gga tat gga gag ggc tgc
gta 3159 Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys
Val 1040 1045 1050 acg atc cat gag atc gaa gac aat aca gac gaa ctg
aaa ttc agc 3204 Thr Ile His Glu Ile Glu Asp Asn Thr Asp Glu Leu
Lys Phe Ser 1055 1060 1065 aac tgt gta gaa gag gaa gta tat cca aac
aac aca gta acg tgt 3249 Asn Cys Val Glu Glu Glu Val Tyr Pro Asn
Asn Thr Val Thr Cys 1070 1075 1080 aat aat tat act ggg act caa gaa
gaa tat gag ggt acg tac act 3294 Asn Asn Tyr Thr Gly Thr Gln Glu
Glu Tyr Glu Gly Thr Tyr Thr 1085 1090 1095 tct cgt aat caa gga tat
gac gaa gcc tat ggt aat aac cct tcc 3339 Ser Arg Asn Gln Gly Tyr
Asp Glu Ala Tyr Gly Asn Asn Pro Ser 1100 1105 1110 gta cca gct gat
tac gct tca gtc tat gaa gaa aaa tcg tat aca 3384 Val Pro Ala Asp
Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr 1115 1120 1125 gat gga
cga aga gag aat cct tgt gaa tct aac aga ggc tat ggg 3429 Asp Gly
Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly 1130 1135 1140
gat tac aca cca cta ccg gct ggt tat gta aca aag gat tta gag 3474
Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu 1145
1150 1155 tac ttc cca gag acc gat aag gta tgg att gag atc gga gaa
aca 3519 Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu
Thr 1160 1165 1170 gaa gga aca ttc atc gtg gat agc gtg gaa tta ctc
ctt atg gag 3564 Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu
Leu Met Glu 1175 1180 1185 gaa 3567 Glu 10 1189 PRT Artificial
Sequence Recombinant Delta Endotoxin 10 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 Leu 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 Gly Asn Asn Phe Asn Ile
Tyr Val Glu Ala 100 105 110 Phe Lys Glu Trp Glu Val Asp Pro Asn Asn
Pro Gly 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 1010 1015 1020 Glu Val Ser Gln Glu Val
Arg Val Cys Pro Gly Arg Gly Tyr Ile 1025 1030 1035 Leu Arg Val Thr
Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val 1040 1045 1050 Thr Ile
His Glu Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser 1055 1060 1065
Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys 1070
1075 1080 Asn Asn Tyr Thr Gly Thr Gln Glu Glu Tyr Glu Gly Thr Tyr
Thr 1085 1090 1095 Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr Gly Asn
Asn Pro Ser 1100 1105 1110 Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu
Glu Lys Ser Tyr Thr 1115 1120 1125 Asp Gly Arg Arg Glu Asn Pro Cys
Glu Ser Asn Arg Gly Tyr Gly 1130 1135 1140 Asp Tyr Thr Pro Leu Pro
Ala Gly Tyr Val Thr Lys Asp Leu Glu 1145 1150 1155 Tyr Phe Pro Glu
Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr 1160 1165 1170 Glu Gly
Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu 1175 1180 1185
Glu 11 3567 DNA Artificial Sequence Recombinant Delta Endotoxin 11
atg gag gaa aat aat caa aat caa tgc ata cct tac aat tgt tta agt 48
Met Glu Glu Asn Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser 1 5
10 15 aat cct gaa gaa gta ctt ttg gat gga gaa cgg ata tca act ggt
aat 96 Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly
Asn 20 25 30 tca tca att gat att tct ctg tca ctt gtt cag ttt ctg
gta tct aac 144 Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu
Val Ser Asn 35 40 45 ttt gta cca ggg gga gga ttt tta gtt gga tta
ata gat ttt gta tgg 192 Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu
Ile Asp Phe Val Trp 50 55 60 gga ata gtt ggc cct tct caa tgg gat
gca ttt cta gta caa att gaa 240 Gly Ile Val Gly Pro Ser Gln Trp Asp
Ala Phe Leu Val Gln Ile Glu 65 70 75 80 caa tta att aat gaa aga ata
gct gaa ttt gct agg aat gct gct att 288 Gln Leu Ile Asn Glu Arg Ile
Ala Glu Phe Ala Arg Asn Ala Ala Ile 85 90 95 gct aat tta gaa gga
tta gga aac aat ttc aat ata tat gtg gaa gca 336 Ala Asn Leu Glu Gly
Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala 100 105 110 ttt aaa gaa
tgg gaa gaa gat ccc cat aat cca gca acc agg acc aga 384 Phe Lys Glu
Trp Glu Glu Asp Pro His Asn Pro Ala Thr Arg Thr Arg 115 120 125 gta
att gat cgc ttt cgt ata ctt gat ggg cta ctt gaa agg gac att 432 Val
Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile 130 135
140 cct tcg ttt cga att tct gga ttt gaa gta ccc ctt tta tcc gtt tat
480 Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr
145 150 155 160 gct caa gcg gcc aat ctg cat cta gct ata tta aga gat
tct gta att 528 Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp
Ser Val Ile 165 170 175 ttt gga gaa aga tgg gga ttg aca acg ata aat
gtc aat gaa aac tat 576 Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn
Val Asn Glu Asn Tyr 180 185 190 aat aga cta att agg cat att gat gaa
tat gct gat cac tgt gca aat 624 Asn Arg Leu Ile Arg His Ile Asp Glu
Tyr Ala Asp His Cys Ala Asn 195 200 205 acg tat aat cgg gga tta aat
aat tta ccg aaa tct acg tat caa gat 672 Thr Tyr Asn Arg Gly Leu Asn
Asn Leu Pro Lys Ser Thr Tyr Gln Asp 210 215 220 tgg ata aca tat aat
cga tta cgg aga gac tta aca ttg act gta tta 720 Trp Ile Thr Tyr Asn
Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu 225 230 235 240 gat atc
gcc gct ttc ttt cca aac tat gac aat agg aga tat cca att 768 Asp Ile
Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile 245 250 255
cag cca gtt ggt caa cta aca agg gaa gtt tat acg gac cca tta att 816
Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile 260
265 270 aat ttt aat cca cag tta cag tct gta gct caa tta cct act ttt
aac 864 Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe
Asn 275 280 285 gtt atg gag agc agc gca att aga aat cct cat tta ttt
gat ata ttg 912 Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe
Asp Ile Leu 290 295 300 aat aat ctt aca atc ttt acg gat tgg ttt agt
gtt gga cgc aat ttt 960 Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser
Val Gly Arg Asn Phe 305 310 315 320 tat tgg gga gga cat cga gta ata
tct agc ctt ata gga ggt ggt aac 1008 Tyr Trp Gly Gly His Arg Val
Ile Ser Ser Leu Ile Gly Gly Gly Asn 325 330 335 ata aca tct cct ata
tat gga aga gag gcg aac cag gag cct cca aga 1056 Ile Thr Ser Pro
Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg 340 345 350 tcc ttt
act ttt aat gga ccg gta ttt agg act tta tca aat cct act 1104 Ser
Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr 355 360
365 tta cga tta tta cag caa cct tgg cca gcg cca cca ttt aat tta cgt
1152 Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu
Arg 370 375 380 ggt gtt gaa gga gta gaa ttt tct aca cct aca aat agc
ttt acg tat 1200 Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn
Ser Phe Thr Tyr 385 390 395 400 cga gga aga ggt acg gtt gat tct tta
act gaa tta ccg cct gag gat 1248 Arg Gly Arg Gly Thr Val Asp Ser
Leu Thr Glu Leu Pro Pro Glu Asp 405 410 415 aat agt gtg cca cct cgc
gaa gga tat agt cat cgt tta tgt cat gca 1296 Asn Ser Val Pro Pro
Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala 420 425 430 act ttt gtt
caa aga tct gga aca cct ttt tta aca act ggt gta gta 1344 Thr Phe
Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val 435 440 445
ttt tct tgg acg cat cgt agt gca act ctt aca aat aca att gat cca
1392 Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp
Pro 450 455 460 gag aga att aat caa ata cct tta gtg aaa gga ttt aga
gtt tgg ggg 1440 Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe
Arg Val Trp Gly 465 470 475 480 ggc acc tct gtc att aca gga cca gga
ttt aca gga ggg gat atc ctt 1488 Gly Thr Ser Val Ile Thr Gly Pro
Gly Phe Thr Gly Gly Asp Ile Leu 485 490 495 cga aga aat acc ttt ggt
gat ttt gta tct cta caa gtc aat att aat 1536 Arg Arg Asn Thr Phe
Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn 500 505 510 tca cca att
acc caa aga tac cgt tta aga ttt cgt tac gct tcc agt 1584 Ser Pro
Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser 515 520 525
agg gat gca cga gtt ata gta tta aca gga gcg gca tcc aca gga gtg
1632 Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly
Val 530 535 540 gga ggc caa gtt agt gta aat atg cct ctt cag aaa act
atg gaa ata 1680 Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys
Thr Met Glu Ile 545 550 555 560 ggg gag aac tta aca tct aga aca ttt
aga tat acc gat ttt agt aat 1728 Gly Glu Asn Leu Thr Ser Arg Thr
Phe Arg Tyr Thr Asp Phe Ser Asn 565 570 575 cct ttt tca ttt aga gct
aat cca gat ata att ggg ata agt gaa caa 1776 Pro Phe Ser Phe Arg
Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln 580 585 590 cct cta ttt
ggt gca ggt tct att agt agc ggt gaa ctt tat ata gat 1824 Pro Leu
Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp 595 600 605
aaa att gaa att att cta gca gat gca aca ttt gaa gca gaa tct gat
1872 Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser
Asp 610 615 620 tta gaa aga gca caa aag gcg gtg aat gcc ctg ttt act
tct tcc aat 1920 Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe
Thr Ser Ser Asn 625 630 635 640 caa atc ggg tta aaa acc gat gtg acg
gat tat cat att gat caa gta 1968 Gln Ile Gly Leu Lys Thr Asp Val
Thr Asp Tyr His Ile Asp Gln Val 645 650 655 tcc aat tta gtg gat tgt
tta tca gat gaa ttt tgt ctg gat gaa aag 2016 Ser Asn Leu Val Asp
Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys 660 665 670 cga gaa ttg
tcc gag aaa gtc aaa cat gcg aag cga ctc agt gat gag 2064 Arg Glu
Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu 675 680 685
cgg aat tta ctt caa gat cca aac ttc aga ggg atc aat aga caa cca
2112 Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln
Pro 690 695 700 gac cgt ggc tgg aga gga agt aca gat att acc atc caa
gga gga gat 2160 Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile
Gln Gly Gly Asp 705 710 715 720 gac gta ttc aaa gag aat tac gtc aca
cta ccg ggt acc gtt gat gag 2208 Asp Val Phe Lys Glu Asn Tyr Val
Thr Leu Pro Gly Thr Val Asp Glu 725 730 735 tgc tat cca acg tat tta
tat cag aaa ata gat gag tcg aaa tta aaa 2256 Cys Tyr Pro Thr Tyr
Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys 740 745 750 gct tat acc
cgt tat gaa tta aga ggg tat atc gaa gat agt caa gac 2304 Ala Tyr
Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp 755 760 765
tta gaa atc tat ttg atc cgt tac aat gca aaa cac gaa ata gta aat
2352 Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val
Asn 770 775 780 gtg cca ggc acg ggt tcc tta tgg ccg ctt tca gcc caa
agt cca atc 2400 Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala
Gln Ser Pro Ile 785 790 795 800 gga aag tgt gga gaa ccg aat cga tgc
gcg cca cac ctt gaa tgg aat 2448 Gly Lys Cys Gly Glu Pro Asn Arg
Cys Ala Pro His Leu Glu Trp Asn 805 810 815 cct gat cta gat tgt tcc
tgc aga gac ggg gaa aaa tgt gca cat cat 2496 Pro Asp Leu Asp Cys
Ser Cys Arg Asp Gly Glu Lys Cys Ala His His 820 825 830 tcc cat cat
ttc acc ttg gat att gat gtt gga tgt aca gac tta aat 2544 Ser His
His Phe Thr Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn 835 840 845
gag gac tta ggt gta tgg gtg ata ttc aag att aag acg caa gat ggc
2592 Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp
Gly 850 855 860 cat gca aga cta ggg aat cta gag ttt ctc gaa gag aaa
cca tta tta 2640 His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu
Lys Pro Leu Leu 865 870 875 880 ggg gaa gca cta gct cgt gtg aaa aga
gcg gag aag aag tgg aga gac 2688 Gly Glu
Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp 885 890 895
aaa cga gag aaa ctg cag ttg gaa aca aat att gtt tat aaa gag gca
2736 Lys Arg Glu Lys Leu Gln Leu Glu Thr Asn Ile Val Tyr Lys Glu
Ala 900 905 910 aaa gaa tct gta gat gct tta ttt gta aac tct caa tat
gat aga tta 2784 Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln
Tyr Asp Arg Leu 915 920 925 caa gtg gat acg aac atc gca atg att cat
gcg gca gat aaa cgc gtt 2832 Gln Val Asp Thr Asn Ile Ala Met Ile
His Ala Ala Asp Lys Arg Val 930 935 940 cat aga atc cgg gaa gcg tat
ctg cca gag ttg tct gtg att cca ggt 2880 His Arg Ile Arg Glu Ala
Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly 945 950 955 960 gtc aat gcg
gcc att ttc gaa gaa tta gag gga cgt att ttt aca gcg 2928 Val Asn
Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala 965 970 975
tat tcc tta tat gat gcg aga aat gtc att aaa aat ggc gat ttc aat
2976 Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe
Asn 980 985 990 aat ggc tta tta tgc tgg aac gtg aaa ggt cat gta gat
gta gaa gag 3024 Asn Gly Leu Leu Cys Trp Asn Val Lys Gly His Val
Asp Val Glu Glu 995 1000 1005 caa aac aac cac cgt tcg gtc ctt gtt
atc cca gaa tgg gag gca 3069 Gln Asn Asn His Arg Ser Val Leu Val
Ile Pro Glu Trp Glu Ala 1010 1015 1020 gaa gtg tca caa gag gtt cgt
gtc tgt cca ggt cgt ggc tat atc 3114 Glu Val Ser Gln Glu Val Arg
Val Cys Pro Gly Arg Gly Tyr Ile 1025 1030 1035 ctt cgt gtc aca gca
tat aaa gag gga tat gga gag ggc tgc gta 3159 Leu Arg Val Thr Ala
Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val 1040 1045 1050 acg atc cat
gag atc gaa gac aat aca gac gaa ctg aaa ttc agc 3204 Thr Ile His
Glu Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser 1055 1060 1065 aac
tgt gta gaa gag gaa gta tat cca aac aac aca gta acg tgt 3249 Asn
Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys 1070 1075
1080 aat aat tat act ggg act caa gaa gaa tat gag ggt acg tac act
3294 Asn Asn Tyr Thr Gly Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr
1085 1090 1095 tct cgt aat caa gga tat gac gaa gcc tat ggt aat aac
cct tcc 3339 Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr Gly Asn Asn
Pro Ser 1100 1105 1110 gta cca gct gat tac gct tca gtc tat gaa gaa
aaa tcg tat aca 3384 Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu
Lys Ser Tyr Thr 1115 1120 1125 gat gga cga aga gag aat cct tgt gaa
tct aac aga ggc tat ggg 3429 Asp Gly Arg Arg Glu Asn Pro Cys Glu
Ser Asn Arg Gly Tyr Gly 1130 1135 1140 gat tac aca cca cta ccg gct
ggt tat gta aca aag gat tta gag 3474 Asp Tyr Thr Pro Leu Pro Ala
Gly Tyr Val Thr Lys Asp Leu Glu 1145 1150 1155 tac ttc cca gag acc
gat aag gta tgg att gag atc gga gaa aca 3519 Tyr Phe Pro Glu Thr
Asp Lys Val Trp Ile Glu Ile Gly Glu Thr 1160 1165 1170 gaa gga aca
ttc atc gtg gat agc gtg gaa tta ctc ctt atg gag 3564 Glu Gly Thr
Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu 1175 1180 1185 gaa
3567 Glu 12 1189 PRT Artificial Sequence Recombinant Delta
Endotoxin 12 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 Leu 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 Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala 100 105
110 Phe Lys Glu Trp Glu Glu Asp Pro His Asn Pro Ala 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 1010 1015 1020 Glu Val Ser Gln Glu Val Arg Val Cys
Pro Gly Arg Gly Tyr Ile 1025 1030 1035 Leu Arg Val Thr Ala Tyr Lys
Glu Gly Tyr Gly Glu Gly Cys Val 1040 1045 1050 Thr Ile His Glu Ile
Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser 1055 1060 1065 Asn Cys Val
Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys 1070 1075 1080 Asn
Asn Tyr Thr Gly Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr 1085 1090
1095 Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr Gly Asn Asn Pro Ser
1100 1105 1110 Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser
Tyr Thr 1115 1120 1125 Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn
Arg Gly Tyr Gly 1130 1135 1140 Asp Tyr Thr Pro Leu Pro Ala Gly Tyr
Val Thr Lys Asp Leu Glu 1145 1150 1155 Tyr Phe Pro Glu Thr Asp Lys
Val Trp Ile Glu Ile Gly Glu Thr 1160 1165 1170 Glu Gly Thr Phe Ile
Val Asp Ser Val Glu Leu Leu Leu Met Glu 1175 1180 1185 Glu 13 49
DNA Artificial Sequence Synthetic oligonucleotide 13 gcatttaaag
aatgggaaga agataataat ccagcaacca ggaccagag 49 14 55 DNA Artificial
Sequence Synthetic oligonucleotide 14 gcatttaaag aatgggaaga
agatcctaat gcaaatccag caaccaggac cagag 55 15 17 DNA Artificial
Sequence Synthetic oligonucleotide 15 cccgatcggc cgcatgc 17 16 51
DNA Artificial Sequence Synthetic oligonucleotide 16 gcatttaaag
aatgggaagg gatcctagga atccagcaac caggaccaga g 51 17 30 DNA
Artificial Sequence Synthetic oligonucleotide 17 gagctcttgt
taaaaaaggt gttccagatc 30 18 63 DNA Artificial Sequence Synthetic
oligonucleotide 18 gcatttaaag aatgggaann nnnnnnnnnn nnnnnnnnna
ccaggaccag agtaattgat 60 cgc 63 19 55 DNA Artificial Sequence
Synthetic oligonucleotide 19 gggctacttg aaagggacat tccttcgttt
gcaatttctg gatttgaagt acccc 55 20 39 DNA Artificial Sequence
Synthetic oligonucleotide 20 ccaagaaaat actagagctc ttgttaaaaa
aggtgttcc 39 21 50 DNA Artificial Sequence Synthetic
oligonucleotide 21 gagattctgt aatttttgga gaagcatggg ggttgacaac
gataaatgtc 50 22 63 DNA Artificial Sequence Synthetic
oligonucleotide 22 gcatttaaag aatgggaaga agatcctaat aatccagcaa
ccaggaccag agtaattgat 60 cgc 63 23 7 PRT Artificial Sequence
Recombinant Delta Endotoxin 23 Glu Asp Pro Asn Asn Pro Ala 1 5 24
51 DNA Artificial Sequence Recombinant Delta Endotoxin 24
gcatttaaag aatgggaagg gatcctagga atccagcaac caggaccaga g 51 25 63
DNA Artificial Sequence Recombinant Delta Endotoxin 25 gcatttaaag
aatgggaaga tgatcctcat aatcccacaa ccaggaccag agtaattgat 60 cgc 63 26
7 PRT Artificial Sequence Recombinant Delta Endotoxin 26 Asp Asp
Pro His Asn Pro Thr 1 5 27 7 PRT Artificial Sequence Recombinant
Delta Endotoxin 27 Val Asp Pro Asn Asn Pro Gly 1 5 28 50 PRT
Artificial Sequence Recombinant Delta Endotoxin 28 Thr Asn Pro Ala
Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met 1 5 10 15 Asn Ser
Ala Leu Thr Thr Ala Ile Pro Leu Leu Ala Val Gln Asn Tyr 20 25 30
Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu 35
40 45 Ser Val 50 29 50 PRT Artificial Sequence Recombinant Delta
Endotoxin 29 Thr Asn Pro Ala Leu Thr Glu Glu Met Arg Ile Gln Phe
Asn Asp Met 1 5 10 15 Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe
Thr Val Gln Asn Tyr 20 25 30 Gln Val Pro Leu Leu Ser Val Tyr Val
Gln Ala Ala Asn Leu His Leu 35 40 45 Ser Val 50 30 50 PRT
Artificial Sequence Recombinant Delta Endotoxin 30 Thr Asn Pro Ala
Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met 1 5 10 15 Asn Ser
Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr 20 25 30
Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu 35
40 45 Ser Val 50 31 50 PRT Artificial Sequence Recombinant Delta
Endotoxin 31 Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe
Asn Asp Met 1 5 10 15 Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe
Thr Val Gln Asn Tyr 20 25 30 Gln Val Pro Leu Leu Ser Val Tyr Val
Gln Ala Val Asn Leu His Leu 35 40 45 Ser Val 50 32 50 PRT
Artificial Sequence Recombinant Delta Endotoxin 32 Thr Asn Pro Ala
Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met 1 5 10 15 Asn Ser
Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr 20 25 30
Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu 35
40 45 Ser Val 50 33 50 PRT Artificial Sequence Recombinant Delta
Endotoxin 33 Asn Asn Ala Gln Leu Arg Glu Asp Val Arg Ile Arg Phe
Ala Asn Thr 1 5 10 15 Asp Asp Ala Leu Ile Thr Ala Ile Asn Asn Phe
Thr Leu Thr Ser Phe 20 25 30 Glu Ile Pro Leu Leu Ser Val Tyr Val
Gln Ala Ala Asn Leu His Leu 35 40 45 Ser Leu 50 34 50 PRT
Artificial Sequence Recombinant Delta Endotoxin 34 Asn Asn Ala Gln
Leu Arg Glu Asp Val Arg Ile Arg Phe Ala Asn Thr 1 5 10 15 Asp Asp
Ala Leu Ile Thr Ala Ile Asn Asn Phe Thr Leu Thr Ser Phe 20 25 30
Glu Ile Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu 35
40 45 Ser Leu 50 35 50 PRT Artificial Sequence Recombinant Delta
Endotoxin 35 Asn Asn Pro Ala Ser Gln Glu Arg Val Arg Thr Arg Phe
Arg Leu Thr 1 5 10 15 Asp Asp Ala Ile Val Thr Gly Leu Pro Thr Leu
Ala Ile Arg
Asn Leu 20 25 30 Glu Val Val Asn Leu Ser Val Tyr Thr Gln Ala Ala
Asn Leu His Leu 35 40 45 Ser Leu 50 36 50 PRT Artificial Sequence
Recombinant Delta Endotoxin 36 Asn Asn Pro Glu Thr Arg Thr Arg Val
Ile Asp Arg Phe Arg Ile Leu 1 5 10 15 Asp Gly Leu Leu Glu Arg Asp
Ile Pro Ser Phe Arg Ile Ser Gly Phe 20 25 30 Glu Val Pro Leu Leu
Ser Val Tyr Ala Gln Ala Ala Asn Leu His Leu 35 40 45 Ala Ile 50 37
50 PRT Artificial Sequence Recombinant Delta Endotoxin 37 Asp Asn
Pro Val Thr Arg Thr Arg Val Val Asp Arg Phe Arg Ile Leu 1 5 10 15
Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile Ala Gly Phe 20
25 30 Glu Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Ala Asn Leu His
Leu 35 40 45 Ala Ile 50 38 50 PRT Artificial Sequence Recombinant
Delta Endotoxin 38 Thr Asn Pro Ala Leu Lys Glu Glu Met Arg Thr Gln
Phe Asn Asp Met 1 5 10 15 Asn Ser Ile Leu Val Thr Ala Ile Pro Leu
Phe Ser Val Gln Asn Tyr 20 25 30 Gln Val Pro Phe Leu Ser Val Tyr
Val Gln Ala Ala Asn Leu His Leu 35 40 45 Ser Val 50 39 50 PRT
Artificial Sequence Recombinant Delta Endotoxin 39 Thr Asn Pro Ala
Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met 1 5 10 15 Asn Ser
Ala Leu Thr Thr Ala Ile Pro Leu Phe Ser Val Gln Gly Tyr 20 25 30
Glu Ile Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu 35
40 45 Ser Val 50 40 50 PRT Artificial Sequence Recombinant Delta
Endotoxin 40 Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe
Asn Asp Met 1 5 10 15 Asn Ser Ala Leu Ile Thr Ala Ile Pro Leu Phe
Arg Val Gln Asn Tyr 20 25 30 Glu Val Ala Leu Leu Ser Val Tyr Val
Gln Ala Ala Asn Leu His Leu 35 40 45 Ser Ile 50 41 50 PRT
Artificial Sequence Recombinant Delta Endotoxin 41 Ser Asn Pro Ala
Leu Arg Glu Glu Met Arg Thr Gln Phe Asn Val Met 1 5 10 15 Asn Ser
Ala Leu Ile Ala Ala Ile Pro Leu Leu Arg Val Arg Asn Tyr 20 25 30
Glu Val Ala Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu 35
40 45 Ser Val 50 42 50 PRT Artificial Sequence Recombinant Delta
Endotoxin 42 Asn Asn Glu Ala Leu Gln Gln Asp Val Arg Asn Arg Phe
Ser Asn Thr 1 5 10 15 Asp Asn Ala Leu Ile Thr Ala Ile Pro Ile Leu
Arg Glu Gln Gly Phe 20 25 30 Glu Ile Pro Leu Leu Ser Val Tyr Val
Gln Ala Ala Asn Leu His Leu 35 40 45 Ser Leu 50 43 50 PRT
Artificial Sequence Recombinant Delta Endotoxin 43 Asn Asn Glu Ser
Leu Gln Gln Asp Val Arg Asn Arg Phe Ser Asn Thr 1 5 10 15 Asp Asn
Ala Leu Ile Thr Ala Ile Pro Ile Leu Arg Glu Gln Gly Phe 20 25 30
Glu Ile Pro Leu Leu Thr Val Tyr Val Gln Ala Ala Asn Leu His Leu 35
40 45 Ser Leu 50 44 50 PRT Artificial Sequence Recombinant Delta
Endotoxin 44 Asp Asn Glu Ala Ala Lys Ser Arg Val Ile Asp Arg Phe
Arg Ile Leu 1 5 10 15 Asp Gly Leu Ile Glu Ala Asn Ile Pro Ser Phe
Arg Ile Ile Gly Phe 20 25 30 Glu Val Pro Leu Leu Ser Val Tyr Val
Gln Ala Ala Asn Leu His Leu 35 40 45 Ala Leu 50 45 50 PRT
Artificial Sequence Recombinant Delta Endotoxin 45 Asp Asn Thr Ala
Ala Arg Ser Arg Val Thr Glu Arg Phe Arg Ile Ile 1 5 10 15 Asp Ala
Gln Ile Glu Ala Asn Ile Pro Ser Phe Arg Ile Pro Gly Phe 20 25 30
Glu Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Ala Asn Leu His Leu 35
40 45 Ala Leu 50 46 50 PRT Artificial Sequence Recombinant Delta
Endotoxin 46 Asp Asp Ala Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr
Ile Ala Leu 1 5 10 15 Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe
Ala Ile Arg Asn Gln 20 25 30 Glu Val Pro Leu Leu Met Val Tyr Ala
Gln Ala Ala Asn Leu His Leu 35 40 45 Leu Leu 50 47 50 PRT
Artificial Sequence Recombinant Delta Endotoxin 47 Asn Asp Ala Arg
Ser Arg Ser Ile Ile Leu Glu Arg Tyr Val Ala Leu 1 5 10 15 Glu Leu
Asp Ile Thr Thr Ala Ile Pro Leu Phe Arg Ile Arg Asn Glu 20 25 30
Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His Leu 35
40 45 Leu Leu 50 48 50 PRT Artificial Sequence Recombinant Delta
Endotoxin 48 Asn Asp Ala Arg Ser Arg Ser Ile Ile Leu Glu Arg Tyr
Val Ala Leu 1 5 10 15 Glu Leu Asp Ile Thr Thr Ala Ile Pro Leu Phe
Arg Ile Arg Asn Glu 20 25 30 Glu Val Pro Leu Leu Met Val Tyr Ala
Gln Ala Ala Asn Leu His Leu 35 40 45 Leu Leu 50 49 50 PRT
Artificial Sequence Recombinant Delta Endotoxin 49 Asn Asp Ala Arg
Ser Arg Ser Ile Ile Arg Glu Arg Tyr Ile Ala Leu 1 5 10 15 Glu Leu
Asp Ile Thr Thr Ala Ile Pro Leu Phe Ser Ile Arg Asn Glu 20 25 30
Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His Leu 35
40 45 Leu Leu 50 50 50 PRT Artificial Sequence Recombinant Delta
Endotoxin 50 Asn Asn Thr Arg Ala Arg Ser Val Val Lys Ser Gln Tyr
Ile Ala Leu 1 5 10 15 Glu Leu Met Phe Val Gln Lys Leu Pro Ser Phe
Ala Val Ser Gly Glu 20 25 30 Glu Val Pro Leu Leu Pro Ile Tyr Ala
Gln Ala Ala Asn Leu His Leu 35 40 45 Leu Leu 50 51 50 PRT
Artificial Sequence Recombinant Delta Endotoxin 51 Asn Asn Thr Arg
Ala Arg Ser Val Val Lys Asn Gln Tyr Ile Ala Leu 1 5 10 15 Glu Leu
Met Phe Val Gln Lys Leu Pro Ser Phe Ala Val Ser Gly Glu 20 25 30
Glu Val Pro Leu Leu Pro Ile Tyr Ala Gln Ala Ala Asn Leu His Leu 35
40 45 Leu Leu 50 52 22 DNA Artificial Sequence Synthetic
oligonucleotide 52 ggatccctcg agctgcagga gc 22 53 55 DNA Artificial
Sequence Synthetic oligonucleotide 53 gggctacttg aaagggacat
tccttcgttt nnnatttctg gatttgaagt acccc 55 54 63 DNA Artificial
Sequence Recombinant Delta Endotoxin 54 gcatttaaag aatgggaagt
agatcctaat aatcctggaa ccaggaccag agtaattgat 60 cgc 63 55 7 PRT
Artificial Sequence Recombinant Delta Endotoxin 55 Val Asp Pro Asn
Asn Pro Gly 1 5 56 63 DNA Artificial Sequence Recombinant Delta
Endotoxin 56 gcatttaaag aatgggaaga agatccccat aatccagcaa ccaggaccag
agtaattgat 60 cgc 63 57 7 PRT Artificial Sequence Recombinant Delta
Endotoxin 57 Glu Asp Pro His Asn Pro Ala 1 5 58 3567 DNA Artificial
Sequence Recombinant Delta Endotoxin 58 atg gag gaa aat aat caa aat
caa tgc ata cct tac aat tgt tta agt 48 Met Glu Glu Asn Asn Gln Asn
Gln Cys Ile Pro Tyr Asn Cys Leu Ser 1 5 10 15 aat cct gaa gaa gta
ctt ttg gat gga gaa cgg ata tca act ggt aat 96 Asn Pro Glu Glu Val
Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn 20 25 30 tca tca att
gat att tct ctg tca ctt gtt cag ttt ctg gta tct aac 144 Ser Ser Ile
Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn 35 40 45 ttt
gta cca ggg gga gga ttt tta gtt gga tta ata gat ttt gta tgg 192 Phe
Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp 50 55
60 gga ata gtt ggc cct tct caa tgg gat gca ttt cta gta caa att gaa
240 Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu
65 70 75 80 caa tta att aat gaa aga ata gct gaa ttt gct agg aat gct
gct att 288 Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala
Ala Ile 85 90 95 gct aat tta gaa gga tta gga aac aat ttc aat ata
tat gtg gaa gca 336 Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile
Tyr Val Glu Ala 100 105 110 ttt aaa gaa tgg gaa gaa gat cct aat aat
cca gca acc agg acc aga 384 Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn
Pro Ala Thr Arg Thr Arg 115 120 125 gta att gat cgc ttt cgt ata ctt
gat ggg cta ctt gaa agg gac att 432 Val Ile Asp Arg Phe Arg Ile Leu
Asp Gly Leu Leu Glu Arg Asp Ile 130 135 140 cct tcg ttt gca att tct
gga ttt gaa gta ccc ctt tta tcc gtt tat 480 Pro Ser Phe Ala Ile Ser
Gly Phe Glu Val Pro Leu Leu Ser Val Tyr 145 150 155 160 gct caa gcg
gcc aat ctg cat cta gct ata tta aga gat tct gta att 528 Ala Gln Ala
Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile 165 170 175 ttt
gga gaa aga tgg gga ttg aca acg ata aat gtc aat gaa aac tat 576 Phe
Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr 180 185
190 aat aga cta att agg cat att gat gaa tat gct gat cac tgt gca aat
624 Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn
195 200 205 acg tat aat cgg gga tta aat aat tta ccg gct agc acg tat
caa gat 672 Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Ala Ser Thr Tyr
Gln Asp 210 215 220 tgg ata aca tat aat cga tta cgg aga gac tta aca
ttg act gta tta 720 Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr
Leu Thr Val Leu 225 230 235 240 gat atc gcc gct ttc ttt cca aac tat
gac aat agg aga tat cca att 768 Asp Ile Ala Ala Phe Phe Pro Asn Tyr
Asp Asn Arg Arg Tyr Pro Ile 245 250 255 cag cca gtt ggt caa cta aca
agg gaa gtt tat acg gac cca tta att 816 Gln Pro Val Gly Gln Leu Thr
Arg Glu Val Tyr Thr Asp Pro Leu Ile 260 265 270 aat ttt aat cca cag
tta cag tct gta gct caa tta cct act ttt aac 864 Asn Phe Asn Pro Gln
Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn 275 280 285 gtt atg gag
agc agc gca att aga aat cct cat tta ttt gat ata ttg 912 Val Met Glu
Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu 290 295 300 aat
aat ctt aca atc ttt acg gat tgg ttt agt gtt gga cgc aat ttt 960 Asn
Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe 305 310
315 320 tat tgg gga gga cat cga gta ata tct agc ctt ata gga ggt ggt
aac 1008 Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly
Gly Asn 325 330 335 ata aca tct cct ata tat gga aga gag gcg aac cag
gag cct cca aga 1056 Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn
Gln Glu Pro Pro Arg 340 345 350 tcc ttt act ttt aat gga ccg gta ttt
agg act tta tca aat cct act 1104 Ser Phe Thr Phe Asn Gly Pro Val
Phe Arg Thr Leu Ser Asn Pro Thr 355 360 365 tta cga tta tta cag caa
cct tgg cca gcg cca cca ttt aat tta cgt 1152 Leu Arg Leu Leu Gln
Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg 370 375 380 ggt gtt gaa
gga gta gaa ttt tct aca cct aca aat agc ttt acg tat 1200 Gly Val
Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr 385 390 395
400 cga gga aga ggt acg gtt gat tct tta act gaa tta ccg cct gag gat
1248 Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu
Asp 405 410 415 aat agt gtg cca cct cgc gaa gga tat agt cat cgt tta
tgt cat gca 1296 Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg
Leu Cys His Ala 420 425 430 act ttt gtt caa aga tct gga aca cct ttt
tta aca act ggt gta gta 1344 Thr Phe Val Gln Arg Ser Gly Thr Pro
Phe Leu Thr Thr Gly Val Val 435 440 445 ttt tct tgg acg cat cgt agt
gca act ctt aca aat aca att gat cca 1392 Phe Ser Trp Thr His Arg
Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro 450 455 460 gag aga att aat
caa ata cct tta gtg aaa gga ttt aga gtt tgg ggg 1440 Glu Arg Ile
Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly 465 470 475 480
ggc acc tct gtc att aca gga cca gga ttt aca gga ggg gat atc ctt
1488 Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile
Leu 485 490 495 cga aga aat acc ttt ggt gat ttt gta tct cta caa gtc
aat att aat 1536 Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln
Val Asn Ile Asn 500 505 510 tca cca att acc caa aga tac cgt tta aga
ttt cgt tac gct tcc agt 1584 Ser Pro Ile Thr Gln Arg Tyr Arg Leu
Arg Phe Arg Tyr Ala Ser Ser 515 520 525 agg gat gca cga gtt ata gta
tta aca gga gcg gca tcc aca gga gtg 1632 Arg Asp Ala Arg Val Ile
Val Leu Thr Gly Ala Ala Ser Thr Gly Val 530 535 540 gga ggc caa gtt
agt gta aat atg cct ctt cag aaa act atg gaa ata 1680 Gly Gly Gln
Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile 545 550 555 560
ggg gag aac tta aca tct aga aca ttt aga tat acc gat ttt agt aat
1728 Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser
Asn 565 570 575 cct ttt tca ttt aga gct aat cca gat ata att ggg ata
agt gaa caa 1776 Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly
Ile Ser Glu Gln 580 585 590 cct cta ttt ggt gca ggt tct att agt agc
ggt gaa ctt tat ata gat 1824 Pro Leu Phe Gly Ala Gly Ser Ile Ser
Ser Gly Glu Leu Tyr Ile Asp 595 600 605 aaa att gaa att att cta gca
gat gca aca ttt gaa gca gaa tct gat 1872 Lys Ile Glu Ile Ile Leu
Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp 610 615 620 tta gaa aga gca
caa aag gcg gtg aat gcc ctg ttt act tct tcc aat 1920 Leu Glu Arg
Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn 625 630 635 640
caa atc ggg tta aaa acc gat gtg acg gat tat cat att gat caa gta
1968 Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln
Val 645 650 655 tcc aat tta gtg gat tgt tta tca gat gaa ttt tgt ctg
gat gaa aag 2016 Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys
Leu Asp Glu Lys 660 665 670 cga gaa ttg tcc gag aaa gtc aaa cat gcg
aag cga ctc agt gat gag 2064 Arg Glu Leu Ser Glu Lys Val Lys His
Ala Lys Arg Leu Ser Asp Glu 675 680 685 cgg aat tta ctt caa gat cca
aac ttc aga ggg atc aat aga caa cca 2112 Arg Asn Leu Leu Gln Asp
Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro 690 695 700 gac cgt ggc tgg
aga gga agt aca gat att acc atc caa gga gga gat 2160 Asp Arg Gly
Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp 705 710 715 720
gac gta ttc aaa gag aat tac gtc aca cta ccg ggt acc gtt gat gag
2208 Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Val Asp
Glu 725 730 735 tgc tat cca acg tat tta tat cag aaa ata gat gag tcg
aaa tta aaa 2256 Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu
Ser Lys Leu Lys 740 745 750 gct tat acc cgt tat gaa tta aga ggg tat
atc gaa gat agt caa gac 2304 Ala Tyr Thr Arg Tyr Glu Leu Arg Gly
Tyr Ile Glu Asp Ser Gln Asp 755 760 765 tta gaa atc tat ttg atc cgt
tac aat gca aaa cac gaa ata gta aat 2352 Leu Glu Ile Tyr Leu Ile
Arg Tyr Asn Ala Lys His Glu Ile Val Asn 770 775 780 gtg cca ggc acg
ggt tcc tta tgg ccg ctt tca gcc caa agt cca atc 2400 Val Pro Gly
Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile 785 790 795 800
gga aag tgt gga gaa ccg aat cga tgc gcg cca cac ctt gaa tgg aat
2448 Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp
Asn 805 810 815 cct gat cta gat tgt tcc tgc aga gac ggg gaa aaa tgt
gca cat cat 2496 Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys
Cys Ala His His 820 825 830 tcc cat cat ttc acc ttg gat att gat gtt
gga tgt aca gac tta aat 2544 Ser His His Phe Thr Leu Asp
Ile Asp Val Gly Cys Thr Asp Leu Asn 835 840 845 gag gac tta ggt gta
tgg gtg ata ttc aag att aag acg caa gat ggc 2592 Glu Asp Leu Gly
Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly 850 855 860 cat gca
aga cta ggg aat cta gag ttt ctc gaa gag aaa cca tta tta 2640 His
Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu 865 870
875 880 ggg gaa gca cta gct cgt gtg aaa aga gcg gag aag aag tgg aga
gac 2688 Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp
Arg Asp 885 890 895 aaa cga gag aaa ctg cag ttg gaa aca aat att gtt
tat aaa gag gca 2736 Lys Arg Glu Lys Leu Gln Leu Glu Thr Asn Ile
Val Tyr Lys Glu Ala 900 905 910 aaa gaa tct gta gat gct tta ttt gta
aac tct caa tat gat aga tta 2784 Lys Glu Ser Val Asp Ala Leu Phe
Val Asn Ser Gln Tyr Asp Arg Leu 915 920 925 caa gtg gat acg aac atc
gca atg att cat gcg gca gat aaa cgc gtt 2832 Gln Val Asp Thr Asn
Ile Ala Met Ile His Ala Ala Asp Lys Arg Val 930 935 940 cat aga atc
cgg gaa gcg tat ctg cca gag ttg tct gtg att cca ggt 2880 His Arg
Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly 945 950 955
960 gtc aat gcg gcc att ttc gaa gaa tta gag gga cgt att ttt aca gcg
2928 Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr
Ala 965 970 975 tat tcc tta tat gat gcg aga aat gtc att aaa aat ggc
gat ttc aat 2976 Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn
Gly Asp Phe Asn 980 985 990 aat ggc tta tta tgc tgg aac gtg aaa ggt
cat gta gat gta gaa gag 3024 Asn Gly Leu Leu Cys Trp Asn Val Lys
Gly His Val Asp Val Glu Glu 995 1000 1005 caa aac aac cac cgt tcg
gtc ctt gtt atc cca gaa tgg gag gca 3069 Gln Asn Asn His Arg Ser
Val Leu Val Ile Pro Glu Trp Glu Ala 1010 1015 1020 gaa gtg tca caa
gag gtt cgt gtc tgt cca ggt cgt ggc tat atc 3114 Glu Val Ser Gln
Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile 1025 1030 1035 ctt cgt
gtc aca gca tat aaa gag gga tat gga gag ggc tgc gta 3159 Leu Arg
Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val 1040 1045 1050
acg atc cat gag atc gaa gac aat aca gac gaa ctg aaa ttc agc 3204
Thr Ile His Glu Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser 1055
1060 1065 aac tgt gta gaa gag gaa gta tat cca aac aac aca gta acg
tgt 3249 Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr
Cys 1070 1075 1080 aat aat tat act ggg act caa gaa gaa tat gag ggt
acg tac act 3294 Asn Asn Tyr Thr Gly Thr Gln Glu Glu Tyr Glu Gly
Thr Tyr Thr 1085 1090 1095 tct cgt aat caa gga tat gac gaa gcc tat
ggt aat aac cct tcc 3339 Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr
Gly Asn Asn Pro Ser 1100 1105 1110 gta cca gct gat tac gct tca gtc
tat gaa gaa aaa tcg tat aca 3384 Val Pro Ala Asp Tyr Ala Ser Val
Tyr Glu Glu Lys Ser Tyr Thr 1115 1120 1125 gat gga cga aga gag aat
cct tgt gaa tct aac aga ggc tat ggg 3429 Asp Gly Arg Arg Glu Asn
Pro Cys Glu Ser Asn Arg Gly Tyr Gly 1130 1135 1140 gat tac aca cca
cta ccg gct ggt tat gta aca aag gat tta gag 3474 Asp Tyr Thr Pro
Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu 1145 1150 1155 tac ttc
cca gag acc gat aag gta tgg att gag atc gga gaa aca 3519 Tyr Phe
Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr 1160 1165 1170
gaa gga aca ttc atc gtg gat agc gtg gaa tta ctc ctt atg gag 3564
Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu 1175
1180 1185 gaa 3567 Glu 59 1189 PRT Artificial Sequence Recombinant
Delta Endotoxin 59 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 Leu 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 Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala 100
105 110 Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Ala 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 Ala 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 Ala 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 1010 1015 1020 Glu Val Ser Gln Glu Val Arg Val
Cys Pro Gly Arg Gly Tyr Ile 1025 1030 1035 Leu Arg Val Thr Ala Tyr
Lys Glu Gly Tyr Gly Glu Gly Cys Val 1040 1045 1050 Thr Ile His Glu
Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser 1055 1060 1065 Asn Cys
Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys 1070 1075 1080
Asn Asn Tyr Thr Gly Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr 1085
1090 1095 Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr Gly Asn Asn Pro
Ser 1100 1105 1110 Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys
Ser Tyr Thr 1115 1120 1125 Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser
Asn Arg Gly Tyr Gly 1130 1135 1140 Asp Tyr Thr Pro Leu Pro Ala Gly
Tyr Val Thr Lys Asp Leu Glu 1145 1150 1155 Tyr Phe Pro Glu Thr Asp
Lys Val Trp Ile Glu Ile Gly Glu Thr 1160 1165 1170 Glu Gly Thr Phe
Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu 1175 1180 1185 Glu 60
3567 DNA Artificial Sequence Recombinant Delta Endotoxin 60 atg gag
gaa aat aat caa aat caa tgc ata cct tac aat tgt tta agt 48 Met Glu
Glu Asn Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser 1 5 10 15
aat cct gaa gaa gta ctt ttg gat gga gaa cgg ata tca act ggt aat 96
Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn 20
25 30 tca tca att gat att tct ctg tca ctt gtt cag ttt ctg gta tct
aac 144 Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser
Asn 35 40 45 ttt gta cca ggg gga gga ttt tta gtt gga tta ata gat
ttt gta tgg 192 Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp
Phe Val Trp 50 55 60 gga ata gtt ggc cct tct caa tgg gat gca ttt
cta gta caa att gaa 240 Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe
Leu Val Gln Ile Glu 65 70 75 80 caa tta att aat gaa aga ata gct gaa
ttt gct agg aat gct gct att 288 Gln Leu Ile Asn Glu Arg Ile Ala Glu
Phe Ala Arg Asn Ala Ala Ile 85 90 95 gct aat tta gaa gga tta gga
aac aat ttc aat ata tat gtg gaa gca 336 Ala Asn Leu Glu Gly Leu Gly
Asn Asn Phe Asn Ile Tyr Val Glu Ala 100 105 110 ttt aaa gaa tgg gaa
gaa gat cct aat aat cca gca acc agg acc aga 384 Phe Lys Glu Trp Glu
Glu Asp Pro Asn Asn Pro Ala Thr Arg Thr Arg 115 120 125 gta att gat
cgc ttt cgt ata ctt gat ggg cta ctt gaa agg gac att 432 Val Ile Asp
Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile 130 135 140 cct
tcg ttt gac att tct gga ttt gaa gta ccc ctt tta tcc gtt tat 480 Pro
Ser Phe Asp Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr 145 150
155 160 gct caa gcg gcc aat ctg cat cta gct ata tta aga gat tct gta
att 528 Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val
Ile 165 170 175 ttt gga gaa aga tgg gga ttg aca acg ata aat gtc aat
gaa aac tat 576 Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn
Glu Asn Tyr 180 185 190 aat aga cta att agg cat att gat gaa tat gct
gat cac tgt gca aat 624 Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala
Asp His Cys Ala Asn 195 200 205 acg tat aat cgg gga tta aat aat tta
ccg gct agc acg tat caa gat 672 Thr Tyr Asn Arg Gly Leu Asn Asn Leu
Pro Ala Ser Thr Tyr Gln Asp 210 215 220 tgg ata aca tat aat cga tta
cgg aga gac tta aca ttg act gta tta 720 Trp Ile Thr Tyr Asn Arg Leu
Arg Arg Asp Leu Thr Leu Thr Val Leu 225 230 235 240 gat atc gcc gct
ttc ttt cca aac tat gac aat agg aga tat cca att 768 Asp Ile Ala Ala
Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile 245 250 255 cag cca
gtt ggt caa cta aca agg gaa gtt tat acg gac cca tta att 816 Gln Pro
Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile 260 265 270
aat ttt aat cca cag tta cag tct gta gct caa tta cct act ttt aac 864
Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn 275
280 285 gtt atg gag agc agc gca att aga aat cct cat tta ttt gat ata
ttg 912 Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile
Leu 290 295 300 aat aat ctt aca atc ttt acg gat tgg ttt agt gtt gga
cgc aat ttt 960 Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly
Arg Asn Phe 305 310 315 320 tat tgg gga gga cat cga gta ata tct agc
ctt ata gga ggt ggt aac 1008 Tyr Trp Gly Gly His Arg Val Ile Ser
Ser Leu Ile Gly Gly Gly Asn 325 330 335 ata aca tct cct ata tat gga
aga gag gcg aac cag gag cct cca aga 1056 Ile Thr Ser Pro Ile Tyr
Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg 340 345 350 tcc ttt act ttt
aat gga ccg gta ttt agg act tta tca aat cct act 1104 Ser Phe Thr
Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr 355 360 365 tta
cga tta tta cag caa cct tgg cca gcg cca cca ttt aat tta cgt 1152
Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg 370
375 380 ggt gtt gaa gga gta gaa ttt tct aca cct aca aat agc ttt acg
tat 1200 Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe
Thr Tyr 385 390 395 400 cga gga aga ggt acg gtt gat tct tta act gaa
tta ccg cct gag gat 1248 Arg Gly Arg Gly Thr Val Asp Ser Leu
Thr
Glu Leu Pro Pro Glu Asp 405 410 415 aat agt gtg cca cct cgc gaa gga
tat agt cat cgt tta tgt cat gca 1296 Asn Ser Val Pro Pro Arg Glu
Gly Tyr Ser His Arg Leu Cys His Ala 420 425 430 act ttt gtt caa aga
tct gga aca cct ttt tta aca act ggt gta gta 1344 Thr Phe Val Gln
Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val 435 440 445 ttt tct
tgg acg cat cgt agt gca act ctt aca aat aca att gat cca 1392 Phe
Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro 450 455
460 gag aga att aat caa ata cct tta gtg aaa gga ttt aga gtt tgg ggg
1440 Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp
Gly 465 470 475 480 ggc acc tct gtc att aca gga cca gga ttt aca gga
ggg gat atc ctt 1488 Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr
Gly Gly Asp Ile Leu 485 490 495 cga aga aat acc ttt ggt gat ttt gta
tct cta caa gtc aat att aat 1536 Arg Arg Asn Thr Phe Gly Asp Phe
Val Ser Leu Gln Val Asn Ile Asn 500 505 510 tca cca att acc caa aga
tac cgt tta aga ttt cgt tac gct tcc agt 1584 Ser Pro Ile Thr Gln
Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser 515 520 525 agg gat gca
cga gtt ata gta tta aca gga gcg gca tcc aca gga gtg 1632 Arg Asp
Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val 530 535 540
gga ggc caa gtt agt gta aat atg cct ctt cag aaa act atg gaa ata
1680 Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu
Ile 545 550 555 560 ggg gag aac tta aca tct aga aca ttt aga tat acc
gat ttt agt aat 1728 Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr
Thr Asp Phe Ser Asn 565 570 575 cct ttt tca ttt aga gct aat cca gat
ata att ggg ata agt gaa caa 1776 Pro Phe Ser Phe Arg Ala Asn Pro
Asp Ile Ile Gly Ile Ser Glu Gln 580 585 590 cct cta ttt ggt gca ggt
tct att agt agc ggt gaa ctt tat ata gat 1824 Pro Leu Phe Gly Ala
Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp 595 600 605 aaa att gaa
att att cta gca gat gca aca ttt gaa gca gaa tct gat 1872 Lys Ile
Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp 610 615 620
tta gaa aga gca caa aag gcg gtg aat gcc ctg ttt act tct tcc aat
1920 Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser
Asn 625 630 635 640 caa atc ggg tta aaa acc gat gtg acg gat tat cat
att gat caa gta 1968 Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr
His Ile Asp Gln Val 645 650 655 tcc aat tta gtg gat tgt tta tca gat
gaa ttt tgt ctg gat gaa aag 2016 Ser Asn Leu Val Asp Cys Leu Ser
Asp Glu Phe Cys Leu Asp Glu Lys 660 665 670 cga gaa ttg tcc gag aaa
gtc aaa cat gcg aag cga ctc agt gat gag 2064 Arg Glu Leu Ser Glu
Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu 675 680 685 cgg aat tta
ctt caa gat cca aac ttc aga ggg atc aat aga caa cca 2112 Arg Asn
Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro 690 695 700
gac cgt ggc tgg aga gga agt aca gat att acc atc caa gga gga gat
2160 Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly
Asp 705 710 715 720 gac gta ttc aaa gag aat tac gtc aca cta ccg ggt
acc gtt gat gag 2208 Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro
Gly Thr Val Asp Glu 725 730 735 tgc tat cca acg tat tta tat cag aaa
ata gat gag tcg aaa tta aaa 2256 Cys Tyr Pro Thr Tyr Leu Tyr Gln
Lys Ile Asp Glu Ser Lys Leu Lys 740 745 750 gct tat acc cgt tat gaa
tta aga ggg tat atc gaa gat agt caa gac 2304 Ala Tyr Thr Arg Tyr
Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp 755 760 765 tta gaa atc
tat ttg atc cgt tac aat gca aaa cac gaa ata gta aat 2352 Leu Glu
Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val Asn 770 775 780
gtg cca ggc acg ggt tcc tta tgg ccg ctt tca gcc caa agt cca atc
2400 Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro
Ile 785 790 795 800 gga aag tgt gga gaa ccg aat cga tgc gcg cca cac
ctt gaa tgg aat 2448 Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro
His Leu Glu Trp Asn 805 810 815 cct gat cta gat tgt tcc tgc aga gac
ggg gaa aaa tgt gca cat cat 2496 Pro Asp Leu Asp Cys Ser Cys Arg
Asp Gly Glu Lys Cys Ala His His 820 825 830 tcc cat cat ttc acc ttg
gat att gat gtt gga tgt aca gac tta aat 2544 Ser His His Phe Thr
Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn 835 840 845 gag gac tta
ggt gta tgg gtg ata ttc aag att aag acg caa gat ggc 2592 Glu Asp
Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly 850 855 860
cat gca aga cta ggg aat cta gag ttt ctc gaa gag aaa cca tta tta
2640 His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu
Leu 865 870 875 880 ggg gaa gca cta gct cgt gtg aaa aga gcg gag aag
aag tgg aga gac 2688 Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu
Lys Lys Trp Arg Asp 885 890 895 aaa cga gag aaa ctg cag ttg gaa aca
aat att gtt tat aaa gag gca 2736 Lys Arg Glu Lys Leu Gln Leu Glu
Thr Asn Ile Val Tyr Lys Glu Ala 900 905 910 aaa gaa tct gta gat gct
tta ttt gta aac tct caa tat gat aga tta 2784 Lys Glu Ser Val Asp
Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu 915 920 925 caa gtg gat
acg aac atc gca atg att cat gcg gca gat aaa cgc gtt 2832 Gln Val
Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val 930 935 940
cat aga atc cgg gaa gcg tat ctg cca gag ttg tct gtg att cca ggt
2880 His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro
Gly 945 950 955 960 gtc aat gcg gcc att ttc gaa gaa tta gag gga cgt
att ttt aca gcg 2928 Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly
Arg Ile Phe Thr Ala 965 970 975 tat tcc tta tat gat gcg aga aat gtc
att aaa aat ggc gat ttc aat 2976 Tyr Ser Leu Tyr Asp Ala Arg Asn
Val Ile Lys Asn Gly Asp Phe Asn 980 985 990 aat ggc tta tta tgc tgg
aac gtg aaa ggt cat gta gat gta gaa gag 3024 Asn Gly Leu Leu Cys
Trp Asn Val Lys Gly His Val Asp Val Glu Glu 995 1000 1005 caa aac
aac cac cgt tcg gtc ctt gtt atc cca gaa tgg gag gca 3069 Gln Asn
Asn His Arg Ser Val Leu Val Ile Pro Glu Trp Glu Ala 1010 1015 1020
gaa gtg tca caa gag gtt cgt gtc tgt cca ggt cgt ggc tat atc 3114
Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile 1025
1030 1035 ctt cgt gtc aca gca tat aaa gag gga tat gga gag ggc tgc
gta 3159 Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys
Val 1040 1045 1050 acg atc cat gag atc gaa gac aat aca gac gaa ctg
aaa ttc agc 3204 Thr Ile His Glu Ile Glu Asp Asn Thr Asp Glu Leu
Lys Phe Ser 1055 1060 1065 aac tgt gta gaa gag gaa gta tat cca aac
aac aca gta acg tgt 3249 Asn Cys Val Glu Glu Glu Val Tyr Pro Asn
Asn Thr Val Thr Cys 1070 1075 1080 aat aat tat act ggg act caa gaa
gaa tat gag ggt acg tac act 3294 Asn Asn Tyr Thr Gly Thr Gln Glu
Glu Tyr Glu Gly Thr Tyr Thr 1085 1090 1095 tct cgt aat caa gga tat
gac gaa gcc tat ggt aat aac cct tcc 3339 Ser Arg Asn Gln Gly Tyr
Asp Glu Ala Tyr Gly Asn Asn Pro Ser 1100 1105 1110 gta cca gct gat
tac gct tca gtc tat gaa gaa aaa tcg tat aca 3384 Val Pro Ala Asp
Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr 1115 1120 1125 gat gga
cga aga gag aat cct tgt gaa tct aac aga ggc tat ggg 3429 Asp Gly
Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly 1130 1135 1140
gat tac aca cca cta ccg gct ggt tat gta aca aag gat tta gag 3474
Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu 1145
1150 1155 tac ttc cca gag acc gat aag gta tgg att gag atc gga gaa
aca 3519 Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu
Thr 1160 1165 1170 gaa gga aca ttc atc gtg gat agc gtg gaa tta ctc
ctt atg gag 3564 Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu
Leu Met Glu 1175 1180 1185 gaa 3567 Glu 61 1189 PRT Artificial
Sequence Recombinant Delta Endotoxin 61 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 Leu 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 Gly Asn Asn Phe Asn Ile
Tyr Val Glu Ala 100 105 110 Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn
Pro Ala 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 Asp 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 Ala 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 1010 1015 1020 Glu Val Ser
Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile 1025 1030 1035 Leu
Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val 1040 1045
1050 Thr Ile His Glu Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser
1055 1060 1065 Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val
Thr Cys 1070 1075 1080 Asn Asn Tyr Thr Gly Thr Gln Glu Glu Tyr Glu
Gly Thr Tyr Thr 1085 1090 1095 Ser Arg Asn Gln Gly Tyr Asp Glu Ala
Tyr Gly Asn Asn Pro Ser 1100 1105 1110 Val Pro Ala Asp Tyr Ala Ser
Val Tyr Glu Glu Lys Ser Tyr Thr 1115 1120 1125 Asp Gly Arg Arg Glu
Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly 1130 1135 1140 Asp Tyr Thr
Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu 1145 1150 1155 Tyr
Phe Pro Glu Thr Asp Lys Val Trp
Ile Glu Ile Gly Glu Thr 1160 1165 1170 Glu Gly Thr Phe Ile Val Asp
Ser Val Glu Leu Leu Leu Met Glu 1175 1180 1185 Glu 62 47 DNA
Artificial Sequence Synthetic oligonucleotide 62 cggggattaa
ataatttacc ggctagcacg tatcaagatt ggataac 47 63 47 DNA Artificial
Sequence Synthetic oligonucleotide 63 cggggattaa ataatttacc
gaaasvaacg tatcaagatt ggataac 47 64 23 DNA Artificial Sequence
Synthetic oligonucleotide 64 ggatagcact catcaaaggt acc 23 65 47 DNA
Artificial Sequence Synthetic oligonucleotide 65 cggggattaa
ataatsvacc gaaaagcacg tatcaagatt ggataac 47 66 47 DNA Artificial
Sequence Synthetic oligonucleotide 66 cggggattaa ataatttasv
aaaaagcacg tatcaagatt ggataac 47 67 47 DNA Artificial Sequence
Synthetic oligonucleotide 67 cggggattaa ataatttacc gsvaagcacg
tatcaagatt ggataac 47 68 53 DNA Artificial Sequence Synthetic
oligonucleotide 68 ggattaaata atttaccgaa aagcsvatat caagattgga
taacatataa tcg 53 69 53 DNA Artificial Sequence Synthetic
oligonucleotide 69 ggattaaata atttaccgaa aagcacgsva caagattgga
taacatataa tcg 53 70 52 DNA Artificial Sequence Synthetic
oligonucleotide 70 gattctgtaa tttttsvaga aagatgggga ttgacaacga
taaatgtcaa tg 52 71 52 DNA Artificial Sequence Synthetic
oligonucleotide 71 gattctgtaa tttttggasv aagatgggga ttgacaacga
taaatgtcaa tg 52 72 52 DNA Artificial Sequence Synthetic
oligonucleotide 72 gattctgtaa tttttggaga asvatgggga ttgacaacga
taaatgtcaa tg 52 73 54 DNA Artificial Sequence Synthetic
oligonucleotide 73 tctgtaattt ttggagaaag asvaggattg acaacgataa
atgtcaatga aaac 54 74 51 DNA Artificial Sequence Synthetic
oligonucleotide 74 gtaatttttg gagaaagatg gsvattgaca acgataaatg
tcaatgaaaa c 51 75 51 DNA Artificial Sequence Synthetic
oligonucleotide 75 gtaatttttg gagaaagatg gggasnaaca acgataaatg
tcaatgaaaa c 51 76 51 DNA Artificial Sequence Synthetic
oligonucleotide 76 gtaatttttg gagaaagatg gggattgsna acgataaatg
tcaatgaaaa c 51
* * * * *
References