U.S. patent application number 12/467777 was filed with the patent office on 2009-10-29 for lepidopteran-active bacillus thuringiensis delta-endotoxin compositions and methods of use.
Invention is credited to James A. Baum, Chih-Rei Chu, William P. Donovan, Amy J. Gilmer, Mark J. Rupar.
Application Number | 20090270327 12/467777 |
Document ID | / |
Family ID | 22549588 |
Filed Date | 2009-10-29 |
United States Patent
Application |
20090270327 |
Kind Code |
A1 |
Baum; James A. ; et
al. |
October 29, 2009 |
Lepidopteran-Active Bacillus Thuringiensis Delta-Endotoxin
Compositions and Methods of Use
Abstract
Disclosed are Bacillus thuringiensis strains comprising novel
crystal proteins which exhibit insecticidal activity against
lepidopteran insects. Also disclosed are novel B. thuringiensis
genes and their encoded crystal proteins, as well as methods of
making and using transgenic cells comprising the novel nucleic acid
sequences of the invention.
Inventors: |
Baum; James A.; (Webster
Groves, MO) ; Chu; Chih-Rei; (Exton, PA) ;
Donovan; William P.; (Levittown, PA) ; Gilmer; Amy
J.; (Langhorne, PA) ; Rupar; Mark J.;
(Wilmington, DE) |
Correspondence
Address: |
HOWREY LLP
C/O IP DOCKETING DEPARTMENT, 2941 FAIRVIEW PARK DRIVE SUITE 200
FALLS CHURCH
VA
22042
US
|
Family ID: |
22549588 |
Appl. No.: |
12/467777 |
Filed: |
May 18, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11487813 |
Jul 17, 2006 |
7534939 |
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12467777 |
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10428961 |
May 2, 2003 |
7078509 |
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11487813 |
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09661322 |
Sep 13, 2000 |
6593293 |
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10428961 |
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60153995 |
Sep 15, 1999 |
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Current U.S.
Class: |
514/2.1 ;
435/6.15; 435/7.1; 530/387.9; 536/23.5; 800/302 |
Current CPC
Class: |
Y02A 40/146 20180101;
Y02A 40/162 20180101; C07K 14/325 20130101; C12N 15/8286
20130101 |
Class at
Publication: |
514/12 ; 800/302;
536/23.5; 435/6; 530/387.9; 435/7.1 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A01H 5/00 20060101 A01H005/00; C07H 21/04 20060101
C07H021/04; C12Q 1/68 20060101 C12Q001/68; C07K 16/18 20060101
C07K016/18; G01N 33/53 20060101 G01N033/53 |
Claims
1-52. (canceled)
53. An isolated nucleic acid segment having utility as a probe or
primer, characterized as: (a) a nucleic acid segment comprises 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:45 or SEQ ID
NO:47; or (b) a nucleic acid segment comprising at least a 14
nucleotide long contiguous sequence that specifically hybridizes to
SEQ ID NO: 1, SEQ ID NO:45 or SEQ ID NO:47, or the complement
thereof under stringent conditions.
54. The isolated nucleic acid segment of claim 53 consisting of the
sequence as set forth in SEQ ID NO:60 or SEQ ID NO:61.
55. A method for detecting a nucleic acid sequence encoding a
Lepidopteran toxic crystal protein, comprising the steps of: (a)
obtaining sample nucleic acids suspected of encoding a Lepidopteran
toxic crystal protein; (b) contacting said sample nucleic acids
with the isolated nucleic acid segment of claim 53 or 54 under
conditions effective to allow hybridization of complementary
nucleic acids; and (c) detecting the hybridized complementary
nucleic acids thus formed.
56. The method of claim 55, wherein said nucleic acid segment
comprises a detectable label and said hybridized complementary
nucleic acids are detected by detecting said label.
57. A nucleic acid detection kit comprising, in suitable container
means, the nucleic acid segment of claim 53 or 54 and a detection
reagent.
58. A purified antibody that specifically binds to the polypeptide
of SEQ ID NO:2 or an insecticidal fragment thereof.
59. The purified antibody of claim 58 generated by using a
polypeptide according to SEQ ID NO:2 as an immunogen.
60. The purified antibody of claim 59 produced by a hybridoma,
wherein a polypeptide according to SEQ ID NO:2 is used to generate
the hybridoma producing the antibody.
61. A method for detecting a Lepidopteran toxic crystal protein in
a biological sample, comprising the steps of: (a) obtaining a
biological sample suspected of containing the protein; (b)
contacting the sample with an antibody in accordance with any one
of claims 58 to 60 under conditions effective to allow formation of
complexes; and (c) detecting the complexes so formed.
62. An immunodetection kit comprising, in suitable container means,
an antibody in accordance with any one of claims 58 to 60, and an
immunodetection reagent.
63. A transgenic plant event expressing the polypeptide of SEQ ID
NO:2 or an insecticidal fragment thereof.
64. The transgenic plant event of claim 63, further defined as a
monocotyledonous plant, progeny or seed thereof.
65. The transgenic plant event of claim 64, further defined as a
corn, wheat, oat, rice, barley, turf grass, or pasture grass plant,
progeny or seed thereof.
66. The transgenic plant event of claim 63, further defined as a
dicotyledonous plant, progeny or seed thereof.
67. The transgenic plant event of claim 66, further defined as a
legume, soybean, tobacco, tomato, potato, cotton, fruit, berry,
vegetable or tree plant, progeny or seed thereof.
68. An artificial diet for Lepidopteran insects, comprising an
insecticidal protein as set forth in SEQ ID NO:2 or an insecticidal
fragment thereof.
69. The artificial diet of claim 68, wherein said insecticidal
protein is prepared by a process comprising the steps of: a)
culturing a Bacillus thuringiensis cell having the accession number
NRRL B-21921 under conditions effective to produce an insecticidal
protein; and b) obtaining from said cell the insecticidal protein
so produced.
70. A method for killing Lepidopteran insects, comprising providing
the artificial diet of claim 68 or 69 to Lepidopteran insect
larvae.
Description
[0001] This application claims the benefit of priority from U.S.
Provisional Application No. 60/153,995, filed Sep. 15, 1999, the
entire of contents of which is hereby specifically incorporated 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
molecular biology. More particularly, certain embodiments concern
methods and compositions comprising DNA segments, and proteins
derived from bacterial species. More particularly, it concerns
novel genes from Bacillus thuringiensis encoding lepidopteran-toxic
crystal proteins. Various methods for making and using these DNA
segments, DNA segments encoding synthetically-modified Cry
proteins, and native and synthetic crystal proteins are disclosed,
such as, for example, the use of DNA segments as diagnostic probes
and templates for protein production, and the use of proteins,
fusion protein carriers and peptides in various immunological and
diagnostic applications. Also disclosed are methods of making and
using nucleic acid segments in the development of transgenic plant
cells containing the DNA segments disclosed herein.
[0004] 1.2 Description of the Related Art
[0005] Almost all field crops, plants, and commercial farming areas
are susceptible to attack by one or more insect pests. Particularly
problematic are Coleopteran and Lepidoptern pests. For example,
vegetable and cole crops such as artichokes, kohlrabi, arugula,
leeks, asparagus, lentils, beans, lettuce (e.g., head, leaf,
romaine), beets, bok choy, malanga, broccoli, melons (e.g.,
muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brussels
sprouts, cabbage, cardoni, carrots, napa, cauliflower, okra,
onions, celery, parsley, chick peas, parsnips, chicory, peas,
chinese cabbage, peppers, collards, potatoes, cucumber, pumpkins,
cucurbits, radishes, dry bulb onions, rutabaga, eggplant, salsify,
escarole, shallots, endive, soybean, garlic, spinach, green onions,
squash, greens, sugar beets, sweet potatoes, turnip, swiss chard,
horseradish, tomatoes, kale, turnips, and a variety of spices are
sensitive to infestation by one or more of the following insect
pests: alfalfa looper, armyworm, beet armyworm, artichoke plume
moth, cabbage budworm, cabbage looper, cabbage webworm, corn
earworm, celery leafeater, cross-striped cabbageworm, european corn
borer, diamondback moth, green cloverworm, imported cabbageworm,
melonworm, omnivorous leafroller, pickleworm, rindworm complex,
saltmarsh caterpillar, soybean looper, tobacco budworm, tomato
fruitworm, tomato hornworm, tomato pinworm, velvetbean caterpillar,
and yellowstriped armyworm. Likewise, pasture and hay crops such as
alfalfa, pasture grasses and silage are often attacked by such
pests as armyworm, beef armyworm, alfalfa caterpillar, European
skipper, a variety of loopers and webworms, as well as
yellowstriped armyworms.
[0006] Fruit and vine crops such as apples, apricots, cherries,
nectarines, peaches, pears, plums, prunes, quince almonds,
chestnuts, filberts, pecans, pistachios, walnuts, citrus,
blackberries, blueberries, boysenberries, cranberries, currants,
loganberries, raspberries, strawberries, grapes, avocados, bananas,
kiwi, persimmons, pomegranate, pineapple, tropical fruits are often
susceptible to attack and defoliation by achema sphinx moth,
amorbia, armyworm, citrus cutworm, banana skipper, blackheaded
fireworm, blueberry leafroller, cankerworm, cherry fruitworm,
citrus cutworm, cranberry girdler, eastern tent caterpillar, fall
webworm, fall webworm, filbert leafroller, filbert webworm, fruit
tree leafroller, grape berry moth, grape leaffolder, grapeleaf
skeletonizer, green fruitworm, gummosos-batrachedra commosae, gypsy
moth, hickory shuckworm, hornworms, loopers, navel orangeworm,
obliquebanded leafroller, omnivorous leafroller. omnivorous looper,
orange tortrix, orangedog, oriental fruit moth, pandemis
leafroller, peach twig borer, pecan nut casebearer, redbanded
leafroller, redhumped caterpillar, roughskinned cutworm, saltmarsh
caterpillar, spanworm, tent caterpillar, thecla-thecla basillides,
tobacco budworm, tortrix moth, tufted apple budmoth, variegated
leafroller, walnut caterpillar, western tent caterpillar, and
yellowstriped armyworm.
[0007] Field crops such as canola/rape seed, evening primrose,
meadow foam, corn (field, sweet, popcorn), cotton, hops, jojoba,
peanuts, rice, safflower, small grains (barley, oats, rye, wheat,
etc.), sorghum, soybeans, sunflowers, and tobacco are often targets
for infestation by insects including armyworm, asian and other corn
borers, banded sunflower moth, beet armyworm, bollworm, cabbage
looper, corn rootworm (including southern and western varieties),
cotton leaf perforator, diamondback moth, european corn borer,
green cloverworm, headmoth, headworm, imported cabbageworm, loopers
(including Anacamptodes spp.), obliquebanded leafroller, omnivorous
leaftier, podworm, podworm, saltmarsh caterpillar, southwestern
corn borer, soybean looper, spotted cutworm, sunflower moth,
tobacco budworm, tobacco hornworm, velvetbean caterpillar.
[0008] Bedding plants, flowers, ornamentals, vegetables and
container stock are frequently fed upon by a host of insect pests
such as armyworm, azalea moth, beet armyworm, diamondback moth,
ello moth (hornworm), Florida fern caterpillar, Io moth, loopers,
oleander moth, omnivorous leafroller, omnivorous looper, and
tobacco budworm.
[0009] Forests, fruit, ornamental, and nut-bearing trees, as well
as shrubs and other nursery stock are often susceptible to attack
from diverse insects such as bagworm, blackheaded budworm,
browntail moth, california oakworm, douglas fir tussock moth, elm
spanworm, fall webworm, fruittree leafroller, greenstriped
mapleworm, gypsy moth, jack pine budworm, mimosa webworm, pine
butterfly, redhumped caterpillar, saddleback caterpillar, saddle
prominent caterpillar, spring and fall cankerworm, spruce budworm,
tent caterpillar, tortrix, and western tussock moth. Likewise, turf
grasses are often attacked by pests such as armyworm, sod webworm,
and tropical sod webworm.
[0010] Because crops of commercial interest are often the target of
insect attack, environmentally-sensitive methods for controlling or
eradicating insect infestation are desirable in many instances.
This is particularly true for farmers, nurserymen, growers, and
commercial and residential areas which seek to control insect
populations using eco-friendly compositions.
[0011] The most widely used environmentally-sensitive insecticidal
formulations developed in recent years have been composed of
microbial pesticides derived from the bacterium Bacillus
thuringiensis. B. thuringiensis is a Gram-positive bacterium that
produces crystal proteins or inclusion bodies 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.
1.2.1 B. thuringiensis Crystal Proteins
1.2.1 .delta.-Endotoxins
[0012] .delta.-endotoxins are used to control a wide range of
leaf-eating caterpillars and beetles, as well as mosquitoes. These
proteinaceous parasporal crystals, also referred to as insecticidal
crystal proteins, crystal proteins, Bt inclusions, crystalline
inclusions, inclusion bodies, and Bt toxins, are a large collection
of insecticidal proteins produced by B. thuringiensis that are
toxic upon ingestion by a susceptible insect host. 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).
[0013] One of the unique features of B. thuringiensis is its
production of crystal proteins during sporulation 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 proteins having insecticidal
activity against lepidopteran and dipteran insects 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.
[0014] The mechanism of insecticidal activity of the B.
thuringiensis crystal proteins has been studied extensively in the
past decade. It has been shown that the crystal proteins are toxic
to the insect only after ingestion of the protein by the insect.
The alkaline pH and proteolytic enzymes in the insect mid-gut
solubilize the proteins, thereby allowing the release of components
which are toxic to the insect. These toxic components disrupt the
mid-gut cells, cause the insect to cease feeding, and, eventually,
bring about insect death. For this reason, B. thuringiensis has
proven to be an effective and environmentally safe insecticide in
dealing with various insect pests.
[0015] As noted by Hofte and Whiteley (1989), the majority of
insecticidal B. thuringiensis strains are active against insects of
the order Lepidoptera, i.e., caterpillar insects. Other B.
thuringiensis strains are insecticidally active against insects of
the order Diptera, i.e., flies and mosquitoes, or against both
lepidopteran and dipteran insects. In recent years, a few B.
thuringiensis strains have been reported as producing crystal
proteins that are toxic to insects of the order Coleoptera, i.e.,
beetles (Krieg et al., 1983; Sick et al., 1990; Donovan et al.,
1992; Lambert et al., 1992a; 1992b).
1.2.2 Genes Encoding Crystal Proteins
[0016] 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.
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.
[0017] Recently, a new nomenclature was developed which
systematically classified the Cry proteins based upon amino acid
sequence homology rather than upon insect target specificities
(Crickmore et al., 1998). The classification scheme for many known
toxins, including allelic variations in individual proteins, is
summarized and regularly updated at
http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/. The
information was most recently updated as of Apr. 27, 1999 and is
herein incorporated by reference.
1.2.3 Crystal Proteins Toxic to Lepidopteran Insects
2.0 SUMMARY OF THE INVENTION
[0018] The recent review by Schnepf et al. (1998) describes the
enormous diversity of insecticidal crystal proteins derived from B.
thuringiensis. Cry proteins of the Cry1, Cry2, and Cry9 classes are
particularly known for their toxicity towards lepidopteran larvae,
however, the degree of toxicity varies significantly depending on
the target lepidopteran pest (Hofte and Whiteley, 1989). For
instance, Cry1Ac shows poor toxicity towards the armyworm,
Spodoptera littoralis, but strong toxicity towards the tobacco
budworm, Heliothis virescens. In addition, slight variations in
amino acid sequence within a Cry protein class can dramatically
impact insecticidal activity (see Schnepf et al., 1998 and
references therein). The Cry3Ba and Cry3Bb genes, for instance,
share 94% amino acid sequence identity, but only Cry3Bb exhibits
significant toxicity towards the Southern corn rootworm, Diabrotica
undecimpunctata howardi (Donovan et al., 1992). Similarly, Cry2Aa
and Cry2Ab share 87% amino acid sequence identity, yet only Cry2Aa
displays toxicity towards mosquitoes (Widner and Whiteley, 1990).
Von Tersch et al. (1991) demonstrated that Cry1Ac proteins varying
by only seven amino acids (>99% sequence identity) nevertheless
show significant differences in insecticidal activity. Lee et al.
(1996) reported that Cry1Ab alleles differing at only two amino
acid positions exhibited a 10-fold difference in toxicity towards
the gypsy moth, Lymantria dispar. Thus, even Cry proteins that are
considered to be alleles of known Cry proteins or to belong to a
Cry protein subclass (Crickmore et al., 1998) may have unique and
useful insecticidal properties. International Patent Application
Publication No. WO 98/00546 and WO 98/40490 describe a variety of
Cry1-, Cry2-, and Cry9-related crystal proteins obtained from B.
thuringiensis.
2.1 Cry DNA Segments
[0019] The present invention concerns nucleic acid segments, that
can be isolated from virtually any source, that are free from total
genomic DNA and that encode the novel peptides disclosed herein.
Nucleic acid segments encoding these polypeptides may encode active
proteins, peptides or peptide fragments, polypeptide subunits,
functional domains, or the like of one or more crystal proteins. In
addition the invention encompasses nucleic acid segments which may
be synthesized entirely in vitro using methods that are well-known
to those of skill in the art which encode the novel Cry
polypeptides, peptides, peptide fragments, subunits, or functional
domains disclosed herein.
[0020] As used herein, the term "nucleic acid segment" refers to a
polynucleotide molecule that has been isolated free of total
genomic DNA of a particular species. Therefore, a nucleic acid
segment encoding an endotoxin polypeptide refers to a nucleic acid
segment that comprises one or more crystal protein-encoding
sequences yet is isolated away from, or purified free from, total
genomic DNA of the species from which the nucleic acid 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 "nucleic acid segment", are polynucleotide segments and
smaller fragments of such segments, and also recombinant vectors,
including, for example, plasmids, cosmids, phagemids, phages,
viruses, and the like.
[0021] 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. Also, the term
includes an expression cassette comprising at least a promoter
operably linked to one or more protein coding sequences, operably
linked to at least a transcriptional termination sequence.
[0022] "Isolated substantially away from other coding sequences"
means that the gene of interest, in this case, a nucleic acid
segment or gene encoding all or part of a bacterial insecticidal
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 nucleic acid segments or
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.
[0023] 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:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID
NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ
ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,
SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID
NO:48, SEQ ID NO:50 and SEQ ID NO. 63.
[0024] The term "a sequence essentially as set forth in SEQ ID
NO:2, SEQ ID NO:4, or SEQ ID NO:6," for example, means that the
sequence substantially corresponds to a portion of the sequence of
SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 and has relatively few
amino acids that are not identical with, 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 from about 70% to about 80%, or more preferably about 81, 82,
83, 84, 85, 86, 87, 88, 89, or about 90%, or even more preferably
about 91, 92, 93, 94, 95, 96, 97, 98, or 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:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID
NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ
ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,
SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID
NO:48, SEQ ID NO:50 and SEQ ID NO. 63 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:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ
ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34,
SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:
63."
[0025] In addition, sequences that have from about 70% to about
80%, or more preferably about 81, 82, 83, 84, 85, 86, 87, 88, 89,
or about 90%, or even more preferably about 91, 92, 93, 94, 95, 96,
97, 98, or about 99% nucleic acid sequence identity or functional
equivalence to the nucleic acids 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:13, SEQ
ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,
SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID
NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ
ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62
will be sequences that are "essentially as 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:1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,
SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID
NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ
ID NO:49 and SEQ ID NO:62."
[0026] 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.
[0027] 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 any of the peptide
sequences 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:14, SEQ ID NO:16,
SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID
NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ
ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44,
SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO: 63, or that
are identical with or complementary to DNA sequences which encode
any of the peptides 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:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ
ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34,
SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO: 63,
and particularly those 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:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,
SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID
NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ
ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49
and SEQ ID NO:62. For example, DNA sequences such as about 18
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.
[0028] It will be readily understood that "intermediate lengths",
in these contexts, means any length between the quoted ranges, such
as 18, 19, 20, 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 in the ranges of from about 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 10,00 or so nucleotides and the like.
[0029] 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
sequences 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:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ
ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36,
SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID
NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO: 63, including
those 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:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,
SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID
NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ
ID NO:49 and SEQ ID NO:62. 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.
[0030] The DNA segments of the present invention encompass
biologically-functional, equivalent peptides. Such sequences may
arise as a consequence of codon degeneracy 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.
[0031] 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).
[0032] 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.
2.2 Cry DNA Segments as Hybridization Probes and Primers
[0033] 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:13, SEQ ID
NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ
ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,
SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID
NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62
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 bp, etc. (including all intermediate
lengths and up to and including the full-length gene sequences
encoding each polypeptide will also be of use in certain
embodiments.
[0034] 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.
[0035] Nucleic acid molecules having sequence regions consisting of
contiguous nucleotide stretches of about 14 to about 17 or so,
18-25, 26-35, 36-50, or even up to and including sequences of about
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:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,
SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62, 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 to 200 or so nucleotides, but larger
contiguous complementarity stretches may be used, according to the
length complementary sequences one wishes to detect.
[0036] 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.
[0037] 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. to 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., 1990; Maloy 1994; Segal, 1976;
Prokop, 1991; and Kuby, 1991, are particularly relevant.
[0038] 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. 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.
[0039] 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
environmentally 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.
[0040] 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.
2.3 Vectors and Methods for Recombinant Expression of Cry
Polypeptides
[0041] 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).
[0042] 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
sequences 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:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ
ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36,
SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID
NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO: 63.
2.4 Cry Transgenes and Transgenic Plants Expressing Cry
Polypeptides
[0043] In yet another aspect, the present invention provides
methods for producing a transgenic plant which expresses a nucleic
acid segment encoding the novel polypeptides and endotoxins 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 a DNA segment which contains
a promoter operatively linked to a coding region that encodes one
or more CryET31, CryET40, CryET43, CryET44, CryET45, CryET46,
CryET47, CryET49, CryET51, CryET52, CryET53, CryET54, CryET55,
CryET56, CryET57, CryET59, CryET60, CryET61, CryET62, CryET63,
CryET64, CryET66, CryET67, CryET68, CryET72, CryET73, and CryET83
polypeptides. 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
polypeptide 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.
[0044] Another aspect of the invention comprises transgenic plants
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.
[0045] It is contemplated that in some instances either the nuclear
or plastidic genome, or both, of a transgenic plant of the present
invention will have been augmented through the stable introduction
of one or more cryET31, cryET40, cryET43, cryET44, cryET45,
cryET46, cryET47, cryET49, cryET51, cryET52, cryET53, cryET54,
cryET55, cryET56, cryET57, cryET59, cryET60, cryET61, cryET62,
cryET63, cryET64, cryET66, cryET67, cryET68, cryET72, cryET73, and
cryET83 transgenes, either native, synthetically modified, or
mutated. In some instances, more than one transgene will be
incorporated into one or more genomes 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.
[0046] A preferred gene which may be introduced includes, for
example, a crystal protein-encoding 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.
[0047] 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.
[0048] 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 one or more CryET31, CryET40, CryET43, CryET44, CryET45,
CryET46, CryET47, CryET49, CryET51, CryET52, CryET53, CryET54,
CryET55, CryET56, CryET57, CryET59, CryET60, CryET61, CryET62,
CryET63, CryET64, CryET66, CryET67, CryET68, CryET72, CryET73, and
CryET83 polypeptides which are toxic to a lepidopteran insect.
Particularly preferred plants include turf grasses, kapok, sorghum,
cotton, corn, soybeans, oats, rye, wheat, flax, tobacco, rice,
tomatoes, potatoes, or other vegetables, ornamental plants, fruit
trees, and the like.
[0049] 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 a crystal protein-encoding transgene stably
incorporated into their 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 CryET31, CryET40, CryET43, CryET44, CryET45, CryET46,
CryET47, CryET49, CryET51, CryET52, CryET53, CryET54, CryET55,
CryET56, CryET57, CryET59, CryET60, CryET61, CryET62, CryET63,
CryET64, CryET66, CryET67, CryET68, CryET72, CryET73, and CryET83
crystal proteins or polypeptides are aspects of this invention. As
well-known to those of skill in the art, a progeny of a plant is
understood to mean any offspring or any descendant from such a
plant, but in this case means any offspring or any descendant which
also contains the transgene.
2.5 Site-Specific Mutagenesis
[0050] 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. The technique of
site-specific mutagenesis is well known in the art, as exemplified
by various publications.
[0051] 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 appropriate cells, such as E. coli cells, and clones are
selected which include recombinant vectors bearing the mutated
sequence arrangement.
[0052] The preparation of sequence variants of the
endotoxin-encoding nucleic acid 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.
2.6 Antibody Compositions and Methods of Making
[0053] In particular embodiments, the inventors contemplate the use
of antibodies, either monoclonal (mAbs) or polyclonal which bind to
one or more of the polypeptides disclosed herein. Means for
preparing and characterizing antibodies are well known in the art
(See, e.g., Harlow and Lane, 1988; incorporated herein by
reference). mAbs may be readily prepared through use of well-known
techniques, such as those exemplified in U.S. Pat. No. 4,196,265,
incorporated herein by reference.
2.7 ELISAs and Immunoprecipitation
[0054] ELISAs may be used in conjunction with the invention. Many
different protocols exist for performing ELISAs. These are well
known to those of ordinary skill in the art. Examples of basic
ELISA protocols may be found in any standard molecular biology
laboratory manual (e.g. Sambrook, Fritsch, and Maniatis, eds.
Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y.:
Cold Spring Harbor Laboratory, 1989).
2.8 Western Blots
[0055] The compositions of the present invention will find great
use in immunoblot or western blot analysis. Methods of performing
immunoblot and western blot analysis are well known to those of
skill in the are (see Sambrook, et al, ibid). Immunologically-based
detection methods for use in conjunction with Western blotting
include enzymatically-, radiolabel-, or fluorescently-tagged
secondary antibodies against the toxin moiety are considered to be
of particular use in this regard.
2.9 Crystal Protein Screening and Detection Kits
[0056] The present invention contemplates methods and kits for
screening samples suspected of containing crystal protein
polypeptides or crystal protein-related polypeptides, or cells
producing such polypeptides. A kit may contain one or more
antibodies of the present invention, and may also contain
reagent(s) for detecting an interaction between a sample and an
antibody of the present invention. The provided reagent(s) can be
radio-, fluorescently- or enzymatically-labeled or even epitope or
ligand tagged. The kit can contain a known radiolabeled agent
capable of binding or interacting with a nucleic acid or antibody
of the present invention.
[0057] The reagent(s) of the kit can be provided as a liquid
solution, attached to a solid support or as a dried powder.
Preferably, when the reagent(s) are provided in a liquid solution,
the liquid solution is an aqueous solution. Preferably, when the
reagent(s) provided are attached to a solid support, the solid
support can be chromatograph media, a test plate having a plurality
of wells, or a microscope slide. When the reagent(s) provided are a
dry powder, the powder can be reconstituted by the addition of a
suitable solvent, that may be provided.
[0058] In still further embodiments, the present invention concerns
immunodetection methods and associated kits. It is proposed that
the crystal proteins or peptides of the present invention may be
employed to detect antibodies having reactivity therewith, or,
alternatively, antibodies prepared in accordance with the present
invention, may be employed to detect crystal proteins or crystal
protein-related epitope-containing peptides. In general, these
methods will include first obtaining a sample suspected of
containing such a protein, peptide or antibody, contacting the
sample with an antibody or peptide in accordance with the present
invention, as the case may be, under conditions effective to allow
the formation of an immunocomplex, and then detecting the presence
of the immunocomplex.
[0059] In general, the detection of immunocomplex formation is
quite well known in the art and may be achieved through the
application of numerous approaches. For example, the present
invention contemplates the application of ELISA, RIA, immunoblot
(e.g., dot blot), indirect immunofluorescence techniques and the
like. One may find additional advantages through the use of a
secondary binding ligand such as a second antibody or a
biotin/avidin ligand binding arrangement, as is known in the
art.
[0060] For assaying purposes, it is proposed that virtually any
sample suspected of comprising either a crystal protein or peptide
or a crystal protein-related peptide or antibody sought to be
detected, as the case may be, may be employed. It is contemplated
that such embodiments may have application in the tittering of
antigen or antibody samples, in the selection of hybridomas, and
the like. In related embodiments, the present invention
contemplates the preparation of kits that may be employed to detect
the presence of crystal proteins or related peptides and/or
antibodies in a sample. Samples may include cells, cell
supernatants, cell suspensions, cell extracts, enzyme fractions,
protein extracts, or other cell-free compositions suspected of
containing crystal proteins or peptides.
[0061] Generally speaking, kits in accordance with the present
invention will include a suitable crystal protein, peptide or an
antibody directed against such a protein or peptide, together with
an immunodetection reagent and a means for containing the antibody
or antigen and reagent. The immunodetection reagent will typically
comprise a label associated with the antibody or antigen, or
associated with a secondary binding ligand. Exemplary ligands might
include a secondary antibody directed against the first antibody or
antigen or a biotin or avidin (or streptavidin) ligand having an
associated label. Of course, as noted above, a number of exemplary
labels are known in the art and all such labels may be employed in
connection with the present invention.
[0062] The container will generally include a vial into which the
antibody, antigen or detection reagent may be placed, and
preferably suitably aliquoted. The kits of the present invention
will also typically include a means for containing the antibody,
antigen, and reagent containers in close confinement for commercial
sale. Such containers may include injection or blow-molded plastic
containers into which the desired vials are retained.
2.10 Epitopic Core Sequences
[0063] The present invention is also directed to protein or peptide
compositions, free from total cells and other peptides, which
comprise a purified protein or peptide which incorporates an
epitope that is immunologically cross-reactive with one or more
anti-crystal protein antibodies. In particular, the invention
concerns epitopic core sequences derived from Cry proteins or
peptides.
[0064] As used herein, the term "incorporating an epitope(s) that
is immunologically cross-reactive with one or more anti-crystal
protein antibodies" is intended to refer to a peptide or protein
antigen which includes a primary, secondary or tertiary structure
similar to an epitope located within a crystal protein or
polypeptide. The level of similarity will generally be to such a
degree that monoclonal or polyclonal antibodies directed against
the crystal protein or polypeptide will also bind to, react with,
or otherwise recognize, the cross-reactive peptide or protein
antigen. Various immunoassay methods may be employed in conjunction
with such antibodies, such as, for example, Western blotting,
ELISA, RIA, and the like, all of which are known to those of skill
in the art. The identification of Cry immunodominant epitopes,
and/or their functional equivalents, suitable for use in vaccines
is a relatively straightforward matter (e.g. U.S. Pat. No.
4,554,101; Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Pat. No.
4,554,101). The amino acid sequence of these "epitopic core
sequences" may then be readily incorporated into peptides, either
through the application of peptide synthesis or recombinant
technology.
[0065] Preferred peptides for use in accordance with the present
invention will generally be on the order of about 8 to about 20
amino acids in length, and more preferably about 8 to about 15
amino acids in length. It is proposed that particular advantages of
the present invention may be realized through the preparation of
synthetic peptides which include modified and/or extended
epitopic/immunogenic core sequences which result in a "universal"
epitopic peptide directed to crystal proteins, and in particular
CryET31, CryET40, CryET43, CryET44, CryET45, CryET46, CryET47,
CryET49, CryET51, CryET52, CryET53, CryET54, CryET55, CryET56,
CryET57, CryET59, CryET60, CryET61, CryET62, CryET63, CryET64,
CryET66, CryET67, CryET68, CryET72, CryET73, CryET83 and related
sequences. These epitopic core sequences are identified herein in
particular aspects as hydrophilic regions of the particular
polypeptide antigen.
[0066] Computerized peptide sequence analysis programs (e.g.,
DNAStar.RTM. software, DNAStar, Inc., Madison, Wis.) may also be
useful in designing synthetic peptides in accordance with the
present disclosure.
[0067] Syntheses of epitopic sequences, or peptides which include
an antigenic epitope within their sequence, are readily achieved
using conventional synthetic techniques such as the solid phase
method (e.g., through the use of commercially available peptide
synthesizer such as an Applied Biosystems Model 430A Peptide
Synthesizer).
2.11 Biological Functional Equivalents
[0068] 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 1.
TABLE-US-00001 TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG
GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic
acid Glu B 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
[0069] 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.
[0070] 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.
[0071] 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 (-0.4); threonine
(-0.7); serine (-0.8); 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).
[0072] 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.
[0073] 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.
[0074] 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).
[0075] 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.
[0076] 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.
2.12 Insecticidal Compositions and Methods of Use
[0077] 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. In a
preferred embodiment, the bioinsecticide composition comprises an
oil flowable suspension of bacterial cells which expresses a novel
crystal protein disclosed herein. Preferably the cells are B.
thuringiensis NRRL B-21921, NRRL B-21922, NRRL B-21923, NRRL
B-21924, NRRL B-21925, NRRL B-21926, NRRL B-21927, NRRL B-21928,
NRRL B-21929, NRRL B-21930, NRRL B-21931, NRRL B-21932, NRRL
B-21933, NRRL B-21934, NRRL B-21935, NRRL B-21936, NRRL B-21937,
NRRL B-21938, NRRL B-21939, NRRL B-21940, NRRL B-21941, NRRL
B-21942, NRRL B-21943, and NRRL B-21944, 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 B. thuringiensis, B. megaterium, B. subtilis, E.
coli, or Pseudomonas spp.
[0078] In another important embodiment, the bioinsecticide
composition comprises a water dispersible granule. This granule
comprises bacterial cells which expresses a novel crystal protein
disclosed herein. Preferred bacterial cells are B. thuringiensis
NRRL B-21921, NRRL B-21922, NRRL B-21923, NRRL B-21924, NRRL
B-21925, NRRL B-21926, NRRL B-21927, NRRL B-21928, NRRL B-21929,
NRRL B-21930, NRRL B-21931, NRRL B-21932, NRRL B-21933, NRRL
B-21934, NRRL B-21935, NRRL B-21936, NRRL B-21937, NRRL B-21938,
NRRL B-21939, NRRL B-21940, NRRL B-21941, NRRL B-21942, NRRL
B-21943, and NRRL B-21944, however, bacteria such as B.
thuringiensis, B. megaterium, B. subtilis, E. coli, or Pseudomonas
spp. cells transformed with a DNA segment disclosed herein and
expressing the crystal protein are also contemplated to be
useful.
[0079] In a third important embodiment, the bioinsecticide
composition comprises a wettable powder, dust, pellet, or colloidal
concentrate. This powder comprises bacterial cells which expresses
a novel crystal protein disclosed herein. Preferred bacterial cells
are B. thuringiensis NRRL B-21921, NRRL B-21922, NRRL B-21923, NRRL
B-21924, NRRL B-21925, NRRL B-21926, NRRL B-21927, NRRL B-21928,
NRRL B-21929, NRRL B-21930, NRRL B-21931, NRRL B-21932, NRRL
B-21933, NRRL B-21934, NRRL B-21935, NRRL B-21936, NRRL B-21937,
NRRL B-21938, NRRL B-21939, NRRL B-21940, NRRL B-21941, NRRL
B-21942, NRRL B-21943, and NRRL B-21944 cells, however, bacteria
such as B. thuringiensis, B. megaterium, B. subtilis, 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.
[0080] In a fourth important embodiment, the bioinsecticide
composition comprises an aqueous suspension of bacterial cells such
as those described above which express the crystal protein. Such
aqueous suspensions may be provided as a concentrated stock
solution which is diluted prior to application, or alternatively,
as a diluted solution ready-to-apply.
[0081] 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.
[0082] When the insecticidal compositions comprise intact B.
thuringiensis cells expressing the protein of interest, such
bacteria may be formulated in a variety of ways. They may be
employed as wettable powders, granules or dusts, by mixing with
various diluents, 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.
[0083] Alternatively, the novel insecticidal polypeptides 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 colloidal
preparations of such crystals and/or spores as the active
bioinsecticidal composition.
[0084] Regardless of the method of application, the amount of the
active component(s) is applied at an insecticidally-effective
amount, which will vary depending on such factors as, for example,
the specific coleopteran 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.
[0085] 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, desiccated, 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. coli, 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. coli, by homogeneously
mixing, blending and/or grinding the insecticidal composition with
suitable adjuvants using conventional formulation techniques.
[0086] 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.
[0087] Other application techniques, including 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.
[0088] 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.
[0089] 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 polypeptide compositions may be
from about 1% to about 99% or more by weight of the protein
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 110 to about 110 cells/mg.
[0090] 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 50 g to about 500 g of active
ingredient, or of from about 500 g to about 1000 g, or of from
about 1000 g to about 5000 g or more of active ingredient.
5.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
5.1 Some Advantages of the Invention
[0091] The use of B. thuringiensis insecticidal crystal protein
genes for in planta production of insecticidal proteins, thereby
conferring insect resistance on important agronomic plants, is
rapidly gaining commercial acceptance in the United States and
abroad. The need for new insecticidal traits does not diminish,
however, with the successful deployment of a handful of cry genes
in plants. Concerns over the potential for insect resistance
development, for instance, makes it imperative that an arsenal of
insecticidal proteins (i.e. cry genes) be assembled to provide the
genetic material necessary for tomorrow's insecticidal traits. In
addition, transgenic plants producing a B. thuringiensis Cry
protein may still be susceptible to damage from secondary insect
pests, thus prompting the search for additional Cry proteins with
improved efficacy towards these pests. The B. thuringiensis crystal
proteins of the present invention represent a diverse collection of
insecticidal proteins, including several that are toxic towards a
lepidopteran colony exhibiting resistance to certain types of Cry1
proteins. Bioassays against a wide range of lepidopteran pests
confirm that these proteins possess insecticidal activity and,
furthermore, that these proteins vary in efficacy against this
array of target insects. This variation in the spectrum of insects
affected by the toxin proteins suggests differing modes of action
that may be important for future insect resistance management
strategies. In planta expression of the cry genes of the present
invention can confer insect resistance to the host plant as has
been demonstrated for other cry genes from B. thuringiensis.
5.2 Probes and Primers
[0092] 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:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID
NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ
ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39,
SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID
NO:49 and SEQ ID NO:62. The ability of such DNAs and 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.
[0093] 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.
[0094] 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:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,
SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID
NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ
ID NO:49 and SEQ ID NO:62. A size of at least about 14 or so
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 about 14 or so 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 about 14 to about 20 or so 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.
5.3 Expression Vectors
[0095] 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.
[0096] 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.
[0097] 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. 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 the
cryET31, cryET40, cryET43, cryET44, cryET45, cryET46, cryET47,
cryET49, cryET51, cryET5Z cryET53, cryET54, cryET55, cryET56,
cryET57, cryET59, cryET60, cryET61, cryET62, cryET63, cryET64,
cryET66, cryET67, cryET68, cryET7Z cryET73, and cryET83 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.
[0098] 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.
[0099] 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).
[0100] 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.
[0101] 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.
[0102] 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.)
[0103] 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.
[0104] 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.
[0105] 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 include a cauliflower mosaic virus (CaMV 35S) promoter, a
S-E9 small subunit RuBP carboxylase promoter, a rice actin
promoter, a maize histone promoter, a fused CAMV 35S-Arabidopsis
histone promoter, a CaMV 35S promoter, a CaMV 19S promoter, a nos
promoter, an Adh promoter, an actin promoter, a histone promoter, a
ribulose bisphosphate carboxylase promoter, an R-allele promoter, a
root cell promoter, an .alpha.-tubulin promoter, an ABA-inducible
promoter, a turgor-inducible promoter, a rbcS promoter, a corn
sucrose synthetase 1 promoter, a corn alcohol dehydrogenase 1
promoter, a corn light harvesting complex promoter, a corn heat
shock protein promoter, a pea small subunit RuBP carboxylase
promoter, a Ti plasmid mannopine synthase promoter, a Ti plasmid
nopaline synthase promoter, a petunia chalcone isomerase promoter,
a bean glycine rich protein 1 promoter, a CaMV 35S transcript
promoter, a potato patatin promoter, a cab promoter, a
PEP-Carboxylase promoter and an S-E9 small subunit RuBP carboxylase
promoter.
[0106] 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.
[0107] 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.
[0108] 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).
[0109] 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).
[0110] 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.
[0111] 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.
[0112] A coding region that encodes a polypeptide having the
ability to confer insecticidal activity to a cell is preferably a
CryET31, CryET40, CryET43, CryET44, CryET45, CryET46, CryET47,
CryET49, CryET51, CryET52, CryET53, CryET54, CryET55, CryET56,
CryET57, CryET59, CryET60, CryET61, CryET62, CryET63, CryET64,
CryET66, CryET67, CryET68, CryET72, CryET73, and CryET83
polypeptide-encoding gene.
5.7 Nomenclature of the Novel Polypeptides
[0113] The inventors have arbitrarily assigned the designation
CryET31, CryET40, CryET43, CryET44, CryET45, CryET46, CryET47,
CryET49, CryET51, CryET52, CryET53, CryET54, CryET56, CryET57,
CryET59, CryET60, CryET61, CryET62, CryET63, CryET64, CryET66,
CryET67, CryET68, CryET72, CryET73, and CryET83 to the polypeptides
of this invention. Likewise, the arbitrary designations of cryET31,
cryET40, cryET43, cryET44, cryET45, cryET46, cryET47, cryET49,
cryET5, cryET52, cryET53, cryET54, cryET56, cryET57, cryET59,
cryET60, cryET61, cryET62, cryET63, cryET64, cryET66, cryET67,
cryET68, cryET72, cryET73, and cryET83 have been assigned to the
novel nucleic acid sequence which encodes these polypeptides,
respectively. Formal assignment of gene and protein designations
based on the revised nomenclature of crystal protein endotoxins
will be assigned by a committee on the nomenclature of B.
thuringiensis, formed to systematically classify B. thuringiensis
crystal proteins. The inventors contemplate that the arbitrarily
assigned designations of the present invention will be superceded
by the official nomenclature assigned to these sequences.
5.8 Transformed Host Cells and Transgenic Plants
[0114] Methods and compositions for transforming a bacterium, a
yeast cell, a plant cell, or an entire plant with one or more
expression vectors comprising a crystal protein-encoding gene
segment are further aspects of this disclosure. A transgenic
bacterium, yeast cell, plant cell or plant derived from such a
transformation process or the progeny and seeds from such a
transgenic plant are also further embodiments of the invention.
[0115] 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. 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.
[0116] 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.
[0117] 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; U.S. Pat. No. 5,384,253) 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., 1991;
1992; Wagner et al., 1992).
5.8.3 Agrobacterium-Mediated Transfer
[0118] 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 al., 1986; Jorgensen et al., 1987).
[0119] Modern 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.
[0120] 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).
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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).
[0125] 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).
5.8.4 Other Transformation Methods
[0126] 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).
[0127] 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).
5.8.5 Gene Expression in Plants
[0128] Although great progress has been made in recent years with
respect to preparation of transgenic plants which express bacterial
proteins such as B. thuringiensis crystal proteins, the results of
expressing native bacterial genes in plants are often
disappointing. In recent years, however, several potential factors
have been implicated as responsible in varying degrees for the
level of protein expression from a particular coding sequence. For
example, scientists now know that maintaining a significant level
of a particular mRNA in the cell is indeed 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) insufficient 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. 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.
[0129] 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.
[0130] 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.
[0131] 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., 1988). 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.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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 2 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.
TABLE-US-00002 TABLE 2 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 ''
[0136] 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.
[0137] 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.
[0138] 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.
[0139] 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.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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.
5.8.6 Synthetic Oligonucleotides for Mutagenesis
[0144] When oligonucleotides are used in the mutagenesis, it is
desirable to maintain the proper amino acid sequence and reading
frame, without introducing common restriction sites such as BglII,
HindIII, SacI, KpnI, EcoRI, NcoI, PstI and SalI into the modified
gene. These restriction sites are found in poly-linker insertion
sites of many cloning vectors. Of course, the introduction of new
polyadenylation signals, ATTTA sequences or consecutive stretches
of more than five A+T or G+C, should also be avoided. The preferred
size for the oligonucleotides is about 40 to about 50 bases, but
fragments ranging from about 18 to about 100 bases have been
utilized. In most cases, a minimum of about 5 to about 8 base pairs
of homology to the template DNA on both ends of the synthesized
fragment are maintained to insure proper hybridization of the
primer to the template. The oligonucleotides should avoid sequences
longer than five base pairs A+T or G+C. Codons used in the
replacement of wild-type codons should preferably avoid the TA or
CG doublet wherever possible. Codons are selected from a plant
preferred codon table (such as Table 3 below) so as to avoid codons
which are rarely found in plant genomes, and efforts should be made
to select codons to preferably adjust the G+C content to about
50%.
[0145] Regions with many consecutive A+T bases or G+C bases are
predicted to have a higher likelihood to form hairpin structures
due to self-complementarity. Disruption of these regions by the
insertion of heterogeneous base pairs is preferred and should
reduce the likelihood of the formation of self-complementary
secondary structures such as hairpins which are known in some
organisms to inhibit transcription (transcriptional terminators)
and translation (attenuators).
[0146] Alternatively, a completely synthetic gene for a given amino
acid sequence can be prepared, with regions of five or more
consecutive A+T or G+C nucleotides being avoided. Codons are
selected avoiding the TA and CG doublets in codons whenever
possible. Codon usage can be normalized against a plant preferred
codon usage table (such as Table 3) and the G+C content preferably
adjusted to about 50%. The resulting sequence should be examined to
ensure that there are minimal putative plant polyadenylation
signals and ATTTA sequences.
TABLE-US-00003 TABLE 3 PREFERRED CODON USAGE IN PLANTS Amino Acid
Codon Percent Usage in Plants ARG CGA 7 CGC 11 CGG 5 CGU 25 AGA 29
AGG 23 SER UCA 14 UCC 26 UCG 3 UCU 21 AGC 21 AGU 15 THR ACA 21 ACC
41 ACG 7 ACU 31 PRO CCA 45 CCC 19 CCG 9 CCU 26 HIS CAC 65 CAU 35
GLU GAA 48 GAG 52 ASP GAC 48 GAU 52 TYR UAC 68 UAU 32 CYS UGC 78
UGU 22 LEU CUA 8 CUC 20 CUG 10 CUU 28 UUA 5 UUG 30 ALA GCA 23 GCC
32 GCG 3 GCU 41 GLY GGA 32 GGC 20 GGG 11 GGU 37 ILE AUA 12 AUC 45
AUU 43 VAL GUA 9 GUC 20 GUG 28 GUU 43 LYS AAA 36 AAG 64 ASN AAC 72
AAU 28 GLN CAA 64 CAG 36 PHE UUC 56 UUU 44 MET AUC 100 TRP UCC
100
[0147] Restriction sites found in commonly used cloning vectors are
also preferably avoided. However, placement of several unique
restriction sites throughout the gene is useful for analysis of
gene expression or construction of gene variants.
5.8.7 "Plantized" Gene Constructs
[0148] 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.
[0149] A number of promoters which are active in plant cells have
been described in the literature. These include the nopaline
synthase (NOS) and octopine synthase (OCS) promoters (which are
carried on tumor-inducing plasmids of A. tumefaciens), the
Cauliflower Mosaic Virus (CaMV) 19S and 35S promoters, the
light-inducible promoter from the small subunit of ribulose
bis-phosphate carboxylase (ssRUBISCO, a very abundant plant
polypeptide) and the mannopine synthase (MAS) promoter (Velten et
al., 1984; Velten and Schell, 1985). All of these promoters have
been used to create various types of DNA constructs which have been
expressed in plants (see e.g., Intl. Pat. Appl. Publ. Ser. No. WO
84/02913).
[0150] Promoters which are known or are found to cause
transcription of RNA in plant cells can be used in the present
invention. Such promoters may be obtained from plants or plant
viruses and include, but are not limited to, the CaMV35S promoter
and promoters isolated from plant genes such as ssRUBISCO genes. As
described below, it is preferred that the particular promoter
selected should be capable of causing sufficient expression to
result in the production of an effective amount of protein.
[0151] 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.
[0152] 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 eukaryotic genes, or from a synthetic gene
sequence. The present invention is not limited to constructs, as
presented in the following examples. Rather, the non-translated
leader sequence can be part of the 5' end of the non-translated
region of the coding sequence for the virus coat protein, or part
of the promoter sequence, or can be derived from an unrelated
promoter or coding sequence. In any case, it is preferred that the
sequence flanking the initiation site conform to the translational
consensus sequence rules for enhanced translation initiation
reported by Kozak (1984).
[0153] The cry DNA constructs of the present invention may also
contain one or more modified or fully-synthetic structural coding
sequences which have been changed to enhance the performance of the
cry gene in plants. The structural genes of the present invention
may optionally encode a fusion protein comprising an amino-terminal
chloroplast transit peptide or secretory signal sequence.
[0154] The DNA construct also contains a 3' non-translated region.
The 3' non-translated region contains a polyadenylation signal
which functions in plants to cause the addition of polyadenylate
nucleotides to the 3' end of the viral RNA. Examples of suitable 3'
regions are (1) the 3' transcribed, non-translated regions
containing the polyadenylation signal of Agrobacterium
tumor-inducing (Ti) plasmid genes, such as the nopaline synthase
(NOS) gene, and (2) plant genes like the soybean storage protein
(7S) genes and the small subunit of the RuBP carboxylase (E9)
gene.
5.9 Methods for Producing Insect-Resistant Transgenic Plants
[0155] By transforming a suitable host cell, such as a plant cell,
with a recombinant cryET31, cryET40, cryET43, cryET44, cryET45,
cryET46, cryET47, cryET49, cryET51, cryET52, cryET53, cryET54,
cryET56, cryET57, cryET59, cryET60, cryET61, cryET62, cryET63,
cryET64, cryET66, cryET67, cryET68, cryET72, cryET73, and cryET83
gene-containing segment, the expression of the encoded crystal
protein (i.e., a bacterial crystal protein or polypeptide having
insecticidal activity against coleopterans) can result in the
formation of insect-resistant plants.
[0156] 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.
[0157] 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).
[0158] 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.
[0159] 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).
[0160] 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.
[0161] 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.
[0162] A transgenic plant of this invention thus has an increased
amount of a coding region (e.g., a cryET31, cryET40, cryET43,
cryET44, cryET45, cryET46, cryET47, cryET49, cryET51, cryET52,
cryET53, cryET54, cryET56, cryET57, cryET59, cryET60, cryET61,
cryET62, cryET63, cryET64, cryET66, cryET67, cryET68, cryET72,
cryET73, and cryET83 gene) that encodes one or more CryET31,
CryET40, CryET43, CryET44, CryET45, CryET46, CryET47, CryET49,
CryET51, CryET52, CryET53, CryET54, CryET56, CryET57, CryET59,
CryET60, CryET61, CryET62, CryET63, CryET64, CryET66, CryET67,
CryET68, CryET72, CryET73, and CryET83 polypeptides. 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 coleopteran 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.
5.10 Definitions
[0163] The following words and phrases have the meanings set forth
below.
[0164] 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.
[0165] Identity or percent identity: refers to the degree of
similarity between two nucleic acid or protein sequences. An
alignment of the two sequences is performed by a suitable computer
program. A widely used and accepted computer program for performing
sequence alignments is CLUSTALW v1.6 (Thompson, et al. Nucl. Acids
Res., 22: 4673-4680, 1994). The number of matching bases or amino
acids is divided by the total number of bases or amino acids, and
multiplied by 100 to obtain a percent identity. For example, if two
580 base pair sequences had 145 matched bases, they would be 25
percent identical. If the two compared sequences are of different
lengths, the number of matches is divided by the shorter of the two
lengths. For example, if there were 100 matched amino acids between
200 and a 400 amino acid proteins, they are 50 percent identical
with respect to the shorter sequence. If the shorter sequence is
less than 150 bases or 50 amino acids in length, the number of
matches are divided by 150 (for nucleic acid bases) or 50 (for
amino acids), and multiplied by 100 to obtain a percent
identity.
[0166] 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.
[0167] Regeneration: The process of growing a plant from a plant
cell (e.g., plant protoplast or explant).
[0168] Structural gene: A polynucleotide sequence that encodes a
polypeptide, that is expressed to produce a polypeptide, or which
is cryptic or incapable of expression in its natural host cell but
which can be isolated and purified and operably linked to at least
a promoter functional in one or more host cell types to express the
encoded polypeptide.
[0169] 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.
[0170] Transformed cell: A cell whose DNA has been altered by the
introduction of an exogenous DNA molecule into that cell.
[0171] 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.
[0172] 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.
[0173] 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. A plasmid is an
exemplary vector.
5.11 Isolating Homologous Gene and Gene Fragments
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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.
6.0 EXAMPLES
[0182] 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.
6.1 Example 1
Identification of B. thuringiensis Strains Containing Novel
.delta.-Endotoxins
[0183] Wild-type B. thuringiensis strains containing novel
insecticidal protein genes were identified by Southern blot
hybridization studies employing specific DNA probes. Twenty-four
unique cry genes were discovered that are related to B.
thuringiensis genes in the cry1, cry2, or cry9 classes of toxin
genes.
[0184] Various methods were employed to clone the novel genes and
express them in a crystal protein-negative (Cry-) strain of B.
thuringiensis. These methods include PCR.TM. amplification of the
region of cry1-related genes that encodes the active portion of the
toxin gene. The PCR.TM. product is then joined to a fragment from
the cry1Ac gene encoding the C-terminal region of the protoxin.
This gene fusion was then expressed in a B. thuringiensis
recombinant strain to produce a hybrid protoxin. In this instance,
it is recognized that the sequence of the amplified DNA can be used
to design hybridization probes to isolate the entire coding
sequence of the novel cry gene from the wild-type B. thuringiensis
strain.
[0185] Wild-type B. thuringiensis strains were screened in a
bioassay to identify strains that are toxic to larvae of
lepidopteran insects (procedure described in Example 10). Active
strains were then examined genetically to determine if they contain
novel toxin genes. The method used to make this determination is
described below and includes isolation of genomic DNA from the B.
thuringiensis strain, restriction enzyme digestion, Southern blot
hybridization, and analysis of the hybridizing restriction
fragments to determine which genes are present in a strain.
[0186] Total genomic DNA was extracted by the following procedure.
Vegetative cells were resuspended in a lysis buffer containing 50
mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 4 mg/ml
lysozyme. The suspension was incubated at 37.degree. C. for 1 h.
Following incubation, the suspension was extracted once with an
equal volume of phenol, then once with an equal volume of
phenol:chloroform:isoamyl alcohol (50:48:2), and once with an equal
volume of chloroform:isoamyl alcohol (24:1). The DNA was
precipitated from the aqueous phase by the addition of one-tenth
volume 3 M sodium acetate and two volumes of 100% ethanol. The
precipitated DNA was collected by centrifugation, washed with 70%
ethanol and resuspended in distilled water.
[0187] The DNA samples were digested with the restriction enzymes
ClaI and PstI. The combination of these two enzymes give a
digestion pattern of fragments that, when hybridized with the probe
wd207 (described below), allows the identification of many of the
known cry1-related toxin genes. Hybridizing fragments that did not
correspond to the fragment sizes expected for the known genes were
classified as unknown and were candidates for cloning and
characterization.
[0188] The digested DNA was size fractionated by electrophoresis
through a 1.0% agarose gel in 1.times.TBE (0.089 M Tris-borate,
0.089 M boric acid, 0.002 M EDTA) overnight at 2 V/cm of gel
length. The fractionated DNA fragments were then transferred to a
Millipore Immobilon-NC.RTM. nitrocellulose filter (Millipore Corp.,
Bedford, Mass.) according to the method of Southern (1975). The DNA
fragments were fixed to the nitrocellulose by baking the filter at
80.degree. C. in a vacuum oven.
[0189] To identify the DNA fragment(s) containing the sequences
related to cry1 genes, the oligonucleotide wd207 was radioactively
labeled at the 5' end and used as a hybridization probe. To
radioactively label the probe, 1-5 pmoles of wd207 were added to a
reaction (20 ul total volume) containing 3 ul [.gamma.-.sup.32P]ATP
(3,000 Ci/mmole at 10 mCi/ml), 70 mM Tris-HCl, pH 7.8, 10 mM
MgCl.sub.2, 5 mM DTT, and 10 units T4 polynucleotide kinase
(Promega Corp., Madison, Wis.). The reaction was incubated for 20
min at 37.degree. C. to allow the transfer of the radioactive
phosphate to the 5'-end of the oligonucleotide, thus making it
useful as a hybridization probe.
[0190] The oligonucleotide probe used in this analysis, designated
wd207, has the following sequence:
TABLE-US-00004 (SEQ ID NO: 51)
5'-TGGATACTTGATCAATATGATAATCCGTCACATCTGTTTTTA-3'
[0191] This oligonucleotide was designed to specifically hybridize
to a conserved region of cry1 genes downstream from the proteolytic
activation site in the protoxin. Table 4 lists some of the B.
thuringiensis toxin genes and their identities with wd207. The
orientation of the wd207 sequence is inverted and reversed relative
to the coding sequences of the cry genes.
TABLE-US-00005 TABLE 4 cry Gene % Identity to wd207 Nucleotide
Position in CDS cry1Aa 100% 1903-1944 cry1Ba 95.2% 1991-2032 cry1Ca
97.6% 1930-1971 cry1Da 97.6% 1858-1899 cry1Ea 97.6% 1885-1926
[0192] The labeled probe was then incubated with the nitrocellulose
filter overnight at 45.degree. C. in 3.times.SSC (1.times.SSC=0.15
M NaCl, 0.015 M sodium citrate), 0.1% SDS, 10.times.Denhardt's
reagent (0.2% BSA, 0.2% polyvinylpyrrolidone, 0.2% Ficoll), and 0.2
mg/ml heparin. Following this incubation period, the filter was
washed in several changes of 3.times.SSC, 0.1% SDS at 45.degree. C.
The filter was blotted dry and exposed to Kodak X-OMAT AR X-ray
film (Eastman Kodak Co., Rochester, N.Y.) overnight at -70.degree.
C. with an intensifying screen to obtain an autoradiogram.
[0193] The autoradiograms were analyzed to determine which
wild-type B. thuringiensis strains contained cry1 genes that could
be novel. Since the probe was only 42 nucleotides, it is unlikely
that recognition sites for the restriction endonucleases ClaI and
PstI would occur within the hybridizing region of the cry1-related
genes. Therefore, it was assumed that each hybridizing restriction
fragment represented one cry1-related gene. The sizes, in kilobases
(kb), of the hybridizing restriction fragments were determined
based on the migration of the fragment in the agarose gel relative
to DNA fragments of known size. The size of a fragment could be
used to determine if that fragment represented a known cry1 gene.
For example, from the DNA sequence of the cry1Ac gene it was known
that wd207 would hybridize to a 0.43 kb fragment after digestion of
cry1Ac DNA with ClaI and PstI. If the Southern blot analysis of a
strain showed a 0.43 kb hybridizing fragment, that strain was
assigned a probable genotype of cry1Ac. Fragments that could not be
easily assigned a probable genotype were selected as candidates for
further analysis. Because many cry1-containing strains have more
than one cry1-related gene, all fragments were given a putative
designation.
TABLE-US-00006 TABLE 5 SUMMARY OF GENES AND PROTEINS Polypeptide
Polypeptide Polynucleotide WT- Recomb. Gene Cloning DNA Cloning
Designation Seq. ID No.: Seq ID No.: Strain Strain Family
Method.sup.1 Probe.sup.2 Vector Plasmid Cry ET31 2 1 EG6701 EG11562
cry2 Mbol cry2a pHT315 pEG1331 Cry ET40 4 3 EG5476 EG11901 cry1 PCR
.TM. -- pEG1064 pEG1901 Cry ET43 6 5 EG2878 EG7692 cry1 PCR .TM. --
pEG1064 pEG1806 Cry ET44 8 7 EG3114 EG11629 cry1 PCR .TM. --
pEG1064 pEG1807 Cry ET45 10 9 EG3114 EG7694 cry1 PCR .TM. --
pEG1064 pEG1808 Cry ET46 12 11 EG6451 EG7695 cry1 PCR .TM. --
pEG1064 pEG1809 Cry ET47 14 13 EG6451 EG7696 cry1 PCR .TM. --
pEG1064 pEG1810 Cry ET49 16 15 EG6451 EG11630 cry1 PCR .TM. --
pEG1064 pEG1812 Cry ET51 18 17 EG5391 EG11921 cry1 Mbol wd207
pHT315 pEG1912 Cry ET52 20 19 EG10475 EG11584 cry1 BamHI wd207
pEG290 pEG1340 Cry ET53 22 21 EG3874 EG11906 cry1 Mbol cry1Aa
pHT315 pEG1904 Cry ET54 EG3874 EG11907 cry1 Mbol cry1Aa pHT315
pEG1905 Cry ET56 24 23 EG3874 EG11909 cry1 Mbol cry1Aa pHT315
pEG1907 Cry ET57 26 25 EG3874 EG11910 cry1 Mbol cry1Aa pHT315
pEG1908 Cry ET59 28 27 EG9290 EG12102 cry9 Mbol pr56, pHT315 pEG945
cryET59 Cry ET60 30 29 EG9290 EG12103 cry9 Mbol pr56, pHT315 pEG946
cryET59 Cry ET61 32 31 EG4612 EG11634 cry1 Mbol wd207 pHT315
pEG1813 Cry ET62 34 33 EG6831 EG11635 cry1 Mbol wd207 pHT315
pEG1814 Cry ET63 36 35 EG4623 EG11636 cry1 Mbol wd207 pHT315
pEG1815 Cry ET64 38 37 EG4612 EG11638 cry1 Mbol wd207 pHT315
pEG1816 Cry ET66 40 39 EG5020 EG11640 cry1 Mbol wd207 pHT315
pEG1817 Cry ET67 42 41 EG4869 EG11642 cry1 Mbol wd207 pHT315
pEG1818 Cry ET68 44 43 EG5020 EG11644 cry1 Mbol wd207 pHT315
pEG1819 Cry ET72 46 45 EG4420 EG11440 cry2 HindIII cry2Aa pEG597
pEG1260 Cry ET73 48 47 EG3874 EG11465 cry2 HindIII cry2Aa pEG597
pEG1279 Cry ET83 50 49 EG6346 EG11785 cry9 Mbol cryET59, pHT315
pEG397 cryET83 .sup.1Methods include the construction of genomic
libraries containing partial Mbol fragments (Example 4), the
construction of genomic libraries containing size-selected BamHI or
HindIII restriction fragments (Example 5), the amplification of
novel cry sequences by PCR .TM. and the construction of novel cry
gene fusions (Example 6). .sup.2Hybridization probes included the
700 base pair EcoRI fragment obtained from digestion of the cry1Aa
gene, gene fragments from the cry2Aa, cryET59, and cryET83 genes,
and synthetic oligonucleotides (wd207, pr56).
6.2 Example 2
Identification of B. thuringiensis Strains Containing Novel
Cry2-Related Genes
[0194] Proteins encoded by the cry2 class of B. thuringiensis class
of toxin genes have activity on the larvae of lepidopteran and
diopteran insects. Southern blot hybridization analysis of DNA
extracted from lepidopteran-active strains was utilized to identify
novel cry2-related genes. Total genomic DNA was isolated as
described in Section 6.1. The DNA was digested with the restriction
endonuclease Sau3A and run on a 1.2% agarose gel as described. The
digested DNA was transferred to nitrocellulose filters to be probed
with a DNA fragment containing the cry2Aa gene. Hybridizations were
performed at 55.degree. C. and the filters washed and exposed to
X-ray film to obtain an autoradiogram.
[0195] Sau3A digestion followed by hybridization with the cry2Aa
gene gave characteristic patterns of hybridizing fragments allowing
the identification of the cry2Aa, cry2Ab, and cry2Ac genes.
Hybridizing fragments that differed from these patterns indicated
the presence of a novel cry2-related gene in that strain.
[0196] Once a strain was identified as containing one or more novel
cry2-related genes, an additional Southern blot hybridization was
performed. The procedures were the same as those already described
above, except another restriction enzyme, usually HindIII, was
used. Since an enzyme like HindIII (a "six base cutter") cuts DNA
less frequently than does Sau3A or MboI, it was more likely to
generate a restriction fragment containing the entire cry2-related
gene which could then be readily cloned.
6.3 Example 3
Identification of B. thuringiensis Strains Containing Novel
Cry9-Type Genes
[0197] A cry9-specific oligonucleotide, designated pr56, was
designed to facilitate the identification of strains harboring
cry9-type genes. This oligonucleotide corresponds to nucleotides
4349-4416 of the gene (GenBank Accession No. Z37527). The sequence
of pr56 was as follows:
TABLE-US-00007 (SEQ ID NO: 52)
5'-AGTAACGGTGTTACTATTAGCGAGGGCGGTCCATTCTTTAAAGGTCG
TGCACTTCAGTTAGC-3'.
[0198] B. thuringiensis isolates were spotted or "patched" on SGNB
plates, with no more than 50 isolates per plate, and grown
overnight at 25.degree. C. The B. thuringiensis colonies were
transferred to nitrocellulose filters and the filters placed,
colony side up, on fresh SGNB plates for overnight growth at
30.degree. C. Subsequently, the filters were placed, colony side
up, on Whatman paper soaked in denaturing solution (1.5 M NaCl, 0.5
N NaOH) for 20 min. After denaturation, the filters were placed on
Whatman paper soaked in neutralizing solution (3 M NaCl, 1.5 M
Tris-HCl, pH 7.0) for 20 min. Finally, the filters were washed in
3.times.SSC (1.times.SSC=0.15 M NaCl and 0.015 M sodium citrate) to
remove cellular debris and baked in a vacuum oven at 80.degree. C.
for 90 min.
[0199] The cry9-specific oligonucleotide pr56 (.about.10 pmoles)
was end-labeled with [.gamma.-.sup.32P]ATP using T4 polynucleotide
kinase. The labeling reaction was carried out at 37.degree. C. for
20 min and terminated by incubating the reaction at 100 C for 3
min. After ethanol precipitation, the labeled oligonucleotide was
resuspended in 100 .mu.l distilled H.sub.2O.
[0200] The filters were incubated with the cry9-specific probe in
6.times.SSC, 10.times.Denhardt's solution, 0.5% glycine, 0.2% SDS
at 47.degree. C. overnight. The filters were washed twice in
3.times.SSC, 0.1% SDS for 15 min at 47.degree. C. and twice in
1.times.SSC, 0.1% SDS for 15 min at 47.degree. C. The dried filters
were exposed to X-OMAT XAR-5 film (Eastman Kodak Co.) at
-70.degree. C. using an intensifying screen. The developed
autoradiogram revealed 24 isolates of B. thuringiensis containing
DNA that hybridized to the cry9 probe.
[0201] To identify cry9C-type genes among these strains, two
opposing oligonucleotide primers specific for the cry9C gene
(GenBank Accession No. Z37527) were designed for polymerase chain
reaction (PCR.TM.) analyses. The sequence of pr58 is:
TABLE-US-00008 5'-CGACTTCTCCTGCTAATGGAGG-3'. (SEQ ID NO: 53)
The sequence of pr59 is:
TABLE-US-00009 (SEQ ID NO: 54)
5'-CTCGCTAATAGTAACACCGTTACTTGCC-3'.
Plasmid DNAs were isolated from the isolates of B. thuringiensis
believed to contain cry9-type genes. B. thuringiensis isolates were
grown overnight at 30.degree. C. on Luria agar plates and 2
loopfuls of cells from each isolate were suspended in 50 mM
glucose, 10 mM Tris-HCl, 1 mM EDTA (1.times.GTE) containing 4 mg/ml
lysozyme. After a 10 min incubation at room temperature, plasmid
DNAs were extracted using a standard alkaline lysis procedure
(Maniatis et al., 1982). The plasmid DNAs were resuspended in 20
.mu.l of 1.times.TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Two
microliters of the plasmid DNA preparations were used in the
PCR.TM. reactions. Amplifications were performed in 100 .mu.l
volumes with a Perkin-Elmer DNA Thermocycler (Perkin-Elmer Cetus,
Foster City, Calif.) using materials and methods provided in the
Perkin-Elmer GeneAmp.TM. kit. Conditions for the PCR.TM. were as
follows: 95.degree. C. for 30 sec, 46.degree. C. for 30 sec,
70.degree. C. for 1 min; 30 cycles. A PCR.TM. using these primers
and the cry9C gene as a template should yield a DNA fragment of
.about.970 bp. Of twenty-four strains found to hybridize to the
cry9 probe (SEQ ID NO:XX), only one strain, EG9290, yielded the
predicted amplified DNA fragment.
6.4 Example 4
Cloning of B. thuringiensis Toxin Genes by Constructing MboI
Partial Digest Libraries
[0202] The restriction endonuclease MboI was utilized in the
construction of genomic DNA libraries because it has a recognition
sequence of four base pairs which occurs frequently in long
stretches of DNA. Total genomic DNA was isolated from B.
thuringiensis strains as described in Section 6.1. The DNA was
digested under conditions allowing limited cleavage of a DNA
strand. The method of establishing these conditions has been
described (Maniatis et al, 1982). Digestion of DNA in this manner
created a set of essentially randomly cleaved, overlapping
fragments which were used to create a library representative of the
entire genome.
[0203] The digested DNA fragments were separated, according to
size, by agarose gel electrophoresis through a 0.6% agarose,
1.times.TBE gel, overnight at 2 volts/cm of gel length. The gel was
stained with ethidium bromide so that the digested DNA could be
visualized when exposed to long-wave UV light. A razor blade was
used to excise a gel slice containing DNA fragments of
approximately 9-kb to 12-kb in size. The DNA fragments were removed
from the agarose by placing the slice in a dialysis bag with enough
TE (10 mM Tris-HCl, 1 mM EDTA) to cover the slice. The bag was then
closed and placed in a horizontal electrophoresis apparatus filled
with 1.times.TBE buffer. The DNA was electroeluted from the slice
into the TE at 100 volts for 2 h. The TE was removed from the bag,
extracted with phenol:chloroform (1:1), followed by extraction with
chloroform. The DNA fragments are then collected by the standard
technique of ethanol precipitation (see Maniatis et al., 1982).
[0204] To create a library in E. coli of the partially-digested
DNA, the fragments were ligated into the shuttle vector, pHT315
(Arantes and Lereclus, 1991). This plasmid contains replication
origins for E. coli and B. thuringiensis, genes for resistance to
the antibiotics erythromycin and ampicillin, and a multiple cloning
site. The MboI fragments were mixed with BamHI-digested pHT315 that
had been treated with calf intestinal, or bacterial, alkaline
phosphatase (GibcoBRL, Gaithersburg, Md.) to remove the
5'-phosphates from the digested plasmid, preventing re-ligation of
the vector to itself. After purification, T4 ligase and a ligation
buffer (Promega Corp., Madison, Wis.) were added to the reaction
containing the digested vector and the MboI fragments. These were
incubated overnight at 15.degree. C., or at room temperature for 1
h, to allow the insertion and ligation of the MboI fragments into
the pHT315 vector DNA.
[0205] The ligation mixture was then introduced into
transformation-competent E. coli SURE.RTM. cells (Stratagene
Cloning Systems, La Jolla, Calif.), following procedures described
by the manufacturer. The transformed E. coli cells were then plated
on LB agar plates containing 50-75 .mu.g/ml ampicillin and
incubated overnight at 37.degree. C. The growth of several hundred
ampicillin-resistant colonies on each plate indicated the presence
of recombinant plasmid in the cells of each of those colonies.
[0206] To isolate the colonies harboring sequences encoding toxin
genes, the colonies were first transferred to nitrocellulose
filters. This was accomplished by simply placing a circular
nitrocellulose filter (Millipore HATF 08525, Millipore Corp.,
Bedford, Mass.) directly on top of the LB-ampicillin agar plates
containing the transformed colonies. When the filter was slowly
peeled off of the plate the colonies stick to the filter giving an
exact replica of the pattern of colonies from the original plate.
Enough cells from each colony were left on the plate that 5 to 6 h
of growth at 37.degree. C. restored the colonies. The plates were
then stored at 4.degree. C. until needed. The nitrocellulose
filters with the transferred colonies are then placed, colony-side
up, on fresh LB-ampicillin agar plates and allowed to grow at
37.degree. C. until they reached an approximate 1 mm diameter.
[0207] To release the DNA from the recombinant E. coli cells the
nitrocellulose filters were placed, colony-side up, on 2-sheets of
Whatman 3MM chromatography paper (Whatman International Ltd.,
Maidstone, England) soaked with 0.5 N NaOH, 1.5 M NaCl for 15 min.
This treatment lysed the cells and denatured the released DNA
allowing it to stick to the nitrocellulose filter. The filters were
then neutralized by placing the filters, colony-side up, on 2
sheets of Whatman paper soaked with 1 M NH.sub.4-acetate, 0.02 M
NaOH for 10 min. The filters were rinsed in 3.times.SSC, air dried,
and baked for 1 h at 80.degree. C. in a vacuum oven. The filters
were then ready for use in hybridization studies using probes to
identify different classes of B. thuringiensis genes, as described
in the above examples.
[0208] In order to identify colonies containing cloned cry1-related
genes, the cry1-specific oligonucleotide wd207 was labeled at the
5'-end with [.gamma.-.sup.32P]ATP and T4 polynucleotide kinase. The
labeled probe was added to the filters in 3.times.SSC, 0.1% SDS,
10.times.Denhardt's reagent (0.2% BSA, 0.2% polyvinylpyrrolidone,
0.2% Ficoll), 0.2 mg/ml heparin and incubated overnight at
47.degree. C. These conditions allowed hybridization of the labeled
oligonucleotide to related sequences present on the nitrocellulose
blots of the transformed E. coli colonies. Following incubation the
filters were washed in several changes of 3.times.SSC, 0.1% SDS at
45.degree. C. The filters were blotted dry and exposed to Kodak
X-OMAT AR X-ray film (Eastman Kodak Co., Rochester, N.Y.) overnight
at -70.degree. C. with an intensifying screen.
[0209] Colonies that contain cloned cry1-related sequences were
identified by aligning signals on the autoradiogram with the
colonies on the original transformation plates. The isolated
colonies were then grown in LB-ampicillin liquid medium from which
the cells could be harvested and recombinant plasmid prepared by
the standard alkaline-lysis miniprep procedure (Maniatis et al.,
1982). The plasmid DNA was then used as a template for DNA
sequencing reactions necessary to confirm that the cloned gene was
novel. If the cloned gene was novel, the plasmid was then
introduced into a crystal protein-negative strain of B.
thuringiensis (Cry) so that the encoded protein could be expressed
and characterized. These procedures are described in detail in the
following sections.
6.5 Example 5
Cloning of Specific Endonuclease Restriction Fragments
[0210] The identification of a specific restriction fragment
containing a novel B. thuringiensis gene has been described for
cry2-related genes in Section 2. The procedure for cloning a
restriction fragment of known size was essentially the same as
described for cloning an MboI fragment. The DNA was digested with a
restriction enzyme (e.g., HindIII), and run through an agarose gel
to separate the fragments by size. Fragments of the proper size,
identified by Southern blot analysis (Example 2), were excised with
a razor blade and electroeluted from the gel slice into TE buffer
from which they could be precipitated. The isolated restriction
fragments were then ligated into an E. coli/B. thuringiensis
shuttle vector and transformed into E. coli to construct a
size-selected library. The library could then be hybridized with a
specific gene probe, as described in Example 4, to isolate the
colony containing the cloned novel gene.
6.6 Example 6
Cloning of PCR.TM.-Amplified Fragments
[0211] A rapid method for cloning and expressing novel cry1 gene
fragments from B. thuringiensis was developed using the polymerase
chain reaction. Flanking primers were designed to anneal to
conserved regions 5' to and within cry1 genes. With the exception
of certain cry3 genes, most B. thuringiensis cry genes are
transcriptionally regulated, at least in part, by RNA polymerases
containing the mother cell-specific .sigma..sup.E or sigE, sigma
factor. These .sigma..sup.Y-regulated cry genes possess 5' promoter
sequences that are recognized by .sigma..sup.E. Alignment of these
promoter sequences reveals considerable sequence variation,
although a consensus sequence can be identified (Baum and Malvar,
1995). A primer, designated "sigE", containing a sequence identical
to the cry1Ac .sigma..sup.E promoter sequence, was designed that
would anneal to related GE promoter sequences 5' to uncharacterized
cry genes. The sigE primer also includes a BbuI site (isoschizimer:
SphI) to facilitate cloning of amplified fragments. The sequence of
the sigE primer is shown below:
TABLE-US-00010 (SEQ ID NO: 55)
5'-ATTTAGTAGCATGCGTTGCACTTTGTGCATTTTTTCATAAGATGAGT
CATATGTTTTAAAT-3'.
[0212] The opposing primer, designated KpnR, anneals to a
3'-proximal region of the cry1 gene that is generally conserved.
This primer incorporates an Asp718 site (isoschizimer: KpnI)
conserved among the cry1A genes to facilitate cloning of the
amplified fragment and to permit the construction of fusion
proteins containing a carboxyl-terminal portion of the Cry1Ac
protein. The sequence of the KpnR primer is shown below:
TABLE-US-00011 5'-GGATAGCACTCATCAAAGGTACC-3' (SEQ ID NO: 56)
[0213] PCR.TM.s were carried out using a Perkin Elmer DNA
thermocycler and the following parameters: 94.degree. C., 2 min.;
40 cycles consisting of 94.degree. C., 30 sec; 40.degree. C., 2
min; 72.degree. C., 3 min; and a 10 second extension added to the
72.degree. C. incubation after 20 cycles. The standard PCR.TM.
buffer (100 .mu.l volume) was modified to include 1.times.Taq
Extender buffer, 25 .mu.M each of the sigE and KpnR primers, and
0.5-1.0 .mu.l of Taq Extender (Stratagene Inc.) in addition to
0.5-1.0 .mu.l of Taq polymerase. Typically, 1-2 .mu.l of the DNA
preparations from novel B. thuringiensis isolates were included in
the PCR.TM.s. PCR.TM.s with cry genes incorporating these primers
resulted in the amplification of a .about.2.3-kb DNA fragment
flanked by restriction sites for BbuI and Asp718.
[0214] For the cloning and expression of these gene fragments, the
cry1Ac shuttle vector pEG1064 was used. This plasmid is derived
from the cry1Ac shuttle vector pEG857 (Baum et al., 1990), with the
following modifications. A frameshift mutation was generated at a
unique NcoI site within the cry1Ac coding region by cleaving pEG857
with the restriction endonuclease NcoI, blunt-ending the
NcoI-generated ends with Klenow polymerase and ligating the blunt
ends with T4 ligase. In similar fashion, an Asp718 site located in
the multiple cloning site 3' to the cry1Ac gene was removed,
leaving only the single Asp718 site contained within the cry1Ac
coding sequence. The resulting plasmid, pEG1064, cannot direct the
production of crystal protein when introduced into an
acrystalliferous (Cry.sup.-) strain of B. thuringiensis because of
the frameshift mutation. For cloning and expression of unknown cry
genes, pEG1064 was cleaved with BbuI and Asp718 and the vector
fragment purified following gel electrophoresis. Amplified
fragments of unknown cry genes, obtained by PCR.TM. amplification
of total B. thuringiensis DNA, were digested with the restriction
endonucleases BbuI and Asp718 and ligated into the BbuI and Asp718
sites of the pEG1064 vector fragment. The ligation mixture was used
to transform the Cry.sup.- B. thuringiensis strains, EG10368 or
EG10650, to chloramphenicol resistance using an electroporation
protocol previously described (Mettus and Macaluso, 1990)
Chloramphenicol-resistant (Cm.sup.R) isolates were evaluated for
crystal protein production by phase-contrast microscopy. Crystal
forming (Cry+) isolates were subsequently grown in C2 liquid broth
medium (Donovan et al., 1988) to obtain crystal protein for
SDS-PAGE analysis and insect bioassay.
[0215] Because of the frameshift mutation within the cry1Ac gene,
the crystal proteins obtained from the transformants could not be
derived from the vector pEG1064. The Cry.sup.+ transformants thus
contained unknown cry gene fragments fused, at the Asp718 site, to
a 3'-portion of the cry1Ac gene. Transcription of these gene
fusions in B. thuringiensis was presumably directed from the
.sigma..sup.E promoter incorporated into the amplified cry gene
fragment. The fusion proteins, containing the entire active toxin
region of the unknown Cry protein, were capable of producing
crystals in B. thuringiensis.
6.7 Example 7
Cloning of Cry9-Related Genes
[0216] Total DNA was isolated from B. thuringiensis strain EG9290
for cloning studies. EG9290 was grown overnight at 30.degree. C. in
1.times. brain heart infusion, 0.5% glycerol (BHIG). In the
morning, 500 .mu.l of the overnight growth was suspended in 50 ml
BHIG and the culture incubated at 30.degree. C. with agitation
until the culture reached a Klett reading of 150 (red filter). The
cells were harvested by centrifugation, suspended in 5 ml
1.times.GTE buffer containing 4 mg/ml lysozyme and 100 .mu.g/ml
Rnase A, and incubated at 37.degree. C. for 20 min. The cells were
lysed by the addition of 0.5 ml of 20% SDS. The released DNA was
precipitated by the addition of 2.5 ml 7.5 M ammonium acetate and 7
ml of isopropanol. The precipitated DNA was spooled out of the
mixture using a glass micropipette and washed in 80% ethanol. The
DNA was resuspended in 10 ml 1.times.TE, extracted with one volume
each of buffered phenol and chloroform:isoamyl alcohol (24:1), and
precipitated as before. The spooled DNA was washed in 80% ethanol,
allowed to air dry for several min, and suspended in 600 .mu.l
1.times.TE. The DNA concentration was estimated at 500
.mu.g/ml.
[0217] A library of EG9290 total DNA was constructed using
partially digested MboI fragments of EG9290 DNA and the general
methods described herein. The partial MboI fragments were inserted
into the unique BamHI site of cloning vector pHT315. The ligation
mixture was used to transform E. coli Sure.TM. cells to ampicillin
resistance by electroporation employing electrocompetent cells and
protocols provided by Stratagene (La Jolla, Calif.) and the BioRad
Gene Pulser.TM. apparatus (Bio-Rad Laboratories, Hercules, Calif.).
Recombinant clones harboring cry9-type genes were identified by
colony blot hybridization using a .sup.32P-labeled probe consisting
of the putative cry9C fragment generated by amplification of EG9290
DNA with primers pr58 and pr59. Plasmid DNAs were extracted from
the E. coli clones using a standard alkaline lysis procedure.
[0218] Plasmid DNAs from the E. coli recombinant clones were used
to transform B. thuringiensis strain EG10368 to erythromycin
resistance using the electroporation procedure described by Mettus
and Macaluso (1990). Cells were plated onto starch agar plates
containing 20 .mu.g/ml erythromycin and incubated at 30.degree. C.
After six days, colonies with a more opaque appearance were
recovered from the plates and streaked out onto fresh starch agar
plates containing 20 .mu.g/ml erythromycin to isolate single
colonies. Colonies exhibiting a more opaque appearance were
observed to produce large parasporal inclusions/crystals by
phase-contrast microscopy.
[0219] Recombinant EG10368 clones producing parasporal
inclusion/crystals were evaluated for crystal protein production in
broth culture. Single colonies were inoculated into C2 medium
containing 10 .mu.g/ml erythromycin and grown at 30.degree. C. for
3 days at 28-30.degree. C., at which time the cultures were fully
sporulated and lysed. Spores and crystals were pelleted by
centrifugation and resuspended in 20 mM Tris-HCl, 1 mM EDTA, pH
7.0. Aliquots of this material were analyzed by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE). Two EG10368 recombinant clones,
initially identified as 9290-2 and 9290-3, were observed to produce
distinct proteins of 130 kDa. 9290-2 was designated EG12102 and
9290-3 was designated EG12103. The EG12102 protein was designated
CryET59 while the EG12103 protein was designated CryET60.
[0220] Plasmid DNAs were prepared from EG12102 and EG12103 using a
standard alkaline lysis procedure. Digestion of the plasmids with
the restriction endonuclease XbaI confirmed that the two strains
harbored distinct cry genes. The cry plasmids of EG12102 and
EG12103, designated pEG945 and pEG946, respectively, were used to
transform E. coli Sure.TM. cells to ampicillin resistance by
electroporation, employing electrocompetent cells and protocols
provided by Stratagene Inc. The E. coli recombinant strain
containing pEG945 was designated EG12132, and the E. coli
recombinant strain containing pEG946 was designated EG12133. pEG945
and pEG946 were purified from the E. coli recombinant strains using
the QIAGEN midi-column plasmid purification kit and protocols
(QIAGEN Inc., Valencia, Calif.).
[0221] The cryET83 gene was cloned from B. thuringiensis strain
EG6346 subspecies aizawai using similar methods. Southern blot
analysis of genomic DNA from EG6346 revealed a unique restriction
fragment that hybridized to the cryET59 probe. A series of
degenerate oligonucleotide primers, pr95, pr97, and pr98, were
designed to amplify cry9-related sequences from genomic DNA. The
sequences of these primers are as shown:
TABLE-US-00012 pr95: (SEQ ID NO: 57) 5'-GTWTGGACSCRTCGHGATGTGG-3'
pr97: (SEQ ID NO: 58)
5'-TAATTTCTGCTAGCCCWATTTCTGGATTTAATTGTTGATC-3' pr98: (SEQ ID NO:
59) 5'-ATWACNCAAMTWCCDTTRG-3' where D = A, G; H = A, C, T; M = A,
C; N = A, C, G, T; R = A, G; S = C, G; and W = A, T.
[0222] A PCR.TM. using Taq polymerase, Taq Extender.TM.
(Stratagene, La Jolla, Calif.), the opposing primers pr95 and pr97,
and total EG6346 DNA yielded a DNA fragment that was faintly
visible on an ethidium bromide-stained agarose gel. This DNA served
as the template for a second round of PCR.TM. using the opposing
primers pr97 and pr98. The resulting amplified DNA fragment was
suitable for cloning and served as a hybridization probe for
subsequent cloning experiments. A library of EG6346 total DNA was
constructed using partially digested 9-12 kb MboI fragments of
EG6346 DNA ligated into the unique BamHI site of cloning vector
pHT315. E. coli recombinant clones harboring the cryET83 gene were
identified by colony blot hybridization using the EG6346-specific
DNA fragment as a chemiluminescent hybridization probe and the
CDP-Star.TM. nucleic acid chemiluminescent reagent kit from NEN.TM.
Life Science Products (Boston, Mass.) to prepare the hybridization
probe. The recombinant plasmid harboring the cryET83 gene was
designated pEG397. The E. coli recombinant stain containing pEG397
was designated EG11786. The B. thuringiensis recombinant strain
containing pEG397 was designated EG11785.
6.8 Example 8
Sequencing of Cloned B. thuringiensis Toxin Genes
[0223] Partial sequences for the cloned toxin genes were determined
following established dideoxy chain-termination DNA sequencing
procedures (Sanger et al., 1977). Preparation of the Double
Stranded Plasmid Template DNA was Accomplished Using a standard
alkaline lysis procedure or using a QIAGEN plasmid purification kit
(QIAGEN Inc., Valencia, Calif.). The sequencing reactions were
performed using the Sequenase.TM. Version 2.0 DNA Sequencing Kit
(United States Biochemical/Amersham Life Science Inc., Cleveland,
Ohio) following the manufacturer's procedures and using
.sup.35S-dATP as the labeling isotope (obtained from DuPont
NEN.RTM. Research Products, Boston, Mass.). Denaturing gel
electrophoresis of the reactions is done on a 6% (wt./vol.)
acrylamide, 42% (wt./vol.) urea sequencing gel. The dried gels are
exposed to Kodak X-OMAT AR X-ray film (Eastman Kodak Company,
Rochester, N.Y.) overnight at room temperature. Alternatively, some
cry genes were sequenced using automated sequencing methods. DNA
samples were sequenced using the ABI PRISM.TM. DyeDeoxy sequencing
chemistry kit (Applied Biosystems, Foster City, Calif.) according
to the manufacturer's suggested protocol. The completed reactions
were run on as ABI 377 automated DNA sequencer. DNA sequence data
were analyzed using Sequencher.TM. v3.0 DNA analysis software (Gene
Codes Corp., Ann Arbor, Mich.). Successive oligonucleotides to be
used for priming sequencing reactions were designed from the
sequencing data of the previous set of reactions.
[0224] The sequence determination for the cry1-related genes
involved the use of the oligonucleotide probe wd207, described in
Example 2, as the initial sequencing primer. This oligonucleotide
anneals to a conserved region of cry1 genes, but because of the
inverted and reversed orientation of wd207, it generates sequence
towards the 5'-end of the coding region allowing sequence of the
variable region of the gene to be read. A typical sequencing run of
250-300 nucleotides was usually sufficient to determine the
identity of the gene. If additional data were necessary, one or
more additional oligonucleotides could be synthesized to continue
the sequence until it could be determined if the sequence was
unique. In cases where wd207 did not function well as a primer,
other oligonucleotides, designed to anneal to conserved regions of
cry1 genes, were used. One such oligonucleotide was the KpnR primer
described herein above.
[0225] The sequencing of the cloned cry2-related genes followed the
same general procedures as those described for the cry1 genes,
except that oligonucleotides specific for conserved regions in cry2
genes were used as sequencing primers. The two primers used in
these examples were wd268 and wd269, shown below.
TABLE-US-00013 Primer wd268 corresponds to cry2Aa nucleotides
579-597 5'-AATGCAGATGAATGGGG-3'. (SEQ ID NO: 60) Primer wd269
corresponds to cry2Aa 1740-1757 5'-TGATAATGGAGCTCGTT-3' (SEQ ID NO:
61)
[0226] The sequencing of cryET59 and cryET60 commenced with the use
of primer pr56. The sequencing of cryET83 commenced with the use of
primer pr98. Successive oligonucleotides to be used for priming
sequencing reactions were designed from the sequencing data of the
previous set of reactions.
[0227] The derived sequences were compared to sequences of known
cry genes using the FSTNSCAN program in the PC/GENE sequence
analysis package (Intelligenetics, Mountain View, Calif.). This
analysis permitted a preliminary classification of the cloned cry
genes with respect to previously-known cry genes (Table 11).
TABLE-US-00014 TABLE 6 HOMOLOGY COMPARISON OF DNA SEQUENCES.sup.1
Cloned Gene DNA Sequence Identity cryET31 90% identity with SEQ ID
NO: 4 of WO 98/40490 cryET40 99% identity with cry1Aa cryET43 88%
identity with cry1Bd1 cryET44 90% identity with cry1Da/1Db cryET45
91% identity with cry1Da/1Db cryET46 98% identity with cry1Ga
cryET47 99% identity with cry1Ab cryET49 95% identity with cry1Ja
cryET51 85% identity with cry1Ac cryET52 84% identity with
cry1Da/1Db cryET53 99% identity with SEQ ID NO: 8 of U.S. Pat. No.
5,723,758 cryET54 99.8% identity with cry1Be cryET56 80% identity
with cry1Ac cryET57 98% identity with cry1Da cryET59 95% identity
with cry9Ca cryET60 99.6% identity with cry9Aa cryET61 97% identity
with cry1Ha cryET62 99% identity with cry1Ad cryET63 93% identity
with cry1Ac cryET64 91% identity with SEQ ID NO: 9 of U.S. Pat. No.
5,723,758 cryET66 76% identity with cryIGa cryET67 99% identity
with SEQ ID NO: 10 of U.S. Pat. No. 5,723,758 cryET72 98% identity
with SEQ ID NO: 4 of WO 98/40490 cryET73 99% identity with SEQ ID
NO: 6 of WO 98/40490 cryET83 .sup.1Ktup value set at 2 for
FSTNSCAN. The cryET59 and cryET60 sequences were compared using the
FASTA program (Ktup = 6) in the PC/GENE sequence analysis
package.
6.9 Example 9
Expression of Cloned Toxin Genes in a B. thuringiensis Host
[0228] Plasmid DNA was isolated from E. coli colonies identified by
hybridization to a gene-specific probe. The isolated plasmid was
then introduced into a crystal protein-negative (Cry-) strain of B.
thuringiensis using the electroporation protocol of Mettus and
Macaluso (1990). Each of the cloning vectors used (see Table 5) has
a gene to confer antibiotic resistance on the cells harboring that
plasmid. B. thuringiensis transformants were selected by growth on
agar plates containing 25 mg/ml erythromycin (pHT315) or 5 mg/ml
chloramphenicol (pEG597 and pEG1064). Antibiotic-resistant colonies
were then evaluated for crystal protein production by
phase-contrast microscopy. Crystal producing colonies were then
grown in C2 medium (Donovan et al., 1988) to obtain cultures which
were analyzed by SDS-PAGE and insect bioassay.
[0229] C2 cultures were inoculated with cells from Cry.sup.+
colonies and grown for three days at 25-30.degree. C. in the
presence of the appropriate antibiotic. During this time the
culture grew to stationary phase, sporulated and lysed, releasing
the protein inclusions into the medium. The cultures are harvested
by centrifugation, which pellets the spores and crystals. The
pellets were washed in a solution of 0.005% Triton X-100.RTM., 2 mM
EDTA and centrifuged again. The washed pellets were resuspended at
one-tenth the original volume in 0.005% Triton X-100.RTM., 2 mM
EDTA.
[0230] Crystal protein were solubilized from the spores-crystal
suspension by incubating the suspension in a solubilization buffer
[0.14 M Tris-HCl pH 8.0, 2% (wt./vol.) sodium dodecyl sulfate
(SDS), 5% (vol./vol.) 2-mercaptoethanol, 10% (vol./vol.) glycerol,
and 0.1% bromphenol blue] at 100.degree. C. for 5 min. The
solubilized crystal proteins were size-fractionated by SDS-PAGE
using a gel with an acrylamide concentration of 10%. After size
fractionation the proteins were visualized by staining with
Coomassie Brilliant Blue R-250.
[0231] The expected size for Cry1- and Cry9-related crystal
proteins was approximately 130 kDa. The expected size for
Cry2-related proteins was approximately 65 kDa.
6.10 Example 10
Insecticidal Activity of the Cloned B. thuringiensis Toxin
Genes
[0232] B. thuringiensis recombinant strains producing individual
cloned cry genes were grown in C2 medium until the cultures were
fully sporulated and lysed. These C2 cultures were used to evaluate
the insecticidal activity of the crystal proteins produced. Each
culture was diluted with 0.005% Triton.RTM. X-100 to achieve the
appropriate dilution for two-dose bioassay screens. Fifty
microliters of each dilution were topically applied to 32 wells
containing 1.0 ml artificial diet per well (surface area of 175
mm.sup.2). A single lepidopteran larvae was placed in each of the
treated wells and the tray was covered by a clear perforated mylar
sheet. With the exception of the P. xylostella bioassays, that
employed 3rd instar larvae, all the bioassays were performed with
neonate larvae. Larval mortality was scored after 7 days of feeding
at 28-30.degree. C. and percent mortality was expressed as ratio of
the number of dead larvae to the total number of larvae treated
(Table 12). In some instances, severe stunting of larval growth was
observed after 7 days, and the ratio of stunted/unstunted larva was
also recorded. The bioassay results shown in Table 7 demonstrate
that the crystal proteins produced by the recombinant B.
thuringiensis strains do exhibit insecticidal activity and,
furthermore,
TABLE-US-00015 TABLE 7A Bioassay evaluations with ET crystal
proteins Spodoptera exigua Spodoptera frugiperda 250 nl/well 2500
nl/well # stunted/ # stunted/ % mortality % mortality # treated 250
nl/well % 2500 nl/well # treated Cry1Ac 0 5 4/32 16 53 1/32 ET31 5
12 17/32 9 6 4/32 ET40 0 5 0 3 3 0 ET43 0 8 0 3 3 2/32 ET44 0 2 0 6
0 1/32 ET45 0 0 0 0 0 1/32 ET46 0 12 0 0 6 0 ET47 19 49 11/32 31 81
6/32 ET49 0 8 0 0 3 0 ET51 0 0 0 0 0 0 ET52 0 0 0 3 3 0 ET53 0 0 0
3 0 0 ET54 0 66 3/32 6 34 9/32 ET56 0 0 0 0 6 0 ET57 2 15 18/32 3
94 0 ET59 0 0 0 0 3 0 ET60 0 0 0 0 3 0 ET61 2 5 2/32 0 3 0 ET62 2
59 12/32 0 13 0 ET63 0 12 5/32 3 0 0 ET64 0 0 0 3 6 0 ET66 0 12
1/32 3 0 1/31 ET67 29 90 0 13 61 0 ET72 0 0 0 3 94 5/31 ET73 0 2 0
0 0 0 Control 8 8 0 0 0 0
TABLE-US-00016 TABLE 7B Bioassay evaluations with ET crystal
proteins Plutella xylostella Ostrinia nubilalis 2500 nl/well #
stunted/ 250 nl/well 2500 nl/well # stunted/ 250 nl/well % %
mortality # treated % mortality % mortality # treated Cry1Ac 100
100 0 100 100 0 ET31 0 2 0 100 100 0 ET40 0 68 0 0 0 2/32 ET43 5
100 0 46 100 0 ET44 0 0 0 0 0 3/32 ET45 0 0 0 0 0 4/32 ET46 0 8 0 0
0 0 ET47 100 100 0 100 100 0 ET49 0 5 0 0 0 0 ET51 0 0 0 0 0 0 ET52
2 43 0 0 14 16/32 ET53 8 97 0 4 46 5/32 ET54 14 100 0 25 89 1/32
ET56 0 0 0 0 0 0 ET57 0 97 0 0 7 0 ET59 100 100 0 96 100 0 ET60 100
100 0 100 96 0 ET61 0 11 0 0 0 2/32 ET62 97 100 0 100 100 0 ET63
100 100 0 100 100 0 ET64 40 100 0 68 100 0 ET66 100 100 0 86 100 0
ET67 87 100 0 0 79 1/32 ET72 0 0 0 0 0 0 ET73 2 2 0 93 100 0
Control 2 2 0 0 0 0
TABLE-US-00017 TABLE 7C Bioassay evaluations with ET crystal
proteins Heliothis virescens Helicoverpa zea 250 nl/ 2500 nl/well #
stunted/ 250 nl/well 2500 nl/well well % % mortality # treated %
mortality % mortality Cry1Ac 100 100 0 100 100 ET31 97 97 1/32 8 81
ET40 2 5 2/32 2 5 ET43 87 97 1/32 0 2 ET44 8 5 1/32 5 8 ET45 0 11 0
8 18 ET46 12 25 0 0 8 ET47 87 100 0 83 100 ET49 8 2 0 11 15 ET51 2
15 0 5 5 ET52 0 31 1/32 93 11 ET53 22 64 2/32 90 61 ET54 15 64 5/32
2 5 ET56 0 11 0 8 0 ET57 2 0 0 11 28 ET59 28 84 4/32 2 2 ET60 56 97
1/32 31 28 ET61 5 5 0 8 5 ET62 44 87 4/32 21 64 ET63 100 100 0 100
100 ET64 0 21 0 5 0 ET66 0 8 1/32 0 5 ET67 18 93 1/32 0 68 ET72 34
64 11/32 8 2 ET73 42 90 2/32 8 48 Control 5 5 0 5 5
TABLE-US-00018 TABLE 7D Bioassay evaluations with ET crystal
proteins Agrotis ipsilon Trichoplusia ni 2500 nl/well # stunted/
250 nl/well 2500 nl/well # stunted/ 250 nl/well % % mortality #
treated % mortality % mortality # treated Cry1Ac 94 100 100 100 0
ET31 6 6 90 100 0 ET40 0 6 13 32 0 ET43 0 45 100 100 0 ET44 6 13 16
26 0 ET45 0 6 13 39 0 ET46 0 0 29 74 0 ET47 0 34 97 100 0 ET49 3 0
13 81 0 ET51 0 0 3 19 0 ET52 0 28 81 100 0 ET53 25 81 74 100 0 ET54
3 6 100 100 0 ET56 3 3 16 26 0 ET57 13 74 19 100 0 ET59 3 3 10 84 0
ET60 3 0 97 100 0 ET61 6 28 29 52 0 ET62 23 58 100 100 0 ET63 3 0
100 100 0 ET64 0 0 87 100 0 ET66 13 91 26 81 0 ET67 3 0 6 100 0
ET72 0 0 23 74 8/32 ET73 13 6 94 100 0 Control 0 0 3 3 0
that the crystal proteins exhibit differential activity towards the
lepidopteran species tested.
[0233] Additional bioassays were performed with the crystal
proteins designated CryET59, CryET60, CryET66, and CryET83. Crystal
proteins produced in C2 medium were quantified by SDS-PAGE and
densitometry using the method described by Brussock, S. M. and
Currier, T. C., 1990, "Use of Sodium Dodecyl Sulfate-Polyacrylamide
Gel Electrophoresis to Quantify Bacillus thuringiensis
.delta.-Endotoxins", in Analytical Chemistry of Bacillus
thuringiensis (L. A. Hickle and W. L. Fitch, eds.), The American
Chemical Society, pp. 78-87.
TABLE-US-00019 TABLE 8 Bioassay Evaluation of CryET59 and CryET60
Percent mortality.sup.1 Dose Toxin ng/well AI HV HZ ON PX rPX SE TN
Control.sup.2 -- 2 6 0 0 2 0 2 0 CryET59 100 2 37 0 94 100 100 2 13
CryET59 500 11 80 3 100 100 100 0 63 CryET59 5000 62 100 6 100 100
100 71 100 CryET60 500 0 93 22 100 100 100 0 100 CryET60 5000 2 100
25 100 100 100 14 100 .sup.1AI = Agrotis ipsilon, HV = Heliothis
virescens, HZ = Helicoverpa zea, ON = Ostrinia nubilalis, PX =
Plutella xylostella, rPX = Plutella xylostella colony resistant to
Cry1A and Cry IF toxins, SE = Spodoptera exigua, TN = Trichoplusia
ni. .sup.2Control = no toxin added.
The procedure was modified to eliminate the neutralization step
with 3M HEPES. Crystal proteins resolved by SDS-PAGE were
quantified by densitometry using a Molecular Dynamics model 300A
computing densitometer and purified bovine serum albumin (Pierce,
Rockford, Ill.) as a standard.
[0234] The bioassay results shown in Table 8 demonstrate that
CryET59 and CryET60 are toxic to a number of lepidopteran species,
including a colony of P. xylostella that is resistant to Cry1A and
Cry1F crystal proteins. Eight-dose assays with CryET66 also
demonstrated excellent toxicity towards both the susceptible and
resistant colonies of P. xylostella (Table 14). In this instance,
eight crystal protein concentrations were prepared by serial
dilution of the crystal protein suspensions in 0.005% Triton.RTM.
X-100 and 50 ul of each concentration was topically applied to
wells containing 1.0 ml of artificial diet. After the wells had
dried, a single larvae was placed in each of the treated wells and
the tray was covered by a clear perforated mylar sheet (32 larvae
for each crystal protein concentration). Larval mortality was
scored after 7 days of feeding at 28-30.degree. C. Mortality data
was expressed as LC.sub.50 and LC.sub.95 values, the concentration
of crystal protein (ng/175 mm.sup.2 diet well) causing 50% and 95%
mortality, respectively (Daum, 1970).
TABLE-US-00020 TABLE 9 Toxin LC.sub.50.sup.1 95% C.I.
LC.sub.95.sup.2 Slope Toxicity of CryET66 towards Plutella
xylostella Cry1Ac 8.05 5.0-15.2 52.94 2.01 Cry1C 25.06 15.7-40.6
117.07 2.46 CryET66 0.42 0.4-0.5 1.4 3.13 Toxicity of CryET66
towards Cry1A-resistant Plutella xylostella Cry1Ac *No significant
mortality Cry1C 27.32 15.4-51.1 156.13 2.17 CryET66 1.65 1.3-2.0
6.41 2.79 .sup.1the concentration of crystal protein, in nanograms
of crystal protein per well, required to achieve 50% mortality
.sup.2the concentration of crystal protein, in nanograms of crystal
protein per well, required to achieve 95% mortality.
Table 15 shows that the CryET83 protein exhibits toxicity towards a
wide variety of lepidopteran pests and may exhibit improved
toxicity towards S. exigua and H. virescens when compared to the
other Cry9-type proteins CryET59 and CryET60.
TABLE-US-00021 TABLE 10 Toxicity of CryET83 towards lepidopteran
larvae.sup.1 Dose.sup.2 AI.sup.3 HV HZ ON PX SE SF TN 5 5 10 9 50
53 75 69 100 91 500 0 100 67 100 5000 32 100 10000 84 100
.sup.1Toxicity calculated as percent mortality among treated
larvae. .sup.2ng CryET83 crystal protein/175 mm.sup.2 diet well
.sup.3Abbreviations described in Table 8; SF = Spodoptera
frugiperda
The recombinant B. thuringiensis strains listed in Table 5 were
deposited with the ARS Patent Culture Collection and had been
assigned the NRRL deposit numbers shown in Table 11.
TABLE-US-00022 TABLE 11 Biological Deposits Polypeptide Polypeptide
Polynucleotide Recomb. NRRL Deposit Designation Seq. ID No.: Seq ID
No.: Strain No.: Cry ET31 2 1 EG11562 B-21921 Cry ET40 4 3 EG11901
B-21922 Cry ET43 6 5 EG7692 B-21923 Cry ET44 8 7 EG11629 B-21924
Cry ET45 10 9 EG7694 B-21925 Cry ET46 12 11 EG7695 B-21926 Cry ET47
14 13 EG7696 B-21927 Cry ET49 16 15 EG11630 B-21928 Cry ET51 18 17
EG11921 B-21929 Cry ET52 20 19 EG11584 B-21930 Cry ET53 22 21
EG11906 B-21931 Cry ET54 63 62 EG11907 B-21932 Cry ET56 24 23
EG11909 B-21933 Cry ET57 26 25 EG11910 B-21934 Cry ET59 28 27
EG12102 B-21935 Cry ET60 30 29 EG12103 B-21936 Cry ET61 32 31
EG11634 B-21937 Cry ET62 34 33 EG11635 B-21938 Cry ET63 36 35
EG11636 B-21939 Cry ET64 38 37 EG11638 B-21940 Cry ET66 40 39
EG11640 B-21941 Cry ET67 42 41 EG11642 B-21942 Cry ET68 44 43
EG11644 B-30137 Cry ET72 46 45 EG11440 B-21943 Cry ET73 48 47
EG11465 B-21944 CryET83 50 49 EG11785 B-30138
6.11 Example 11
Modification of Cry Genes for Expression in Plants
[0235] Wild-type cry genes are known to be expressed poorly in
plants as a full length gene or as a truncated gene. Typically, the
G+C content of a cry gene is low (37%) and often contains many A+T
rich regions, potential polyadenylation sites and numerous ATTTA
sequences. Table 12 shows a list of potential polyadenylation
sequences which should be avoided when preparing the "plantized"
gene construct.
TABLE-US-00023 TABLE 12 List of Sequences of Potential
Polyadenylation Signals AATAAA* AAGCAT AATAAT* ATTAAT AACCAA ATACAT
ATATAA AAAATA AATCAA ATTAAA** ATACTA AATTAA** ATAAAA AATACA**
ATGAAA CATAAA** *indicates a potential major plant polyadenylation
site. **indicates a potential minor animal polyadenylation
site.
[0236] All others are potential minor plant polyadenylation
sites.
[0237] The regions for mutagenesis may be selected in the following
manner. All regions of the DNA sequence of the cry gene are
identified which contained five or more consecutive base pairs
which were A or T. These were ranked in terms of length and highest
percentage of A+T in the surrounding sequence over a 20-30 base
pair region. The DNA is analysed for regions which might contain
polyadenylation sites or ATTTA sequences. Oligonucleotides are then
designed which maximize the elimination of A+T consecutive regions
which contained one or more polyadenylation sites or ATTTA
sequences. Two potential plant polyadenylation sites have been
shown to be more critical based on published reports. Codons are
selected which increase G+C content, but do not generate
restriction sites for enzymes useful for cloning and assembly of
the modified gene (e.g., BamHI, BglII, SacI, NcoI, EcoRV, etc.).
Likewise condons are avoided which contain the doublets TA or GC
which have been reported to be infrequently-found codons in
plants.
[0238] Although the CaMV35S promoter is generally a high level
constitutive promoter in most plant tissues, the expression level
of genes driven the CaMV35S promoter is low in floral tissue
relative to the levels seen in leaf tissue. Because the
economically important targets damaged by some insects are the
floral parts or derived from floral parts (e.g., cotton squares and
bolls, tobacco buds, tomato buds and fruit), it is often
advantageous to increase the expression of crystal proteins in
these tissues over that obtained with the CaMV35S promoter.
[0239] The 35S promoter of Figwort Mosaic Virus (FMV) is analogous
to the CaMV35S promoter. This promoter has been isolated and
engineered into a plant transformation vector. Relative to the CaMV
promoter, the FMV 35S promoter is highly expressed in the floral
tissue, while still providing similar high levels of gene
expression in other tissues such as leaf. A plant transformation
vector, may be constructed in which the full length synthetic cry
gene is driven by the FMV 35S promoter. Tobacco plants may be
transformed with the vector and compared for expression of the
crystal protein by Western blot or ELISA immunoassay in leaf and
floral tissue. The FMV promoter has been used to produce relatively
high levels of crystal protein in floral tissue compared to the
CaMV promoter.
6.12 Example 12
Expression of Synthetic Cry Genes with ssRUBISCO Promoters and
Chloroplast Transit Peptides
[0240] The genes in plants encoding the small subunit of
RUBISCO(SSU) are often highly expressed, light regulated and
sometimes show tissue specificity. These expression properties are
largely due to the promoter sequences of these genes. It has been
possible to use SSU promoters to express heterologous genes in
transformed plants. Typically a plant will contain multiple SSU
genes, and the expression levels and tissue specificity of
different SSU genes will be different. The SSU proteins are encoded
in the nucleus and synthesized in the cytoplasm as precursors that
contain an N-terminal extension known as the chloroplast transit
peptide (CTP). The CTP directs the precursor to the chloroplast and
promotes the uptake of the SSU protein into the chloroplast. In
this process, the CTP is cleaved from the SSU protein. These CTP
sequences have been used to direct heterologous proteins into
chloroplasts of transformed plants.
[0241] The SSU promoters might have several advantages for
expression of heterologous genes in plants. Some SSU promoters are
very highly expressed and could give rise to expression levels as
high or higher than those observed with the CaMV35S promoter. The
tissue distribution of expression from SSU promoters is different
from that of the CaMV35S promoter, so for control of some insect
pests, it may be advantageous to direct the expression of crystal
proteins to those cells in which SSU is most highly expressed. For
example, although relatively constitutive, in the leaf the CaMV35S
promoter is more highly expressed in vascular tissue than in some
other parts of the leaf, while most SSU promoters are most highly
expressed in the mesophyll cells of the leaf. Some SSU promoters
also are more highly tissue specific, so it could be possible to
utilize a specific SSU promoter to express the protein of the
present invention in only a subset of plant tissues, if for example
expression of such a protein in certain cells was found to be
deleterious to those cells. For example, for control of Colorado
potato beetle in potato, it may be advantageous to use SSU
promoters to direct crystal protein expression to the leaves but
not to the edible tubers.
[0242] Utilizing SSU CTP sequences to localize crystal proteins to
the chloroplast might also be advantageous. Localization of the B.
thuringiensis crystal proteins to the chloroplast could protect
these from proteases found in the cytoplasm. This could stabilize
the proteins and lead to higher levels of accumulation of active
toxin. cry genes containing the CTP may be used in combination with
the SSU promoter or with other promoters such as CaMV35S.
6.13 Example 13
Targeting of Cry Proteins to the Extracellular Space or Vacuole
Through the Use of Signal Peptides
[0243] The B. thuringiensis proteins produced from the synthetic
genes described here are localized to the cytoplasm of the plant
cell, and this cytoplasmic localization results in plants that are
insecticidally effective. It may be advantageous for some purposes
to direct the B. thuringiensis proteins to other compartments of
the plant cell. Localizing B. thuringiensis proteins in
compartments other than the cytoplasm may result in less exposure
of the B. thuringiensis proteins to cytoplasmic proteases leading
to greater accumulation of the protein yielding enhanced
insecticidal activity. Extracellular localization could lead to
more efficient exposure of certain insects to the B. thuringiensis
proteins leading to greater efficacy. If a B. thuringiensis protein
were found to be deleterious to plant cell function, then
localization to a noncytoplasmic compartment could protect these
cells from the protein.
[0244] In plants as well as other eukaryotes, proteins that are
destined to be localized either extracellularly or in several
specific compartments are typically synthesized with an N-terminal
amino acid extension known as the signal peptide. This signal
peptide directs the protein to enter the compartmentalization
pathway, and it is typically cleaved from the mature protein as an
early step in compartmentalization. For an extracellular protein,
the secretory pathway typically involves cotranslational insertion
into the endoplasmic reticulum with cleavage of the signal peptide
occurring at this stage. The mature protein then passes through the
Golgi body into vesicles that fuse with the plasma membrane thus
releasing the protein into the extracellular space. Proteins
destined for other compartments follow a similar pathway. For
example, proteins that are destined for the endoplasmic reticulum
or the Golgi body follow this scheme, but they are specifically
retained in the appropriate compartment. In plants, some proteins
are also targeted to the vacuole, another membrane bound
compartment in the cytoplasm of many plant cells. Vacuole targeted
proteins diverge from the above pathway at the Golgi body where
they enter vesicles that fuse with the vacuole.
[0245] A common feature of this protein targeting is the signal
peptide that initiates the compartmentalization process. Fusing a
signal peptide to a protein will in many cases lead to the
targeting of that protein to the endoplasmic reticulum. The
efficiency of this step may depend on the sequence of the mature
protein itself as well. The signals that direct a protein to a
specific compartment rather than to the extracellular space are not
as clearly defined. It appears that many of the signals that direct
the protein to specific compartments are contained within the amino
acid sequence of the mature protein. This has been shown for some
vacuole targeted proteins, but it is not yet possible to define
these sequences precisely. It appears that secretion into the
extracellular space is the "default" pathway for a protein that
contains a signal sequence but no other compartmentalization
signals. Thus, a strategy to direct B. thuringiensis proteins out
of the cytoplasm is to fuse the genes for synthetic B.
thuringiensis genes to DNA sequences encoding known plant signal
peptides. These fusion genes will give rise to B. thuringiensis
proteins that enter the secretory pathway, and lead to
extracellular secretion or targeting to the vacuole or other
compartments.
[0246] Signal sequences for several plant genes have been
described. One such sequence is for the tobacco pathogenesis
related protein PR1b has been previously described (Cornelissen et
al., 1986). The PR1b protein is normally localized to the
extracellular space. Another type of signal peptide is contained on
seed storage proteins of legumes. These proteins are localized to
the protein body of seeds, which is a vacuole like compartment
found in seeds. A signal peptide DNA sequence for the
.beta.-subunit of the 7S storage protein of common bean (Phaseolus
vulgaris), PvuB has been described (Doyle et al., 1986). Based on
the published these published sequences, genes may be synthesized
chemically using oligonucleotides that encode the signal peptides
for PR1b and PvuB. In some cases to achieve secretion or
compartmentalization of heterologous proteins, it may be necessary
to include some amino acid sequence beyond the normal cleavage site
of the signal peptide. This may be necessary to insure proper
cleavage of the signal peptide.
6.14 Example 14
Isolation of Transgenic Plants Resistant to Insects Using
Crytransgenes
6.64.1 Plant Gene Construction
[0247] 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.
[0248] 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).
[0249] 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.
[0250] 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.
[0251] 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.
[0252] 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.
[0253] 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).
6.14.2 Plant Transformation and Expression
[0254] A plant gene containing a structural coding sequence of the
present invention can be inserted into the genome of a plant by any
suitable method. 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).
6.14.3 Construction of Monocot Plant Expression Vectors for Cry
Genes
[0255] For efficient expression of cry genes in transgenic plants,
the gene must have a suitable sequence composition (Diehn et al.,
1996). To place the 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),
a vector such as pMON19469 may be used. Such a vector is
conveniently digested with NcoI and EcoRI restriction enzymes. The
larger vector band of approximately 4.6 kb is then electrophoresed,
purified, and ligated with T4 DNA ligase to an NcoI-EcoRI fragment
which contains the synthetic 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 is then subjected to restriction endonuclease analysis with
enzymes such as NcoI and EcoRI (together), NotI, and/or PstI
individually or in combination, to identify clones containing the
cry coding sequence fused to an intron such as the hsp70 intron,
placed under the control of the enhanced CaMV35S promoter.
[0256] To place the gene in a vector suitable for recovery of
stably transformed and insect resistant plants, the 3.75-kb NotI
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 is then ligated with a vector such
as pMON30460 which has been previously 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 desired plasmid may be identified by restriction
endonuclease digestion of plasmid miniprep DNAs. Restriction
enzymes such as NotI, EcoRV, HindIII, NcoI, EcoRI, and BglII may be
used to identify the appropriate clones in which the orientation of
both genes are in tandem (i.e. the 3' end of the cry expression
cassette is linked to the 5' end of the nptII expression cassette).
Expression of the Cry protein by the resulting plasmid in corn
protoplasts may be confirmed by electroporation of the vector DNA
into protoplasts followed by protein blot and ELISA analysis. This
vector may be introduced into the genomic DNA of corn embryos 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.
[0257] As an example, the vector may be introduced via
cobombardment with a hygromycin resistance conferring plasmid into
immature embryo scutella (IES) of maize, followed by hygromycin
selection, and regeneration. Transgenic corn lines expressing the
cry protein may then be identified by ELISA analysis. Progeny seed
from these events may then be subsequently tested for protection
from insect feeding.
7.0 REFERENCES
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[0434] 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 composition, 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. Accordingly, the exclusive rights sought to be
patented are as described in the claims below.
Sequence CWU 1
1
6311899DNABacillus thuringiensisCDS(1)..(1899) 1atg aat aat gta tta
aat aac gga aga act act att tgt gat gcg tat 48Met Asn Asn Val Leu
Asn Asn Gly Arg Thr Thr Ile Cys Asp Ala Tyr1 5 10 15aat gta gtg gcc
cat gat cca ttt agt ttt gag cat aaa tca tta gat 96Asn Val Val Ala
His Asp Pro Phe Ser Phe Glu His Lys Ser Leu Asp 20 25 30acc atc cga
aaa gaa tgg atg gag tgg aaa aga aca gat cat agt tta 144Thr Ile Arg
Lys Glu Trp Met Glu Trp Lys Arg Thr Asp His Ser Leu 35 40 45tat gta
gct cct ata gtc gga act gtt tct agc ttt ctg cta aag aag 192Tyr Val
Ala Pro Ile Val Gly Thr Val Ser Ser Phe Leu Leu Lys Lys 50 55 60gtg
ggg agt ctt att gga aaa agg ata ttg agt gaa tta tgg ggg tta 240Val
Gly Ser Leu Ile Gly Lys Arg Ile Leu Ser Glu Leu Trp Gly Leu65 70 75
80ata ttt cct agt ggt agc aca aat cta atg caa gat att tta agg gag
288Ile Phe Pro Ser Gly Ser Thr Asn Leu Met Gln Asp Ile Leu Arg Glu
85 90 95aca gaa caa ttc cta aat caa aga ctt aat aca gac act ctt gcc
cgt 336Thr Glu Gln Phe Leu Asn Gln Arg Leu Asn Thr Asp Thr Leu Ala
Arg 100 105 110gta aat gcg gaa ttg gaa ggg ctg caa gcg aat ata agg
gag ttt aat 384Val Asn Ala Glu Leu Glu Gly Leu Gln Ala Asn Ile Arg
Glu Phe Asn 115 120 125caa caa gta gat aat ttt tta aat cct act caa
aac cct gtt cct tta 432Gln Gln Val Asp Asn Phe Leu Asn Pro Thr Gln
Asn Pro Val Pro Leu 130 135 140tca ata act tct tca gtt aat aca atg
cag caa tta ttt cta aat aga 480Ser Ile Thr Ser Ser Val Asn Thr Met
Gln Gln Leu Phe Leu Asn Arg145 150 155 160tta ccc cag ttc cgt gtg
caa gga tac caa ctg tta tta tta cct tta 528Leu Pro Gln Phe Arg Val
Gln Gly Tyr Gln Leu Leu Leu Leu Pro Leu 165 170 175ttt gca cag gca
gcc aat atg cat ctt tct ttt att aga gat gtt gtt 576Phe Ala Gln Ala
Ala Asn Met His Leu Ser Phe Ile Arg Asp Val Val 180 185 190ctc aat
gca gat gaa tgg gga att tca gca gca aca tta cgt acg tat 624Leu Asn
Ala Asp Glu Trp Gly Ile Ser Ala Ala Thr Leu Arg Thr Tyr 195 200
205caa aat tat ctg aaa aat tat aca aca gag tac tct aat tat tgt ata
672Gln Asn Tyr Leu Lys Asn Tyr Thr Thr Glu Tyr Ser Asn Tyr Cys Ile
210 215 220aat acg tat caa act gcg ttt aga ggt tta aac acc cgt tta
cac gat 720Asn Thr Tyr Gln Thr Ala Phe Arg Gly Leu Asn Thr Arg Leu
His Asp225 230 235 240atg tta gaa ttt aga aca tat atg ttt tta aat
gta ttt gaa tat gta 768Met Leu Glu Phe Arg Thr Tyr Met Phe Leu Asn
Val Phe Glu Tyr Val 245 250 255tct atc tgg tcg ttg ttt aaa tat caa
agc ctt cta gta tct tct ggc 816Ser Ile Trp Ser Leu Phe Lys Tyr Gln
Ser Leu Leu Val Ser Ser Gly 260 265 270gct aat tta tat gca agc ggt
agt gga cca cag cag act caa tca ttt 864Ala Asn Leu Tyr Ala Ser Gly
Ser Gly Pro Gln Gln Thr Gln Ser Phe 275 280 285act tca caa gac tgg
cca ttt tta tat tct ctt ttc caa gtt aat tca 912Thr Ser Gln Asp Trp
Pro Phe Leu Tyr Ser Leu Phe Gln Val Asn Ser 290 295 300aat tat gtg
tta aat ggc ttt agt ggc gct aga ctt acg cag act ttc 960Asn Tyr Val
Leu Asn Gly Phe Ser Gly Ala Arg Leu Thr Gln Thr Phe305 310 315
320cct aat att ggt ggt tta cct ggt act act aca act cac gca ttg ctt
1008Pro Asn Ile Gly Gly Leu Pro Gly Thr Thr Thr Thr His Ala Leu Leu
325 330 335gcg gca agg gtc aat tac agt gga gga gtt tcg tct ggt gat
ata ggc 1056Ala Ala Arg Val Asn Tyr Ser Gly Gly Val Ser Ser Gly Asp
Ile Gly 340 345 350gct gtg ttt aat caa aat ttt agt tgt agc aca ttt
ctc cca cct ttg 1104Ala Val Phe Asn Gln Asn Phe Ser Cys Ser Thr Phe
Leu Pro Pro Leu 355 360 365tta aca cca ttt gtt agg agt tgg cta gat
tca ggt tca gat cga ggg 1152Leu Thr Pro Phe Val Arg Ser Trp Leu Asp
Ser Gly Ser Asp Arg Gly 370 375 380ggt gtt aat acc gtt aca aat tgg
caa aca gaa tcg ttt gag tca act 1200Gly Val Asn Thr Val Thr Asn Trp
Gln Thr Glu Ser Phe Glu Ser Thr385 390 395 400tta ggt tta agg tgt
ggt gct ttt aca gct cgt ggt aat tca aac tat 1248Leu Gly Leu Arg Cys
Gly Ala Phe Thr Ala Arg Gly Asn Ser Asn Tyr 405 410 415ttc cca gat
tat ttt atc cgt aat att tca gga gtt cct tta gtt gtt 1296Phe Pro Asp
Tyr Phe Ile Arg Asn Ile Ser Gly Val Pro Leu Val Val 420 425 430aga
aat gaa gat tta aga aga ccg tta cac tat aat gaa ata aga aat 1344Arg
Asn Glu Asp Leu Arg Arg Pro Leu His Tyr Asn Glu Ile Arg Asn 435 440
445ata gaa agt cct tca gga aca cct ggt gga tta cga gct tat atg gta
1392Ile Glu Ser Pro Ser Gly Thr Pro Gly Gly Leu Arg Ala Tyr Met Val
450 455 460tct gtg cat aat aga aaa aat aat atc tat gcc gtg cat gaa
aat ggt 1440Ser Val His Asn Arg Lys Asn Asn Ile Tyr Ala Val His Glu
Asn Gly465 470 475 480act atg att cat tta gcg ccg gaa gat tat aca
gga ttc acc ata tcg 1488Thr Met Ile His Leu Ala Pro Glu Asp Tyr Thr
Gly Phe Thr Ile Ser 485 490 495ccg ata cat gca act caa gtg aat aat
caa acg cga aca ttt att tct 1536Pro Ile His Ala Thr Gln Val Asn Asn
Gln Thr Arg Thr Phe Ile Ser 500 505 510gaa aaa ttt gga aat caa ggt
gat tcc tta aga ttt gaa caa agc aac 1584Glu Lys Phe Gly Asn Gln Gly
Asp Ser Leu Arg Phe Glu Gln Ser Asn 515 520 525acg aca gca cgt tat
aca ctt aga gga aat gga aat agt tac aat ctt 1632Thr Thr Ala Arg Tyr
Thr Leu Arg Gly Asn Gly Asn Ser Tyr Asn Leu 530 535 540tat tta aga
gta tct tca cta gga aat tcc act att cga gtt act ata 1680Tyr Leu Arg
Val Ser Ser Leu Gly Asn Ser Thr Ile Arg Val Thr Ile545 550 555
560aac ggt agg gtt tat act gct tca aat gtt aat act act aca aat aac
1728Asn Gly Arg Val Tyr Thr Ala Ser Asn Val Asn Thr Thr Thr Asn Asn
565 570 575gat gga gtt aat gat aat ggc gct cgt ttt tta gat att aat
atg ggt 1776Asp Gly Val Asn Asp Asn Gly Ala Arg Phe Leu Asp Ile Asn
Met Gly 580 585 590aat gta gta gca agt gat aat act aat gta ccg tta
gat ata aat gtg 1824Asn Val Val Ala Ser Asp Asn Thr Asn Val Pro Leu
Asp Ile Asn Val 595 600 605aca ttt aac tcc ggt act caa ttt gag ctt
atg aat att atg ttt gtt 1872Thr Phe Asn Ser Gly Thr Gln Phe Glu Leu
Met Asn Ile Met Phe Val 610 615 620cca act aat ctt cca cca ata tat
taa 1899Pro Thr Asn Leu Pro Pro Ile Tyr625 6302632PRTBacillus
thuringiensis 2Met Asn Asn Val Leu Asn Asn Gly Arg Thr Thr Ile Cys
Asp Ala Tyr1 5 10 15Asn Val Val Ala His Asp Pro Phe Ser Phe Glu His
Lys Ser Leu Asp 20 25 30Thr Ile Arg Lys Glu Trp Met Glu Trp Lys Arg
Thr Asp His Ser Leu 35 40 45Tyr Val Ala Pro Ile Val Gly Thr Val Ser
Ser Phe Leu Leu Lys Lys 50 55 60Val Gly Ser Leu Ile Gly Lys Arg Ile
Leu Ser Glu Leu Trp Gly Leu65 70 75 80Ile Phe Pro Ser Gly Ser Thr
Asn Leu Met Gln Asp Ile Leu Arg Glu 85 90 95Thr Glu Gln Phe Leu Asn
Gln Arg Leu Asn Thr Asp Thr Leu Ala Arg 100 105 110Val Asn Ala Glu
Leu Glu Gly Leu Gln Ala Asn Ile Arg Glu Phe Asn 115 120 125Gln Gln
Val Asp Asn Phe Leu Asn Pro Thr Gln Asn Pro Val Pro Leu 130 135
140Ser Ile Thr Ser Ser Val Asn Thr Met Gln Gln Leu Phe Leu Asn
Arg145 150 155 160Leu Pro Gln Phe Arg Val Gln Gly Tyr Gln Leu Leu
Leu Leu Pro Leu 165 170 175Phe Ala Gln Ala Ala Asn Met His Leu Ser
Phe Ile Arg Asp Val Val 180 185 190Leu Asn Ala Asp Glu Trp Gly Ile
Ser Ala Ala Thr Leu Arg Thr Tyr 195 200 205Gln Asn Tyr Leu Lys Asn
Tyr Thr Thr Glu Tyr Ser Asn Tyr Cys Ile 210 215 220Asn Thr Tyr Gln
Thr Ala Phe Arg Gly Leu Asn Thr Arg Leu His Asp225 230 235 240Met
Leu Glu Phe Arg Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr Val 245 250
255Ser Ile Trp Ser Leu Phe Lys Tyr Gln Ser Leu Leu Val Ser Ser Gly
260 265 270Ala Asn Leu Tyr Ala Ser Gly Ser Gly Pro Gln Gln Thr Gln
Ser Phe 275 280 285Thr Ser Gln Asp Trp Pro Phe Leu Tyr Ser Leu Phe
Gln Val Asn Ser 290 295 300Asn Tyr Val Leu Asn Gly Phe Ser Gly Ala
Arg Leu Thr Gln Thr Phe305 310 315 320Pro Asn Ile Gly Gly Leu Pro
Gly Thr Thr Thr Thr His Ala Leu Leu 325 330 335Ala Ala Arg Val Asn
Tyr Ser Gly Gly Val Ser Ser Gly Asp Ile Gly 340 345 350Ala Val Phe
Asn Gln Asn Phe Ser Cys Ser Thr Phe Leu Pro Pro Leu 355 360 365Leu
Thr Pro Phe Val Arg Ser Trp Leu Asp Ser Gly Ser Asp Arg Gly 370 375
380Gly Val Asn Thr Val Thr Asn Trp Gln Thr Glu Ser Phe Glu Ser
Thr385 390 395 400Leu Gly Leu Arg Cys Gly Ala Phe Thr Ala Arg Gly
Asn Ser Asn Tyr 405 410 415Phe Pro Asp Tyr Phe Ile Arg Asn Ile Ser
Gly Val Pro Leu Val Val 420 425 430Arg Asn Glu Asp Leu Arg Arg Pro
Leu His Tyr Asn Glu Ile Arg Asn 435 440 445Ile Glu Ser Pro Ser Gly
Thr Pro Gly Gly Leu Arg Ala Tyr Met Val 450 455 460Ser Val His Asn
Arg Lys Asn Asn Ile Tyr Ala Val His Glu Asn Gly465 470 475 480Thr
Met Ile His Leu Ala Pro Glu Asp Tyr Thr Gly Phe Thr Ile Ser 485 490
495Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr Phe Ile Ser
500 505 510Glu Lys Phe Gly Asn Gln Gly Asp Ser Leu Arg Phe Glu Gln
Ser Asn 515 520 525Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly Asn
Ser Tyr Asn Leu 530 535 540Tyr Leu Arg Val Ser Ser Leu Gly Asn Ser
Thr Ile Arg Val Thr Ile545 550 555 560Asn Gly Arg Val Tyr Thr Ala
Ser Asn Val Asn Thr Thr Thr Asn Asn 565 570 575Asp Gly Val Asn Asp
Asn Gly Ala Arg Phe Leu Asp Ile Asn Met Gly 580 585 590Asn Val Val
Ala Ser Asp Asn Thr Asn Val Pro Leu Asp Ile Asn Val 595 600 605Thr
Phe Asn Ser Gly Thr Gln Phe Glu Leu Met Asn Ile Met Phe Val 610 615
620Pro Thr Asn Leu Pro Pro Ile Tyr625 6303729DNABacillus
thuringiensis 3ttcgctagga accaagccat ttctagatta gaaggactaa
gcaatcttta tcaaatttac 60gcagaatctt ttagagagtg ggaagcagat cctactaatc
cagcattaag agaagagatg 120cgtattcaat tcaatgacat gaacagtgcc
cttacaaccg ctattcctct tttggcagtt 180caaaattatc aagttcctct
tttatcagta tatgttcaag ctgcaaattt acatttatca 240gttttgagag
atgtttcagt gtttggacaa aggtggggat ttgatgccgc gactatcaat
300agtcgttata atgatttaac taggcttatt ggcaactata cagattatgc
tgtgcgctgg 360tacaatacgg gattagagcg tgtatgggga ccggattcta
gagattgggt aaggtataat 420caatttagaa gagagctaac acttactgta
ttagatatcg ttgctctatt ctcaaattat 480gatagtcgaa ggtatccaat
tcgaacagtt tcccaattaa caagagaaat ttatacgaac 540ccagtattag
aaaattttga tggtagtttt cgtggaatgg ctcagagaat agaacagaat
600attaggcaac cacatcttat ggatatcctt aatagtataa ccatttatac
tgatgtgcat 660agaggcttta attattggtc agggcatcaa ataacagctt
ctcctgtagg gttttcagga 720ccagaattc 7294243PRTBacillus thuringiensis
4Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu1 5
10 15Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro
Thr 20 25 30Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp
Met Asn 35 40 45Ser Ala Leu Thr Thr Ala Ile Pro Leu Leu Ala Val Gln
Asn Tyr Gln 50 55 60Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn
Leu His Leu Ser65 70 75 80Val Leu Arg Asp Val Ser Val Phe Gly Gln
Arg Trp Gly Phe Asp Ala 85 90 95Ala Thr Ile Asn Ser Arg Tyr Asn Asp
Leu Thr Arg Leu Ile Gly Asn 100 105 110Tyr Thr Asp Tyr Ala Val Arg
Trp Tyr Asn Thr Gly Leu Glu Arg Val 115 120 125Trp Gly Pro Asp Ser
Arg Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg 130 135 140Glu Leu Thr
Leu Thr Val Leu Asp Ile Val Ala Leu Phe Ser Asn Tyr145 150 155
160Asp Ser Arg Arg Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu
165 170 175Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe
Arg Gly 180 185 190Met Ala Gln Arg Ile Glu Gln Asn Ile Arg Gln Pro
His Leu Met Asp 195 200 205Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp
Val His Arg Gly Phe Asn 210 215 220Tyr Trp Ser Gly His Gln Ile Thr
Ala Ser Pro Val Gly Phe Ser Gly225 230 235 240Pro Glu
Phe51959DNABacillus thuringiensis 5gaaaatgaga atgaaattat aaatgcctta
tcgattccag ctgtatcgaa tcattccgca 60caaatggatc tatcgctaga tgctcgtatt
gaggattctt tgtgtatagc cgaggggaat 120aatatcaatc cacttgttag
cgcatcaaca gtccaaacgg gtataaacat agctggtaga 180atattgggcg
tattaggtgt gccgtttgct ggacaactag ctagttttta tagttttctt
240gttggggaat tatggcctag tggtagagat ccatgggaaa ttttcctgga
atatgtagaa 300caacttataa gacaacaagt aacagaaaat actaggaata
cggctattgc tcgattagaa 360ggtctaggaa gaggctatag atcttaccag
caggctcttg aaacttggtt agataaccga 420aatgatgcaa gatcaagaag
cattattctt gagcgctatg ttgctttaga acttgacatt 480actactgcta
taccgctttt cagaatacga aatgaagaag ttccattatt aatggtatat
540gctcaagctg caaatttaca cctattatta ttgagagacg catccctttt
tggtagtgaa 600tgggggatgg catcttccga tgttaaccaa tattaccagg
aacaaatcag atatacagag 660gaatattcta accattgcgt acaatggtat
aatacagggc taaataactt aagagggaca 720aatgctgaaa gttggttgcg
gtataatcaa ttccgtagag acctaacgtt aggggtatta 780gatttagtag
ccctattccc aagctatgat actcgcactt atccaatcaa tacgagtgct
840cagttaacaa gagaaattta tacagatcca attgggagaa caaatgcacc
ttcaggattt 900gcaagtacga attggtttaa taataatgca ccatcgtttt
ctgccataga ggctgccatt 960ttcaggcctc cgcatctact tgattttcca
gaacaactta caatttacag tgcatcaagc 1020cgttggagta gcactcaaca
tatgaattat tgggtgggac ataggcttaa cttccgccca 1080ataggaggga
cattaaatac ctcaacacaa ggacttacta ataatacttc aattaatcct
1140gtaacattac attacgtttc gtctcgtgac gtttatagaa cagaatcaaa
tgcagggaca 1200aatatactat ttactactcc tgtgaatgga gtaccttggg
ctagatttaa ttttataacc 1260ctcagaatat ttatgaaaga ggcgccacta
cctacagtca accgtatcag ggagttggga 1320ttcaattatt tgattcagaa
actgaattac caccagaaac aacagaacga ccaaattatg 1380aatcatatag
tcatagatat ctcatataga ctaatcatag gaaacacttt gagagcacca
1440gtctattctt ggacgcatcg tagtgcagat cgtacgaata cgattggacc
aaatagaatt 1500actcaaattc ctgcagtgaa gggaagattt ctttttaatg
gttctgtgat ttcaggacca 1560ggatttactg gtggagacgt agttagattg
aataggaata atggtaatat ccaaaataga 1620gggtatattg aagttccaat
tcaattcacg tcgacatcta ccagatatcg agttcgagta 1680cgttatgctt
ctgtaacctc gattgagctc aatgttaatt tgggcaattc atcaattttt
1740acgaacacat taccagcaac agctgcatca ttagataatc tacaatcagg
ggattttggt 1800tatgttgaaa tcaacaatgc ttttacatcc gcaacaggta
atatagtagg tgctagaaat 1860tttagtgcaa atgcagaagt aataatagac
agatttgaat ttatcccagt tactgcaacc 1920ttcgaggtag aatatgattt
agaaagagca caaaaggcg 19596653PRTBacillus thuringiensis 6Glu Asn Glu
Asn Glu Ile Ile Asn Ala Leu Ser Ile Pro Ala Val Ser1 5 10 15Asn His
Ser Ala Gln Met Asp Leu Ser Leu Asp Ala Arg Ile Glu Asp 20 25 30Ser
Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn Pro Leu Val Ser Ala 35 40
45Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly Val
50 55 60Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe
Leu65 70 75 80Val Gly Glu Leu Trp Pro Ser Gly Arg Asp Pro Trp Glu
Ile Phe Leu 85 90 95Glu Tyr Val Glu Gln Leu Ile Arg Gln Gln Val Thr
Glu Asn
Thr Arg 100 105 110Asn Thr Ala Ile Ala Arg Leu Glu Gly Leu Gly Arg
Gly Tyr Arg Ser 115 120 125Tyr Gln Gln Ala Leu Glu Thr Trp Leu Asp
Asn Arg Asn Asp Ala Arg 130 135 140Ser Arg Ser Ile Ile Leu Glu Arg
Tyr Val Ala Leu Glu Leu Asp Ile145 150 155 160Thr Thr Ala Ile Pro
Leu Phe Arg Ile Arg Asn Glu Glu Val Pro Leu 165 170 175Leu Met Val
Tyr Ala Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg 180 185 190Asp
Ala Ser Leu Phe Gly Ser Glu Trp Gly Met Ala Ser Ser Asp Val 195 200
205Asn Gln Tyr Tyr Gln Glu Gln Ile Arg Tyr Thr Glu Glu Tyr Ser Asn
210 215 220His Cys Val Gln Trp Tyr Asn Thr Gly Leu Asn Asn Leu Arg
Gly Thr225 230 235 240Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe
Arg Arg Asp Leu Thr 245 250 255Leu Gly Val Leu Asp Leu Val Ala Leu
Phe Pro Ser Tyr Asp Thr Arg 260 265 270Thr Tyr Pro Ile Asn Thr Ser
Ala Gln Leu Thr Arg Glu Ile Tyr Thr 275 280 285Asp Pro Ile Gly Arg
Thr Asn Ala Pro Ser Gly Phe Ala Ser Thr Asn 290 295 300Trp Phe Asn
Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Ile305 310 315
320Phe Arg Pro Pro His Leu Leu Asp Phe Pro Glu Gln Leu Thr Ile Tyr
325 330 335Ser Ala Ser Ser Arg Trp Ser Ser Thr Gln His Met Asn Tyr
Trp Val 340 345 350Gly His Arg Leu Asn Phe Arg Pro Ile Gly Gly Thr
Leu Asn Thr Ser 355 360 365Thr Gln Gly Leu Thr Asn Asn Thr Ser Ile
Asn Pro Val Thr Leu His 370 375 380Tyr Val Ser Ser Arg Asp Val Tyr
Arg Thr Glu Ser Asn Ala Gly Thr385 390 395 400Asn Ile Leu Phe Thr
Thr Pro Val Asn Gly Val Pro Trp Ala Arg Phe 405 410 415Asn Phe Ile
Thr Leu Arg Ile Phe Met Lys Glu Ala Pro Leu Pro Thr 420 425 430Val
Asn Arg Ile Arg Glu Leu Gly Phe Asn Tyr Leu Ile Gln Lys Leu 435 440
445Asn Tyr His Gln Lys Gln Gln Asn Asp Gln Ile Met Asn His Ile Val
450 455 460Ile Asp Ile Ser Tyr Arg Leu Ile Ile Gly Asn Thr Leu Arg
Ala Pro465 470 475 480Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg
Thr Asn Thr Ile Gly 485 490 495Pro Asn Arg Ile Thr Gln Ile Pro Ala
Val Lys Gly Arg Phe Leu Phe 500 505 510Asn Gly Ser Val Ile Ser Gly
Pro Gly Phe Thr Gly Gly Asp Val Val 515 520 525Arg Leu Asn Arg Asn
Asn Gly Asn Ile Gln Asn Arg Gly Tyr Ile Glu 530 535 540Val Pro Ile
Gln Phe Thr Ser Thr Ser Thr Arg Tyr Arg Val Arg Val545 550 555
560Arg Tyr Ala Ser Val Thr Ser Ile Glu Leu Asn Val Asn Leu Gly Asn
565 570 575Ser Ser Ile Phe Thr Asn Thr Leu Pro Ala Thr Ala Ala Ser
Leu Asp 580 585 590Asn Leu Gln Ser Gly Asp Phe Gly Tyr Val Glu Ile
Asn Asn Ala Phe 595 600 605Thr Ser Ala Thr Gly Asn Ile Val Gly Ala
Arg Asn Phe Ser Ala Asn 610 615 620Ala Glu Val Ile Ile Asp Arg Phe
Glu Phe Ile Pro Val Thr Ala Thr625 630 635 640Phe Glu Val Glu Tyr
Asp Leu Glu Arg Ala Gln Lys Ala 645 6507328DNABacillus
thuringiensis 7ctttacagga agattaccac aaagttatta tatcgtttcc
gttatgcttc gggagcaaat 60aggagtggtt cattaagtta ttcacagcaa acttcgtatg
taatttcatt tccaaaaact 120atggacgcag gtgaaccact aacatctcgt
tcgttcgctt ttacaacaac cgtcactcca 180atagccttta cacgagctca
agaagaattt gatttataca tccaacagaa tgtttatata 240gatagagttg
aatttatccc agtagatgca acatttgagg caaaatctga tttagaaaga
300gcgaaaaagg cggtgaatgc cttgttta 3288109PRTBacillus thuringiensis
8Leu Tyr Arg Lys Ile Thr Thr Lys Leu Leu Tyr Arg Phe Arg Tyr Ala1 5
10 15Ser Gly Ala Asn Arg Ser Gly Ser Leu Ser Tyr Ser Gln Gln Thr
Ser 20 25 30Tyr Val Ile Ser Phe Pro Lys Thr Met Asp Ala Gly Glu Pro
Leu Thr 35 40 45Ser Arg Ser Phe Ala Phe Thr Thr Thr Val Thr Pro Ile
Ala Phe Thr 50 55 60Arg Ala Gln Glu Glu Phe Asp Leu Tyr Ile Gln Gln
Asn Val Tyr Ile65 70 75 80Asp Arg Val Glu Phe Ile Pro Val Asp Ala
Thr Phe Glu Ala Lys Ser 85 90 95Asp Leu Glu Arg Ala Lys Lys Ala Val
Asn Ala Leu Phe 100 1059340DNABacillus thuringiensis 9ttacgagtaa
cctttacagg aagattacca caaagttatt atatacgttt ccgttatgct 60tcgggagcaa
ataggagtgg ttcattaagt tattcacagc aaacttcgta tgtaatttca
120tttccaaaaa ctatggacgc aggtgaacca ctaacatctc gttcgttcgc
ttttacaaca 180accgtcactc caataacctt tacacgagct caagaagaat
ttgatttata catccaacag 240aatgtttata tagatagagt tgaatttatc
ccagtagatg caacatttga ggcaaaatct 300gatttagaaa gagcgaaaaa
ggcggtgaat gccttgttta 34010113PRTBacillus thuringiensis 10Leu Arg
Val Thr Phe Thr Gly Arg Leu Pro Gln Ser Tyr Tyr Ile Arg1 5 10 15Phe
Arg Tyr Ala Ser Gly Ala Asn Arg Ser Gly Ser Leu Ser Tyr Ser 20 25
30Gln Gln Thr Ser Tyr Val Ile Ser Phe Pro Lys Thr Met Asp Ala Gly
35 40 45Glu Pro Leu Thr Ser Arg Ser Phe Ala Phe Thr Thr Thr Val Thr
Pro 50 55 60Ile Thr Phe Thr Arg Ala Gln Glu Glu Phe Asp Leu Tyr Ile
Gln Gln65 70 75 80Asn Val Tyr Ile Asp Arg Val Glu Phe Ile Pro Val
Asp Ala Thr Phe 85 90 95Glu Ala Lys Ser Asp Leu Glu Arg Ala Lys Lys
Ala Val Asn Ala Leu 100 105 110Phe11306DNABacillus thuringiensis
11gtatcgcgtg agatcgtatg ctctacgaca gatttacaat tctatacgaa tattaatgga
60actactatta atattggtaa tttctcgagc actatggaca gtggggatga tttacagtac
120ggaagattca gggttgcagg ttttactact ccatttacct tttcagatgc
aaacagcaca 180ttcacaatag gtgcttttgg cttctctcca aacaacgaag
tttatataga tcgaattgaa 240tttgtcccgg cagaagtaac atttgaggca
gaatatgatt tagagaaagc tcagaaagcg 300gtgaat 30612102PRTBacillus
thuringiensis 12Val Ser Arg Glu Ile Val Cys Ser Thr Thr Asp Leu Gln
Phe Tyr Thr1 5 10 15Asn Ile Asn Gly Thr Thr Ile Asn Ile Gly Asn Phe
Ser Ser Thr Met 20 25 30Asp Ser Gly Asp Asp Leu Gln Tyr Gly Arg Phe
Arg Val Ala Gly Phe 35 40 45Thr Thr Pro Phe Thr Phe Ser Asp Ala Asn
Ser Thr Phe Thr Ile Gly 50 55 60Ala Phe Gly Phe Ser Pro Asn Asn Glu
Val Tyr Ile Asp Arg Ile Glu65 70 75 80Phe Val Pro Ala Glu Val Thr
Phe Glu Ala Glu Tyr Asp Leu Glu Lys 85 90 95Ala Gln Lys Ala Val Asn
10013279DNABacillus thuringiensis 13caattccata catcaattga
cggaagacct attaatcagg ggaatttttc agcaactatg 60agtagtggga gtaatttaca
gtccggaagc tttaggactg taggttttac tactccgttt 120aacttttcaa
atggatcaag tgtatttacg ttaagtgctc atgtcttcaa ttcaggcaat
180gaagtttata tagatcgaat tgaatttatt ccggcagaag taacctttga
ggcagaatat 240gatttagaaa gagcacaaaa ggcggtgaat gagctgttt
2791493PRTBacillus thuringiensis 14Gln Phe His Thr Ser Ile Asp Gly
Arg Pro Ile Asn Gln Gly Asn Phe1 5 10 15Ser Ala Thr Met Ser Ser Gly
Ser Asn Leu Gln Ser Gly Ser Phe Arg 20 25 30Thr Val Gly Phe Thr Thr
Pro Phe Asn Phe Ser Asn Gly Ser Ser Val 35 40 45Phe Thr Leu Ser Ala
His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile 50 55 60Asp Arg Ile Glu
Phe Ile Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr65 70 75 80Asp Leu
Glu Arg Ala Gln Lys Ala Val Asn Glu Leu Phe 85 9015397DNABacillus
thuringiensis 15aggaccaggt tttacaggtg ggatatcctt cgaagaacga
atgttggtag ctttggagat 60atgcgtgtaa acattactgc accactatca caaagatatc
gcgtaagaat tcgctatgct 120tctacgacag atttacaatt tttcacgaga
atcaatggaa cttctgtaaa tcaaggtaat 180ttccaaagaa ctatgaatag
agggggtaat ttagaatctg gaaactttag gactgcagga 240tttagtacgc
cttttagttt tttcaaatgc gcaaagtaca ttcacattgg gtactcaggc
300ttttcaaatc aggaagttta tatagatcga attgaatttg tcccggcaga
agtaacattc 360gaggcagaat ctgatttgga aagagcgcaa aaggcgg
39716132PRTBacillus thuringiensis 16Arg Thr Arg Phe Tyr Arg Trp Asp
Ile Leu Arg Arg Thr Asn Val Gly1 5 10 15Ser Phe Gly Asp Met Arg Val
Asn Ile Thr Ala Pro Leu Ser Gln Arg 20 25 30Tyr Arg Val Arg Ile Arg
Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe 35 40 45Thr Arg Ile Asn Gly
Thr Ser Val Asn Gln Gly Asn Phe Gln Arg Thr 50 55 60Met Asn Arg Gly
Gly Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly65 70 75 80Phe Ser
Thr Pro Phe Ser Phe Phe Lys Cys Ala Lys Tyr Ile His Ile 85 90 95Gly
Tyr Ser Gly Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu 100 105
110Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg
115 120 125Ala Gln Lys Ala 13017123DNABacillus thuringiensis
17ataatctaca atcaggggga ttttggttat gttgaaatca acaatgcttt tacatccgca
60acaggtaata tagtaggtgc tagaaatttt acgtgcaaat gcagaagtaa taatagacag
120att 1231841PRTBacillus thuringiensis 18Ile Ile Tyr Asn Gln Gly
Asp Phe Gly Tyr Val Glu Ile Asn Asn Ala1 5 10 15Phe Thr Ser Ala Thr
Gly Asn Ile Val Gly Ala Arg Asn Phe Thr Cys 20 25 30Lys Cys Arg Ser
Asn Asn Arg Gln Ile 35 4019192DNABacillus thuringiensis
19agttattata tacgtttccg ttatgcttcc gtagctaata ggagtggtat atttagctat
60tcacagccaa cttcatatgg aatttccttt ccaaaaacta tggatgcaga tgaatcatta
120acatctcgtt catttgcact tgctacactt gctacaccgc taacctttag
aaggcaagaa 180gaattaaatc ta 1922064PRTBacillus thuringiensis 20Ser
Tyr Tyr Ile Arg Phe Arg Tyr Ala Ser Val Ala Asn Arg Ser Gly1 5 10
15Ile Phe Ser Tyr Ser Gln Pro Thr Ser Tyr Gly Ile Ser Phe Pro Lys
20 25 30Thr Met Asp Ala Asp Glu Ser Leu Thr Ser Arg Ser Phe Ala Leu
Ala 35 40 45Thr Leu Ala Thr Pro Leu Thr Phe Arg Arg Gln Glu Glu Leu
Asn Leu 50 55 60213507DNABacillus thuringiensisCDS(1)..(3507) 21atg
gag ata aat aat cag aac caa tgc ata cca tat aat tgc tta agt 48Met
Glu Ile Asn Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser1 5 10
15aat cct gag gaa gta ttt ttg gat ggg gag agg ata tta cct gat atc
96Asn Pro Glu Glu Val Phe Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile
20 25 30gat cca ctc gaa gtt tct ttg tcg ctt ttg caa ttt ctt ttg aat
aac 144Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn
Asn 35 40 45ttt gtt cca ggg ggg ggg ttt att tca gga tta ctt gat aaa
ata tgg 192Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Leu Asp Lys
Ile Trp 50 55 60ggg gct ttg aga cca tct gat tgg gaa tta ttt ctt gca
cag att gaa 240Gly Ala Leu Arg Pro Ser Asp Trp Glu Leu Phe Leu Ala
Gln Ile Glu65 70 75 80cag ttg att gat cga aga ata gaa aga aca gta
aga gca aaa gca atc 288Gln Leu Ile Asp Arg Arg Ile Glu Arg Thr Val
Arg Ala Lys Ala Ile 85 90 95gct gaa tta gaa ggt tta ggg aga agt tat
caa cta tat gga gag gca 336Ala Glu Leu Glu Gly Leu Gly Arg Ser Tyr
Gln Leu Tyr Gly Glu Ala 100 105 110ttt aaa gag tgg gaa aaa act cca
gat aac aca gcg gct cgg tct aga 384Phe Lys Glu Trp Glu Lys Thr Pro
Asp Asn Thr Ala Ala Arg Ser Arg 115 120 125gta act gag aga ttt cgt
ata att gat gct caa att gaa gca aat atc 432Val Thr Glu Arg Phe Arg
Ile Ile Asp Ala Gln Ile Glu Ala Asn Ile 130 135 140cct tcg ttt cgg
gtt tcc gga ttt gaa gtg cca ctt cta ttg gtt tat 480Pro Ser Phe Arg
Val Ser Gly Phe Glu Val Pro Leu Leu Leu Val Tyr145 150 155 160acc
caa gca gct aat ttg cat ctc gct cta tta aga gat tct gtt gtt 528Thr
Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Val 165 170
175ttt gga gag aga tgg gga ttg acg act aca aat gtc aat gat atc tat
576Phe Gly Glu Arg Trp Gly Leu Thr Thr Thr Asn Val Asn Asp Ile Tyr
180 185 190aat aga caa gtt aat aga att ggt gaa tat agc aag cat tgt
gta gat 624Asn Arg Gln Val Asn Arg Ile Gly Glu Tyr Ser Lys His Cys
Val Asp 195 200 205acg tat aaa aca gaa tta gaa cgt cta gga ttt aga
tct ata gcg caa 672Thr Tyr Lys Thr Glu Leu Glu Arg Leu Gly Phe Arg
Ser Ile Ala Gln 210 215 220tgg aga ata tat aat cag ttt aga agg gaa
ttg aca cta acg gta tta 720Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu
Leu Thr Leu Thr Val Leu225 230 235 240gat att gtc gct gtt ttc ccg
aac tat gat agt aga ctg tat ccg att 768Asp Ile Val Ala Val Phe Pro
Asn Tyr Asp Ser Arg Leu Tyr Pro Ile 245 250 255cga aca att tct caa
ttg aca aga gaa att tat aca tcc cca gta agc 816Arg Thr Ile Ser Gln
Leu Thr Arg Glu Ile Tyr Thr Ser Pro Val Ser 260 265 270gaa ttt tat
tat ggt gtc att aat agt aat aat ata att ggt acc ctt 864Glu Phe Tyr
Tyr Gly Val Ile Asn Ser Asn Asn Ile Ile Gly Thr Leu 275 280 285act
gaa cag caa ata agg cga cca cat ctt atg gac ttc ttt aac tcc 912Thr
Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295
300atg atc atg tat acg tca gat aat aga cga gaa cat tat tgg tca gga
960Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser
Gly305 310 315 320ctt gaa atg acg gct act aat act gag gga cat caa
agg tca ttc cct 1008Leu Glu Met Thr Ala Thr Asn Thr Glu Gly His Gln
Arg Ser Phe Pro 325 330 335tta gct ggg act ata ggg aat tca gct cca
cca gta act gtt aga aat 1056Leu Ala Gly Thr Ile Gly Asn Ser Ala Pro
Pro Val Thr Val Arg Asn 340 345 350aat ggt gag gga att tat aga ata
tta tcg gaa cca ttt tat tca gca 1104Asn Gly Glu Gly Ile Tyr Arg Ile
Leu Ser Glu Pro Phe Tyr Ser Ala 355 360 365cct ttt cta ggc aca agt
gtg cta gga agt cgt ggg gaa gaa ttt gct 1152Pro Phe Leu Gly Thr Ser
Val Leu Gly Ser Arg Gly Glu Glu Phe Ala 370 375 380ttt gca tct aat
act act aca agt ctg cca tct aca ata tat aga aat 1200Phe Ala Ser Asn
Thr Thr Thr Ser Leu Pro Ser Thr Ile Tyr Arg Asn385 390 395 400cgt
gga aca gta gat tca tta gtc agc ata ccg cca cag gat tat agc 1248Arg
Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp Tyr Ser 405 410
415gta cca ccg cac agg ggg tat agt cat tta tta agt cac gtt acg atg
1296Val Pro Pro His Arg Gly Tyr Ser His Leu Leu Ser His Val Thr Met
420 425 430cgc aat agt tct cct ata ttc cac tgg aca cat cgt agt gca
acc cct 1344Arg Asn Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala
Thr Pro 435 440 445aga aat aca att gat cca gat agt atc act caa att
cca gca gtt aag 1392Arg Asn Thr Ile Asp Pro Asp Ser Ile Thr Gln Ile
Pro Ala Val Lys 450 455 460gga gcg tat att ttt aat agt cca gtc att
act ggg cca gga cat aca 1440Gly Ala Tyr Ile Phe Asn Ser Pro Val Ile
Thr Gly Pro Gly His Thr465 470 475 480ggt ggg gat ata ata agg ttt
aac cct aat act cag aac aac ata aga 1488Gly Gly Asp Ile Ile Arg Phe
Asn Pro Asn Thr Gln Asn Asn Ile Arg 485 490 495att cca ttt caa tca
aat gcg gta cag cgt tat cga att aga atg cgt 1536Ile Pro Phe Gln Ser
Asn Ala Val Gln Arg Tyr Arg Ile Arg Met Arg 500 505 510tat gcg gca
gaa gct gat tgt att tta gaa agt gga gta aac att gtt 1584Tyr Ala Ala
Glu Ala Asp Cys Ile Leu Glu Ser Gly Val Asn Ile Val
515 520 525act ggg gca ggg gtc acc ttt agg cca att cct att aaa gct
aca atg 1632Thr Gly Ala Gly Val Thr Phe Arg Pro Ile Pro Ile Lys Ala
Thr Met 530 535 540act cct gga agt cct tta aca tat tac agc ttc cag
tat gca gat tta 1680Thr Pro Gly Ser Pro Leu Thr Tyr Tyr Ser Phe Gln
Tyr Ala Asp Leu545 550 555 560aat ata aat ctt act gcg ccg ata aga
cct aat aat ttt gta tct att 1728Asn Ile Asn Leu Thr Ala Pro Ile Arg
Pro Asn Asn Phe Val Ser Ile 565 570 575aga cgt tca aac caa cca gga
aac ctt tat ata gat aga att gaa ttc 1776Arg Arg Ser Asn Gln Pro Gly
Asn Leu Tyr Ile Asp Arg Ile Glu Phe 580 585 590att cca att gac cca
atc cgt gag gca gaa cat gat tta gaa aga gcg 1824Ile Pro Ile Asp Pro
Ile Arg Glu Ala Glu His Asp Leu Glu Arg Ala 595 600 605caa aag gcg
gtg aat gcg ctg ttt act tct tcc aat caa cta gga tta 1872Gln Lys Ala
Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Leu Gly Leu 610 615 620aaa
aca gat gtg acg gat tat cat att gat caa gtg tcc aat tta gtt 1920Lys
Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val625 630
635 640gcg tgt tta tcg gat gaa ttc tgc ctg gat gaa aag cga gaa ttg
tcc 1968Ala Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu
Ser 645 650 655gag aaa gtt aaa cat gcg aag cga ctc agt gat gag aga
aat tta ctc 2016Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg
Asn Leu Leu 660 665 670caa gat caa aac ttt aca ggc atc aat agg caa
gta gac cgt ggg tgg 2064Gln Asp Gln Asn Phe Thr Gly Ile Asn Arg Gln
Val Asp Arg Gly Trp 675 680 685aga gga agt acg gat att acc atc caa
gga ggg aat gat gta ttc aaa 2112Arg Gly Ser Thr Asp Ile Thr Ile Gln
Gly Gly Asn Asp Val Phe Lys 690 695 700gag aat tac gtc aca cta cca
ggt acc ttt gat gag tgt tac cca acg 2160Glu Asn Tyr Val Thr Leu Pro
Gly Thr Phe Asp Glu Cys Tyr Pro Thr705 710 715 720tat ttg tat caa
aaa ata gat gag tca aaa tta aaa cct tat act cgc 2208Tyr Leu Tyr Gln
Lys Ile Asp Glu Ser Lys Leu Lys Pro Tyr Thr Arg 725 730 735tat gaa
tta aga ggg tat att gaa gat agt caa gac tta gaa gtc tat 2256Tyr Glu
Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr 740 745
750ttg atc cgt tac aat gca aaa cac gaa acg tta aat gtg cca ggt acg
2304Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Leu Asn Val Pro Gly Thr
755 760 765ggt tcc tta tgg cca ctt gca gcc gaa agt tca atc ggg agg
tgc ggc 2352Gly Ser Leu Trp Pro Leu Ala Ala Glu Ser Ser Ile Gly Arg
Cys Gly 770 775 780gaa ccg aat cga tgc gcg cca cat att gaa tgg aat
cct gaa cta gat 2400Glu Pro Asn Arg Cys Ala Pro His Ile Glu Trp Asn
Pro Glu Leu Asp785 790 795 800tgt tcg tgt agg gat gga gaa aaa tgt
gca cat cat tct cat cat ttc 2448Cys Ser Cys Arg Asp Gly Glu Lys Cys
Ala His His Ser His His Phe 805 810 815tcc ttg gat att gat gtt gga
tgt aca gac tta aat gag gat tta ggt 2496Ser Leu Asp Ile Asp Val Gly
Cys Thr Asp Leu Asn Glu Asp Leu Gly 820 825 830gta tgg gtg ata ttt
aag att aag acg caa gat ggc tat gca aga cta 2544Val Trp Val Ile Phe
Lys Ile Lys Thr Gln Asp Gly Tyr Ala Arg Leu 835 840 845gga aat tta
gag ttt ctc gaa gag aaa cca ttg tta gga gaa gcg cta 2592Gly Asn Leu
Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu 850 855 860gct
cgt gtg aag aga gcg gag aaa aaa tgg aga gac aaa cgc gac aaa 2640Ala
Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Asp Lys865 870
875 880ttg gaa tgg gaa aca aat att gtt tat aaa gag gca aaa gaa tct
gta 2688Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser
Val 885 890 895gat gct tta ttc gta gat tct caa tat aat aga tta caa
acg gat acg 2736Asp Ala Leu Phe Val Asp Ser Gln Tyr Asn Arg Leu Gln
Thr Asp Thr 900 905 910aac att gcg atg att cat gtg gca gat aaa cgc
gtt cat cga atc cga 2784Asn Ile Ala Met Ile His Val Ala Asp Lys Arg
Val His Arg Ile Arg 915 920 925gaa gcg tat ttg cca gag tta tct gtg
att ccg ggt gtc aat gcg gct 2832Glu Ala Tyr Leu Pro Glu Leu Ser Val
Ile Pro Gly Val Asn Ala Ala 930 935 940att ttc gaa gaa tta gaa ggt
ctt att ttc act gca ttc tcc cta tat 2880Ile Phe Glu Glu Leu Glu Gly
Leu Ile Phe Thr Ala Phe Ser Leu Tyr945 950 955 960gat gcg aga aat
gtc att aaa aac gga gat ttc aat cat ggt tta tca 2928Asp Ala Arg Asn
Val Ile Lys Asn Gly Asp Phe Asn His Gly Leu Ser 965 970 975tgc tgg
aac gtg aaa ggg cat gta gat gta gaa gaa caa aat aac cac 2976Cys Trp
Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His 980 985
990cgt tcg gtc ctt gtt gtt ccg gaa tgg gaa gca gaa gtg tca caa gaa
3024Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu
995 1000 1005gtc cgc gta tgt cca gga cgt ggc tat atc ctg cgt gtt
aca gcg 3069Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr
Ala 1010 1015 1020tac aaa gag ggc tac gga gaa gga tgc gta acg atc
cat gaa att 3114Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His
Glu Ile 1025 1030 1035gaa gat cat aca gac gaa ctg aaa ttt aga aac
tgt gaa gaa gag 3159Glu Asp His Thr Asp Glu Leu Lys Phe Arg Asn Cys
Glu Glu Glu 1040 1045 1050gaa gtg tat ccg aat aac acg gta acg tgt
aat gat tat cca gca 3204Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn
Asp Tyr Pro Ala 1055 1060 1065aat caa gaa gaa tac agg gct gcg gaa
act tcc cgt aat cgt gga 3249Asn Gln Glu Glu Tyr Arg Ala Ala Glu Thr
Ser Arg Asn Arg Gly 1070 1075 1080tat ggc gaa tct tat gaa agt aat
tct tcc ata cca gct gag tat 3294Tyr Gly Glu Ser Tyr Glu Ser Asn Ser
Ser Ile Pro Ala Glu Tyr 1085 1090 1095gcg cca att tat gag aaa gca
tat aca gat gga aga aaa gag aat 3339Ala Pro Ile Tyr Glu Lys Ala Tyr
Thr Asp Gly Arg Lys Glu Asn 1100 1105 1110tct tgt gaa tct aac aga
gga tat gga aat tac aca ccg tta cca 3384Ser Cys Glu Ser Asn Arg Gly
Tyr Gly Asn Tyr Thr Pro Leu Pro 1115 1120 1125gca ggt tat gtg aca
aaa gaa tta gag tac ttc cca gaa acc gat 3429Ala Gly Tyr Val Thr Lys
Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1130 1135 1140aag gta tgg ata
gag att gga gaa acg gaa gga aca ttc atc gta 3474Lys Val Trp Ile Glu
Ile Gly Glu Thr Glu Gly Thr Phe Ile Val 1145 1150 1155gac agt gtg
gaa tta ctc ctc atg gag gaa tag 3507Asp Ser Val Glu Leu Leu Leu Met
Glu Glu 1160 1165221168PRTBacillus thuringiensis 22Met Glu Ile Asn
Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser1 5 10 15Asn Pro Glu
Glu Val Phe Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile 20 25 30Asp Pro
Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn 35 40 45Phe
Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Leu Asp Lys Ile Trp 50 55
60Gly Ala Leu Arg Pro Ser Asp Trp Glu Leu Phe Leu Ala Gln Ile Glu65
70 75 80Gln Leu Ile Asp Arg Arg Ile Glu Arg Thr Val Arg Ala Lys Ala
Ile 85 90 95Ala Glu Leu Glu Gly Leu Gly Arg Ser Tyr Gln Leu Tyr Gly
Glu Ala 100 105 110Phe Lys Glu Trp Glu Lys Thr Pro Asp Asn Thr Ala
Ala Arg Ser Arg 115 120 125Val Thr Glu Arg Phe Arg Ile Ile Asp Ala
Gln Ile Glu Ala Asn Ile 130 135 140Pro Ser Phe Arg Val Ser Gly Phe
Glu Val Pro Leu Leu Leu Val Tyr145 150 155 160Thr Gln Ala Ala Asn
Leu His Leu Ala Leu Leu Arg Asp Ser Val Val 165 170 175Phe Gly Glu
Arg Trp Gly Leu Thr Thr Thr Asn Val Asn Asp Ile Tyr 180 185 190Asn
Arg Gln Val Asn Arg Ile Gly Glu Tyr Ser Lys His Cys Val Asp 195 200
205Thr Tyr Lys Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln
210 215 220Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr
Val Leu225 230 235 240Asp Ile Val Ala Val Phe Pro Asn Tyr Asp Ser
Arg Leu Tyr Pro Ile 245 250 255Arg Thr Ile Ser Gln Leu Thr Arg Glu
Ile Tyr Thr Ser Pro Val Ser 260 265 270Glu Phe Tyr Tyr Gly Val Ile
Asn Ser Asn Asn Ile Ile Gly Thr Leu 275 280 285Thr Glu Gln Gln Ile
Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295 300Met Ile Met
Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly305 310 315
320Leu Glu Met Thr Ala Thr Asn Thr Glu Gly His Gln Arg Ser Phe Pro
325 330 335Leu Ala Gly Thr Ile Gly Asn Ser Ala Pro Pro Val Thr Val
Arg Asn 340 345 350Asn Gly Glu Gly Ile Tyr Arg Ile Leu Ser Glu Pro
Phe Tyr Ser Ala 355 360 365Pro Phe Leu Gly Thr Ser Val Leu Gly Ser
Arg Gly Glu Glu Phe Ala 370 375 380Phe Ala Ser Asn Thr Thr Thr Ser
Leu Pro Ser Thr Ile Tyr Arg Asn385 390 395 400Arg Gly Thr Val Asp
Ser Leu Val Ser Ile Pro Pro Gln Asp Tyr Ser 405 410 415Val Pro Pro
His Arg Gly Tyr Ser His Leu Leu Ser His Val Thr Met 420 425 430Arg
Asn Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala Thr Pro 435 440
445Arg Asn Thr Ile Asp Pro Asp Ser Ile Thr Gln Ile Pro Ala Val Lys
450 455 460Gly Ala Tyr Ile Phe Asn Ser Pro Val Ile Thr Gly Pro Gly
His Thr465 470 475 480Gly Gly Asp Ile Ile Arg Phe Asn Pro Asn Thr
Gln Asn Asn Ile Arg 485 490 495Ile Pro Phe Gln Ser Asn Ala Val Gln
Arg Tyr Arg Ile Arg Met Arg 500 505 510Tyr Ala Ala Glu Ala Asp Cys
Ile Leu Glu Ser Gly Val Asn Ile Val 515 520 525Thr Gly Ala Gly Val
Thr Phe Arg Pro Ile Pro Ile Lys Ala Thr Met 530 535 540Thr Pro Gly
Ser Pro Leu Thr Tyr Tyr Ser Phe Gln Tyr Ala Asp Leu545 550 555
560Asn Ile Asn Leu Thr Ala Pro Ile Arg Pro Asn Asn Phe Val Ser Ile
565 570 575Arg Arg Ser Asn Gln Pro Gly Asn Leu Tyr Ile Asp Arg Ile
Glu Phe 580 585 590Ile Pro Ile Asp Pro Ile Arg Glu Ala Glu His Asp
Leu Glu Arg Ala 595 600 605Gln Lys Ala Val Asn Ala Leu Phe Thr Ser
Ser Asn Gln Leu Gly Leu 610 615 620Lys Thr Asp Val Thr Asp Tyr His
Ile Asp Gln Val Ser Asn Leu Val625 630 635 640Ala Cys Leu Ser Asp
Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser 645 650 655Glu Lys Val
Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu 660 665 670Gln
Asp Gln Asn Phe Thr Gly Ile Asn Arg Gln Val Asp Arg Gly Trp 675 680
685Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys
690 695 700Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr
Pro Thr705 710 715 720Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu
Lys Pro Tyr Thr Arg 725 730 735Tyr Glu Leu Arg Gly Tyr Ile Glu Asp
Ser Gln Asp Leu Glu Val Tyr 740 745 750Leu Ile Arg Tyr Asn Ala Lys
His Glu Thr Leu Asn Val Pro Gly Thr 755 760 765Gly Ser Leu Trp Pro
Leu Ala Ala Glu Ser Ser Ile Gly Arg Cys Gly 770 775 780Glu Pro Asn
Arg Cys Ala Pro His Ile Glu Trp Asn Pro Glu Leu Asp785 790 795
800Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe
805 810 815Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp
Leu Gly 820 825 830Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly
Tyr Ala Arg Leu 835 840 845Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro
Leu Leu Gly Glu Ala Leu 850 855 860Ala Arg Val Lys Arg Ala Glu Lys
Lys Trp Arg Asp Lys Arg Asp Lys865 870 875 880Leu Glu Trp Glu Thr
Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val 885 890 895Asp Ala Leu
Phe Val Asp Ser Gln Tyr Asn Arg Leu Gln Thr Asp Thr 900 905 910Asn
Ile Ala Met Ile His Val Ala Asp Lys Arg Val His Arg Ile Arg 915 920
925Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala
930 935 940Ile Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser
Leu Tyr945 950 955 960Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe
Asn His Gly Leu Ser 965 970 975Cys Trp Asn Val Lys Gly His Val Asp
Val Glu Glu Gln Asn Asn His 980 985 990Arg Ser Val Leu Val Val Pro
Glu Trp Glu Ala Glu Val Ser Gln Glu 995 1000 1005Val Arg Val Cys
Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala 1010 1015 1020Tyr Lys
Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile 1025 1030
1035Glu Asp His Thr Asp Glu Leu Lys Phe Arg Asn Cys Glu Glu Glu
1040 1045 1050Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr
Pro Ala 1055 1060 1065Asn Gln Glu Glu Tyr Arg Ala Ala Glu Thr Ser
Arg Asn Arg Gly 1070 1075 1080Tyr Gly Glu Ser Tyr Glu Ser Asn Ser
Ser Ile Pro Ala Glu Tyr 1085 1090 1095Ala Pro Ile Tyr Glu Lys Ala
Tyr Thr Asp Gly Arg Lys Glu Asn 1100 1105 1110Ser Cys Glu Ser Asn
Arg Gly Tyr Gly Asn Tyr Thr Pro Leu Pro 1115 1120 1125Ala Gly Tyr
Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1130 1135 1140Lys
Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val 1145 1150
1155Asp Ser Val Glu Leu Leu Leu Met Glu Glu 1160
116523348DNABacillus thuringiensis 23aataatagag gtcatcttcc
aattccaatc caattttctt cgcgttctac cagatatcga 60gttcgtgtac gttatgcttc
tgcaaccccc attcaagtca atgttcattg ggaaaatagc 120tcgttttttt
caggtacagt accagctacg gctcagtcat tagataatct acaatcaaac
180aattttggtt actttgagac cgctaatact atttcatctt cattagatgg
tatagtaggt 240attagaaatt ttagtgcaaa tgcagatttg ataatagaca
gatttgaatt tatcccagtg 300gatgcaacct ccgaggcaga acatgattta
gaaagagcgc aaaaggcg 34824116PRTBacillus thuringiensis 24Asn Asn Arg
Gly His Leu Pro Ile Pro Ile Gln Phe Ser Ser Arg Ser1 5 10 15Thr Arg
Tyr Arg Val Arg Val Arg Tyr Ala Ser Ala Thr Pro Ile Gln 20 25 30Val
Asn Val His Trp Glu Asn Ser Ser Phe Phe Ser Gly Thr Val Pro 35 40
45Ala Thr Ala Gln Ser Leu Asp Asn Leu Gln Ser Asn Asn Phe Gly Tyr
50 55 60Phe Glu Thr Ala Asn Thr Ile Ser Ser Ser Leu Asp Gly Ile Val
Gly65 70 75 80Ile Arg Asn Phe Ser Ala Asn Ala Asp Leu Ile Ile Asp
Arg Phe Glu 85 90 95Phe Ile Pro Val Asp Ala Thr Ser Glu Ala Glu His
Asp Leu Glu Arg 100 105 110Ala Gln Lys Ala 11525186DNABacillus
thuringiensis 25ccactaacat ctcgttcgtt cgctcataca acactcttca
ctccaataac cttttcacga 60gctcaagaag aatttgatct atacatccaa tcgggtgttt
atatagatcg aattgaattt 120attccagtta ctgcaacatt tgaggcagaa
tatgatttag aaagagcgca aagggcggtg 180aatgcc 1862662PRTBacillus
thuringiensis 26Pro Leu Thr Ser Arg Ser Phe Ala His Thr Thr Leu Phe
Thr Pro Ile1 5 10 15Thr Phe Ser Arg Ala Gln Glu
Glu Phe Asp Leu Tyr Ile Gln Ser Gly 20 25 30Val Tyr Ile Asp Arg Ile
Glu Phe Ile Pro Val Thr Ala Thr Phe Glu 35 40 45Ala Glu Tyr Asp Leu
Glu Arg Ala Gln Arg Ala Val Asn Ala 50 55 60273471DNABacillus
thuringiensis 27atgaatcgaa ataatcaaaa tgaatatgaa attattgatg
ccccccattg tgggtgtcca 60tcagatgacg atgtgaggta tcctttggca agtgacccaa
atgcagcgtt acaaaatatg 120aactataaag attacttaca aatgacagat
gaggactaca ctgattctta tataaatcct 180agtttatcta ttagtggtag
agatgcagtt cagactgcgc ttactgttgt tgggagaata 240ctcggggctt
taggtgttcc gttttctgga caaatagtga gtttttatca attcctttta
300aatacactgt ggccagttaa tgatacagct atatgggaag ctttcatgcg
acaggtggag 360gaacttgtca atcaacaaat aacagaattt gcaagaaatc
aggcacttgc aagattgcaa 420ggattaggag actcttttaa tgtatatcaa
cgttcccttc aaaattggtt ggctgatcga 480aatgatacac gaaatttaag
tgttgttcgt gctcaattta tagctttaga ccttgatttt 540gttaatgcta
ttccattgtt tgcagtaaat ggacagcagg ttccattact gtcagtatat
600gcacaagctg tgaatttaca tttgttatta ttaaaagatg catctctttt
tggagaagga 660tggggattca cacaggggga aatttccaca tattatgacc
gtcaattgga actaaccgct 720aagtacacta attactgtga aacttggtat
aatacaggtt tagatcgttt aagaggaaca 780aatactgaaa gttggttaag
atatcatcaa ttccgtagag aaatgacttt agtggtatta 840gatgttgtgg
cgctatttcc atattatgat gtacgacttt atccaacggg atcaaaccca
900cagcttacac gtgaggtata tacagatccg attgtattta atccaccagc
taatgttgga 960ctttgccgac gttggggtac taatccctat aatacttttt
ctgagctcga aaatgccttc 1020attcgcccac cacatctttt tgataggctg
aatagcttaa caatcagcag taatcgattt 1080ccagtttcat ctaattttat
ggattattgg tcaggacata cgttacgccg tagttatctg 1140aacgattcag
cagtacaaga agatagttat ggcctaatta caaccacaag agcaacaatt
1200aatcctggag ttgatggaac aaaccgcata gagtcaacgg cagtagattt
tcgttctgca 1260ttgataggta tatatggcgt gaatagagct tcttttgtcc
caggaggctt gtttaatggt 1320acgacttctc ctgctaatgg aggatgtaga
gatctctatg atacaaatga tgaattacca 1380ccagatgaaa gtaccggaag
ttctacccat agactatctc atgttacctt ttttagtttt 1440caaactaatc
aggctggatc tatagctaat gcaggaagtg tacctactta tgtttggacc
1500cgtcgtgatg tggaccttaa taatacgatt accccaaata gaattacaca
attaccattg 1560gtaaaggcat ctgcacctgt ttcgggtact acggtcttaa
aaggtccagg atttacagga 1620gggggtatac tccgaagaac aactaatggc
acatttggaa cgttaagagt aacagttaat 1680tcaccattaa cacaaagata
tcgcgtaaga gttcgttttg cttcatcagg aaatttcagc 1740ataaggatac
tgcgtggaaa tacctctata gcttatcaaa gatttgggag tacaatgaac
1800agaggacagg aactaactta cgaatcattt gtcacaagtg agttcactac
taatcagagc 1860gatctgcctt ttacatttac acaagctcaa gaaaatttaa
caatccttgc agaaggtgtt 1920agcaccggta gtgaatattt tatagataga
attgaaatca tccctgtgaa cccggcacga 1980gaagcagaag aggatttaga
agcagcgaag aaagcggtgg cgaacttgtt tacacgtaca 2040agggacggat
tacaggtaaa tgtgacagat tatcaagtgg accaagcggc aaatttagtg
2100tcatgcttat ccgatgaaca atatgggcat gacaaaaaga tgttattgga
agcggtaaga 2160gcggcaaaac gcctcagccg cgaacgcaac ttacttcaag
atccagattt taatacaatc 2220aatagtacag aagagaatgg ctggaaggca
agtaacggtg ttactattag cgagggcggt 2280ccattcttta aaggtcgtgc
acttcagtta gcaagcgcaa gagaaaatta tccaacatac 2340atttatcaaa
aagtagatgc atcggtgtta aagccttata cacgctatag actagatgga
2400tttgtgaaga gtagtcaaga tttagaaatt gatctcatcc accatcataa
agtccatctt 2460gtaaaaaatg taccagataa tttagtatct gatacttact
cagatggttc ttgcagcgga 2520atcaaccgtt gtgatgaaca gcatcaggta
gatatgcagc tagatgcgga gcatcatcca 2580atggattgct gtgaagcggc
tcaaacacat gagttttctt cctatattaa tacaggggat 2640ctaaatgcaa
gtgtagatca gggcatttgg gttgtattaa aagttcgaac aacagatggg
2700tatgcgacgt taggaaatct tgaattggta gaggttgggc cattatcggg
tgaatctcta 2760gaacgggaac aaagagataa tgcgaaatgg aatgcagagc
taggaagaaa acgtgcagaa 2820atagatcgtg tgtatttagc tgcgaaacaa
gcaattaatc atctgtttgt agactatcaa 2880gatcaacaat taaatccaga
aattgggcta gcagaaatta atgaagcttc aaatcttgta 2940gagtcaattt
cgggtgtata tagtgataca ctattacaga ttcctgggat taactacgaa
3000atttacacag agttatccga tcgcttacaa caagcatcgt atctgtatac
gtctagaaat 3060gcggtgcaaa atggagactt taacagtggt ctagatagtt
ggaatacaac tatggatgca 3120tcggttcagc aagatggcaa tatgcatttc
ttagttcttt cgcattggga tgcacaagtt 3180tcccaacaat tgagagtaaa
tccgaattgt aagtatgtct tacgtgtgac agcaagaaaa 3240gtaggaggcg
gagatggata cgtcacaatc cgagatggcg ctcatcacca agaaactctt
3300acatttaatg catgtgacta cgatgtaaat ggtacgtatg tcaatgacaa
ttcgtatata 3360acagaagaag tggtattcta cccagagaca aaacatatgt
gggtagaggt gagtgaatcc 3420gaaggttcat tctatataga cagtattgag
tttattgaaa cacaagagta g 3471281156PRTBacillus thuringiensis 28Met
Asn Arg Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Pro His1 5 10
15Cys Gly Cys Pro Ser Asp Asp Asp Val Arg Tyr Pro Leu Ala Ser Asp
20 25 30Pro Asn Ala Ala Leu Gln Asn Met Asn Tyr Lys Asp Tyr Leu Gln
Met 35 40 45Thr Asp Glu Asp Tyr Thr Asp Ser Tyr Ile Asn Pro Ser Leu
Ser Ile 50 55 60Ser Gly Arg Asp Ala Val Gln Thr Ala Leu Thr Val Val
Gly Arg Ile65 70 75 80Leu Gly Ala Leu Gly Val Pro Phe Ser Gly Gln
Ile Val Ser Phe Tyr 85 90 95Gln Phe Leu Leu Asn Thr Leu Trp Pro Val
Asn Asp Thr Ala Ile Trp 100 105 110Glu Ala Phe Met Arg Gln Val Glu
Glu Leu Val Asn Gln Gln Ile Thr 115 120 125Glu Phe Ala Arg Asn Gln
Ala Leu Ala Arg Leu Gln Gly Leu Gly Asp 130 135 140Ser Phe Asn Val
Tyr Gln Arg Ser Leu Gln Asn Trp Leu Ala Asp Arg145 150 155 160Asn
Asp Thr Arg Asn Leu Ser Val Val Arg Ala Gln Phe Ile Ala Leu 165 170
175Asp Leu Asp Phe Val Asn Ala Ile Pro Leu Phe Ala Val Asn Gly Gln
180 185 190Gln Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Val Asn Leu
His Leu 195 200 205Leu Leu Leu Lys Asp Ala Ser Leu Phe Gly Glu Gly
Trp Gly Phe Thr 210 215 220Gln Gly Glu Ile Ser Thr Tyr Tyr Asp Arg
Gln Leu Glu Leu Thr Ala225 230 235 240Lys Tyr Thr Asn Tyr Cys Glu
Thr Trp Tyr Asn Thr Gly Leu Asp Arg 245 250 255Leu Arg Gly Thr Asn
Thr Glu Ser Trp Leu Arg Tyr His Gln Phe Arg 260 265 270Arg Glu Met
Thr Leu Val Val Leu Asp Val Val Ala Leu Phe Pro Tyr 275 280 285Tyr
Asp Val Arg Leu Tyr Pro Thr Gly Ser Asn Pro Gln Leu Thr Arg 290 295
300Glu Val Tyr Thr Asp Pro Ile Val Phe Asn Pro Pro Ala Asn Val
Gly305 310 315 320Leu Cys Arg Arg Trp Gly Thr Asn Pro Tyr Asn Thr
Phe Ser Glu Leu 325 330 335Glu Asn Ala Phe Ile Arg Pro Pro His Leu
Phe Asp Arg Leu Asn Ser 340 345 350Leu Thr Ile Ser Ser Asn Arg Phe
Pro Val Ser Ser Asn Phe Met Asp 355 360 365Tyr Trp Ser Gly His Thr
Leu Arg Arg Ser Tyr Leu Asn Asp Ser Ala 370 375 380Val Gln Glu Asp
Ser Tyr Gly Leu Ile Thr Thr Thr Arg Ala Thr Ile385 390 395 400Asn
Pro Gly Val Asp Gly Thr Asn Arg Ile Glu Ser Thr Ala Val Asp 405 410
415Phe Arg Ser Ala Leu Ile Gly Ile Tyr Gly Val Asn Arg Ala Ser Phe
420 425 430Val Pro Gly Gly Leu Phe Asn Gly Thr Thr Ser Pro Ala Asn
Gly Gly 435 440 445Cys Arg Asp Leu Tyr Asp Thr Asn Asp Glu Leu Pro
Pro Asp Glu Ser 450 455 460Thr Gly Ser Ser Thr His Arg Leu Ser His
Val Thr Phe Phe Ser Phe465 470 475 480Gln Thr Asn Gln Ala Gly Ser
Ile Ala Asn Ala Gly Ser Val Pro Thr 485 490 495Tyr Val Trp Thr Arg
Arg Asp Val Asp Leu Asn Asn Thr Ile Thr Pro 500 505 510Asn Arg Ile
Thr Gln Leu Pro Leu Val Lys Ala Ser Ala Pro Val Ser 515 520 525Gly
Thr Thr Val Leu Lys Gly Pro Gly Phe Thr Gly Gly Gly Ile Leu 530 535
540Arg Arg Thr Thr Asn Gly Thr Phe Gly Thr Leu Arg Val Thr Val
Asn545 550 555 560Ser Pro Leu Thr Gln Arg Tyr Arg Val Arg Val Arg
Phe Ala Ser Ser 565 570 575Gly Asn Phe Ser Ile Arg Ile Leu Arg Gly
Asn Thr Ser Ile Ala Tyr 580 585 590Gln Arg Phe Gly Ser Thr Met Asn
Arg Gly Gln Glu Leu Thr Tyr Glu 595 600 605Ser Phe Val Thr Ser Glu
Phe Thr Thr Asn Gln Ser Asp Leu Pro Phe 610 615 620Thr Phe Thr Gln
Ala Gln Glu Asn Leu Thr Ile Leu Ala Glu Gly Val625 630 635 640Ser
Thr Gly Ser Glu Tyr Phe Ile Asp Arg Ile Glu Ile Ile Pro Val 645 650
655Asn Pro Ala Arg Glu Ala Glu Glu Asp Leu Glu Ala Ala Lys Lys Ala
660 665 670Val Ala Asn Leu Phe Thr Arg Thr Arg Asp Gly Leu Gln Val
Asn Val 675 680 685Thr Asp Tyr Gln Val Asp Gln Ala Ala Asn Leu Val
Ser Cys Leu Ser 690 695 700Asp Glu Gln Tyr Gly His Asp Lys Lys Met
Leu Leu Glu Ala Val Arg705 710 715 720Ala Ala Lys Arg Leu Ser Arg
Glu Arg Asn Leu Leu Gln Asp Pro Asp 725 730 735Phe Asn Thr Ile Asn
Ser Thr Glu Glu Asn Gly Trp Lys Ala Ser Asn 740 745 750Gly Val Thr
Ile Ser Glu Gly Gly Pro Phe Phe Lys Gly Arg Ala Leu 755 760 765Gln
Leu Ala Ser Ala Arg Glu Asn Tyr Pro Thr Tyr Ile Tyr Gln Lys 770 775
780Val Asp Ala Ser Val Leu Lys Pro Tyr Thr Arg Tyr Arg Leu Asp
Gly785 790 795 800Phe Val Lys Ser Ser Gln Asp Leu Glu Ile Asp Leu
Ile His His His 805 810 815Lys Val His Leu Val Lys Asn Val Pro Asp
Asn Leu Val Ser Asp Thr 820 825 830Tyr Ser Asp Gly Ser Cys Ser Gly
Ile Asn Arg Cys Asp Glu Gln His 835 840 845Gln Val Asp Met Gln Leu
Asp Ala Glu His His Pro Met Asp Cys Cys 850 855 860Glu Ala Ala Gln
Thr His Glu Phe Ser Ser Tyr Ile Asn Thr Gly Asp865 870 875 880Leu
Asn Ala Ser Val Asp Gln Gly Ile Trp Val Val Leu Lys Val Arg 885 890
895Thr Thr Asp Gly Tyr Ala Thr Leu Gly Asn Leu Glu Leu Val Glu Val
900 905 910Gly Pro Leu Ser Gly Glu Ser Leu Glu Arg Glu Gln Arg Asp
Asn Ala 915 920 925Lys Trp Asn Ala Glu Leu Gly Arg Lys Arg Ala Glu
Ile Asp Arg Val 930 935 940Tyr Leu Ala Ala Lys Gln Ala Ile Asn His
Leu Phe Val Asp Tyr Gln945 950 955 960Asp Gln Gln Leu Asn Pro Glu
Ile Gly Leu Ala Glu Ile Asn Glu Ala 965 970 975Ser Asn Leu Val Glu
Ser Ile Ser Gly Val Tyr Ser Asp Thr Leu Leu 980 985 990Gln Ile Pro
Gly Ile Asn Tyr Glu Ile Tyr Thr Glu Leu Ser Asp Arg 995 1000
1005Leu Gln Gln Ala Ser Tyr Leu Tyr Thr Ser Arg Asn Ala Val Gln
1010 1015 1020Asn Gly Asp Phe Asn Ser Gly Leu Asp Ser Trp Asn Thr
Thr Met 1025 1030 1035Asp Ala Ser Val Gln Gln Asp Gly Asn Met His
Phe Leu Val Leu 1040 1045 1050Ser His Trp Asp Ala Gln Val Ser Gln
Gln Leu Arg Val Asn Pro 1055 1060 1065Asn Cys Lys Tyr Val Leu Arg
Val Thr Ala Arg Lys Val Gly Gly 1070 1075 1080Gly Asp Gly Tyr Val
Thr Ile Arg Asp Gly Ala His His Gln Glu 1085 1090 1095Thr Leu Thr
Phe Asn Ala Cys Asp Tyr Asp Val Asn Gly Thr Tyr 1100 1105 1110Val
Asn Asp Asn Ser Tyr Ile Thr Glu Glu Val Val Phe Tyr Pro 1115 1120
1125Glu Thr Lys His Met Trp Val Glu Val Ser Glu Ser Glu Gly Ser
1130 1135 1140Phe Tyr Ile Asp Ser Ile Glu Phe Ile Glu Thr Gln Glu
1145 1150 1155292407DNABacillus thuringiensis 29atgaatcaaa
ataaacacgg aattattggc gcttccaatt gtggttgtgc atctgatgat 60gttgcgaaat
atcctttagc caacaatcca tattcatctg ctttaaattt aaattcttgt
120caaaatagta gtattctcaa ctggattaac ataataggcg atgcagcaaa
agaagcagta 180tctattggga caaccatagt ctctcttatc acagcacctt
ctcttactgg attaatttca 240atagtatatg accttatagg taaagtacta
ggaggtagta gtggacaatc catatcagat 300ttgtctatat gtgacttatt
atctattatt gatttacggg taagtcagag tgttttaaat 360gatgggattg
cagattttaa tggttctgta ctcttataca ggaactattt agaggctctg
420gatagctgga ataagaatcc taattctgct tctgctgaag aactccgtac
tcgttttaga 480atcgccgact cagaatttga tagaatttta acccgagggt
ctttaacgaa tggtggctcg 540ttagctagac aaaatgccca aatattatta
ttaccttctt ttgcgagcgc tgcatttttc 600catttattac tactaaggga
tgctactaga tatggcacta attgggggct atacaatgct 660acacctttta
taaattatca atcaaaacta gtagagctta ttgaactata tactgattat
720tgcgtacatt gggataatcg aggttcaacc gaactaagac aacgagggcc
tagtgctaca 780gcttggttag aatttcatag atatcggaga gagatgacat
tgatgggatt agaaatagta 840gcatcatttt caagtcttga tattactaat
tacccaatag aaacagattt tcagttgagt 900agggtcattt atacagatcc
aattggtttt gtacatcgta gtagtcttag gggagaaagt 960tggtttagct
ttgttaatag agctaatttc tcagatttag aaaatgcaat acctaatcct
1020agaccgtctt ggtttttaaa taatatgatt atatctactg gttcacttac
attgccggtt 1080agcccaagta ctgatagagc gagggtatgg tatggaagtc
gagatcgaat ttcccctgct 1140aattcacaat ttattactga actaatctct
ggacaacata cgactgctac acaaactatt 1200ttagggcgaa atatatttag
agtagattct caagcttgta atttaaatga taccacatat 1260ggagtgaata
gggcggtatt ttatcatgat gcgagtgaag gttctcaaag atccgtgtac
1320gaggggtata ttcgaacaac tgggatagat aaccctagag ttcaaaatat
taacacttat 1380ttacctggag aaaattcaga tatcccaact ccagaagact
atactcatat attaagcaca 1440acaataaatt taacaggagg acttagacaa
gtagcatcta atcgccgttc atctttagta 1500atgtatggtt ggacacataa
aagtctggct cgtaacaata ccattaatcc agatagaatt 1560acacagatac
cattgacgaa ggttgatacc cgaggcacag gtgtttctta tgtgaatgat
1620ccaggattta taggaggagc tctacttcaa aggactgacc atggttcgct
tggagtattg 1680agggtccaat ttccacttca cttaagacaa caatatcgta
ttagagtccg ttatgcttct 1740acaacaaata ttcgattgag tgtgaatggc
agtttcggta ctatttctca aaatctccct 1800agtacaatga gattaggaga
ggatttaaga tacggatctt ttgctataag agagtttaat 1860acttctatta
gacccactgc aagtccggac caaattcgat tgacaataga accatctttt
1920attagacaag aggtctatgt agatagaatt gagttcattc cagttaatcc
gacgcgagag 1980gcgaaagagg atctagaagc agcaaaaaaa gcggtggcga
gcttgtttac acgcacaagg 2040gacggattac aagtaaatgt gaaagattat
caagtcgatc aagcggcaaa tttagtgtca 2100tgcttatcag atgaacaata
tgggtatgac aaaaagatgt tattggaagc ggtacgtgcg 2160gcaaaacgac
ttagccgaga acgcaactta cttcaggatc cagattttaa tacaatcaat
2220agtacagaag aaaatggatg gaaagcaagt aacggcgtta ctattagtga
gggcgggcca 2280ttctataaag gccgtgcaat tcagctagca agtgcacgag
aaaattaccc aacatacatc 2340tatcaaaaag tagatgcatc ggagttaaag
ccgtatacac gttatagact ggatgggttc 2400gtgaaga 240730802PRTBacillus
thuringiensis 30Met Asn Gln Asn Lys His Gly Ile Ile Gly Ala Ser Asn
Cys Gly Cys1 5 10 15Ala Ser Asp Asp Val Ala Lys Tyr Pro Leu Ala Asn
Asn Pro Tyr Ser 20 25 30Ser Ala Leu Asn Leu Asn Ser Cys Gln Asn Ser
Ser Ile Leu Asn Trp 35 40 45Ile Asn Ile Ile Gly Asp Ala Ala Lys Glu
Ala Val Ser Ile Gly Thr 50 55 60Thr Ile Val Ser Leu Ile Thr Ala Pro
Ser Leu Thr Gly Leu Ile Ser65 70 75 80Ile Val Tyr Asp Leu Ile Gly
Lys Val Leu Gly Gly Ser Ser Gly Gln 85 90 95Ser Ile Ser Asp Leu Ser
Ile Cys Asp Leu Leu Ser Ile Ile Asp Leu 100 105 110Arg Val Ser Gln
Ser Val Leu Asn Asp Gly Ile Ala Asp Phe Asn Gly 115 120 125Ser Val
Leu Leu Tyr Arg Asn Tyr Leu Glu Ala Leu Asp Ser Trp Asn 130 135
140Lys Asn Pro Asn Ser Ala Ser Ala Glu Glu Leu Arg Thr Arg Phe
Arg145 150 155 160Ile Ala Asp Ser Glu Phe Asp Arg Ile Leu Thr Arg
Gly Ser Leu Thr 165 170 175Asn Gly Gly Ser Leu Ala Arg Gln Asn Ala
Gln Ile Leu Leu Leu Pro 180 185 190Ser Phe Ala Ser Ala Ala Phe Phe
His Leu Leu Leu Leu Arg Asp Ala 195 200 205Thr Arg Tyr Gly Thr Asn
Trp Gly Leu Tyr Asn Ala Thr Pro Phe Ile 210 215 220Asn Tyr Gln Ser
Lys Leu Val Glu Leu Ile Glu Leu Tyr Thr Asp Tyr225 230 235 240Cys
Val His Trp Asp Asn Arg Gly Ser Thr Glu Leu Arg Gln Arg Gly 245 250
255Pro Ser Ala Thr Ala Trp Leu Glu Phe His Arg Tyr Arg Arg Glu Met
260 265 270Thr Leu Met Gly Leu Glu Ile Val Ala Ser
Phe Ser Ser Leu Asp Ile 275 280 285Thr Asn Tyr Pro Ile Glu Thr Asp
Phe Gln Leu Ser Arg Val Ile Tyr 290 295 300Thr Asp Pro Ile Gly Phe
Val His Arg Ser Ser Leu Arg Gly Glu Ser305 310 315 320Trp Phe Ser
Phe Val Asn Arg Ala Asn Phe Ser Asp Leu Glu Asn Ala 325 330 335Ile
Pro Asn Pro Arg Pro Ser Trp Phe Leu Asn Asn Met Ile Ile Ser 340 345
350Thr Gly Ser Leu Thr Leu Pro Val Ser Pro Ser Thr Asp Arg Ala Arg
355 360 365Val Trp Tyr Gly Ser Arg Asp Arg Ile Ser Pro Ala Asn Ser
Gln Phe 370 375 380Ile Thr Glu Leu Ile Ser Gly Gln His Thr Thr Ala
Thr Gln Thr Ile385 390 395 400Leu Gly Arg Asn Ile Phe Arg Val Asp
Ser Gln Ala Cys Asn Leu Asn 405 410 415Asp Thr Thr Tyr Gly Val Asn
Arg Ala Val Phe Tyr His Asp Ala Ser 420 425 430Glu Gly Ser Gln Arg
Ser Val Tyr Glu Gly Tyr Ile Arg Thr Thr Gly 435 440 445Ile Asp Asn
Pro Arg Val Gln Asn Ile Asn Thr Tyr Leu Pro Gly Glu 450 455 460Asn
Ser Asp Ile Pro Thr Pro Glu Asp Tyr Thr His Ile Leu Ser Thr465 470
475 480Thr Ile Asn Leu Thr Gly Gly Leu Arg Gln Val Ala Ser Asn Arg
Arg 485 490 495Ser Ser Leu Val Met Tyr Gly Trp Thr His Lys Ser Leu
Ala Arg Asn 500 505 510Asn Thr Ile Asn Pro Asp Arg Ile Thr Gln Ile
Pro Leu Thr Lys Val 515 520 525Asp Thr Arg Gly Thr Gly Val Ser Tyr
Val Asn Asp Pro Gly Phe Ile 530 535 540Gly Gly Ala Leu Leu Gln Arg
Thr Asp His Gly Ser Leu Gly Val Leu545 550 555 560Arg Val Gln Phe
Pro Leu His Leu Arg Gln Gln Tyr Arg Ile Arg Val 565 570 575Arg Tyr
Ala Ser Thr Thr Asn Ile Arg Leu Ser Val Asn Gly Ser Phe 580 585
590Gly Thr Ile Ser Gln Asn Leu Pro Ser Thr Met Arg Leu Gly Glu Asp
595 600 605Leu Arg Tyr Gly Ser Phe Ala Ile Arg Glu Phe Asn Thr Ser
Ile Arg 610 615 620Pro Thr Ala Ser Pro Asp Gln Ile Arg Leu Thr Ile
Glu Pro Ser Phe625 630 635 640Ile Arg Gln Glu Val Tyr Val Asp Arg
Ile Glu Phe Ile Pro Val Asn 645 650 655Pro Thr Arg Glu Ala Lys Glu
Asp Leu Glu Ala Ala Lys Lys Ala Val 660 665 670Ala Ser Leu Phe Thr
Arg Thr Arg Asp Gly Leu Gln Val Asn Val Lys 675 680 685Asp Tyr Gln
Val Asp Gln Ala Ala Asn Leu Val Ser Cys Leu Ser Asp 690 695 700Glu
Gln Tyr Gly Tyr Asp Lys Lys Met Leu Leu Glu Ala Val Arg Ala705 710
715 720Ala Lys Arg Leu Ser Arg Glu Arg Asn Leu Leu Gln Asp Pro Asp
Phe 725 730 735Asn Thr Ile Asn Ser Thr Glu Glu Asn Gly Trp Lys Ala
Ser Asn Gly 740 745 750Val Thr Ile Ser Glu Gly Gly Pro Phe Tyr Lys
Gly Arg Ala Ile Gln 755 760 765Leu Ala Ser Ala Arg Glu Asn Tyr Pro
Thr Tyr Ile Tyr Gln Lys Val 770 775 780Asp Ala Ser Glu Leu Lys Pro
Tyr Thr Arg Tyr Arg Leu Asp Gly Phe785 790 795 800Val
Lys31192DNABacillus thuringiensis 31catttacgca acctcgtatg
gatttcattt ccaagaacta tgggaacaga tgacccatta 60acttctcgtt cgtttgctct
tacaactctt ttcacaccaa taaccttaac acgagcacaa 120gaagaattta
atctaacaat accacggggt gtttatatag acagaattga attcgtccca
180gttatgccac at 1923264PRTBacillus thuringiensis 32His Leu Arg Asn
Leu Val Trp Ile Ser Phe Pro Arg Thr Met Gly Thr1 5 10 15Asp Asp Pro
Leu Thr Ser Arg Ser Phe Ala Leu Thr Thr Leu Phe Thr 20 25 30Pro Ile
Thr Leu Thr Arg Ala Gln Glu Glu Phe Asn Leu Thr Ile Pro 35 40 45Arg
Gly Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Val Met Pro His 50 55
6033246DNABacillus thuringiensis 33gcttctacta caaatttaca attccataca
tcaattgacg gaagacctat taatcagggg 60aatttttcag caactatgag tagtgggggt
aatttacagt ccggaagctt taggactgca 120ggctttacta ctccgtttaa
cttttcaaat ggatcaagta tatttacgtt aagtgctcat 180gtcttcaatt
caggcaatga agtttatata gatcgaattg aatttgttcc ggcagaagta 240acattt
2463482PRTBacillus thuringiensis 34Ala Ser Thr Thr Asn Leu Gln Phe
His Thr Ser Ile Asp Gly Arg Pro1 5 10 15Ile Asn Gln Gly Asn Phe Ser
Ala Thr Met Ser Ser Gly Gly Asn Leu 20 25 30Gln Ser Gly Ser Phe Arg
Thr Ala Gly Phe Thr Thr Pro Phe Asn Phe 35 40 45Ser Asn Gly Ser Ser
Ile Phe Thr Leu Ser Ala His Val Phe Asn Ser 50 55 60Gly Asn Glu Val
Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu Val65 70 75 80Thr
Phe35177DNABacillus thuringiensis 35ctctttccag attatattca
gcctcgagtg ttgcagtaac tggaataaat tcaaatctgt 60ctattatcac tcctgcagtc
ccactaaaat ttctaacacc tactatatta cctaatgaag 120atgtaaaagc
attggcactt caaaatcact tgattgtaga ttatctaatg acgtagc
1773657PRTBacillus thuringiensis 36Leu Ser Arg Leu Tyr Ser Ala Ser
Ser Val Ala Val Thr Gly Ile Asn1 5 10 15Ser Asn Leu Ser Ile Ile Thr
Pro Ala Val Pro Leu Lys Phe Leu Thr 20 25 30Pro Thr Ile Leu Pro Asn
Glu Asp Val Lys Ala Leu Ala Leu Gln Asn 35 40 45His Leu Ile Val Asp
Tyr Leu Met Thr 50 55374173DNABacillus thuringiensisCDS(1)..(3687)
37ttg act tca aat agg aaa aat gag aat gaa att ata aat gct tta tcg
48Leu Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser1
5 10 15att cca gct gta tcg aat cat tcc aca caa atg gat cta tca cca
gat 96Ile Pro Ala Val Ser Asn His Ser Thr Gln Met Asp Leu Ser Pro
Asp 20 25 30gct cgt att gag gat tct ttg tgt ata gcc gag ggg aat aat
atc aat 144Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn
Ile Asn 35 40 45cca ctt gtt agc gca tca aca gtc caa acg ggt att aac
ata gct ggt 192Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn
Ile Ala Gly 50 55 60aga ata cta ggt gta tta ggc gta ccg ttt gct gga
caa ata gct agt 240Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly
Gln Ile Ala Ser65 70 75 80ttt tat agt ttt ctt gtt ggt gaa tta tgg
ccc cgc ggc aga gat cag 288Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp
Pro Arg Gly Arg Asp Gln 85 90 95tgg gaa att ttc cta gaa cat gtc gaa
caa ctt ata aat caa caa ata 336Trp Glu Ile Phe Leu Glu His Val Glu
Gln Leu Ile Asn Gln Gln Ile 100 105 110aca gaa aat gct agg aat acg
gca ctt gct cga tta caa ggt tta gga 384Thr Glu Asn Ala Arg Asn Thr
Ala Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125gat tcc ttt aga gcc
tat caa cag tca ctt gaa gat tgg cta gaa aac 432Asp Ser Phe Arg Ala
Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140cgt gat gat
gca aga acg aga agt gtt ctt tat acc caa tat ata gcc 480Arg Asp Asp
Ala Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala145 150 155
160tta gaa ctt gat ttt ctt aat gcg atg ccg ctt ttc gca att aga aac
528Leu Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn
165 170 175caa gaa gtt cca tta tta atg gta tat gct caa gct gca aat
tta cac 576Gln Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn
Leu His 180 185 190cta tta tta ttg aga gat gcc tct ctt ttt ggt agt
gaa ttt ggg ctt 624Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser
Glu Phe Gly Leu 195 200 205aca tcg cag gaa att caa cgt tat tat gag
cgc caa gtg gaa caa acg 672Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu
Arg Gln Val Glu Gln Thr 210 215 220aga gat tat tcc gac tat tgc gta
gaa tgg tat aat aca ggt cta aat 720Arg Asp Tyr Ser Asp Tyr Cys Val
Glu Trp Tyr Asn Thr Gly Leu Asn225 230 235 240agc ttg aga ggg aca
aat gcc gca agt tgg gtg cgt tat aat caa ttc 768Ser Leu Arg Gly Thr
Asn Ala Ala Ser Trp Val Arg Tyr Asn Gln Phe 245 250 255cgt aga gat
cta acg tta ggg gta tta gat cta gtg gca cta ttc cca 816Arg Arg Asp
Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270agc
tat gac act cgc act tat cca ata aat acg agt gct cag tta aca 864Ser
Tyr Asp Thr Arg Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280
285agg gaa gtt tat aca gac gca att gga gca aca ggg gta aat atg gca
912Arg Glu Val Tyr Thr Asp Ala Ile Gly Ala Thr Gly Val Asn Met Ala
290 295 300agt atg aat tgg tat aat aat aat gca cct tcg ttt tcc gct
ata gag 960Ser Met Asn Trp Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala
Ile Glu305 310 315 320act gcg gtt atc cga agc ccg cat cta ctt gat
ttt cta gaa caa ctt 1008Thr Ala Val Ile Arg Ser Pro His Leu Leu Asp
Phe Leu Glu Gln Leu 325 330 335aca att ttt agc act tca tca cga tgg
agt gct act agg cat atg act 1056Thr Ile Phe Ser Thr Ser Ser Arg Trp
Ser Ala Thr Arg His Met Thr 340 345 350tac tgg cgg ggg cac aca att
caa tct cgg cca ata gga ggc gga tta 1104Tyr Trp Arg Gly His Thr Ile
Gln Ser Arg Pro Ile Gly Gly Gly Leu 355 360 365aat acc tca acg cat
ggg tct acc aat act tct att aat cct gta aga 1152Asn Thr Ser Thr His
Gly Ser Thr Asn Thr Ser Ile Asn Pro Val Arg 370 375 380tta tca ttc
ttc tct cga gac gta tat tgg act gaa tca tat gca gga 1200Leu Ser Phe
Phe Ser Arg Asp Val Tyr Trp Thr Glu Ser Tyr Ala Gly385 390 395
400gtg ctt cta tgg gga att tac ctt gaa cct att cat ggt gtc cct act
1248Val Leu Leu Trp Gly Ile Tyr Leu Glu Pro Ile His Gly Val Pro Thr
405 410 415gtt aga ttt aat ttt agg aac cct cag aat act ttt gaa aga
ggt act 1296Val Arg Phe Asn Phe Arg Asn Pro Gln Asn Thr Phe Glu Arg
Gly Thr 420 425 430gct aac tat agt caa ccc tat gag tca cct ggg ctt
caa tta aaa gat 1344Ala Asn Tyr Ser Gln Pro Tyr Glu Ser Pro Gly Leu
Gln Leu Lys Asp 435 440 445tca gaa act gaa tta cca cca gaa aca aca
gaa cga cca aat tat gaa 1392Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr
Glu Arg Pro Asn Tyr Glu 450 455 460tca tat agt cat agg tta tct cac
ata ggg ctc att tca caa tct agg 1440Ser Tyr Ser His Arg Leu Ser His
Ile Gly Leu Ile Ser Gln Ser Arg465 470 475 480gtg cat gta cca gta
tat tct tgg acg cac cgt agt gca gat cgt aca 1488Val His Val Pro Val
Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr 485 490 495aat acc att
agt tca gat agc ata aca caa ata cca ttg gta aaa tca 1536Asn Thr Ile
Ser Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ser 500 505 510ttc
aac ctt aat tca ggt acc tct gta gtc agt ggc cca gga ttt aca 1584Phe
Asn Leu Asn Ser Gly Thr Ser Val Val Ser Gly Pro Gly Phe Thr 515 520
525gga ggg gat ata atc cga act aac gtt aat ggt agt gta cta agt atg
1632Gly Gly Asp Ile Ile Arg Thr Asn Val Asn Gly Ser Val Leu Ser Met
530 535 540ggt ctt aat ttt aat aat aca tca tta cag cgg tat cgc gtg
aga gtt 1680Gly Leu Asn Phe Asn Asn Thr Ser Leu Gln Arg Tyr Arg Val
Arg Val545 550 555 560cgt tat gct gct tct caa aca atg gtc ctg agg
gta act gtc gga ggg 1728Arg Tyr Ala Ala Ser Gln Thr Met Val Leu Arg
Val Thr Val Gly Gly 565 570 575agt act act ttt gat caa gga ttc cct
agt act atg agt gca aat gag 1776Ser Thr Thr Phe Asp Gln Gly Phe Pro
Ser Thr Met Ser Ala Asn Glu 580 585 590tct ttg aca tct caa tca ttt
aga ttt gca gaa ttt cct gta ggt att 1824Ser Leu Thr Ser Gln Ser Phe
Arg Phe Ala Glu Phe Pro Val Gly Ile 595 600 605agt gca tct ggc agt
caa act gct gga ata agt ata agt aat aat gca 1872Ser Ala Ser Gly Ser
Gln Thr Ala Gly Ile Ser Ile Ser Asn Asn Ala 610 615 620ggt aga caa
acg ttt cac ttt gat aaa att gaa ttc att cca att act 1920Gly Arg Gln
Thr Phe His Phe Asp Lys Ile Glu Phe Ile Pro Ile Thr625 630 635
640gca acc ttc gaa gca gaa tac gat tta gaa agg gcg caa gag gcg gtg
1968Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu Ala Val
645 650 655aat gct ctg ttt act aat acg aat cca aga aga ttg aaa aca
gat gtg 2016Asn Ala Leu Phe Thr Asn Thr Asn Pro Arg Arg Leu Lys Thr
Asp Val 660 665 670aca gat tat cat att gat caa gta tcc aat tta gtg
gcg tgt tta tcg 2064Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val
Ala Cys Leu Ser 675 680 685gat gaa ttc tgc tta gat gaa aag aga gaa
tta ctt gag aaa gtg aaa 2112Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu
Leu Leu Glu Lys Val Lys 690 695 700tat gcg aaa cga ctc agt gat gaa
aga aac tta ctc caa gat cca aac 2160Tyr Ala Lys Arg Leu Ser Asp Glu
Arg Asn Leu Leu Gln Asp Pro Asn705 710 715 720ttc aca tcc atc aat
aag caa cca gac ttc ata tct act aat gag caa 2208Phe Thr Ser Ile Asn
Lys Gln Pro Asp Phe Ile Ser Thr Asn Glu Gln 725 730 735tcg aat ttc
aca tct atc cat gaa caa tct gaa cat gga tgg tgg gga 2256Ser Asn Phe
Thr Ser Ile His Glu Gln Ser Glu His Gly Trp Trp Gly 740 745 750agt
gag aac att aca atc cag gaa gga aat gac gta ttt aaa gag aat 2304Ser
Glu Asn Ile Thr Ile Gln Glu Gly Asn Asp Val Phe Lys Glu Asn 755 760
765tac gtc aca cta ccg ggg act ttt aat gag tgt tat ccg acg tat tta
2352Tyr Val Thr Leu Pro Gly Thr Phe Asn Glu Cys Tyr Pro Thr Tyr Leu
770 775 780tat caa aaa ata gga gag tcg gaa tta aaa gct tat act cgc
tac caa 2400Tyr Gln Lys Ile Gly Glu Ser Glu Leu Lys Ala Tyr Thr Arg
Tyr Gln785 790 795 800tta aga ggg tat att gaa gat agt caa gat tta
gag ata tat ttg att 2448Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu
Glu Ile Tyr Leu Ile 805 810 815cgt tat aat gcg aaa cat gaa aca ttg
gat gtt cca ggt acc gag tcc 2496Arg Tyr Asn Ala Lys His Glu Thr Leu
Asp Val Pro Gly Thr Glu Ser 820 825 830gta tgg ccg ctt tca gtt gaa
agc cca atc gga agg tgc gga gaa ccg 2544Val Trp Pro Leu Ser Val Glu
Ser Pro Ile Gly Arg Cys Gly Glu Pro 835 840 845aat cga tgc gca cca
cat ttt gaa tgg aat cct gat cta gat tgt tcc 2592Asn Arg Cys Ala Pro
His Phe Glu Trp Asn Pro Asp Leu Asp Cys Ser 850 855 860tgc aga gat
gga gaa aaa tgt gcg cat cat tcc cat cat ttc tct ttg 2640Cys Arg Asp
Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu865 870 875
880gat att gat att gga tgc aca gac ttg cat gag aat cta ggc gtg tgg
2688Asp Ile Asp Ile Gly Cys Thr Asp Leu His Glu Asn Leu Gly Val Trp
885 890 895gtg gta ttc aag att aag acg cag gaa ggt cat gca aga cta
ggg aat 2736Val Val Phe Lys Ile Lys Thr Gln Glu Gly His Ala Arg Leu
Gly Asn 900 905 910ctg gaa ttt att gaa gag aaa cca tta tta gga gaa
gca ctg tct cgt 2784Leu Glu Phe Ile Glu Glu Lys Pro Leu Leu Gly Glu
Ala Leu Ser Arg 915 920 925gtg aag aga gca gag aaa aaa tgg aga gac
aaa cgt gaa aaa cta caa 2832Val Lys Arg Ala Glu Lys Lys Trp Arg Asp
Lys Arg Glu Lys Leu Gln 930 935 940ttg gaa aca aaa cga gta tat aca
gag gca aaa gaa gct gtg gat gct 2880Leu Glu Thr Lys Arg Val Tyr Thr
Glu Ala Lys Glu Ala Val Asp Ala945 950 955 960tta ttt gta gat tct
caa tat aat aga tta caa gcg gat aca aac att 2928Leu Phe Val Asp Ser
Gln Tyr Asn Arg Leu Gln Ala Asp Thr Asn Ile 965 970 975ggc atg att
cat gcg gca gat aaa ctt gtt cat cga att cga gag gct 2976Gly Met Ile
His Ala Ala Asp Lys Leu Val His Arg Ile Arg Glu Ala 980 985 990tat
ctg tca gaa tta tct gtt atc ccg ggt gta aat
gcg gaa att ttt 3024Tyr Leu Ser Glu Leu Ser Val Ile Pro Gly Val Asn
Ala Glu Ile Phe 995 1000 1005gaa gaa tta gaa ggt cgc att atc act
gca atc tcc cta tac gat 3069Glu Glu Leu Glu Gly Arg Ile Ile Thr Ala
Ile Ser Leu Tyr Asp 1010 1015 1020gcg aga aat gtc gtt aaa aat ggt
gat ttt aat aat gga tta gca 3114Ala Arg Asn Val Val Lys Asn Gly Asp
Phe Asn Asn Gly Leu Ala 1025 1030 1035tgc tgg aat gta aaa ggg cat
gta gat gta caa cag agc cat cac 3159Cys Trp Asn Val Lys Gly His Val
Asp Val Gln Gln Ser His His 1040 1045 1050cgt tct gtc ctt gtt atc
cca gaa tgg gaa gca gaa gtg tca caa 3204Arg Ser Val Leu Val Ile Pro
Glu Trp Glu Ala Glu Val Ser Gln 1055 1060 1065gca gtt cgc gtc tgt
ccg ggg cgt ggc tat atc ctc cgt gtc aca 3249Ala Val Arg Val Cys Pro
Gly Arg Gly Tyr Ile Leu Arg Val Thr 1070 1075 1080gcg tac aaa gag
gga tat gga gag ggt tgt gta acg atc cat gaa 3294Ala Tyr Lys Glu Gly
Tyr Gly Glu Gly Cys Val Thr Ile His Glu 1085 1090 1095atc gag aac
aat aca gac gaa cta aaa ttt aaa aac tgt gaa gaa 3339Ile Glu Asn Asn
Thr Asp Glu Leu Lys Phe Lys Asn Cys Glu Glu 1100 1105 1110gag gaa
gtg tat cca acg gat aca gga acg tgt aat gat tat act 3384Glu Glu Val
Tyr Pro Thr Asp Thr Gly Thr Cys Asn Asp Tyr Thr 1115 1120 1125gca
cac caa ggt aca gca gca tgt aat tcc cgt aat gct gga tat 3429Ala His
Gln Gly Thr Ala Ala Cys Asn Ser Arg Asn Ala Gly Tyr 1130 1135
1140gag gat gca tat gaa gtt gat act aca gca tct gtt aat tac aaa
3474Glu Asp Ala Tyr Glu Val Asp Thr Thr Ala Ser Val Asn Tyr Lys
1145 1150 1155ccg act tat gaa gaa gaa acg tat aca gat gta cga aga
gat aat 3519Pro Thr Tyr Glu Glu Glu Thr Tyr Thr Asp Val Arg Arg Asp
Asn 1160 1165 1170cat tgt gaa tat gac aga ggg tat gtg aat tat cca
cca cta cca 3564His Cys Glu Tyr Asp Arg Gly Tyr Val Asn Tyr Pro Pro
Leu Pro 1175 1180 1185gct ggt tat gtg aca aag gaa tta gaa tat ttc
cca gaa acc gat 3609Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro
Glu Thr Asp 1190 1195 1200aag gta tgg att gag att gga gaa acg gaa
gga aca ttc atc gtg 3654Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly
Thr Phe Ile Val 1205 1210 1215gac agc ata gaa tta ctc ctt atg gaa
gaa tag gaccgtccga 3697Asp Ser Ile Glu Leu Leu Leu Met Glu Glu 1220
1225gtatagcagt ttaataaatc ttaatcaaaa tagtagtcta acttccgtta
caatttaata 3757agtaaattac agttgtaaaa agaaaacgga catcactcct
aagagagcga tgtccgtttt 3817ctatatggtg tgtgctaacg ataagtgtac
acggaatttc attatccaaa ttaatattta 3877tttgagaaaa ggatcatgtt
atatagagat atttccttat aatatttgtt ccacgttcat 3937aatttttgaa
tgatacatta caacaaaaac tgtcacaaat ttatatgttc tacataaaat
3997atatggttaa gaacctaaga agttatgaat caagtaatag gataaaactg
aaaaaggaag 4057tgtaggtaca atgaataaaa aaataagaaa tgaagatgag
cattcatcga tagaattatc 4117atatagtact tcaaaaaatc aaaagcataa
ggtaccattt tgttgtacaa tttcag 4173381228PRTBacillus thuringiensis
38Leu Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser1
5 10 15Ile Pro Ala Val Ser Asn His Ser Thr Gln Met Asp Leu Ser Pro
Asp 20 25 30Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn
Ile Asn 35 40 45Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn
Ile Ala Gly 50 55 60Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly
Gln Ile Ala Ser65 70 75 80Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp
Pro Arg Gly Arg Asp Gln 85 90 95Trp Glu Ile Phe Leu Glu His Val Glu
Gln Leu Ile Asn Gln Gln Ile 100 105 110Thr Glu Asn Ala Arg Asn Thr
Ala Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125Asp Ser Phe Arg Ala
Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140Arg Asp Asp
Ala Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala145 150 155
160Leu Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn
165 170 175Gln Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn
Leu His 180 185 190Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser
Glu Phe Gly Leu 195 200 205Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu
Arg Gln Val Glu Gln Thr 210 215 220Arg Asp Tyr Ser Asp Tyr Cys Val
Glu Trp Tyr Asn Thr Gly Leu Asn225 230 235 240Ser Leu Arg Gly Thr
Asn Ala Ala Ser Trp Val Arg Tyr Asn Gln Phe 245 250 255Arg Arg Asp
Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270Ser
Tyr Asp Thr Arg Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280
285Arg Glu Val Tyr Thr Asp Ala Ile Gly Ala Thr Gly Val Asn Met Ala
290 295 300Ser Met Asn Trp Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala
Ile Glu305 310 315 320Thr Ala Val Ile Arg Ser Pro His Leu Leu Asp
Phe Leu Glu Gln Leu 325 330 335Thr Ile Phe Ser Thr Ser Ser Arg Trp
Ser Ala Thr Arg His Met Thr 340 345 350Tyr Trp Arg Gly His Thr Ile
Gln Ser Arg Pro Ile Gly Gly Gly Leu 355 360 365Asn Thr Ser Thr His
Gly Ser Thr Asn Thr Ser Ile Asn Pro Val Arg 370 375 380Leu Ser Phe
Phe Ser Arg Asp Val Tyr Trp Thr Glu Ser Tyr Ala Gly385 390 395
400Val Leu Leu Trp Gly Ile Tyr Leu Glu Pro Ile His Gly Val Pro Thr
405 410 415Val Arg Phe Asn Phe Arg Asn Pro Gln Asn Thr Phe Glu Arg
Gly Thr 420 425 430Ala Asn Tyr Ser Gln Pro Tyr Glu Ser Pro Gly Leu
Gln Leu Lys Asp 435 440 445Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr
Glu Arg Pro Asn Tyr Glu 450 455 460Ser Tyr Ser His Arg Leu Ser His
Ile Gly Leu Ile Ser Gln Ser Arg465 470 475 480Val His Val Pro Val
Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr 485 490 495Asn Thr Ile
Ser Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ser 500 505 510Phe
Asn Leu Asn Ser Gly Thr Ser Val Val Ser Gly Pro Gly Phe Thr 515 520
525Gly Gly Asp Ile Ile Arg Thr Asn Val Asn Gly Ser Val Leu Ser Met
530 535 540Gly Leu Asn Phe Asn Asn Thr Ser Leu Gln Arg Tyr Arg Val
Arg Val545 550 555 560Arg Tyr Ala Ala Ser Gln Thr Met Val Leu Arg
Val Thr Val Gly Gly 565 570 575Ser Thr Thr Phe Asp Gln Gly Phe Pro
Ser Thr Met Ser Ala Asn Glu 580 585 590Ser Leu Thr Ser Gln Ser Phe
Arg Phe Ala Glu Phe Pro Val Gly Ile 595 600 605Ser Ala Ser Gly Ser
Gln Thr Ala Gly Ile Ser Ile Ser Asn Asn Ala 610 615 620Gly Arg Gln
Thr Phe His Phe Asp Lys Ile Glu Phe Ile Pro Ile Thr625 630 635
640Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu Ala Val
645 650 655Asn Ala Leu Phe Thr Asn Thr Asn Pro Arg Arg Leu Lys Thr
Asp Val 660 665 670Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val
Ala Cys Leu Ser 675 680 685Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu
Leu Leu Glu Lys Val Lys 690 695 700Tyr Ala Lys Arg Leu Ser Asp Glu
Arg Asn Leu Leu Gln Asp Pro Asn705 710 715 720Phe Thr Ser Ile Asn
Lys Gln Pro Asp Phe Ile Ser Thr Asn Glu Gln 725 730 735Ser Asn Phe
Thr Ser Ile His Glu Gln Ser Glu His Gly Trp Trp Gly 740 745 750Ser
Glu Asn Ile Thr Ile Gln Glu Gly Asn Asp Val Phe Lys Glu Asn 755 760
765Tyr Val Thr Leu Pro Gly Thr Phe Asn Glu Cys Tyr Pro Thr Tyr Leu
770 775 780Tyr Gln Lys Ile Gly Glu Ser Glu Leu Lys Ala Tyr Thr Arg
Tyr Gln785 790 795 800Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu
Glu Ile Tyr Leu Ile 805 810 815Arg Tyr Asn Ala Lys His Glu Thr Leu
Asp Val Pro Gly Thr Glu Ser 820 825 830Val Trp Pro Leu Ser Val Glu
Ser Pro Ile Gly Arg Cys Gly Glu Pro 835 840 845Asn Arg Cys Ala Pro
His Phe Glu Trp Asn Pro Asp Leu Asp Cys Ser 850 855 860Cys Arg Asp
Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu865 870 875
880Asp Ile Asp Ile Gly Cys Thr Asp Leu His Glu Asn Leu Gly Val Trp
885 890 895Val Val Phe Lys Ile Lys Thr Gln Glu Gly His Ala Arg Leu
Gly Asn 900 905 910Leu Glu Phe Ile Glu Glu Lys Pro Leu Leu Gly Glu
Ala Leu Ser Arg 915 920 925Val Lys Arg Ala Glu Lys Lys Trp Arg Asp
Lys Arg Glu Lys Leu Gln 930 935 940Leu Glu Thr Lys Arg Val Tyr Thr
Glu Ala Lys Glu Ala Val Asp Ala945 950 955 960Leu Phe Val Asp Ser
Gln Tyr Asn Arg Leu Gln Ala Asp Thr Asn Ile 965 970 975Gly Met Ile
His Ala Ala Asp Lys Leu Val His Arg Ile Arg Glu Ala 980 985 990Tyr
Leu Ser Glu Leu Ser Val Ile Pro Gly Val Asn Ala Glu Ile Phe 995
1000 1005Glu Glu Leu Glu Gly Arg Ile Ile Thr Ala Ile Ser Leu Tyr
Asp 1010 1015 1020Ala Arg Asn Val Val Lys Asn Gly Asp Phe Asn Asn
Gly Leu Ala 1025 1030 1035Cys Trp Asn Val Lys Gly His Val Asp Val
Gln Gln Ser His His 1040 1045 1050Arg Ser Val Leu Val Ile Pro Glu
Trp Glu Ala Glu Val Ser Gln 1055 1060 1065Ala Val Arg Val Cys Pro
Gly Arg Gly Tyr Ile Leu Arg Val Thr 1070 1075 1080Ala Tyr Lys Glu
Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu 1085 1090 1095Ile Glu
Asn Asn Thr Asp Glu Leu Lys Phe Lys Asn Cys Glu Glu 1100 1105
1110Glu Glu Val Tyr Pro Thr Asp Thr Gly Thr Cys Asn Asp Tyr Thr
1115 1120 1125Ala His Gln Gly Thr Ala Ala Cys Asn Ser Arg Asn Ala
Gly Tyr 1130 1135 1140Glu Asp Ala Tyr Glu Val Asp Thr Thr Ala Ser
Val Asn Tyr Lys 1145 1150 1155Pro Thr Tyr Glu Glu Glu Thr Tyr Thr
Asp Val Arg Arg Asp Asn 1160 1165 1170His Cys Glu Tyr Asp Arg Gly
Tyr Val Asn Tyr Pro Pro Leu Pro 1175 1180 1185Ala Gly Tyr Val Thr
Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1190 1195 1200Lys Val Trp
Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val 1205 1210 1215Asp
Ser Ile Glu Leu Leu Leu Met Glu Glu 1220 1225393504DNABacillus
thuringiensisCDS(1)..(3504) 39atg gag aga aat aat cag gat caa tgc
att cct tat aat tgt tta aat 48Met Glu Arg Asn Asn Gln Asp Gln Cys
Ile Pro Tyr Asn Cys Leu Asn1 5 10 15aat cct gag att gag ata tta gat
gtt gaa aat ttc aat ctc gaa ctt 96Asn Pro Glu Ile Glu Ile Leu Asp
Val Glu Asn Phe Asn Leu Glu Leu 20 25 30gta tcg caa gtc agt gtg gga
ctt aca cgt ttt ctt cta gag tca gct 144Val Ser Gln Val Ser Val Gly
Leu Thr Arg Phe Leu Leu Glu Ser Ala 35 40 45gtc cca gga gcg ggt ttt
gca ctt ggc cta ttc gat atc att tgg gga 192Val Pro Gly Ala Gly Phe
Ala Leu Gly Leu Phe Asp Ile Ile Trp Gly 50 55 60gct cta ggc gtc gat
caa tgg agc tta ttc ctt gcg caa att gag caa 240Ala Leu Gly Val Asp
Gln Trp Ser Leu Phe Leu Ala Gln Ile Glu Gln65 70 75 80tta att aat
gaa agg ata aca aca gtt gaa agg aat aga gca att caa 288Leu Ile Asn
Glu Arg Ile Thr Thr Val Glu Arg Asn Arg Ala Ile Gln 85 90 95aca tta
agt gga cta tcg agt agt tat gaa gta tat att gag gca tta 336Thr Leu
Ser Gly Leu Ser Ser Ser Tyr Glu Val Tyr Ile Glu Ala Leu 100 105
110aga gaa tgg gag aat aat cca gat aat cca gct tca caa gag aga gtg
384Arg Glu Trp Glu Asn Asn Pro Asp Asn Pro Ala Ser Gln Glu Arg Val
115 120 125cgt aca cga ttt cgt aca acg gac gac gct cta ata aca gct
ata cct 432Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Ile Thr Ala
Ile Pro 130 135 140aat tta gcg att cca gat ttt gag ata gct act tta
tca gtg tat gtt 480Asn Leu Ala Ile Pro Asp Phe Glu Ile Ala Thr Leu
Ser Val Tyr Val145 150 155 160caa gca gcc aat cta cat cta tct tta
tta aga gat gct gtt tac ttt 528Gln Ala Ala Asn Leu His Leu Ser Leu
Leu Arg Asp Ala Val Tyr Phe 165 170 175gga gaa aga tgg gga ctc aca
caa gta aat att gaa gat ctt tat acg 576Gly Glu Arg Trp Gly Leu Thr
Gln Val Asn Ile Glu Asp Leu Tyr Thr 180 185 190aga tta aca aga aat
att cat att tat tca gat cat tgt gca agg tgg 624Arg Leu Thr Arg Asn
Ile His Ile Tyr Ser Asp His Cys Ala Arg Trp 195 200 205tat aat caa
ggt tta aat aat att gga gca aca aat acg aga tat ttg 672Tyr Asn Gln
Gly Leu Asn Asn Ile Gly Ala Thr Asn Thr Arg Tyr Leu 210 215 220gaa
ttc caa aga gaa tta aca ctc tct gtc tta gat att gtg gcc ctt 720Glu
Phe Gln Arg Glu Leu Thr Leu Ser Val Leu Asp Ile Val Ala Leu225 230
235 240ttc ccg aat tac gac atc cga aca tat tca att ccg aca caa agt
caa 768Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Ser Ile Pro Thr Gln Ser
Gln 245 250 255tta aca agg gag att tat acc gat ata att gct gca ccc
aat gca tca 816Leu Thr Arg Glu Ile Tyr Thr Asp Ile Ile Ala Ala Pro
Asn Ala Ser 260 265 270aat tta ata gtg gga acg caa ggc cta gtg aga
gca cct cac tta atg 864Asn Leu Ile Val Gly Thr Gln Gly Leu Val Arg
Ala Pro His Leu Met 275 280 285gac ttt tta gtc cgt ttg aat att tat
act ggc ttg gct aga aat att 912Asp Phe Leu Val Arg Leu Asn Ile Tyr
Thr Gly Leu Ala Arg Asn Ile 290 295 300cgt cat tgg gca gga cat gaa
gta ata tct aga aga aca ggt gga gtg 960Arg His Trp Ala Gly His Glu
Val Ile Ser Arg Arg Thr Gly Gly Val305 310 315 320gat tta aat act
ata caa tct cct tta tat gga aca gct gca act aca 1008Asp Leu Asn Thr
Ile Gln Ser Pro Leu Tyr Gly Thr Ala Ala Thr Thr 325 330 335gaa agt
cca cgt tta ata att cct ttt aat gag gat tct tat ctt ggt 1056Glu Ser
Pro Arg Leu Ile Ile Pro Phe Asn Glu Asp Ser Tyr Leu Gly 340 345
350ggt ttt att tat aga aca tta tca tcc cct att tat gta cca cct tct
1104Gly Phe Ile Tyr Arg Thr Leu Ser Ser Pro Ile Tyr Val Pro Pro Ser
355 360 365gga att tcg agt caa aga aca tct tta gtg gag ggt gtg gga
ttt cag 1152Gly Ile Ser Ser Gln Arg Thr Ser Leu Val Glu Gly Val Gly
Phe Gln 370 375 380aca ccg aat aac tca ata ctt caa tac aga caa cgt
gga aca ttg gat 1200Thr Pro Asn Asn Ser Ile Leu Gln Tyr Arg Gln Arg
Gly Thr Leu Asp385 390 395 400tcc ctt gag caa gta cca ctt caa gaa
gag ggg aga cca ggc ggt ttt 1248Ser Leu Glu Gln Val Pro Leu Gln Glu
Glu Gly Arg Pro Gly Gly Phe 405 410 415ggt gct agt cat aga ttg tgt
cat gct aca ttt gct caa tca cct ata 1296Gly Ala Ser His Arg Leu Cys
His Ala Thr Phe Ala Gln Ser Pro Ile 420 425 430ggt act aac tat tat
ata agg gca ccg ttg ttt tct tgg acg cat ctg 1344Gly Thr Asn Tyr Tyr
Ile Arg Ala Pro Leu Phe Ser Trp Thr His Leu 435 440 445agt gca act
ctt act aat gaa gtt cgt gta tct aga att aca caa tta 1392Ser Ala Thr
Leu Thr Asn Glu Val Arg Val Ser Arg Ile Thr Gln Leu 450 455 460ccg
atg gtg aag gcg cat acg ctt cat gcg gga gct act gtt gtt aga 1440Pro
Met Val Lys Ala His Thr Leu His Ala Gly Ala Thr Val Val Arg465
470 475 480gga cca gga ttt aca gga gga gat ata ctc cga aga act act
tca ggc 1488Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Thr
Ser Gly 485 490 495tca ttt ggg gat atg aga ata aca aat ttt tca agt
tca tca tcg agg 1536Ser Phe Gly Asp Met Arg Ile Thr Asn Phe Ser Ser
Ser Ser Ser Arg 500 505 510tat cgt gta aga ata cgt tat gct tct act
aca gat tta caa ttt ttc 1584Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr
Thr Asp Leu Gln Phe Phe 515 520 525ttg aat gtt gga gga acc cct gtc
aat gta gcc gat ttc ccg aaa acc 1632Leu Asn Val Gly Gly Thr Pro Val
Asn Val Ala Asp Phe Pro Lys Thr 530 535 540ata gat aga ggg gaa aac
tta gaa tat gga agc ttt aga acg gca ggt 1680Ile Asp Arg Gly Glu Asn
Leu Glu Tyr Gly Ser Phe Arg Thr Ala Gly545 550 555 560ttt act acc
cct ttt agt ttt gta agt tca aca aat aat ttc aca tta 1728Phe Thr Thr
Pro Phe Ser Phe Val Ser Ser Thr Asn Asn Phe Thr Leu 565 570 575ggt
gtt cag agt gtt tct tca ggt aac gag att ttt gta gat cga att 1776Gly
Val Gln Ser Val Ser Ser Gly Asn Glu Ile Phe Val Asp Arg Ile 580 585
590gaa ttt gtt ccg gca gat gca acc ttt gag gca gaa tat gat tta gaa
1824Glu Phe Val Pro Ala Asp Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu
595 600 605aga gcg caa gag gcg gtg aat gct ctg ttt act tct acg aat
caa aga 1872Arg Ala Gln Glu Ala Val Asn Ala Leu Phe Thr Ser Thr Asn
Gln Arg 610 615 620gga ctg aaa aca gat gtg acg gat tat cat att gat
caa gtg tcc aat 1920Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp
Gln Val Ser Asn625 630 635 640tta gtg gat tgt tta tcc gat gaa ttc
tgt cta gat gaa aaa aga gaa 1968Leu Val Asp Cys Leu Ser Asp Glu Phe
Cys Leu Asp Glu Lys Arg Glu 645 650 655ttg tcc gaa aaa att aaa cat
gca aag cga ctc agt gat gag cgg aat 2016Leu Ser Glu Lys Ile Lys His
Ala Lys Arg Leu Ser Asp Glu Arg Asn 660 665 670tta ctc caa gat tca
aac ttt aga ggc atc aat aga caa cca gat cgt 2064Leu Leu Gln Asp Ser
Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg 675 680 685ggc tgg aga
gga agt acg gat att act atc caa gga gga aat gac gta 2112Gly Trp Arg
Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val 690 695 700ttc
aaa gaa aat tac gtc aca cta cca ggt acc ttt gat gag tgc tat 2160Phe
Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr705 710
715 720cca aca tat ttg tat caa aaa atc gat gaa tca aaa tta aaa gcc
ttt 2208Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala
Phe 725 730 735acc cgt tat caa tta aga ggg tat atc gaa gat agt caa
gac tta gaa 2256Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln
Asp Leu Glu 740 745 750atc tat tta att cgc tac aat gca aaa cat gaa
aca gta aat gtg cca 2304Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu
Thr Val Asn Val Pro 755 760 765ggt acg ggt tcc tta tgg ccg ctt tca
gcc caa agt cca atc gga aag 2352Gly Thr Gly Ser Leu Trp Pro Leu Ser
Ala Gln Ser Pro Ile Gly Lys 770 775 780tgt gga gag ccg aat cga tgc
gcg cca cac ctt gaa tgg aat cct gac 2400Cys Gly Glu Pro Asn Arg Cys
Ala Pro His Leu Glu Trp Asn Pro Asp785 790 795 800tta gat tgt tcg
tgt agg gat gga gaa aag tgt gcc cat cat tcg cat 2448Leu Asp Cys Ser
Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His 805 810 815cat ttc
tcc tta gac att gat gta gga tgt aca gac tta aat gag gac 2496His Phe
Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp 820 825
830cta ggt gta tgg gtg atc ttt aag att aag acg caa gat ggg cac gca
2544Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala
835 840 845aga cta ggg aat cta gag ttt ctc gaa gag aaa cca tta gta
gga gaa 2592Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val
Gly Glu 850 855 860gcg cta gct cgt gtg aaa aga gcg gag aaa aaa tgg
aga gac aaa cgt 2640Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp
Arg Asp Lys Arg865 870 875 880gaa aaa ttg gaa tgg gaa aca aat atc
gtt tat aaa gag gca aaa gaa 2688Glu Lys Leu Glu Trp Glu Thr Asn Ile
Val Tyr Lys Glu Ala Lys Glu 885 890 895tct gta gat gct tta ttt gta
aac tct caa tat gat caa tta caa gcg 2736Ser Val Asp Ala Leu Phe Val
Asn Ser Gln Tyr Asp Gln Leu Gln Ala 900 905 910gat acg aat att gcc
atg att cat gcg gca gat aaa cgt gtt cat agc 2784Asp Thr Asn Ile Ala
Met Ile His Ala Ala Asp Lys Arg Val His Ser 915 920 925att cga gaa
gct tat ctg cct gag ctg tct gtg att ccg ggt gtc aat 2832Ile Arg Glu
Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn 930 935 940gcg
gct att ttt gaa gaa tta gaa ggg cgt att ttc act gca ttc tcc 2880Ala
Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser945 950
955 960cta tat gat gcg aga aat gtc att aaa aat ggt gat ttt aat aat
ggc 2928Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn
Gly 965 970 975tta tcc tgc tgg aac gtg aaa ggg cat gta gat gta gaa
gaa caa aac 2976Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu
Glu Gln Asn 980 985 990aac caa cgt tcg gtc ctt gtt gtt ccg gaa tgg
gaa gca gaa gtg tca 3024Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp
Glu Ala Glu Val Ser 995 1000 1005caa gaa gtt cgt gtc tgt ccg ggt
cgt ggc tat atc ctt cgt gtc 3069Gln Glu Val Arg Val Cys Pro Gly Arg
Gly Tyr Ile Leu Arg Val 1010 1015 1020aca gcg tac aag gag gga tat
gga gaa ggt tgc gta acc att cat 3114Thr Ala Tyr Lys Glu Gly Tyr Gly
Glu Gly Cys Val Thr Ile His 1025 1030 1035gag atc gag aac aat aca
gac gaa ctg aag ttt agc aac tgc gta 3159Glu Ile Glu Asn Asn Thr Asp
Glu Leu Lys Phe Ser Asn Cys Val 1040 1045 1050gaa gag gaa atc tat
cca aat aac acg gta acg tgt aat gat tat 3204Glu Glu Glu Ile Tyr Pro
Asn Asn Thr Val Thr Cys Asn Asp Tyr 1055 1060 1065act gta aat caa
gaa gaa tac gga ggt gcg tac act tct cgt aat 3249Thr Val Asn Gln Glu
Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn 1070 1075 1080cga gga tat
aac gaa gct cct tcc gta cca gct gat tat gcg tca 3294Arg Gly Tyr Asn
Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser 1085 1090 1095gtc tat
gaa gaa aaa tcg tat aca gat gga cga aga gag aat cct 3339Val Tyr Glu
Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro 1100 1105 1110tgt
gaa ttt aac aga ggg tat agg gat tac acg cca cta cca gtt 3384Cys Glu
Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val 1115 1120
1125ggt tat gtg aca aaa gaa tta gaa tac ttc cca gaa acc gat aag
3429Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys
1130 1135 1140gta tgg att gag att gga gaa acg gaa gga aca ttt atc
gtg gac 3474Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val
Asp 1145 1150 1155agc gtg gaa tta ctc ctt atg gag gaa tag 3504Ser
Val Glu Leu Leu Leu Met Glu Glu 1160 1165401167PRTBacillus
thuringiensis 40Met Glu Arg Asn Asn Gln Asp Gln Cys Ile Pro Tyr Asn
Cys Leu Asn1 5 10 15Asn Pro Glu Ile Glu Ile Leu Asp Val Glu Asn Phe
Asn Leu Glu Leu 20 25 30Val Ser Gln Val Ser Val Gly Leu Thr Arg Phe
Leu Leu Glu Ser Ala 35 40 45Val Pro Gly Ala Gly Phe Ala Leu Gly Leu
Phe Asp Ile Ile Trp Gly 50 55 60Ala Leu Gly Val Asp Gln Trp Ser Leu
Phe Leu Ala Gln Ile Glu Gln65 70 75 80Leu Ile Asn Glu Arg Ile Thr
Thr Val Glu Arg Asn Arg Ala Ile Gln 85 90 95Thr Leu Ser Gly Leu Ser
Ser Ser Tyr Glu Val Tyr Ile Glu Ala Leu 100 105 110Arg Glu Trp Glu
Asn Asn Pro Asp Asn Pro Ala Ser Gln Glu Arg Val 115 120 125Arg Thr
Arg Phe Arg Thr Thr Asp Asp Ala Leu Ile Thr Ala Ile Pro 130 135
140Asn Leu Ala Ile Pro Asp Phe Glu Ile Ala Thr Leu Ser Val Tyr
Val145 150 155 160Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp
Ala Val Tyr Phe 165 170 175Gly Glu Arg Trp Gly Leu Thr Gln Val Asn
Ile Glu Asp Leu Tyr Thr 180 185 190Arg Leu Thr Arg Asn Ile His Ile
Tyr Ser Asp His Cys Ala Arg Trp 195 200 205Tyr Asn Gln Gly Leu Asn
Asn Ile Gly Ala Thr Asn Thr Arg Tyr Leu 210 215 220Glu Phe Gln Arg
Glu Leu Thr Leu Ser Val Leu Asp Ile Val Ala Leu225 230 235 240Phe
Pro Asn Tyr Asp Ile Arg Thr Tyr Ser Ile Pro Thr Gln Ser Gln 245 250
255Leu Thr Arg Glu Ile Tyr Thr Asp Ile Ile Ala Ala Pro Asn Ala Ser
260 265 270Asn Leu Ile Val Gly Thr Gln Gly Leu Val Arg Ala Pro His
Leu Met 275 280 285Asp Phe Leu Val Arg Leu Asn Ile Tyr Thr Gly Leu
Ala Arg Asn Ile 290 295 300Arg His Trp Ala Gly His Glu Val Ile Ser
Arg Arg Thr Gly Gly Val305 310 315 320Asp Leu Asn Thr Ile Gln Ser
Pro Leu Tyr Gly Thr Ala Ala Thr Thr 325 330 335Glu Ser Pro Arg Leu
Ile Ile Pro Phe Asn Glu Asp Ser Tyr Leu Gly 340 345 350Gly Phe Ile
Tyr Arg Thr Leu Ser Ser Pro Ile Tyr Val Pro Pro Ser 355 360 365Gly
Ile Ser Ser Gln Arg Thr Ser Leu Val Glu Gly Val Gly Phe Gln 370 375
380Thr Pro Asn Asn Ser Ile Leu Gln Tyr Arg Gln Arg Gly Thr Leu
Asp385 390 395 400Ser Leu Glu Gln Val Pro Leu Gln Glu Glu Gly Arg
Pro Gly Gly Phe 405 410 415Gly Ala Ser His Arg Leu Cys His Ala Thr
Phe Ala Gln Ser Pro Ile 420 425 430Gly Thr Asn Tyr Tyr Ile Arg Ala
Pro Leu Phe Ser Trp Thr His Leu 435 440 445Ser Ala Thr Leu Thr Asn
Glu Val Arg Val Ser Arg Ile Thr Gln Leu 450 455 460Pro Met Val Lys
Ala His Thr Leu His Ala Gly Ala Thr Val Val Arg465 470 475 480Gly
Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Thr Ser Gly 485 490
495Ser Phe Gly Asp Met Arg Ile Thr Asn Phe Ser Ser Ser Ser Ser Arg
500 505 510Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln
Phe Phe 515 520 525Leu Asn Val Gly Gly Thr Pro Val Asn Val Ala Asp
Phe Pro Lys Thr 530 535 540Ile Asp Arg Gly Glu Asn Leu Glu Tyr Gly
Ser Phe Arg Thr Ala Gly545 550 555 560Phe Thr Thr Pro Phe Ser Phe
Val Ser Ser Thr Asn Asn Phe Thr Leu 565 570 575Gly Val Gln Ser Val
Ser Ser Gly Asn Glu Ile Phe Val Asp Arg Ile 580 585 590Glu Phe Val
Pro Ala Asp Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu 595 600 605Arg
Ala Gln Glu Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Arg 610 615
620Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser
Asn625 630 635 640Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp
Glu Lys Arg Glu 645 650 655Leu Ser Glu Lys Ile Lys His Ala Lys Arg
Leu Ser Asp Glu Arg Asn 660 665 670Leu Leu Gln Asp Ser Asn Phe Arg
Gly Ile Asn Arg Gln Pro Asp Arg 675 680 685Gly Trp Arg Gly Ser Thr
Asp Ile Thr Ile Gln Gly Gly Asn Asp Val 690 695 700Phe Lys Glu Asn
Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr705 710 715 720Pro
Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe 725 730
735Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu
740 745 750Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn
Val Pro 755 760 765Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser
Pro Ile Gly Lys 770 775 780Cys Gly Glu Pro Asn Arg Cys Ala Pro His
Leu Glu Trp Asn Pro Asp785 790 795 800Leu Asp Cys Ser Cys Arg Asp
Gly Glu Lys Cys Ala His His Ser His 805 810 815His Phe Ser Leu Asp
Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp 820 825 830Leu Gly Val
Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala 835 840 845Arg
Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu 850 855
860Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys
Arg865 870 875 880Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys
Glu Ala Lys Glu 885 890 895Ser Val Asp Ala Leu Phe Val Asn Ser Gln
Tyr Asp Gln Leu Gln Ala 900 905 910Asp Thr Asn Ile Ala Met Ile His
Ala Ala Asp Lys Arg Val His Ser 915 920 925Ile Arg Glu Ala Tyr Leu
Pro Glu Leu Ser Val Ile Pro Gly Val Asn 930 935 940Ala Ala Ile Phe
Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser945 950 955 960Leu
Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly 965 970
975Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn
980 985 990Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu
Val Ser 995 1000 1005Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr
Ile Leu Arg Val 1010 1015 1020Thr Ala Tyr Lys Glu Gly Tyr Gly Glu
Gly Cys Val Thr Ile His 1025 1030 1035Glu Ile Glu Asn Asn Thr Asp
Glu Leu Lys Phe Ser Asn Cys Val 1040 1045 1050Glu Glu Glu Ile Tyr
Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr 1055 1060 1065Thr Val Asn
Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn 1070 1075 1080Arg
Gly Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser 1085 1090
1095Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro
1100 1105 1110Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu
Pro Val 1115 1120 1125Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro
Glu Thr Asp Lys 1130 1135 1140Val Trp Ile Glu Ile Gly Glu Thr Glu
Gly Thr Phe Ile Val Asp 1145 1150 1155Ser Val Glu Leu Leu Leu Met
Glu Glu 1160 1165412133DNABacillus
thuringiensisCDS(1)..(2133)misc_feature(598)..(600)n = a or g or c
or t, unknown, or other 41atg aaa tct aag aat caa aat atg cat caa
agc ttg tct aac aat gcg 48Met Lys Ser Lys Asn Gln Asn Met His Gln
Ser Leu Ser Asn Asn Ala1 5 10 15aca gtt gat aaa aac ttt aca ggt tca
cta gaa aat aac aca aat acg 96Thr Val Asp Lys Asn Phe Thr Gly Ser
Leu Glu Asn Asn Thr Asn Thr 20 25 30gaa tta caa aac ttt aat cat gaa
ggt ata gag ccg ttt gtt agt gta 144Glu Leu Gln Asn Phe Asn His Glu
Gly Ile Glu Pro Phe Val Ser Val 35 40 45tca aca att caa acg ggt att
ggt att gct ggt aaa atc ctt ggt aac 192Ser Thr Ile Gln Thr Gly Ile
Gly Ile Ala Gly Lys Ile Leu Gly Asn 50 55 60cta ggc gtt cct ttt gct
ggg caa gta gct agc ctc tat agt ttt atc 240Leu Gly Val Pro Phe Ala
Gly Gln Val Ala Ser Leu Tyr Ser Phe Ile65 70 75 80cta ggt gag ctt
tgg ccc aaa ggg aaa agc caa tgg gaa atc ttt atg 288Leu Gly Glu Leu
Trp Pro Lys Gly Lys Ser Gln Trp Glu Ile Phe Met 85 90 95gaa
cat gta gaa gag ctt att aat caa aag ata tcg act tat gca aga 336Glu
His Val Glu Glu Leu Ile Asn Gln Lys Ile Ser Thr Tyr Ala Arg 100 105
110aac aaa gca ctt gca gat tta aaa gga tta gga gat gct ttg gct gtc
384Asn Lys Ala Leu Ala Asp Leu Lys Gly Leu Gly Asp Ala Leu Ala Val
115 120 125tac cat gaa tcg ctg gaa agt tgg att gaa aat cgc aat aac
aca aga 432Tyr His Glu Ser Leu Glu Ser Trp Ile Glu Asn Arg Asn Asn
Thr Arg 130 135 140acc aga agt gtt gtc aag agc caa tac atc acc ttg
gaa ctt atg ttc 480Thr Arg Ser Val Val Lys Ser Gln Tyr Ile Thr Leu
Glu Leu Met Phe145 150 155 160gta caa tca tta cct tct ttt gca gtg
tct gga gag gaa gta cca cta 528Val Gln Ser Leu Pro Ser Phe Ala Val
Ser Gly Glu Glu Val Pro Leu 165 170 175tta cca ata tat gct caa gct
gca aat tta cac tta ttg cta tta cga 576Leu Pro Ile Tyr Ala Gln Ala
Ala Asn Leu His Leu Leu Leu Leu Arg 180 185 190gat gct tct att ttt
gga aaa nnn tgg ggg tta tca gac tca gaa att 624Asp Ala Ser Ile Phe
Gly Lys Xaa Trp Gly Leu Ser Asp Ser Glu Ile 195 200 205tcc aca ttt
tat aat cgc caa tcc gga aaa tcg aaa gaa tat tct gac 672Ser Thr Phe
Tyr Asn Arg Gln Ser Gly Lys Ser Lys Glu Tyr Ser Asp 210 215 220cac
tgc gta aaa tgg tat aat aca ggc cta aat cgc ttg atg ggg aac 720His
Cys Val Lys Trp Tyr Asn Thr Gly Leu Asn Arg Leu Met Gly Asn225 230
235 240aat gcc gaa agt tgg gta cga tat aat caa ttc cgt aga gac atg
act 768Asn Ala Glu Ser Trp Val Arg Tyr Asn Gln Phe Arg Arg Asp Met
Thr 245 250 255tta atg gta cta gat tta gtg gca cta ttt cca agc tat
gat aca caa 816Leu Met Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr
Asp Thr Gln 260 265 270atg tat cca att aaa act aca gcc caa ctt aca
aga gaa gta tat aca 864Met Tyr Pro Ile Lys Thr Thr Ala Gln Leu Thr
Arg Glu Val Tyr Thr 275 280 285gac gca att ggg aca gta cat ccg cat
cca agt ttt aca agt acg act 912Asp Ala Ile Gly Thr Val His Pro His
Pro Ser Phe Thr Ser Thr Thr 290 295 300tgg tat aat aat aat gca cct
tcg ttc tct acc ata gag gct gct gtt 960Trp Tyr Asn Asn Asn Ala Pro
Ser Phe Ser Thr Ile Glu Ala Ala Val305 310 315 320gtt cga aac ccg
cat cta ctc gat ttt cta gaa caa gtt aca att tac 1008Val Arg Asn Pro
His Leu Leu Asp Phe Leu Glu Gln Val Thr Ile Tyr 325 330 335agc tta
tta agt cga tgg agt aac act cag tat atg aat atg tgg gga 1056Ser Leu
Leu Ser Arg Trp Ser Asn Thr Gln Tyr Met Asn Met Trp Gly 340 345
350gga cat aaa cta gaa ttc cga aca ata gga gga acg tta aat acc tca
1104Gly His Lys Leu Glu Phe Arg Thr Ile Gly Gly Thr Leu Asn Thr Ser
355 360 365aca caa gga tct act aat act tct att aat cct gta aca tta
ccg ttc 1152Thr Gln Gly Ser Thr Asn Thr Ser Ile Asn Pro Val Thr Leu
Pro Phe 370 375 380act tct cga gac gtc tat agg act gaa tca ttg gca
ggg ctg aat cta 1200Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Leu Ala
Gly Leu Asn Leu385 390 395 400ttt tta act caa cct gtt aat gga gta
cct agg gtt gat ttt cat tgg 1248Phe Leu Thr Gln Pro Val Asn Gly Val
Pro Arg Val Asp Phe His Trp 405 410 415aaa ttc gtc aca cat ccg atc
gca tct gat aat ttc tat tat cca ggg 1296Lys Phe Val Thr His Pro Ile
Ala Ser Asp Asn Phe Tyr Tyr Pro Gly 420 425 430tat gct gga att ggg
acg caa tta cag gat tca gaa aat gaa tta cca 1344Tyr Ala Gly Ile Gly
Thr Gln Leu Gln Asp Ser Glu Asn Glu Leu Pro 435 440 445cct gaa gca
aca gga cag cca aat tat gaa tct tat agt cat aga tta 1392Pro Glu Ala
Thr Gly Gln Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu 450 455 460tct
cat ata gga ctc att tca gca tca cat gtg aaa gca ttg gta tat 1440Ser
His Ile Gly Leu Ile Ser Ala Ser His Val Lys Ala Leu Val Tyr465 470
475 480tct tgg acg cat cgt agt gca gat cgt aca aat aca att gag cca
aat 1488Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile Glu Pro
Asn 485 490 495agc att aca caa ata cca tta gta aaa gcg ttc aat ctg
tct tca ggt 1536Ser Ile Thr Gln Ile Pro Leu Val Lys Ala Phe Asn Leu
Ser Ser Gly 500 505 510gcc gct gta gtg aga gga cca gga ttt aca ggt
ggg gat atc ctt cga 1584Ala Ala Val Val Arg Gly Pro Gly Phe Thr Gly
Gly Asp Ile Leu Arg 515 520 525aga aag aat act ggt aca ttt ggg gat
ata cga gta aat att aat cca 1632Arg Lys Asn Thr Gly Thr Phe Gly Asp
Ile Arg Val Asn Ile Asn Pro 530 535 540cca ttt gca caa aga tat cgc
gtg agg att cgc tat gct tct acc aca 1680Pro Phe Ala Gln Arg Tyr Arg
Val Arg Ile Arg Tyr Ala Ser Thr Thr545 550 555 560gat tta caa ttc
cat acg tca att aac ggt aaa gct att aat caa ggt 1728Asp Leu Gln Phe
His Thr Ser Ile Asn Gly Lys Ala Ile Asn Gln Gly 565 570 575aat ttt
tca gca act atg aat aga gga gag gac tta gac tat aaa acc 1776Asn Phe
Ser Ala Thr Met Asn Arg Gly Glu Asp Leu Asp Tyr Lys Thr 580 585
590ttt aga act gta ggc ttt acc acc cca ttt agc ttt tca gat gta caa
1824Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Ser Phe Ser Asp Val Gln
595 600 605agt aca ttc aca ata ggt gct tgg aac ttc tct tca ggt aac
gaa gtt 1872Ser Thr Phe Thr Ile Gly Ala Trp Asn Phe Ser Ser Gly Asn
Glu Val 610 615 620tat ata gat aga att gaa ttt gtt ccg gta gaa gta
aca tat gag gca 1920Tyr Ile Asp Arg Ile Glu Phe Val Pro Val Glu Val
Thr Tyr Glu Ala625 630 635 640gaa tat gat ttt gaa aaa gcg caa gag
gag gtt act gca ctg ttt aca 1968Glu Tyr Asp Phe Glu Lys Ala Gln Glu
Glu Val Thr Ala Leu Phe Thr 645 650 655tct acg aat cca aga gga tta
aaa aca gat gta aag gat tat cat att 2016Ser Thr Asn Pro Arg Gly Leu
Lys Thr Asp Val Lys Asp Tyr His Ile 660 665 670gac cag gta tca aat
tta gta gag tct cta tca gat aaa ttc tat ctt 2064Asp Gln Val Ser Asn
Leu Val Glu Ser Leu Ser Asp Lys Phe Tyr Leu 675 680 685gat gaa aag
aga gaa tta ttc gag ata gtt aaa tac gcg aag caa ctc 2112Asp Glu Lys
Arg Glu Leu Phe Glu Ile Val Lys Tyr Ala Lys Gln Leu 690 695 700cat
att gag cgt aac atg tag 2133His Ile Glu Arg Asn Met705
71042710PRTBacillus thuringiensismisc_feature(200)..(200)Xaa =
unknown or other 42Met Lys Ser Lys Asn Gln Asn Met His Gln Ser Leu
Ser Asn Asn Ala1 5 10 15Thr Val Asp Lys Asn Phe Thr Gly Ser Leu Glu
Asn Asn Thr Asn Thr 20 25 30Glu Leu Gln Asn Phe Asn His Glu Gly Ile
Glu Pro Phe Val Ser Val 35 40 45Ser Thr Ile Gln Thr Gly Ile Gly Ile
Ala Gly Lys Ile Leu Gly Asn 50 55 60Leu Gly Val Pro Phe Ala Gly Gln
Val Ala Ser Leu Tyr Ser Phe Ile65 70 75 80Leu Gly Glu Leu Trp Pro
Lys Gly Lys Ser Gln Trp Glu Ile Phe Met 85 90 95Glu His Val Glu Glu
Leu Ile Asn Gln Lys Ile Ser Thr Tyr Ala Arg 100 105 110Asn Lys Ala
Leu Ala Asp Leu Lys Gly Leu Gly Asp Ala Leu Ala Val 115 120 125Tyr
His Glu Ser Leu Glu Ser Trp Ile Glu Asn Arg Asn Asn Thr Arg 130 135
140Thr Arg Ser Val Val Lys Ser Gln Tyr Ile Thr Leu Glu Leu Met
Phe145 150 155 160Val Gln Ser Leu Pro Ser Phe Ala Val Ser Gly Glu
Glu Val Pro Leu 165 170 175Leu Pro Ile Tyr Ala Gln Ala Ala Asn Leu
His Leu Leu Leu Leu Arg 180 185 190Asp Ala Ser Ile Phe Gly Lys Xaa
Trp Gly Leu Ser Asp Ser Glu Ile 195 200 205Ser Thr Phe Tyr Asn Arg
Gln Ser Gly Lys Ser Lys Glu Tyr Ser Asp 210 215 220His Cys Val Lys
Trp Tyr Asn Thr Gly Leu Asn Arg Leu Met Gly Asn225 230 235 240Asn
Ala Glu Ser Trp Val Arg Tyr Asn Gln Phe Arg Arg Asp Met Thr 245 250
255Leu Met Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Gln
260 265 270Met Tyr Pro Ile Lys Thr Thr Ala Gln Leu Thr Arg Glu Val
Tyr Thr 275 280 285Asp Ala Ile Gly Thr Val His Pro His Pro Ser Phe
Thr Ser Thr Thr 290 295 300Trp Tyr Asn Asn Asn Ala Pro Ser Phe Ser
Thr Ile Glu Ala Ala Val305 310 315 320Val Arg Asn Pro His Leu Leu
Asp Phe Leu Glu Gln Val Thr Ile Tyr 325 330 335Ser Leu Leu Ser Arg
Trp Ser Asn Thr Gln Tyr Met Asn Met Trp Gly 340 345 350Gly His Lys
Leu Glu Phe Arg Thr Ile Gly Gly Thr Leu Asn Thr Ser 355 360 365Thr
Gln Gly Ser Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Pro Phe 370 375
380Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Leu Ala Gly Leu Asn
Leu385 390 395 400Phe Leu Thr Gln Pro Val Asn Gly Val Pro Arg Val
Asp Phe His Trp 405 410 415Lys Phe Val Thr His Pro Ile Ala Ser Asp
Asn Phe Tyr Tyr Pro Gly 420 425 430Tyr Ala Gly Ile Gly Thr Gln Leu
Gln Asp Ser Glu Asn Glu Leu Pro 435 440 445Pro Glu Ala Thr Gly Gln
Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu 450 455 460Ser His Ile Gly
Leu Ile Ser Ala Ser His Val Lys Ala Leu Val Tyr465 470 475 480Ser
Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile Glu Pro Asn 485 490
495Ser Ile Thr Gln Ile Pro Leu Val Lys Ala Phe Asn Leu Ser Ser Gly
500 505 510Ala Ala Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile
Leu Arg 515 520 525Arg Lys Asn Thr Gly Thr Phe Gly Asp Ile Arg Val
Asn Ile Asn Pro 530 535 540Pro Phe Ala Gln Arg Tyr Arg Val Arg Ile
Arg Tyr Ala Ser Thr Thr545 550 555 560Asp Leu Gln Phe His Thr Ser
Ile Asn Gly Lys Ala Ile Asn Gln Gly 565 570 575Asn Phe Ser Ala Thr
Met Asn Arg Gly Glu Asp Leu Asp Tyr Lys Thr 580 585 590Phe Arg Thr
Val Gly Phe Thr Thr Pro Phe Ser Phe Ser Asp Val Gln 595 600 605Ser
Thr Phe Thr Ile Gly Ala Trp Asn Phe Ser Ser Gly Asn Glu Val 610 615
620Tyr Ile Asp Arg Ile Glu Phe Val Pro Val Glu Val Thr Tyr Glu
Ala625 630 635 640Glu Tyr Asp Phe Glu Lys Ala Gln Glu Glu Val Thr
Ala Leu Phe Thr 645 650 655Ser Thr Asn Pro Arg Gly Leu Lys Thr Asp
Val Lys Asp Tyr His Ile 660 665 670Asp Gln Val Ser Asn Leu Val Glu
Ser Leu Ser Asp Lys Phe Tyr Leu 675 680 685Asp Glu Lys Arg Glu Leu
Phe Glu Ile Val Lys Tyr Ala Lys Gln Leu 690 695 700His Ile Glu Arg
Asn Met705 71043218DNABacillus thuringiensis 43gtagccgatt
tcccgaaaac catagataga ggggaaaact tagaatatgg aagctttaga 60acggcaggtt
ttactacccc ttttagtttt gtaagttcaa caaataattt cacattaggt
120gttcagagtg tttcttcagg taacgagatt tttgtagatc gaattgaatt
tgttccggca 180gatgcaacct ttgaggcaga atatgattta gaaagagc
2184472PRTBacillus thuringiensis 44Val Ala Asp Phe Pro Lys Thr Ile
Asp Arg Gly Glu Asn Leu Glu Tyr1 5 10 15Gly Ser Phe Arg Thr Ala Gly
Phe Thr Thr Pro Phe Ser Phe Val Ser 20 25 30Ser Thr Asn Asn Phe Thr
Leu Gly Val Gln Ser Val Ser Ser Gly Asn 35 40 45Glu Ile Phe Val Asp
Arg Ile Glu Phe Val Pro Ala Asp Ala Thr Phe 50 55 60Glu Ala Glu Tyr
Asp Leu Glu Arg65 70451908DNABacillus thuringiensisCDS(1)..(1908)
45atg aat aat gta ttg aat agc gga aaa aca act att tgt aat gcg tat
48Met Asn Asn Val Leu Asn Ser Gly Lys Thr Thr Ile Cys Asn Ala Tyr1
5 10 15aat gta gtg gct cac gat cca ttt agt ttt gaa cat aaa tca tta
gat 96Asn Val Val Ala His Asp Pro Phe Ser Phe Glu His Lys Ser Leu
Asp 20 25 30acc atc caa gaa gaa tgg atg gag tgg aaa aga aca gat cat
agt tta 144Thr Ile Gln Glu Glu Trp Met Glu Trp Lys Arg Thr Asp His
Ser Leu 35 40 45tat gta gct cct gta gtc gga act gtg tct agt ttt ctg
cta aag aaa 192Tyr Val Ala Pro Val Val Gly Thr Val Ser Ser Phe Leu
Leu Lys Lys 50 55 60gtg ggg agt cta att gga aaa agg ata ttg agt gaa
tta tgg ggg tta 240Val Gly Ser Leu Ile Gly Lys Arg Ile Leu Ser Glu
Leu Trp Gly Leu65 70 75 80ata ttt cct agt ggt agt aca aat cta atg
caa gat att tta aga gag 288Ile Phe Pro Ser Gly Ser Thr Asn Leu Met
Gln Asp Ile Leu Arg Glu 85 90 95aca gaa caa ttc cta aat caa aga ctt
aat aca gac acc ctt gat cgt 336Thr Glu Gln Phe Leu Asn Gln Arg Leu
Asn Thr Asp Thr Leu Asp Arg 100 105 110gta aat gca gaa ttg gaa ggg
ctc caa gcg aat ata agg gag ttt aat 384Val Asn Ala Glu Leu Glu Gly
Leu Gln Ala Asn Ile Arg Glu Phe Asn 115 120 125caa caa gta gat aat
ttt tta aac cct act caa aac cct gtt cct tta 432Gln Gln Val Asp Asn
Phe Leu Asn Pro Thr Gln Asn Pro Val Pro Leu 130 135 140tca ata act
tct tca gtt aat aca atg cag caa tta ttt cta aat aga 480Ser Ile Thr
Ser Ser Val Asn Thr Met Gln Gln Leu Phe Leu Asn Arg145 150 155
160tta ccc cag ttc cag ata caa gga tac cag ttg tta tta tta cct tta
528Leu Pro Gln Phe Gln Ile Gln Gly Tyr Gln Leu Leu Leu Leu Pro Leu
165 170 175ttt gca cag gca gcc aat atg cat ctt tct ttt att aga gat
gtt att 576Phe Ala Gln Ala Ala Asn Met His Leu Ser Phe Ile Arg Asp
Val Ile 180 185 190ctt aat gca gat gaa tgg ggc att tca gca gca aca
cta cgt acg tat 624Leu Asn Ala Asp Glu Trp Gly Ile Ser Ala Ala Thr
Leu Arg Thr Tyr 195 200 205cga gac tac ctg aga aat tat aca aga gat
tat tct aat tat tgt ata 672Arg Asp Tyr Leu Arg Asn Tyr Thr Arg Asp
Tyr Ser Asn Tyr Cys Ile 210 215 220aat acg tat caa act gcg ttt aga
ggg tta aac acc cgt tta cac gat 720Asn Thr Tyr Gln Thr Ala Phe Arg
Gly Leu Asn Thr Arg Leu His Asp225 230 235 240atg tta gaa ttt aga
aca tat atg ttt tta aat gta ttt gaa tat gta 768Met Leu Glu Phe Arg
Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr Val 245 250 255tcc att tgg
tca ttg ttt aaa tat cag agt ctt atg gta tct tct ggc 816Ser Ile Trp
Ser Leu Phe Lys Tyr Gln Ser Leu Met Val Ser Ser Gly 260 265 270gct
aat tta tat gct agt ggt agt gga cca cag cag aca caa tca ttt 864Ala
Asn Leu Tyr Ala Ser Gly Ser Gly Pro Gln Gln Thr Gln Ser Phe 275 280
285act gca caa aac tgg cca ttt tta tat tct ctt ttc caa gtt aat tcg
912Thr Ala Gln Asn Trp Pro Phe Leu Tyr Ser Leu Phe Gln Val Asn Ser
290 295 300aat tat ata tta tct ggt att agt ggt aat agg ctt tct act
acc ttc 960Asn Tyr Ile Leu Ser Gly Ile Ser Gly Asn Arg Leu Ser Thr
Thr Phe305 310 315 320cct aat att ggt ggt tta ccg ggt agt act aca
att cat tca ttg aac 1008Pro Asn Ile Gly Gly Leu Pro Gly Ser Thr Thr
Ile His Ser Leu Asn 325 330 335agt gcc agg gtt aat tat agc gga gga
gtt tca tct ggt ctc ata ggg 1056Ser Ala Arg Val Asn Tyr Ser Gly Gly
Val Ser Ser Gly Leu Ile Gly 340 345 350gcg act aat ctc aat cac aac
ttt aat tgc agc acg gtc ctc cct cct 1104Ala Thr Asn Leu Asn His Asn
Phe Asn Cys Ser Thr Val Leu Pro Pro 355 360 365tta tca aca cca ttt
gtt aga agt tgg ctg gat tca ggt aca gat cga 1152Leu Ser Thr Pro Phe
Val Arg Ser Trp Leu Asp Ser Gly Thr Asp Arg 370 375 380gag ggc gtt
gct acc tct acg act tgg cag aca gaa tcc ttc caa ata 1200Glu Gly Val
Ala Thr Ser Thr Thr Trp Gln Thr Glu Ser Phe Gln Ile385 390
395 400act tca ggt tta agg tgt ggt gct ttt cct ttt tca gct cgt gga
aat 1248Thr Ser Gly Leu Arg Cys Gly Ala Phe Pro Phe Ser Ala Arg Gly
Asn 405 410 415tca aac tat ttc cca gat tat ttt atc cgt aat att tct
ggg gtt cct 1296Ser Asn Tyr Phe Pro Asp Tyr Phe Ile Arg Asn Ile Ser
Gly Val Pro 420 425 430tta gtt att aga aac gaa gat cta aca aga ccg
tta cac tat aac caa 1344Leu Val Ile Arg Asn Glu Asp Leu Thr Arg Pro
Leu His Tyr Asn Gln 435 440 445ata aga aat ata gaa agt cct tcg gga
aca cct ggt gga tta cga gct 1392Ile Arg Asn Ile Glu Ser Pro Ser Gly
Thr Pro Gly Gly Leu Arg Ala 450 455 460tat atg gta tct gtg cat aac
aga aaa aat aat atc tat gcc gct cat 1440Tyr Met Val Ser Val His Asn
Arg Lys Asn Asn Ile Tyr Ala Ala His465 470 475 480gaa aat ggt act
atg att cat ttg gca ccg gaa gat tat aca gga ttt 1488Glu Asn Gly Thr
Met Ile His Leu Ala Pro Glu Asp Tyr Thr Gly Phe 485 490 495act ata
tca cca ata cat gcc act caa gtg aat aat caa act cga aca 1536Thr Ile
Ser Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr 500 505
510ttt att tct gaa aaa ttt gga aat caa ggt gat tcc tta aga ttt gaa
1584Phe Ile Ser Glu Lys Phe Gly Asn Gln Gly Asp Ser Leu Arg Phe Glu
515 520 525caa agt aac acg aca gct cgt tat acg ctt aga ggg aat gga
aat agt 1632Gln Ser Asn Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly
Asn Ser 530 535 540tac aat ctt tat tta aga gta tct tca ata gga aat
tca act atc cga 1680Tyr Asn Leu Tyr Leu Arg Val Ser Ser Ile Gly Asn
Ser Thr Ile Arg545 550 555 560gtt act ata aac ggt agg gtt tat act
gct tca aat gtt aat act aat 1728Val Thr Ile Asn Gly Arg Val Tyr Thr
Ala Ser Asn Val Asn Thr Asn 565 570 575aca aat aac gat ggg gtt aat
gat aat gga gct cgt ttt tca gat att 1776Thr Asn Asn Asp Gly Val Asn
Asp Asn Gly Ala Arg Phe Ser Asp Ile 580 585 590aat atc ggt aat gta
gta gca agt gat aat act aat gta ccg tta gat 1824Asn Ile Gly Asn Val
Val Ala Ser Asp Asn Thr Asn Val Pro Leu Asp 595 600 605ata aat gtg
aca tta aac tcc ggt act caa ttt gag ctt atg aat att 1872Ile Asn Val
Thr Leu Asn Ser Gly Thr Gln Phe Glu Leu Met Asn Ile 610 615 620atg
ttt gtg cca act aat ctt cca cca ctt tat taa 1908Met Phe Val Pro Thr
Asn Leu Pro Pro Leu Tyr625 630 63546635PRTBacillus thuringiensis
46Met Asn Asn Val Leu Asn Ser Gly Lys Thr Thr Ile Cys Asn Ala Tyr1
5 10 15Asn Val Val Ala His Asp Pro Phe Ser Phe Glu His Lys Ser Leu
Asp 20 25 30Thr Ile Gln Glu Glu Trp Met Glu Trp Lys Arg Thr Asp His
Ser Leu 35 40 45Tyr Val Ala Pro Val Val Gly Thr Val Ser Ser Phe Leu
Leu Lys Lys 50 55 60Val Gly Ser Leu Ile Gly Lys Arg Ile Leu Ser Glu
Leu Trp Gly Leu65 70 75 80Ile Phe Pro Ser Gly Ser Thr Asn Leu Met
Gln Asp Ile Leu Arg Glu 85 90 95Thr Glu Gln Phe Leu Asn Gln Arg Leu
Asn Thr Asp Thr Leu Asp Arg 100 105 110Val Asn Ala Glu Leu Glu Gly
Leu Gln Ala Asn Ile Arg Glu Phe Asn 115 120 125Gln Gln Val Asp Asn
Phe Leu Asn Pro Thr Gln Asn Pro Val Pro Leu 130 135 140Ser Ile Thr
Ser Ser Val Asn Thr Met Gln Gln Leu Phe Leu Asn Arg145 150 155
160Leu Pro Gln Phe Gln Ile Gln Gly Tyr Gln Leu Leu Leu Leu Pro Leu
165 170 175Phe Ala Gln Ala Ala Asn Met His Leu Ser Phe Ile Arg Asp
Val Ile 180 185 190Leu Asn Ala Asp Glu Trp Gly Ile Ser Ala Ala Thr
Leu Arg Thr Tyr 195 200 205Arg Asp Tyr Leu Arg Asn Tyr Thr Arg Asp
Tyr Ser Asn Tyr Cys Ile 210 215 220Asn Thr Tyr Gln Thr Ala Phe Arg
Gly Leu Asn Thr Arg Leu His Asp225 230 235 240Met Leu Glu Phe Arg
Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr Val 245 250 255Ser Ile Trp
Ser Leu Phe Lys Tyr Gln Ser Leu Met Val Ser Ser Gly 260 265 270Ala
Asn Leu Tyr Ala Ser Gly Ser Gly Pro Gln Gln Thr Gln Ser Phe 275 280
285Thr Ala Gln Asn Trp Pro Phe Leu Tyr Ser Leu Phe Gln Val Asn Ser
290 295 300Asn Tyr Ile Leu Ser Gly Ile Ser Gly Asn Arg Leu Ser Thr
Thr Phe305 310 315 320Pro Asn Ile Gly Gly Leu Pro Gly Ser Thr Thr
Ile His Ser Leu Asn 325 330 335Ser Ala Arg Val Asn Tyr Ser Gly Gly
Val Ser Ser Gly Leu Ile Gly 340 345 350Ala Thr Asn Leu Asn His Asn
Phe Asn Cys Ser Thr Val Leu Pro Pro 355 360 365Leu Ser Thr Pro Phe
Val Arg Ser Trp Leu Asp Ser Gly Thr Asp Arg 370 375 380Glu Gly Val
Ala Thr Ser Thr Thr Trp Gln Thr Glu Ser Phe Gln Ile385 390 395
400Thr Ser Gly Leu Arg Cys Gly Ala Phe Pro Phe Ser Ala Arg Gly Asn
405 410 415Ser Asn Tyr Phe Pro Asp Tyr Phe Ile Arg Asn Ile Ser Gly
Val Pro 420 425 430Leu Val Ile Arg Asn Glu Asp Leu Thr Arg Pro Leu
His Tyr Asn Gln 435 440 445Ile Arg Asn Ile Glu Ser Pro Ser Gly Thr
Pro Gly Gly Leu Arg Ala 450 455 460Tyr Met Val Ser Val His Asn Arg
Lys Asn Asn Ile Tyr Ala Ala His465 470 475 480Glu Asn Gly Thr Met
Ile His Leu Ala Pro Glu Asp Tyr Thr Gly Phe 485 490 495Thr Ile Ser
Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr 500 505 510Phe
Ile Ser Glu Lys Phe Gly Asn Gln Gly Asp Ser Leu Arg Phe Glu 515 520
525Gln Ser Asn Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly Asn Ser
530 535 540Tyr Asn Leu Tyr Leu Arg Val Ser Ser Ile Gly Asn Ser Thr
Ile Arg545 550 555 560Val Thr Ile Asn Gly Arg Val Tyr Thr Ala Ser
Asn Val Asn Thr Asn 565 570 575Thr Asn Asn Asp Gly Val Asn Asp Asn
Gly Ala Arg Phe Ser Asp Ile 580 585 590Asn Ile Gly Asn Val Val Ala
Ser Asp Asn Thr Asn Val Pro Leu Asp 595 600 605Ile Asn Val Thr Leu
Asn Ser Gly Thr Gln Phe Glu Leu Met Asn Ile 610 615 620Met Phe Val
Pro Thr Asn Leu Pro Pro Leu Tyr625 630 635471878DNABacillus
thuringiensisCDS(1)..(1878) 47atg aat act gta ttg aat aac gga aga
aat act act tgt cat gca cat 48Met Asn Thr Val Leu Asn Asn Gly Arg
Asn Thr Thr Cys His Ala His1 5 10 15aat gta gtt gct cat gat cca ttt
agt ttt gaa cat aaa tca tta aat 96Asn Val Val Ala His Asp Pro Phe
Ser Phe Glu His Lys Ser Leu Asn 20 25 30acc ata gaa aaa gaa tgg aaa
gaa tgg aaa aga act gat cat agt tta 144Thr Ile Glu Lys Glu Trp Lys
Glu Trp Lys Arg Thr Asp His Ser Leu 35 40 45tat gta gcc cct att gtg
gga act gtg ggt agt ttt cta tta aag aaa 192Tyr Val Ala Pro Ile Val
Gly Thr Val Gly Ser Phe Leu Leu Lys Lys 50 55 60gta ggg agt ctt gtt
gga aaa agg ata ctg agt gag tta cag aat tta 240Val Gly Ser Leu Val
Gly Lys Arg Ile Leu Ser Glu Leu Gln Asn Leu65 70 75 80att ttt cct
agt ggt agt ata gat tta atg caa gag att tta aga gcg 288Ile Phe Pro
Ser Gly Ser Ile Asp Leu Met Gln Glu Ile Leu Arg Ala 85 90 95aca gaa
caa ttc ata aat caa agg ctt aat gca gac acc ctt ggt cgt 336Thr Glu
Gln Phe Ile Asn Gln Arg Leu Asn Ala Asp Thr Leu Gly Arg 100 105
110gta aat gca gaa ttg gca ggt ctt caa gcg aat gtg gca gag ttt aat
384Val Asn Ala Glu Leu Ala Gly Leu Gln Ala Asn Val Ala Glu Phe Asn
115 120 125cga caa gta gat aat ttt tta aac cct aat caa aac cct gtt
cct tta 432Arg Gln Val Asp Asn Phe Leu Asn Pro Asn Gln Asn Pro Val
Pro Leu 130 135 140gca ata att gat tca gtt aat aca ttg cag caa tta
ttt cta agt aga 480Ala Ile Ile Asp Ser Val Asn Thr Leu Gln Gln Leu
Phe Leu Ser Arg145 150 155 160tta cca cag ttc cag ata caa ggc tat
caa ctg tta tta tta cct tta 528Leu Pro Gln Phe Gln Ile Gln Gly Tyr
Gln Leu Leu Leu Leu Pro Leu 165 170 175ttt gca cag gca gcc aat tta
cat ctt tct ttt att aga gat gtc atc 576Phe Ala Gln Ala Ala Asn Leu
His Leu Ser Phe Ile Arg Asp Val Ile 180 185 190ctt aat gca gat gaa
tgg ggc att tca gca gca aca gta cgc aca tat 624Leu Asn Ala Asp Glu
Trp Gly Ile Ser Ala Ala Thr Val Arg Thr Tyr 195 200 205aga gat cac
ctg aga aat ttc aca aga gat tac tct aat tat tgt ata 672Arg Asp His
Leu Arg Asn Phe Thr Arg Asp Tyr Ser Asn Tyr Cys Ile 210 215 220aat
acg tat caa act gca ttt aga ggt tta aac act cgt tta cac gat 720Asn
Thr Tyr Gln Thr Ala Phe Arg Gly Leu Asn Thr Arg Leu His Asp225 230
235 240atg tta gaa ttt aga aca tat atg ttt tta aat gta ttt gaa tat
gtc 768Met Leu Glu Phe Arg Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr
Val 245 250 255tct atc tgg tcg tta ttt aaa tat caa agc ctt cta gta
tct tcc ggc 816Ser Ile Trp Ser Leu Phe Lys Tyr Gln Ser Leu Leu Val
Ser Ser Gly 260 265 270gct aat tta tat gcg agt ggt agt ggt cca aca
caa tca ttt aca gca 864Ala Asn Leu Tyr Ala Ser Gly Ser Gly Pro Thr
Gln Ser Phe Thr Ala 275 280 285caa aac tgg cca ttt tta tat tct ctt
ttc caa gtt aat tct aat tat 912Gln Asn Trp Pro Phe Leu Tyr Ser Leu
Phe Gln Val Asn Ser Asn Tyr 290 295 300gta tta aat ggt ttg agt ggt
gct agg acc acc att act ttc cct aat 960Val Leu Asn Gly Leu Ser Gly
Ala Arg Thr Thr Ile Thr Phe Pro Asn305 310 315 320att ggt ggt ctt
ccc ggt tct acc aca act caa aca ttg cat ttt gcg 1008Ile Gly Gly Leu
Pro Gly Ser Thr Thr Thr Gln Thr Leu His Phe Ala 325 330 335agg att
aat tat aga ggt gga gtg tca tct agc cgc ata ggt caa gct 1056Arg Ile
Asn Tyr Arg Gly Gly Val Ser Ser Ser Arg Ile Gly Gln Ala 340 345
350aat ctt aat caa aac ttt aac att tcc aca ctt ttc aat cct tta caa
1104Asn Leu Asn Gln Asn Phe Asn Ile Ser Thr Leu Phe Asn Pro Leu Gln
355 360 365aca ccg ttt att aga agt tgg cta gat tct ggt aca gat cgg
gag ggc 1152Thr Pro Phe Ile Arg Ser Trp Leu Asp Ser Gly Thr Asp Arg
Glu Gly 370 375 380gtt gcc acc tct aca aac tgg caa tca gga gcc ttt
gag aca act tta 1200Val Ala Thr Ser Thr Asn Trp Gln Ser Gly Ala Phe
Glu Thr Thr Leu385 390 395 400tta cga ttt agc att ttt tca gct cgt
ggt aat tcg aac ttt ttc cca 1248Leu Arg Phe Ser Ile Phe Ser Ala Arg
Gly Asn Ser Asn Phe Phe Pro 405 410 415gat tat ttt atc cgt aat att
tct ggt gtt gtt ggg act att agc aac 1296Asp Tyr Phe Ile Arg Asn Ile
Ser Gly Val Val Gly Thr Ile Ser Asn 420 425 430gca gat tta gca aga
cct cta cac ttt aat gaa ata aga gat ata gga 1344Ala Asp Leu Ala Arg
Pro Leu His Phe Asn Glu Ile Arg Asp Ile Gly 435 440 445acg aca gca
gtc gct agc ctt gta aca gtg cat aac aga aaa aat aat 1392Thr Thr Ala
Val Ala Ser Leu Val Thr Val His Asn Arg Lys Asn Asn 450 455 460atc
tat gac act cat gaa aat ggt act atg att cat tta gcg cca aat 1440Ile
Tyr Asp Thr His Glu Asn Gly Thr Met Ile His Leu Ala Pro Asn465 470
475 480gac tat aca gga ttt acc gta tct cca ata cat gcc act caa gta
aat 1488Asp Tyr Thr Gly Phe Thr Val Ser Pro Ile His Ala Thr Gln Val
Asn 485 490 495aat caa att cga acg ttt att tcc gaa aaa tat ggt aat
cag ggt gat 1536Asn Gln Ile Arg Thr Phe Ile Ser Glu Lys Tyr Gly Asn
Gln Gly Asp 500 505 510tcc ttg aga ttt gag cta agc aac aca acg gct
cga tac aca ctt aga 1584Ser Leu Arg Phe Glu Leu Ser Asn Thr Thr Ala
Arg Tyr Thr Leu Arg 515 520 525ggg aat gga aat agt tac aat ctt tat
tta aga gta tct tca ata gga 1632Gly Asn Gly Asn Ser Tyr Asn Leu Tyr
Leu Arg Val Ser Ser Ile Gly 530 535 540agt tcc aca att cga gtt act
ata aac ggt aga gtt tat act gca aat 1680Ser Ser Thr Ile Arg Val Thr
Ile Asn Gly Arg Val Tyr Thr Ala Asn545 550 555 560gtt aat act acc
aca aat aat gat gga gta ctt gat aat gga gct cgt 1728Val Asn Thr Thr
Thr Asn Asn Asp Gly Val Leu Asp Asn Gly Ala Arg 565 570 575ttt tca
gat att aat atc ggt aat gta gtg gca agt gct aat act aat 1776Phe Ser
Asp Ile Asn Ile Gly Asn Val Val Ala Ser Ala Asn Thr Asn 580 585
590gta cca tta gat ata caa gtg aca ttt aac gac aat cca caa ttt gag
1824Val Pro Leu Asp Ile Gln Val Thr Phe Asn Asp Asn Pro Gln Phe Glu
595 600 605ctt atg aat att atg ttg ttc caa cta atc ttc cac cac ttt
att aag 1872Leu Met Asn Ile Met Leu Phe Gln Leu Ile Phe His His Phe
Ile Lys 610 615 620gtt tga 1878Val62548625PRTBacillus thuringiensis
48Met Asn Thr Val Leu Asn Asn Gly Arg Asn Thr Thr Cys His Ala His1
5 10 15Asn Val Val Ala His Asp Pro Phe Ser Phe Glu His Lys Ser Leu
Asn 20 25 30Thr Ile Glu Lys Glu Trp Lys Glu Trp Lys Arg Thr Asp His
Ser Leu 35 40 45Tyr Val Ala Pro Ile Val Gly Thr Val Gly Ser Phe Leu
Leu Lys Lys 50 55 60Val Gly Ser Leu Val Gly Lys Arg Ile Leu Ser Glu
Leu Gln Asn Leu65 70 75 80Ile Phe Pro Ser Gly Ser Ile Asp Leu Met
Gln Glu Ile Leu Arg Ala 85 90 95Thr Glu Gln Phe Ile Asn Gln Arg Leu
Asn Ala Asp Thr Leu Gly Arg 100 105 110Val Asn Ala Glu Leu Ala Gly
Leu Gln Ala Asn Val Ala Glu Phe Asn 115 120 125Arg Gln Val Asp Asn
Phe Leu Asn Pro Asn Gln Asn Pro Val Pro Leu 130 135 140Ala Ile Ile
Asp Ser Val Asn Thr Leu Gln Gln Leu Phe Leu Ser Arg145 150 155
160Leu Pro Gln Phe Gln Ile Gln Gly Tyr Gln Leu Leu Leu Leu Pro Leu
165 170 175Phe Ala Gln Ala Ala Asn Leu His Leu Ser Phe Ile Arg Asp
Val Ile 180 185 190Leu Asn Ala Asp Glu Trp Gly Ile Ser Ala Ala Thr
Val Arg Thr Tyr 195 200 205Arg Asp His Leu Arg Asn Phe Thr Arg Asp
Tyr Ser Asn Tyr Cys Ile 210 215 220Asn Thr Tyr Gln Thr Ala Phe Arg
Gly Leu Asn Thr Arg Leu His Asp225 230 235 240Met Leu Glu Phe Arg
Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr Val 245 250 255Ser Ile Trp
Ser Leu Phe Lys Tyr Gln Ser Leu Leu Val Ser Ser Gly 260 265 270Ala
Asn Leu Tyr Ala Ser Gly Ser Gly Pro Thr Gln Ser Phe Thr Ala 275 280
285Gln Asn Trp Pro Phe Leu Tyr Ser Leu Phe Gln Val Asn Ser Asn Tyr
290 295 300Val Leu Asn Gly Leu Ser Gly Ala Arg Thr Thr Ile Thr Phe
Pro Asn305 310 315 320Ile Gly Gly Leu Pro Gly Ser Thr Thr Thr Gln
Thr Leu His Phe Ala 325 330 335Arg Ile Asn Tyr Arg Gly Gly Val Ser
Ser Ser Arg Ile Gly Gln Ala 340 345 350Asn Leu Asn Gln Asn Phe Asn
Ile Ser Thr Leu Phe Asn Pro Leu Gln 355 360 365Thr Pro Phe Ile Arg
Ser Trp Leu Asp Ser Gly Thr Asp Arg Glu Gly 370 375 380Val Ala Thr
Ser Thr Asn Trp Gln Ser Gly Ala Phe Glu Thr Thr Leu385 390 395
400Leu Arg Phe Ser Ile Phe Ser Ala Arg Gly Asn Ser Asn Phe Phe Pro
405 410 415Asp Tyr Phe Ile Arg Asn Ile Ser Gly Val Val Gly Thr Ile
Ser Asn 420
425 430Ala Asp Leu Ala Arg Pro Leu His Phe Asn Glu Ile Arg Asp Ile
Gly 435 440 445Thr Thr Ala Val Ala Ser Leu Val Thr Val His Asn Arg
Lys Asn Asn 450 455 460Ile Tyr Asp Thr His Glu Asn Gly Thr Met Ile
His Leu Ala Pro Asn465 470 475 480Asp Tyr Thr Gly Phe Thr Val Ser
Pro Ile His Ala Thr Gln Val Asn 485 490 495Asn Gln Ile Arg Thr Phe
Ile Ser Glu Lys Tyr Gly Asn Gln Gly Asp 500 505 510Ser Leu Arg Phe
Glu Leu Ser Asn Thr Thr Ala Arg Tyr Thr Leu Arg 515 520 525Gly Asn
Gly Asn Ser Tyr Asn Leu Tyr Leu Arg Val Ser Ser Ile Gly 530 535
540Ser Ser Thr Ile Arg Val Thr Ile Asn Gly Arg Val Tyr Thr Ala
Asn545 550 555 560Val Asn Thr Thr Thr Asn Asn Asp Gly Val Leu Asp
Asn Gly Ala Arg 565 570 575Phe Ser Asp Ile Asn Ile Gly Asn Val Val
Ala Ser Ala Asn Thr Asn 580 585 590Val Pro Leu Asp Ile Gln Val Thr
Phe Asn Asp Asn Pro Gln Phe Glu 595 600 605Leu Met Asn Ile Met Leu
Phe Gln Leu Ile Phe His His Phe Ile Lys 610 615
620Val62549143DNABacillus thuringiensismodified_base(8)..(140)N =
A, T, C or G 49gtcgtganag gnccaggatt tacaggaggg gatatactnc
gaagaacggn cggtggtgca 60tttggaacna ttagngctan ggctantgcc ccnttaacac
aacaatatcg nataagatta 120cgctntgctt ctacnacaan ttt
1435047PRTBacillus thuringiensisSITE(3)..(3)X = R, I, K, or T 50Val
Val Xaa Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr1 5 10
15Xaa Gly Gly Ala Phe Gly Thr Ile Xaa Ala Xaa Ala Xaa Ala Pro Leu
20 25 30Thr Gln Gln Tyr Arg Ile Arg Leu Arg Xaa Ala Ser Thr Thr Xaa
35 40 455142DNAArtificialPrimer 51tggatacttg atcaatatga taatccgtca
catctgtttt ta 425261DNAArtificialPrimer 52agtaacggtg ttactattag
cgagggcggt ccattcttta aggtcgtgca cttcagttag 60c
615322DNAArtificialPrimer 53cgacttctcc tgctaatgga gg
225428DNAArtificialPrimer 54ctcgctaata gtaacaccgt tacttgcc
285561DNAArtificialPrimer 55atttagtagc atgcgttgca ctttgtgcat
tttttcataa gatgagtcat atgttttaaa 60t 615623DNAArtificialPrimer
56ggatagcact catcaaaggt acc 235722DNAArtificialPrimer 57gtwtggacsc
rtcghgatgt gg 225840DNAArtificialPrimer 58taatttctgc tagcccwatt
tctggattta attgttgatc 405919DNAArtificialPrimer 59atwacncaam
twccdttrg 196017DNAArtificialPrimer 60aatgcagatg aatgggg
176117DNAArtificialPrimer 61tgataatgga gctcgtt 17623684DNABacillus
thuringiensis 62ttgacttcaa ataggaaaaa tgagaatgaa attataaatg
ctttatcgat tccagctgta 60tcgaatcatt ccgcacaaat gaatctatca accgatgctc
gtattgagga tagcttgtgt 120atagccgagg ggaacaatat cgatccattt
gttagcgcat caacagtcca aacgggtatt 180aacatagctg gtagaatact
aggtgtatta ggcgtaccgt ttgctggaca aatagctagt 240ttttatagtt
ttcttgttgg tgaattatgg ccccgcggca gagatccttg ggaaattttc
300ctagaacatg tcgaacatct tataagacaa caagtaacag aaaatactag
ggatacggct 360cttgctcgat tacaaggttt aggaaattcc tttagagcct
atcaacagtc acttgaagat 420tggctagaaa accgtgatga tgcaagaacg
agaagtgttc tttataccca atatatagcc 480ttagaacttg attttcttaa
tgcgatgccg cttttcgcaa ttagaaacca agaagttcca 540ttattaatgg
tatatgctca agctgcaaat ttacacctat tattattgag agatgcctct
600ctttttggta gtgaatttgg gcttacatcc caagaaattc aacgttatta
tgagcgccaa 660gtggaaaaaa cgagagaata ttctgattat tgcgcaagat
ggtataatac gggtttaaat 720aatttgagag ggacaaatgc tgaaagttgg
ttgcgatata atcaattccg tagagactta 780acgctaggag tattagatct
agtggcacta ttcccaagct atgacacgcg tgtttatcca 840atgaatacca
gtgctcaatt aacaagagaa atttatacag atccaattgg gagaacaaat
900gcaccttcag gatttgcaag tacgaattgg tttaataata atgcaccatc
gttttctgcc 960atagaggctg ccgttattag gcctccgcat ctacttgatt
ttccagaaca gcttacaatt 1020ttcagcgtat taagtcgatg gagtaatact
caatatatga attactgggt gggacataga 1080cttgaatcgc gaacaataag
ggggtcatta agtacctgga cacacggaaa taccaatact 1140tctattaatc
ctgtaacatt acagttcaca tctcgagacg tttatagaac agaatcattt
1200gcagggataa atatacttct aactactcct gtgaatggag taccttgggc
tagatttaat 1260tggagaaatc ccctgaattc tcttagaggt agccttctct
atactatagg gtatactgga 1320gtggggacac aactatttga ttcagaaact
gaattaccac cagaaacaac agaacgacca 1380aattatgaat cttacagtca
tagattatct aatataagac taatatcagg aaacactttg 1440agagcaccag
tatattcttg gacgcaccgt agtgcagatc gtacaaatac cattagttca
1500gatagcataa cacaaatacc attggtaaaa tcattcaacc ttaattcagg
tacctctgta 1560gtcagtggcc caggatttac aggaggggat ataatccgaa
ctaacgttaa tggtagtgta 1620ctaagtatgg gtcttaattt taataataca
tcattacagc ggtatcgcgt gagagttcgt 1680tatgctgctt ctcaaacaat
ggtcctgagg gtaactgtcg gagggagtac tacttttgat 1740caaggattcc
ctagtactat gagtgcaaat gagtctttga catctcaatc atttagattt
1800gcagaatttc ctgtaggtat tagtgcatct ggcagtcaaa ctgctggaat
aagtataagt 1860aataatgcag gtagacaaac gtttcacttt gataaaattg
aattcattcc aattactgca 1920accttcgaag cagaatatga tttagaaaga
gcgcaagagg cggtgaatgc tctgtttact 1980aatacgaatc caagaaggtt
gaaaacaggt gtgacagatt atcatattga tgaagtatcc 2040aatttagtgg
cgtgtttatc ggatgaattc tgcttggatg aaaagagaga attacttgag
2100aaagtgaaat atgcgaaacg actcagtgat gaaagaaact tactccaaga
tccaaacttc 2160acatccatca ataagcaacc agacttcaat tctaataatg
agcaatcgaa tttcacatct 2220atccatgaac aatctgaaca tggatggtgg
ggaagtgaga acattacaat ccaggaagga 2280aatgacgtat ttaaagagaa
ttacgtcaca ctaccgggta cttttaatga gtgttatccg 2340acgtatttat
atcaaaaaat aggggaggcg gaattaaaag cttatactcg ctaccaatta
2400agtggctata ttgaagatag tcaagattta gagatatatt tgattcgtta
caatgcgaaa 2460catgaaacat tggatgttcc aggtaccgag tccgtatggc
cgctttcagt tgaaagccca 2520atcggaaggt gcggagaacc gaatcgatgc
gcaccacatt ttgaatggaa tcctgatcta 2580gattgttcct gcagagatgg
agaaaaatgt gcgcatcatt cccatcattt ctctttggat 2640attgatgttg
gatgcataga cttgcatgag aacctaggcg tgtgggtggt attcaagatt
2700aagacgcagg aaggtcatgc aagactaggg aacctggaat ttattgaaga
gaaaccatta 2760ttaggagaag cactgtctcg tgtgaagaga gcagagaaaa
aatggagaga caaacgtgaa 2820aaactacaat tggaaacaaa acgagtatat
acagaggcaa aagaagctgt ggatgcttta 2880tttgtagatt ctcaatatga
tagattacaa gcggatacaa acattggcat gattcatgcg 2940gcagataaac
ttgttcatcg aattcgagag gcgtatcttt cagaattatc tgttatccca
3000ggtgtaaatg cggaaatttt tgaagaatta gaaggtcgca ttatcactgc
aatctcccta 3060tacgatgcga gaaatgtcgt taaaaatggt gattttaata
atggattagc atgctggaat 3120gtaaaagggc atgtagatgt acaacagagc
catcaccgtt ctgtccttgt tatcccagaa 3180tgggaagcag aagtgtcaca
agcagttcgc gtctgtccgg ggcgtggcta tatcctccgt 3240gtcacagcgt
acaaagaggg atatggagag ggttgtgtaa ctatccatga aatcgagaac
3300aatacagacg aactaaaatt taaaaactgt gaagaagagg aagtgtatcc
aacggataca 3360ggaacgtgta atgattatac tgcacaccaa ggtacagcag
tatgtaattc ccgtaatgct 3420ggatatgagg atgcatatga agttgatact
acagcatctg ttaattacaa accgacttat 3480gaagaagaaa cgtatacaga
tgtacgaaga gataatcatt gtgaatatga cagagggtat 3540gtgaattatc
caccagtacc agctggttat atgacaaaag aattagaata cttcccagaa
3600accgataagg tatggattga gattggagaa acggaaggga agtttattgt
agacagcgtg 3660gaattactcc ttatggagga atag 3684631227PRTBacillus
thuringiensis 63Leu Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn
Ala Leu Ser1 5 10 15Ile Pro Ala Val Ser Asn His Ser Ala Gln Met Asn
Leu Ser Thr Asp 20 25 30Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu
Gly Asn Asn Ile Asp 35 40 45Pro Phe Val Ser Ala Ser Thr Val Gln Thr
Gly Ile Asn Ile Ala Gly 50 55 60Arg Ile Leu Gly Val Leu Gly Val Pro
Phe Ala Gly Gln Ile Ala Ser65 70 75 80Phe Tyr Ser Phe Leu Val Gly
Glu Leu Trp Pro Arg Gly Arg Asp Pro 85 90 95Trp Glu Ile Phe Leu Glu
His Val Glu His Leu Ile Arg Gln Gln Val 100 105 110Thr Glu Asn Thr
Arg Asp Thr Ala Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125Asn Ser
Phe Arg Ala Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135
140Arg Asp Asp Ala Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile
Ala145 150 155 160Leu Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe
Ala Ile Arg Asn 165 170 175Gln Glu Val Pro Leu Leu Met Val Tyr Ala
Gln Ala Ala Asn Leu His 180 185 190Leu Leu Leu Leu Arg Asp Ala Ser
Leu Phe Gly Ser Glu Phe Gly Leu 195 200 205Thr Ser Gln Glu Ile Gln
Arg Tyr Tyr Glu Arg Gln Val Glu Lys Thr 210 215 220Arg Glu Tyr Ser
Asp Tyr Cys Ala Arg Trp Tyr Asn Thr Gly Leu Asn225 230 235 240Asn
Leu Arg Gly Thr Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250
255Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro
260 265 270Ser Tyr Asp Thr Arg Val Tyr Pro Met Asn Thr Ser Ala Gln
Leu Thr 275 280 285Arg Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn
Ala Pro Ser Gly 290 295 300Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn
Ala Pro Ser Phe Ser Ala305 310 315 320Ile Glu Ala Ala Val Ile Arg
Pro Pro His Leu Leu Asp Phe Pro Glu 325 330 335Gln Leu Thr Ile Phe
Ser Val Leu Ser Arg Trp Ser Asn Thr Gln Tyr 340 345 350Met Asn Tyr
Trp Val Gly His Arg Leu Glu Ser Arg Thr Ile Arg Gly 355 360 365Ser
Leu Ser Thr Trp Thr His Gly Asn Thr Asn Thr Ser Ile Asn Pro 370 375
380Val Thr Leu Gln Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser
Phe385 390 395 400Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val Asn
Gly Val Pro Trp 405 410 415Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn
Ser Leu Arg Gly Ser Leu 420 425 430Leu Tyr Thr Ile Gly Tyr Thr Gly
Val Gly Thr Gln Leu Phe Asp Ser 435 440 445Glu Thr Glu Leu Pro Pro
Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser 450 455 460Tyr Ser His Arg
Leu Ser Asn Ile Arg Leu Ile Ser Gly Asn Thr Leu465 470 475 480Arg
Ala Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn 485 490
495Thr Ile Ser Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ser Phe
500 505 510Asn Leu Asn Ser Gly Thr Ser Val Val Ser Gly Pro Gly Phe
Thr Gly 515 520 525Gly Asp Ile Ile Arg Thr Asn Val Asn Gly Ser Val
Leu Ser Met Gly 530 535 540Leu Asn Phe Asn Asn Thr Ser Leu Gln Arg
Tyr Arg Val Arg Val Arg545 550 555 560Tyr Ala Ala Ser Gln Thr Met
Val Leu Arg Val Thr Val Gly Gly Ser 565 570 575Thr Thr Phe Asp Gln
Gly Phe Pro Ser Thr Met Ser Ala Asn Glu Ser 580 585 590Leu Thr Ser
Gln Ser Phe Arg Phe Ala Glu Phe Pro Val Gly Ile Ser 595 600 605Ala
Ser Gly Ser Gln Thr Ala Gly Ile Ser Ile Ser Asn Asn Ala Gly 610 615
620Arg Gln Thr Phe His Phe Asp Lys Ile Glu Phe Ile Pro Ile Thr
Ala625 630 635 640Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln
Glu Ala Val Asn 645 650 655Ala Leu Phe Thr Asn Thr Asn Pro Arg Arg
Leu Lys Thr Gly Val Thr 660 665 670Asp Tyr His Ile Asp Glu Val Ser
Asn Leu Val Ala Cys Leu Ser Asp 675 680 685Glu Phe Cys Leu Asp Glu
Lys Arg Glu Leu Leu Glu Lys Val Lys Tyr 690 695 700Ala Lys Arg Leu
Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe705 710 715 720Thr
Ser Ile Asn Lys Gln Pro Asp Phe Asn Ser Asn Asn Glu Gln Ser 725 730
735Asn Phe Thr Ser Ile His Glu Gln Ser Glu His Gly Trp Trp Gly Ser
740 745 750Glu Asn Ile Thr Ile Gln Glu Gly Asn Asp Val Phe Lys Glu
Asn Tyr 755 760 765Val Thr Leu Pro Gly Thr Phe Asn Glu Cys Tyr Pro
Thr Tyr Leu Tyr 770 775 780Gln Lys Ile Gly Glu Ala Glu Leu Lys Ala
Tyr Thr Arg Tyr Gln Leu785 790 795 800Ser Gly Tyr Ile Glu Asp Ser
Gln Asp Leu Glu Ile Tyr Leu Ile Arg 805 810 815Tyr Asn Ala Lys His
Glu Thr Leu Asp Val Pro Gly Thr Glu Ser Val 820 825 830Trp Pro Leu
Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu Pro Asn 835 840 845Arg
Cys Ala Pro His Phe Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys 850 855
860Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu
Asp865 870 875 880Ile Asp Val Gly Cys Ile Asp Leu His Glu Asn Leu
Gly Val Trp Val 885 890 895Val Phe Lys Ile Lys Thr Gln Glu Gly His
Ala Arg Leu Gly Asn Leu 900 905 910Glu Phe Ile Glu Glu Lys Pro Leu
Leu Gly Glu Ala Leu Ser Arg Val 915 920 925Lys Arg Ala Glu Lys Lys
Trp Arg Asp Lys Arg Glu Lys Leu Gln Leu 930 935 940Glu Thr Lys Arg
Val Tyr Thr Glu Ala Lys Glu Ala Val Asp Ala Leu945 950 955 960Phe
Val Asp Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile Gly 965 970
975Met Ile His Ala Ala Asp Lys Leu Val His Arg Ile Arg Glu Ala Tyr
980 985 990Leu Ser Glu Leu Ser Val Ile Pro Gly Val Asn Ala Glu Ile
Phe Glu 995 1000 1005Glu Leu Glu Gly Arg Ile Ile Thr Ala Ile Ser
Leu Tyr Asp Ala 1010 1015 1020Arg Asn Val Val Lys Asn Gly Asp Phe
Asn Asn Gly Leu Ala Cys 1025 1030 1035Trp Asn Val Lys Gly His Val
Asp Val Gln Gln Ser His His Arg 1040 1045 1050Ser Val Leu Val Ile
Pro Glu Trp Glu Ala Glu Val Ser Gln Ala 1055 1060 1065Val Arg Val
Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala 1070 1075 1080Tyr
Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile 1085 1090
1095Glu Asn Asn Thr Asp Glu Leu Lys Phe Lys Asn Cys Glu Glu Glu
1100 1105 1110Glu Val Tyr Pro Thr Asp Thr Gly Thr Cys Asn Asp Tyr
Thr Ala 1115 1120 1125His Gln Gly Thr Ala Val Cys Asn Ser Arg Asn
Ala Gly Tyr Glu 1130 1135 1140Asp Ala Tyr Glu Val Asp Thr Thr Ala
Ser Val Asn Tyr Lys Pro 1145 1150 1155Thr Tyr Glu Glu Glu Thr Tyr
Thr Asp Val Arg Arg Asp Asn His 1160 1165 1170Cys Glu Tyr Asp Arg
Gly Tyr Val Asn Tyr Pro Pro Val Pro Ala 1175 1180 1185Gly Tyr Met
Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys 1190 1195 1200Val
Trp Ile Glu Ile Gly Glu Thr Glu Gly Lys Phe Ile Val Asp 1205 1210
1215Ser Val Glu Leu Leu Leu Met Glu Glu 1220 1225
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References