U.S. patent application number 10/360899 was filed with the patent office on 2003-12-11 for polypeptide compositions toxic to anthonomus insects, and methods of use.
Invention is credited to Isaac, Barbara, Krieger (f/k/a Joyce), Elysia K., Mettus, Anne-Marie Light, Moshiri, Farhad, Sivasupramanian, Sakuntala.
Application Number | 20030229919 10/360899 |
Document ID | / |
Family ID | 22757619 |
Filed Date | 2003-12-11 |
United States Patent
Application |
20030229919 |
Kind Code |
A1 |
Isaac, Barbara ; et
al. |
December 11, 2003 |
Polypeptide compositions toxic to anthonomus insects, and methods
of use
Abstract
A novel gene encoding a Coleopteran inhibitory Bacillus
thuringiensis insecticidal crystal protein is disclosed. The
protein, tIC851, is insecticidally active and provides plant
protection from at least cotton boll weevil, Anthomomus grandis,
when applied to plants in an insecticidally effective
composition.
Inventors: |
Isaac, Barbara; (St.
Charles, MO) ; Krieger (f/k/a Joyce), Elysia K.;
(Kirkwood, MO) ; Mettus, Anne-Marie Light;
(Feasterville, PA) ; Moshiri, Farhad;
(Chesterfield, MO) ; Sivasupramanian, Sakuntala;
(Chesterfield, MO) |
Correspondence
Address: |
MONSANTO COMPANY
800 N. LINDBERGH BLVD.
ATTENTION: G.P. WUELLNER, IP PARALEGAL, (E2NA)
ST. LOUIS
MO
63167
US
|
Family ID: |
22757619 |
Appl. No.: |
10/360899 |
Filed: |
February 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10360899 |
Feb 7, 2003 |
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09853533 |
May 11, 2001 |
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6541448 |
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60204367 |
May 15, 2000 |
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Current U.S.
Class: |
800/279 ;
435/320.1; 435/419; 435/69.1; 514/2.1; 514/4.5; 530/350; 536/23.6;
800/314 |
Current CPC
Class: |
C07K 14/325 20130101;
C12N 15/8286 20130101; Y02A 40/146 20180101; A01N 63/50 20200101;
A01N 63/50 20200101; A01N 63/23 20200101 |
Class at
Publication: |
800/279 ;
800/314; 530/350; 435/69.1; 435/320.1; 435/419; 514/12;
536/23.6 |
International
Class: |
A01N 063/00; A01H
001/00; A01H 005/00; C12N 015/82; C07H 021/04; C07K 014/195; C12N
005/04 |
Claims
What is claimed is:
1. An isolated and purified polypeptide comprising the amino acid
sequence as set forth in SEQ ID NO: 8.
2. The polypeptide of claim 1 exhibiting insecticidal activity when
provided orally to a susceptible insect larva.
3. The polypeptide of claim 2 exhibiting insecticidal activity when
provided in an orally administrable diet to a Coleopteran insect
larva.
4. The polypeptide of claim 3 wherein said insect larva is a cotton
boll weevil larva.
5. The polypeptide of claim 1 encoded by a nucleic acid sequence
comprising at least the open reading frame as set forth in SEQ ID
NO: 7 from nucleotide position 28 through nucleotide position
1923.
6. A composition comprising an insecticidally effective amount of
the polypeptide of claim 1 wherein said composition is a bacterial
cell comprising a polynucleotide sequence that encodes said
polypeptide, said composition being selected from the group
consisting of a cell extract, cell suspension, cell homogenate,
cell lysate, cell supernatant, cell filtrate, or cell pellet.
7. The composition of claim 6 wherein said bacterial cell is a
bacterial species selected from the group consisting of Bacillus,
Escherichia, Salmonella, Agrobacterium, and Pseudomonas.
8. The composition of claim 7 wherein said bacterial cell is
selected from the group consisting of EG4135 and EG4268.
9. A composition comprising an insecticidally effective amount of
the polypeptide of claim 1 wherein said composition is formulated
as a powder, dust, pellet, granule, spray, emulsion, colloid, or
solution.
10. The composition according to claim 6, prepared by desiccation
lyophilization, homogenization, extraction, filtration,
centrifugation, sedimentation, or concentration.
11. The composition of claim 10 wherein said polypeptide is present
in a concentration of from about 0.001% to about 99% by weight.
12. An isolated and purified polynucleotide sequence encoding the
polypeptide of SEQ ID NO: 8.
13. The polynucleotide sequence of claim 12 wherein said
polypeptide exhibits insecticidal activity when provided orally to
a susceptible insect larva.
14. The polynucleotide sequence of claim 13 wherein said
polypeptide exhibits insecticidal activity when provided in an
orally administrable diet or composition to a Coleopteran insect
larva.
15. The polynucleotide sequence of claim 14 wherein said insect
larva is a cotton boll weevil larva.
16. The polynucleotide sequence which is or is complementary to the
polynucleotide sequence of claim 15 and which hybridizes under
stringent conditions to a polynucleotide sequence complementary to
or encoding the polypeptide as set forth in SEQ ID NO: 8.
17. A method for protecting a cotton plant from boll weevil
infestation comprising providing to a boll weevil in its diet a
plant transformed to express a protein toxic to said weevil wherein
said protein is expressed in sufficient amounts in said plant's
tissues to control boll weevil infestation and wherein said protein
is selected from the group consisting of Cry22Aa, ET70, and
tIC851.
18. A method for protecting a cotton plant from boll weevil
infestation comprising providing to a boll weevil in its diet a
plant or plant tissue transformed to express one or more proteins
toxic to said weevil wherein said proteins are expressed in
sufficient amounts alone or in combination to control boll weevil
infestation and wherein said proteins are selected from the group
consisting of Cry22Aa, ET70, and tIC851.
19. A vector for use in transforming a host cell, wherein said
vector comprises a polynucleotide sequence encoding the polypeptide
as set forth in SEQ ID NO: 8.
20. The vector of claim 19, wherein said vector is plasmid
pIC17501.
21. The vector of claim 19 wherein said host cell is selected from
the group consisting of a plant cell and a bacterial cell.
22. A plant tissue transformed with a polynucleotide sequence which
expresses the polypeptide of claim 1, wherein said tissue is
selected from the group consisting of a plant cell, an embryonic
plant tissue, plant calli, a leaf, a plant stem, a plant root, a
plant flower, a fruit, a fruiting body, a boll, and a plant
seed.
23. The plant tissue of claim 22 wherein said tissue comprises said
polypeptide present in a coleopteran insect inhibitory effective
amount.
24. The plant tissue of claim 23 wherein said coleopteran insect is
a cotton boll weevil.
25. The plant tissue of claim 22 selected from the group of plants
consisting of corn, wheat, cotton, soybean, oat, rice, rye,
sorghum, sugarcane, tomato, tobacco, kapok, flax, potato, barley,
turf grass, pasture grass, berry bush, fruit tree, legume,
vegetable, ornamental plant, shrub, cactus, succulent, deciduous
tree, and evergreen tree.
26. A method of making a transgenic plant resistant to coleopteran
insect infestation comprising incorporating into a genome of a
plant cell a polynucleotide comprising at least a plant functional
promoter operably linked to a nucleotide sequence encoding the
polypeptide of SEQ ID NO: 8, isolating and propagating a plant cell
transformed with said polynucleotide, regenerating a plant from
said plant cell transformed with said polynucleotide, and
propagating said plant from progeny, wherein said plant expresses
an insecticidally effective amount of said polypeptide from said
polynucleotide.
27. The method of claim 26 wherein said plant cell is either a
monocot or a dicot plant cell.
28. The method of claim 27 wherein said monocot plant cell is
selected from the group of plant cells consisting of corn, wheat,
rye, barley, rice, banana, sugarcane, oat, flax, turf grass,
pasture grass, and sorghum cells.
29. The method of claim 27 wherein said dicot plant cell is
selected from the group of plant cells consisting of cotton,
soybean, canola, potato, tomato, fruit tree, shrub, vegetable, and
berry cells.
30. An isolated and purified antibody which specifically binds to
the peptide as set forth in SEQ ID NO: 8 or an epitope therein,
said antibody produced from the immune system of a vertebrate in
response to the exposure of all or an antigenic part of said
peptide to the animal's immune system.
31. A method for detecting the presence of a peptide as set forth
in SEQ ID NO: 8 in a sample comprising obtaining a solution
suspected of containing said peptide, probing said solution with
the antibody of claim 30, and detecting the binding of said
antibody to said peptide.
32. A kit for detecting the presence of the peptide of SEQ ID NO: 8
in a sample comprising, in suitable container means, an antibody
that binds to said peptide, reagents necessary for mixing the
peptide and antibody in a solution, at least a first
immunodetection reagent providing said antibody along with control
antibody, control antigen, and the reagents and instructions
necessary for detecting said binding.
33. A plant cell transformed with a polynucleotide sequence that
expresses one or more of the polypeptides as set forth in SEQ ID
NO: 2, SEQ ID NO: 8, and SEQ ID NO: 10 or insecticidal fragments
thereof, wherein said cell produces an amount of said one or more
polypeptides effective for controlling a Coleopteran insect pest
infestation.
34. The plant cell of claim 33 wherein said Coleopteran insect pest
is a cotton boll weevil and said plant cell is a cotton plant cell.
Description
1.0 BACKGROUND OF THE INVENTION
[0001] 1.1 Field of the Invention
[0002] The present invention relates generally to the fields of
molecular biology. Methods and compositions comprising DNA
sequences, and polypeptides derived from Bacillus thuringiensis for
use in insecticidal formulations and the development of transgenic
insect-resistant plants are provided. Novel nucleic acids obtained
from Bacillus thuringiensis that encode coleopteran-toxic
polypeptides are disclosed. Various methods for making and using
these nucleic acids, synthetically modified DNA sequences encoding
tIC851 polypeptides, and native and synthetic polypeptide
compositions are also disclosed. The use of DNA sequences as
diagnostic probes and templates for protein synthesis, and the use
of polypeptides, fusion proteins, antibodies, and peptide fragments
in various insecticidal, immunological, and diagnostic applications
are also disclosed, as are methods of making and using nucleic acid
sequences in the development of transgenic plant cells comprising
the polynucleotides.
[0003] 1.2 Description of the Related Art
[0004] Environmentally-sensitive methods for controlling or
eradicating insect infestation are desirable in many instances, in
particular when crops of commercial interest are at issue. 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 well known in the art, and is characterized
morphologically as a Gram-positive bacterium that produces crystal
proteins or inclusion bodies which are aggregations of proteins
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.
[0005] There are several toxin categories established based on
primary structure information and the degree of toxin similarities
to another. 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).
[0006] 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 Schnepf et al. (Microbiol. Mol. Biol. Rev.
(1998) 62:775-806) discusses the genes and proteins that were
identified in B. thuringiensis prior to 1998, and sets forth the
most recent nomenclature and classification scheme as applied to B.
thuringiensis insecticidal genes and proteins. Using older
nomenclature classification schemes, cry1 genes were deemed to
encode lepidopteran-toxic Cry1 proteins, cry2 genes were deemed to
encode Cry2 proteins toxic to both lepidopterans and dipterans,
cry3 genes were deemed to encode coleopteran-toxic Cry3 proteins,
and cry4 genes were deemed to encode dipteran-toxic Cry4 proteins.
However, new nomenclature systematically classifies the Cry
proteins based upon amino acid sequence homology rather than upon
insect target specificities. The classification scheme for many
known toxins, not including allelic variations in individual
proteins, including dendograms and full Bacillus thuringiensis
toxin lists is summarized and regularly updated at
http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html
[0007] Most of the nearly 200 Bt crystal protein toxins presently
known have some degree of lepidopteran activity associated with
them. The large majority of Bacillus thuringiensis insecticidal
proteins which have been identified do not have coleopteran
controlling activity. Therefore, it is particularly important at
least for commercial purposes to identify additional coleopteran
specific insecticidal proteins.
[0008] Cry3 proteins generally display coleopteran activity,
however, these generally have limited host range specificity and
are not significantly toxic to target pests unless ingested in very
high doses. The cloning and expression of the cry3Bb gene has been
described (Donovan et al., 1992). This gene codes for a protein of
74 kDa with activity against Coleopteran insects, particularly the
Colorado potato beetle (CPB) and the southern corn root worm
(SCRW). Improved Cry3Bb proteins have been engineered which display
increased toxicity at the same or lower doses than the wild type
protein (U.S. Pat. No. 6,023,013; Feb. 8, 2000).
[0009] A B. thuringiensis strain, PS201T6, was reported to have
activity against WCRW (Diabrotica virgifera virgifera) (U.S. Pat.
No. 5,436,002). This strain also had activity against Musca
domestica, Aedes aegypti, and Liriomyza trifoli. The vip1A gene,
which produces a vegetative, soluble, insecticidal protein, has
been cloned and sequenced (Intl. Pat. AppI. Pub. No. WO 96/10083,
1996). This gene produces a protein of approximately 80 kDa with
activity against both WCRW and Northern Corn Root Worm (NCRW).
Another toxin protein with activity against coleopteran insects,
including WCRW, is Cry1Ia, an 81-kDa polypeptide, the gene encoding
which has been cloned and sequenced (Intl. Pat. Appl. Pub. No. WO
90/13651, 1990).
2.0 SUMMARY OF THE INVENTION
[0010] The polypeptide of the present invention and the novel DNA
sequences that encode the protein represent a new B. thuringiensis
crystal protein and gene, and share only insubstantial sequence
homology with any previously identified coleopteran inhibitory
endotoxins described in the prior art. Similarly, the B.
thuringiensis strains of the present invention comprise novel gene
sequences that express a polypeptide having insecticidal activity
against coleopteran insects, the cotton boll weevil (Anthonomus
grandis Boheman) in particular.
[0011] Disclosed and claimed herein is an isolated Bacillus
thuringiensis .delta.-endotoxin polypeptide comprising SEQ ID NO:
8. The inventors have identified an insecticidally-active
polypeptide comprising the 632 amino acid long sequence of SEQ ID
NO: 8 which displays insecticidal activity against coleopteran
insects. For example, the inventors have shown that a
&endotoxin polypeptide comprising the sequence of SEQ ID NO: 8
has insecticidal activity against boll weevil larvae (BWV), but not
against western corn rootworm larvae.
[0012] The polypeptide of SEQ ID NO: 8 is encoded by a nucleic acid
segment comprising at least the open reading frame as shown in SEQ
ID NO: 7 from nucleotide position 28 through nucleotide position
1923. The invention also discloses compositions and insecticidal
formulations that comprise such a polypeptide. Such composition may
be a cell extract, cell suspension, cell homogenate, cell lysate,
cell supernatant, cell filtrate, or cell pellet of a bacteria cell
that comprises a polynucleotide that encodes such a polypeptide.
Exemplary bacterial cells that produce such a polypeptide include
Bacillus thuringiensis EG4135 and EG4268, deposited with NRRL
respectively on Apr. 28, 2000. The composition as described in
detail below may be formulated as a powder, dust, pellet, granule,
spray, emulsion, colloid, solution, or such like, and may be
preparable by such conventional means as desiccation,
lyophilization, homogenization, extraction, filtration,
centrifugation, sedimentation, or concentration of a culture of
cells comprising the polypeptide. Preferably such compositions are
obtainable from cultures of Bacillus thuringiensis EG4135 and
EG4268 cells. In all such compositions that contain at least one
such insecticidal polypeptide, the polypeptide may be present in a
concentration of from about 0.001% to about 99% by weight.
[0013] An exemplary insecticidal polypeptide formulation may be
prepared by a process comprising the steps of culturing Bacillus
thuringiensis EG4135 and EG4268 cells under conditions effective to
produce the insecticidal polypeptide; and obtaining the
insecticidal polypeptide so produced.
[0014] For example, the invention discloses and claims a method of
preparing a .delta.-endotoxin polypeptide having insecticidal
activity against a coleopteran insect. The method generally
involves isolating from a culture of Bacillus thuringiensis EG4135
and EG4268 cells that have been grown under appropriate conditions,
the .delta.-endotoxin polypeptide produced by the cells. Such
polypeptides may be isolated from the cell culture or supernatant
or from spore suspensions derived from the cell culture and used in
the native form, or may be otherwise purified or concentrated as
appropriate for the particular application.
[0015] A method of controlling a coleopteran insect population is
also provided by the invention. The method generally involves
contacting the population with an insecticidally-effective amount
of a polypeptide comprising the amino acid sequence of SEQ ID NO:
8. Such methods may be used to kill or reduce the numbers of
coleopteran insects in a given area, or may be prophylactically
applied to an environmental area to prevent infestation by a
susceptible insect. Preferably the insect ingests, or is contacted
with, an insecticidally-effective amount of the polypeptide.
[0016] Additionally, the invention provides a purified antibody
that specifically binds to the insecticidal polypeptide. Also
provided are methods of preparing such an antibody, and methods for
using the antibody to isolate, identify, characterize, and/or
purify polypeptides to which such an antibody specifically binds.
Immunological kits and immunodetection methods useful in the
identification of such polypeptides and peptide fragments and/or
epitopes thereof are provided in detail herein, and also represent
important aspects of the present invention.
[0017] Such antibodies may be used to detect the presence of such
polypeptides in a sample, or may be used as described hereinbelow
in a variety of immunological methods. An exemplary method for
detecting a .delta.-endotoxin polypeptide in a biological sample
generally involves obtaining a biological sample suspected of
containing a .delta.-endotoxin polypeptide; contacting the sample
with an antibody that specifically binds to the polypeptide, under
conditions effective to allow the formation of complexes; and
detecting the complexes so formed.
[0018] For such methods, the invention also provides an
immunodetection kit. Such a kit generally contains, in suitable
container means, an antibody that binds to the .delta.-endotoxin
polypeptide, and at least a first immunodetection reagent.
Optionally, the kit may provide additional reagents or instructions
for using the antibody in the detection of .delta.-endotoxin
polypeptides in a sample.
[0019] Preparation of such antibodies may be achieved using the
disclosed polypeptide as an antigen in an animal as described
below. Antigenic epitopes, shorter peptides, peptide fusions,
carrier-linked peptide fragments, and the like may also be
generated from a whole or a portion of the polypeptide sequence
disclosed in SEQ ID NO: 8. Particularly preferred peptides are
those that comprise at least 10 contiguous amino acids from the
sequence disclosed in SEQ ID NO: 8.
[0020] In another embodiment, the present invention also provides
nucleic acid segments that comprise a selected nucleotide sequence
region that comprises the polynucleotide sequence of SEQ ID NO: 7.
In preferred embodiments, this selected nucleotide sequence region
comprises a gene that encodes a polypeptide comprising at least SEQ
ID NO: 8.
[0021] Another aspect of the invention relates to a
biologically-pure culture of a wild-type B. thuringiensis bacterium
selected from the strains EG4135 and EG4268, deposited on Apr. 28,
2000 with the Agricultural Research Culture Collection, Northern
Regional Research Laboratory (NRRL), Peoria, Ill. Also deposited
was strain sIC8501 which is an E. coli DH5a containing plasmid
pIC17501 which contains at least the native B. thuringiensis strain
EG4135 tIC851 coding sequence. These strains were deposited under
the terms of the Budapest Treaty, and viability statements pursuant
to International Receipt Form BP/4 were obtained. B. thuringiensis
strains EG4135 and EG4268 are naturally-occurring strains that
contain at least one sequence region encoding the 632 amino acid
long polypeptide sequence in SEQ ID NO: 8.
[0022] A further embodiment of the invention relates to a vector
comprising a sequence region that encodes a polypeptide comprising
the amino acid sequence of SEQ ID NO: 8, a recombinant host cell
transformed with such a recombinant vector, and biologically-pure
cultures of recombinant bacteria transformed with a polynucleotide
sequence that encodes the polypeptide disclosed in SEQ ID NO: 8.
Exemplary vectors, recombinant host cells, transgenic cell lines,
and transgenic plants comprising at least a first sequence region
that encodes a polypeptide comprising the sequence of SEQ ID NO: 8
are described in detail herein.
[0023] The present invention also provides transformed host cells,
embryonic plant tissue, plant calli, plantlets, and transgenic
plants that comprise a selected sequence region that encodes the
insecticidal polypeptide. Such cells are preferably prokaryotic or
eukaryotic cells such as bacterial, fungal, or plant cells, with
exemplary bacterial cells including Bacillus thuringiensis,
Bacillus subtilis, Bacillus megaterium, Bacillus cereus,
Escherichia, Salmonella, Agrobacterium or Pseudomonas cells.
[0024] The plants and plant host cells are preferably
monocotyledonous or dicotyledonous plant cells such as corn, wheat,
soybean, oat, cotton, rice, rye, sorghum, sugarcane, tomato,
tobacco, kapok, flax, potato, barley, turf grass, pasture grass,
berry, fruit, legume, vegetable, ornamental plant, shrub, cactus,
succulent, and tree cell.
[0025] Transgenic plants of the present invention preferably have
incorporated into their genome or transformed into their
chloroplast or plastid genomes a selected polynucleotide (or
"transgene"), that comprises at least a first sequence region that
encodes the insecticidal polypeptide of SEQ ID NO: 8. Transgenic
plants are also meant to comprise progeny (descendant, offspring,
etc.) of any generation of such a transgenic plant. A seed of any
generation of all such transgenic insect-resistant plants wherein
said seed comprises a DNA sequence encoding the polypeptide of the
present invention is also an important aspect of the invention.
[0026] Insect resistant, crossed fertile transgenic plants
comprising a transgene that encodes the polypeptide of SEQ ID NO: 8
may be prepared by a method that generally involves obtaining a
fertile transgenic plant that contains a chromosomally incorporated
transgene encoding the insecticidal polypeptide of SEQ ID NO: 8;
operably linked to a promoter active in the plant; crossing the
fertile transgenic plant with a second plant lacking the transgene
to obtain a third plant comprising the transgene; and backcrossing
the third plant to obtain a backcrossed fertile plant. In such
cases, the transgene may be inherited through a male parent or
through a female parent. The second plant may be an inbred, and the
third plant may be a hybrid.
[0027] Likewise, an insect resistant hybrid, transgenic plant may
be prepared by a method that generally involves crossing a first
and a second inbred plant, wherein one or both of the first and
second inbred plants comprises a chromosomally incorporated
transgene that encodes the polypeptide of SEQ ID NO: 8 operably
linked to a plant expressible promoter that expresses the
transgene. In illustrative embodiments, the first and second inbred
plants may be monocot plants selected from the group consisting of:
corn, wheat, rice, barley, oats, rye, sorghum, turfgrass and
sugarcane.
[0028] In related embodiment, the invention also provides a method
of preparing an insect resistant plant. The method generally
involves contacting a recipient plant cell with a DNA composition
comprising at least a first transgene that encodes the polypeptide
of SEQ ID NO: 8 under conditions permitting the uptake of the DNA
composition; selecting a recipient cell comprising a chromosomally
incorporated transgene that encodes the polypeptide; regenerating a
plant from the selected cell; and identifying a fertile transgenic
plant that has enhanced insect resistance relative to the
corresponding non-transformed plant.
[0029] A method of producing transgenic seed generally involves
obtaining a fertile transgenic plant comprising a chromosomally
integrated transgene that encodes a polypeptide comprising the
amino acid sequence of SEQ ID NO: 8, operably linked to a promoter
that expresses the transgene in a plant; and growing the plant
under appropriate conditions to produce the transgenic seed.
[0030] A method of producing progeny of any generation of an insect
resistance-enhanced fertile transgenic plant is also provided by
the invention. The method generally involves collecting transgenic
seed from a transgenic plant comprising a chromosomally integrated
transgene that encodes the polypeptide of SEQ ID NO: 8, operably
linked to a promoter that expresses the transgene in the plant;
planting the collected transgenic seed; and growing the progeny
transgenic plants from the seed.
[0031] These methods for creating transgenic plants, progeny and
seed may involve contacting the plant cell with the DNA composition
using one of the processes well-known for plant cell transformation
such as microprojectile bombardment, electroporation or
Agrobacterium-mediated transformation.
[0032] An exemplary method disclosed herein provides for protecting
a plant from cotton boll weevil infestation comprising providing to
a boll weevil in its diet a plant transformed to express a protein
toxic to said weevil wherein said protein is expressed in
sufficient amounts to control boll weevil infestation and wherein
said protein is selected from the group consisting of Cry22Aa,
ET70, and tIC851. In a further embodiment of this method, a plant
expressing two or more of these proteins for the purpose of
reducing boll weevil infestation is contemplated, in particular for
reducing the development of races of boll weevils resistant to any
of these proteins.
[0033] These and other embodiments of the present invention will be
apparent to those of skill in the art from the following examples
and claims, having benefit of the teachings of the Specification
herein.
[0034] 2.1 tIC851 Polynucleotide Sequences
[0035] The present invention provides polynucleotide sequences that
can be isolated from Bacillus thuringiensis strains, that are free
from total genomic DNA, and that encode the novel insecticidal
polypeptides and peptide fragments disclosed herein. The
polynucleotides encoding these peptides and polypeptides may encode
active insecticidal proteins, or peptide fragments, polypeptide
subunits, functional domains, or the like of one or more tIC851or
tIC851-related crystal proteins, such as the polypeptide disclosed
in SEQ ID NO: 8. In addition the invention encompasses nucleic acid
sequences which may be synthesized entirely in vitro using methods
that are well-known to those of skill in the art which encode the
novel tIC851 polypeptide, peptides, peptide fragments, subunits, or
functional domains disclosed herein.
[0036] As used herein, the term "nucleic acid sequence" or
"polynucleotide" refers to a nucleic acid molecule that has been
isolated free of the total genomic DNA or otherwise of a particular
species. Therefore, a nucleic acid sequence or polynucleotide
encoding an endotoxin polypeptide refers to a nucleic acid molecule
that comprises at least a first crystal protein-encoding sequence
yet is isolated away from, or purified free from, total genomic DNA
of the species from which the nucleic acid sequence 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 sequence", are polynucleotide sequences and smaller
fragments of such sequences, and also recombinant vectors,
including, for example, plasmids, cosmids, phagemids, phage,
virions, baculoviruses, artificial chromosomes, viruses, and the
like. Accordingly, polynucleotide sequences that have between about
70% and about 80%, or more preferably between about 81% and about
90%, or even more preferably between about 91% and about 99%
nucleic acid sequence identity or functional equivalence to the
polynucleotide sequence of SEQ ID NO: 7 will be sequences that are
"essentially as set forth in SEQ ID NO: 7." Highly preferred
sequences are those which are preferably from about 91% to about
100% identical or functionally equivalent to the nucleotide
sequence of SEQ ID NO: 7. Other preferred sequences that encode
tIC851- or tIC851-related sequences are those which are from about
81% to about 90% identical or functionally equivalent to the
polynucleotide sequence set forth in SEQ ID NO: 7. Likewise,
sequences that are from about 71% to about 80% identical or
functionally equivalent to the polynucleotide sequence set forth in
SEQ ID NO: 7 are also contemplated to be useful in the practice of
the present invention.
[0037] Similarly, a polynucleotide comprising an isolated,
purified, or selected gene or sequence region refers to a
polynucleotide 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-, or
polypeptide-encoding unit. As will be understood by those in the
art, this functional term includes both genomic sequences, operator
sequences and smaller engineered gene segments that express, or may
be adapted to express, proteins, polypeptides or peptides. In
certain embodiments, a nucleic acid segment will comprise at least
a first gene that encodes a polypeptide comprising the sequence of
SEQ ID NO: 8.
[0038] To permit expression of the gene, and translation of the
mRNA into mature polypeptide, the nucleic acid sequence preferably
also comprises at least a first promoter operably linked to the
gene to express the insecticidal polypeptide in a host cell
transformed with this nucleic acid sequence. The promoter may be an
endogenous promoter, or alternatively, a heterologous promoter
selected for its ability to promote expression of the gene in one
or more particular cell types. For example, in the creation of
transgenic plants and plant cells comprising a tIC851 gene, the
heterologous promoter of choice is one that is plant-expressible,
and in many instances, may preferably be a plant-expressible
promoter that is tissue- or cell cycle-specific. The selection of
plant-expressible promoters is well-known to those skilled in the
art of plant transformation, and exemplary suitable promoters are
described herein. In certain embodiments, the plant-expressible
promoter may be selected from the group consisting of corn sucrose
synthetase 1, corn alcohol dehydrogenase 1, corn light harvesting
complex, corn heat shock protein, pea small subunit RuBP
carboxylase, Ti plasmid mannopine synthase, Ti plasmid nopaline
synthase, petunia chalcone isomerase, bean glycine rich protein 1,
Potato patatin, lectin, CaMV 35S, and the S-E9 small subunit RuBP
carboxylase promoter.
[0039] "Isolated substantially away from other coding sequences"
means that the gene of interest, in this case, a gene encoding a
bacterial crystal protein, forms the significant part of the coding
region of the DNA segment, and that the DNA segment does not
contain large portions of naturally-occurring coding DNA, such as
large chromosomal fragments or other functional genes or operon
coding regions. Of course, this refers to the DNA segment as
originally isolated, and does not exclude genes, recombinant genes,
synthetic linkers, or coding regions later added to the segment by
the hand of man.
[0040] It will also be understood that this invention is not
limited to the particular nucleic acid sequences which encode
peptides of the present invention, or which encode the amino acid
sequence of SEQ ID NO: 8, including the DNA sequence which is
particularly disclosed in SEQ ID NO: 7. Recombinant vectors and
isolated DNA segments may therefore variously include the
polypeptide-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.
[0041] The DNA sequences 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. 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). 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
sequence, whether encoding a full-length insecticidal 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. In many cases, the promoter may
be the native tIC851 promoter, or alternatively, a heterologous
promoter, such as those of bacterial origin (including promoters
from other crystal proteins), fungal origin, viral, phage or
phagemid origin (including promoters such as CaMV35, and its
derivatives, T3, T7, .lambda., and .phi. promoters and the like),
or plant origin (including constitutive, inducible, and/or
tissue-specific promoters and the like).
[0042] In other embodiments, it is contemplated that certain
advantages will be gained by positioning the coding DNA sequence
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
sequence 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 sequence, 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).
[0043] In yet another aspect, the present invention provides
methods for producing a transgenic plant that expresses a selected
nucleic acid sequence comprising a sequence region that encodes the
novel endotoxin polypeptides of the present invention. The process
of producing transgenic plants is well-known in the art. In
general, the method comprises transforming a suitable plant host
cell with a DNA sequence that contains a promoter operatively
linked to a coding region that encodes one or more tIC851
polypeptides. Such a coding region is generally operatively linked
to at least a first transcription-terminatin- g 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, gene sequence, or sequence region that
encodes at least 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 the genome of a
transgenic plant of the present invention will have been augmented
through the stable introduction of one or more transgenes, either
native, synthetically modified, or mutated, that encodes an
insecticidal polypeptide that is identical to, or highly homologous
to the polypeptide disclosed in SEQ ID NO: 8. In some instances,
more than one transgene will be incorporated into the genome of the
transformed host plant cell. Such is the case when more than one
crystal protein-encoding DNA sequence 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. Alternatively, a second transgene may be introduced into the
plant cell to confer additional phenotypic traits to the plant.
Such transgenes may confer resistance to one or more insects,
bacteria, fungi, viruses, nematodes, or other pathogens.
[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
plant cells, and regeneration of a transgenic cell line from a
transformed cell, cell culture, embryo, or callus tissue are
well-known in the art, and are discussed herein. Vectors,
(including plasmids, cosmids, phage, phagemids, baculovirus,
viruses, virions, BACs [bacterial artificial chromosomes], YACs
[yeast artificial chromosomes)) comprising at least a first nucleic
acid segment encoding an insecticidal polypeptide 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
nucleic acid constructs can further include structures such as
promoters, enhancers, polylinkers, introns, terminators, or even
gene sequences which have positively- or negatively-regulating
activity upon the cloned 5-endotoxin gene as desired. The DNA
sequence or gene may encode either a native or modified crystal
protein, which will be expressed in the resultant recombinant
cells, and/or which will confer to a transgenic plant comprising
such a segment, an improved phenotype (in this case, increased
resistance to insect attack, infestation, or colonization).
[0048] The preparation of a transgenic plant that comprises at
least one polynucleotide sequence encoding a tIC851 or tIC85
1-derived polypeptide for the purpose of increasing or enhancing
the resistance of such a plant to attack by a target insect
represents an important aspect of the invention. In particular, the
inventors describe herein the preparation of insect-resistant
monocotyledonous or dicotyledonous plants, by incorporating into
such a plant, a transgenic DNA sequence encoding at least one
tIC851 polypeptide toxic to a coleopteran insect.
[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 sequences encoding
one or more tIC851 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.
[0050] 2.3 Definitions
[0051] The following words and phrases have the meanings set forth
below.
[0052] A, an: In keeping with long-standing patent tradition, "a"
or "an" used throughout this disclosure is intended to mean "one or
more."
[0053] Comprising, comprises: In keeping with long-standing patent
tradition, "comprising" and "comprises" used throughout this
disclosure is intended to mean "including, but not limited to."
[0054] Expression: The combination of intracellular processes,
including at least transcription and often the subsequent
translation of mRNA of a coding DNA molecule such as a structural
gene to produce a polypeptide.
[0055] Promoter: A recognition site on a DNA sequence or group of
DNA sequences that provide an expression control element for a
structural gene or sequence to be transcribed and to which an RNA
polymerase specifically binds and initiates RNA synthesis
(transcription) of that gene or sequence to be transcribed.
[0056] Regeneration: The process of growing a plant from a plant
cell (e.g., plant protoplast or explant).
[0057] Structural gene: A DNA sequence that encodes a messenger RNA
which can be transcribed to produce a polypeptide.
[0058] Transformation: A process of introducing an exogenous DNA
sequence (e.g., a vector, a recombinant DNA molecule) into a cell,
protoplast, or organelle within a cell, in which that exogenous DNA
is incorporated into DNA native to the cell, or is capable of
autonomous replication within the cell.
[0059] Transformed cell: A cell whose genotype has been altered by
the introduction of an exogenous DNA sequence into that cell.
[0060] Transgenic cell: Any cell derived from or regenerated from a
transformed 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.
[0061] Transgenic plant: A plant or a progeny of any generation of
the plant that was derived from a transformed plant cell or
protoplast, wherein the plant nucleic acids contains an exogenous
selected nucleic acid sequence region not originally present in a
native, non-transgenic plant of the same variety. The terms
"transgenic plant" and "transformed plant" have sometimes been used
in the art as synonymous terms to define a plant whose native DNA
has been altered to contain a heterologous 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 cells as being a transgenic plant. Preferably,
transgenic plants of the present invention include those plants
that comprise at least a first selected polynucleotide that encodes
an insecticidal polypeptide. This selected polynucleotide is
preferably a 6-endotoxin coding region (or gene) operably linked to
at least a first promoter that expresses the coding region to
produce the insecticidal polypeptide in the transgenic plant.
Preferably, the transgenic plants of the present invention that
produce the encoded polypeptide demonstrate a phenotype of improved
resistance to target insect pests. Such transgenic plants, their
progeny, descendants, and seed from any such generation are
preferably insect resistant plants.
[0062] Vector: A nucleic acid molecule capable of replication in a
host cell and/or to which another nucleic acid sequence can be
operably linked so as to bring about replication of the attached
segment. Plasmids, phage, phagemids, and cosmids are all exemplary
vectors. In many embodiments, vectors are used as a vehicle to
introduce one or more selected polynucleotides into a host cell,
thereby generating a "transformed" or "recombinant" host cell.
3.0 BRIEF DESCRIPTION OF THE DRAWINGS
[0063] The drawings form part of the present specification and are
included to further demonstrate certain aspects of the present
invention. The invention may be better understood by reference to
one or more of these drawings in combination with the detailed
description of specific embodiments presented herein.
[0064] FIG. 1 illustrates the nucleotide sequence and amino acid
sequence translation of the tIC851 gene as derived from strains
EG4135 and 4268.
[0065] FIG. 2 illustrates an amino acid sequence alignment of the
related proteins CryET70 and Cry22Aa, as well as the bestfit
alignment of tIC851.
4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0066] 4.1 Some Advantages of the Invention
[0067] The present invention provides a novel .delta.-endotoxin,
designated tIC851, which is highly toxic to the cotton boll weevil,
Anthonomus grandis Boheman. This protein has an amino acid sequence
which is substantially unrelated to other .delta.-endotoxins that
are toxic to coleopteran insects. The identification of Cry22Aa and
CryET70 represented a new class of insecticidal crystal proteins.
Unlike other WCRW toxic insecticidal crystal proteins from B.
thuringiensis, CryET70 does not have significant toxicity to SCRW
or CPB. The only known protein that is related to CryET70 is
Cry22Aa, an insecticidal crystal protein that is reported to be
toxic only to hymenopteran insects (GenBank Accession No. 134547).
The inventors herein disclose a novel Bacillus thuringiensis
.delta.-endotoxin displaying only insubstantial similarity to
either CryET70 or to Cry22Aa, and displaying substantial
differences in insecticidal spectrum and activity when compared to
both of these proteins. The inventors also disclose that both
CryET70 and Cry22Aa have significant toxicity to larvae of the
cotton boll weevil.
[0068] 4.2 Insect Pests
[0069] Almost all field crops, plants, and commercial farming areas
are susceptible to attack by one or more insect pests. Particularly
problematic coleopteran pests are identified in Table 1.
1TABLE 1 TAXONOMY OF COLEOPTERAN PESTS IN THE SUBORDERS
ARCHOSTEMATA AND POLYPHAGA Infraorder &/or Superfamily Family
Subfamily Tribe Genus Species Cupedidae (reticulated Priacma P.
serrara beetles) Bostrichiformia Dermestidae (skin and Attagenus A.
pellio larder beetles) Chrysomeliformia Cerambycidae (long-
Agapanthia Agapanthia sp. horned beetles) Lepturinae Leptura
Leptura sp. (flower long-horned beetle) Rhagium Rhagium sp.
Megacyllene M. robiniae Prioninae Derobrachus D. geminatus
Tetraopes T. tetropthalmus Chrysomelidae (leaf Chlamisinae Exema E.
neglecta beetles) Chrysomelinae Chrysomelini Chrysomela C. tremula,
Chrysomela sp. Oreina O. cacaliae Doryphorini Chrysoline Chrysolina
sp. Leptinotarsa L. decemlineata (Colorado potato beetle)
Infraorder Family Subfamily Tribe Genus Species Gonioctenini
Gonioctena G. fornicata, G. holdausi, G. intermedia, G.
interposita, G. kamikawai, G. linnaeana, G. nigroplagiata, G.
occidentalis, G. olivacea, G. pallida, G. quin-quepunctata, G.
rubripennis, G. rufipes, G. tredecim-maculata, G. variabilis, G.
viminalis Timarchini Timarcha Timarcha sp. Criocerinae Oulema
Oulema sp. Galerucinae Galerucini Monoxia M. inornata, Monoxia sp.
Ophraella O. arctica, O. artemisiae, O. bilineata, O. communa, O.
conferta, O. cribrata, O. notata, O. notulata, O. nuda, O. pilosa,
O. sexvittata, O. slobodkini Luperini Cerotoma C. trifurcata
Diabrotica D. barberi (northern corn rootworm), D. undecimpunctata,
(southern corn rootworm), D. virgifera (western corn rootworm)
unclassified Lachnaia Lachnaia sp. Chrysomelidae Epitrix E.
cucumeris (Harris) (potato flea beetle), E. fuscala (eggplant flea
beetle) Curculionidae (weevils) Curculioninae Anthonomus A. grandis
(boll weevil) Entiminae Naupactini Aramigus A. conirostris, A.
globoculus, A. intermedius, A. planioculus, A. tesselatus
Otiorhynchus Otiorhynchus sp. Phyllobiini Diaprepes D. abbreviata
Phyllobius Phyllobius sp. Galapaganus G. galapagoensis Hyperinae
Hypera H. brunneipennis (Egyptian alfalfa weevil), H. postica
(alfalfa weevil), H. punctata (clover leaf weevil) Molytinae
Pissodes P. affinis, P. nemorensis, P. schwarzi, P. strobi, P.
terminalis Rhynchophorinae Sitophilini Sitophilus S. granarius
(granary weevil), S. zeamais (maize weevil) Nemonychidae
Lebanorhinus L. succinus Scolytidae lps I. acuminatus, I. amitinus,
I. cembrae, I. duplicatus, I. mannsfeldi, I. sexdentatus, I.
typographus Orthotomicus O. erosus Tomicus T. minor Cucujiformia
Coccinellidae (ladybird Epilachna E. borealis (squash ladybird
beetle), E. beetles) varivstis (Mexican bean beetle) Cucujidae
(flat bark Cryptolestes C. ferrugineus beetles) Oryzaephilus O.
surinamensis (saw-toothed grain (grain beetles) beetle) Lagriidae
(long-joined Lagria Lagria sp. beetles) Meloidae (blister beetles)
Epicauta E. funebris Meloe M. proscarabaeus Rhipiphoridae
Rhipiphorus R. fasciatus Tenebrionidae (darkling Alphitobius A.
diaperinus ground beetles) (lesser mealworm) Hegeter H. amaroides,
H. brevicollis, H. costipennis, H. fernandezi, H. glaber, H.
gomerensis, H. gran-canariensis, H. impressus, H. intercedens, H.
lateralis, H. plicifrons, H. politus, H. subrotundatus, H.
tenui-punctatus, H. transversus, H. webbianus Misolampus M. goudoti
Palorus P. ficicola, P. ratzeburgi (small-eyed flour beetle), P.
subdepressus (depressed flour beetle) Pimelia P. baetica, P.
canariensis, P. criba, P. elevata, P. estevezi, P.
fernan-deziopezi, P. grandis, P. granulicollis, P. integra, P.
interjecta, P. laevigata, P. lutaria, P. radula, P. sparsa, P.
variolosa Tenebrio T. molitor (yellow mealworm), T. obscurus (dark
mealworm) Tentyria T. schaumi Tribolium T. brevicornis, T.
castaneum (red flour beetle), T. confusum (confused flour beetle),
T. freemani, T. madens Zophobas Z. atratus Z. rugipes Elateriformia
- Octinodes Octinodes sp. Superfamily Elateroidea Pyrophorus P.
plagio-phthalamus Scarabaeiformia Lucanidae (Stag beetles) Dorcus
D. parallelo-pipedus Lucanus L. cervus Scarabaeidae Allomyrina A.
dichotoma (lamellicorn beetles) Cetoniinae (flower Pachnoda P.
marginata beetle) Dynastinae Xyloryctes X. faunus Geotrupinae
(earth- Geotrupes G. stercorosus boring dung beetles)
Melonlonthinae Costelytra C. zealandica (chafers) Holotrichia H.
diomphalia Melolontha M. melolontha (cockchafer) Odontria O.
striata O. variegata Prodontria P. bicolorata, P. capito, P.
lewisi, P. tarsis, P. modesta, P. pinguis, P. praelatella, P.
truncata, Prodontria sp. Scythrodes S. squalidus Rutelinae (shining
Popillia P. japonica (Japanese beetle) leaf chafers) Scarabaeinae
Copris C. lunaris (black dung beetle) Scarabaeus Scarabaeus sp.
(scarab) Staphyliniformia Hydrophilidae Cercyon Cercyon sp.
Silphidae Nicrophorus N. americanus, N. marginatus, N. orbicollis,
N. tomentosus Staphylinidae (rove Carpelimus Carpelimus sp.
beetles) Quedius Q. mesomelinus Tachyporus Tachyporus sp.
Xantholinus Xantholinus sp.
[0070] 4.3 Probes and Primers
[0071] 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-encoding gene sequence, e.g., a sequence such as that shown
in SEQ ID NO: 8 (tIC851), SEQ ID NO: 10 (Cry22Aa), and SEQ ID NO: 2
(CryET70). 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.
[0072] 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 thermal amplification
technology. Sequences of related crystal protein genes from other
species may also be amplified using such primers.
[0073] 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 an about 23 to about 40 or so long
nucleotide stretch of a crystal protein-encoding sequence, such as
that shown in SEQ ID NO: 7 (tIC851), SEQ ID NO: 9 (cry22Aa), or SEQ
ID NO: 1 (cryET70). A size of at least about 14 or 15 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 23 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 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, specifically
incorporated herein by reference, or by excising selected DNA
fragments from recombinant plasmids containing appropriate inserts
and suitable restriction sites.
[0074] 4.4 Expression Vectors
[0075] The present invention contemplates a polynucleotide of the
present invention comprised within one or more expression vectors.
Thus, in one embodiment an expression vector comprises a nucleic
acid segment containing a tIC851 gene operably linked to a promoter
which expresses the gene. Additionally, the coding region may also
be operably linked to a transcription-terminating region, whereby
the promoter drives the transcription of the coding region, and the
transcription-terminating region halts transcription at some point
3' of the coding region.
[0076] 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.
[0077] 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-derived crystal proteins
include any of the known crystal protein gene promoters, including
the tIC851 gene promoter itself. Alternatively, mutagenized or
recombinant promoters may be engineered by the hand of man and used
to promote expression of the novel gene segments disclosed
herein.
[0078] 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.
[0079] 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).
[0080] 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.
[0081] 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.
[0082] 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 storage protein
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.)
[0083] 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.,
1991a). The expression of the polypeptide is directed specifically
to the seeds of the transgenic plant.
[0084] 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.
[0085] Exemplary tissue-specific promoters are corn sucrose
synthetase 1 (Yang et al., 1990), corn alcohol dehydrogenase 1
(Vogel et al., 1989), corn light harvesting complex (Simpson,
1986), corn heat shock protein (Odell et al., 1985), pea small
subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al.,
1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti
plasmid nopaline synthase (Langridge et al., 1989), petunia
chalcone isomerase (Van Tunen et al., 1988), bean glycine rich
protein 1 (Keller et al., 1989), CaMV 35S transcript (Odell et al.,
1985) and Potato patatin (Wenzler et al., 1989). Preferred
promoters are the cauliflower mosaic virus (CaMV 35S) promoter and
the S-E9 small subunit RuBP carboxylase promoter.
[0086] 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.
[0087] 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). pCaMVCN (available from Pharmacia, Piscataway, N.J.)
includes the cauliflower mosaic virus CaMV 35S promoter.
[0088] 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).
[0089] 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).
[0090] 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 specifically incorporated herein by reference in their
entirety. Those vectors can be modified to include a coding
sequence in accordance with the present invention.
[0091] A variety of methods have been developed to operatively
insert a DNA sequence into a vector via complementary cohesive
termini or blunt ends. For instance, complementary homopolymer
tracts can be added to the DNA sequence to be inserted and to the
vector DNA. The vector and DNA sequence are then joined by hydrogen
bonding between the complementary homopolymeric tails to form
recombinant DNA molecules.
[0092] A coding region that encodes a polypeptide having the
ability to confer insecticidal activity to a cell is preferably a
tIC851 B. thuringiensis crystal protein-encoding gene. In preferred
embodiments, such a polypeptide has the amino acid residue sequence
of SEQ ID NO: 8, or a functional equivalent thereof. In accordance
with such embodiments, a coding region comprising the DNA sequence
of SEQ ID NO: 7. is also preferred.
[0093] 4.5 Characteristic of the tIC851 Polypeptide Isolated from
EG4135
[0094] The present invention provides a novel polypeptide that
defines a whole or a portion of a B. thuringiensis tIC851 crystal
protein.
[0095] In a preferred embodiment, the invention discloses and
claims an isolated and purified tIC851 protein. The tIC851 protein
isolated from EG4135 comprises a 632 amino acid sequence, and has a
calculated molecular mass of approximately 69,527 Da. tIC851 has a
calculated isoelectric constant (pI) equal to 5.80. The amino acid
composition of the tIC851 protein is given in Table 2.
2TABLE 2 AMINO ACID COMPOSITION OF tIC851 Amino % % Acid # Residues
Total Amino Acid # Residues Total Ala 45 7.1 Leu 29 4.6 Arg 13 2.1
Lys 51 8.1 Asn 40 6.3 Met 5 0.8 Asp 49 7.8 Phe 22 3.5 Cys 1 0.2 Pro
34 5.4 Gln 13 2.1 Ser 34 5.4 Glu 41 6.5 Thr 57 9.0 Gly 47 7.4 Tro 8
1.3 His 12 1.9 Tyr 25 3.9 Ile 62 9.8 Val 44 6.9 Acidic (Asp + Glu)
90 14 Basic (Arg + Lys) 64 10 Aromatic (Phe + Trp + Tyr) 55 9
Hydrophobic (Aromatic + Ile + Leu + Met + Val) 195 31
[0096] 4.6 Nomenclature of the Novel Proteins
[0097] The inventors have arbitrarily assigned the designation
tIC851 to the novel protein of the invention. Likewise, the
arbitrary designation of tIC851 has been assigned to the novel
nucleic acid sequence which encodes this polypeptide. 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 superseded by the official
nomenclature assigned to these sequences, and that based on the
lack of identity or substantial similarity to other known
insecticidal protein isolated from Bacillus thuringiensis, the
tIC851 protein will be alone in a separate category and class of
proteins.
[0098] 4.7 Tranformed Host Cells and Transgenic Plants
[0099] 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
sequence 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.
[0100] 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. Suitable methods for introducing transforming DNA into a
cell consist of but are not limited to 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. 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, 1988; Eglitis
et al., 1988); and (4) receptor-mediated mechanisms (Curiel et al.,
1991; 1992; Wagner et al., 1992).
[0101] 4.7.1 Microprojectile Bombardment
[0102] A particularly advantageous method for delivering
transforming DNA sequences into plant cells is microprojectile
bombardment. In this method, particles may be coated with nucleic
acids and delivered into cells by a propelling force. Exemplary
particles include those comprised of tungsten, gold, platinum, and
the like.
[0103] 4.7.2 Agrobacterium-Mediated Transfer
[0104] 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).
[0105] 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.
[0106] 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.
[0107] 4.7.3 Gene Expression in Plants
[0108] To overcome limitations in foreign gene expression in
plants, particular sequences and signals in RNAs that have the
potential for having a specific effect on RNA stability have been
identified. 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.
[0109] 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 destabilizing elements or whether
any of these effects are likely to be seen in plants. Table 3 lists
some of the more common AT rich sequences identified as problematic
when present in a coding sequence for which high levels of
expression are desired.
[0110] 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.
3TABLE 3 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 "
[0111] 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.
[0112] 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.
[0113] 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.
[0114] Typically, to obtain high-level expression of the
.delta.-endotoxin genes in plants, existing structural coding
sequence ("structural gene") which codes for the .delta.-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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 4.7.4 Synthetic Oligonucleotides for Mutagenesis
[0120] 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 4 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%.
4TABLE 4 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 LEU CUA
8 CUC 20 CUG 10 CUU 28 UUA 5 UUG 30 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 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 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 PHE UUC 56 UUU 44 MET AUG 100 TRP UGG 100
[0121] 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).
[0122] 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 4) 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. 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.
[0123] 4.8 Methods for Producing Insect-Resistant Transgenic
Plants
[0124] By transforming a suitable host cell, such as a plant cell,
with a recombinant tIC851 gene sequence, 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.
[0125] A transgenic plant of this invention thus has an increased
amount of a coding region (e.g., a gene) that encodes a polypeptide
in accordance with SEQ ID NO: 8. 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 upon
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.
[0126] Transgenic plants comprising one or more transgenes that
encode a polypeptide in accordance with SEQ ID NO: 8 will
preferably exhibit a phenotype of improved or enhanced insect
resistance to the target coleopteran insects as described herein.
These plants will preferably provide transgenic seeds, which will
be used to create lineages of transgenic plants (i.e. progeny or
advanced generations of the original transgenic plant) that may be
used to produce seed, or used as animal or human foodstuffs, or to
produce fibers, oil, fruit, grains, or other commercially-important
plant products or plant-derived components. In such instances, the
progeny and seed obtained from any generation of the transformed
plants will contain the selected and stably integrated transgene
that encodes the .delta.-endotoxin of the present invention. The
transgenic plants of the present invention may be crossed to
produce hybrid or inbred lines with one or more plants that have
desirable properties. In certain circumstances, it may also be
desirable to create transgenic plants, seed, and progeny that
contain one or more additional transgenes incorporated into their
genome in addition to the transgene encoding the polypeptide of the
invention. For example, the transgenic plants may contain a second
gene encoding the same, or a different insect-resistance
polypeptide, or alternatively, the plants may comprise one or more
additional transgenes such as those conferring herbicide
resistance, fungal resistance, bacterial resistance, stress, salt,
or drought tolerance, improved stalk or root lodging, increased
starch, grain, oil, carbohydrate, amino acid, protein production,
and the like.
[0127] 4.9 Isolating Homologous Gene and Gene Fragments
[0128] 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.
[0129] 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. 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.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 4.10 Recombinant Host Cells
[0135] The nucleotide sequences of the subject invention may be
introduced into a wide variety of microbial and eukaryotic hosts.
As hosts for recombinant expression of tIC851 polypeptides, of
particular interest will be the prokaryotes and the lower
eukaryotes, such as fungi. Illustrative prokaryotes, both
Gram-negative and Gram-positive, include Enterobacteriaceae, such
as Escherichia, Erwinia, Shigella, Salmonella, and Proteus;
Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as
photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,
Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such
as Pseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales,
and Nitrobacteraceae. Among eukaryotes are fungi, such as
Phycomycetes and Ascomycetes, which includes yeast, such as
Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast,
such as Rhodotorula, Aureobasidium, Sporobolomyces, and the
like.
[0136] Characteristics of particular interest in selecting a host
cell for purposes of production include ease of introducing the
genetic constructs of the present invention into the host cell,
availability of expression systems, efficiency of expression,
stability of the gene of interest in the host, and the presence of
auxiliary genetic capabilities.
[0137] A large number of microorganisms known to inhabit the
phylloplane (the surface of the plant leaves) and/or the
rhizosphere (the soil surrounding plant roots) of a wide variety of
important crops may also be desirable host cells for manipulation,
propagation, storage, delivery and/or mutagenesis of the disclosed
genetic constructs. These microorganisms include bacteria, algae,
and fungi. Of particular interest are microorganisms, such as
bacteria, e.g., genera Bacillus (including the species and
subspecies B. thuringiensis kurstaki HD-1, B. thuringiensis
kurstaki HD-73, B. thuringiensis sotto, B. thuringiensis berliner,
B. thuringiensis thuringiensis, B. thuringiensis tolworthi, B.
thuringiensis dendrolimus, B. thuringiensis alesti, B.
thuringiensis galleriae, B. thuringiensis aizawai, B. thuringiensis
subtoxicus, B. thuringiensis entomocidus, B. thuringiensis
tenebrionis and B. thuringiensis san diego); Pseudomonas, Erwinia,
Serratia, Klebsiella, Zanthomonas, Streptomyces, Rhizobium,
Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,
Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and
Alcaligenes; fungi, particularly yeast, e.g., genera Saccharomyces,
Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and
Aureobasidium. Of particular interest are such phytosphere
bacterial species as Pseudomonas syringae, Pseudomonas fluorescens,
Serratia marcescens, Acetobacter xylinum, Agrobacterium
tumefaciens, Rhodobacter sphaeroides, Xanthomonas campestris,
Rhizobium melioti, Alcaligenes eutrophus, and Azotobacter
vinlandii; and phytosphere yeast species such as Rhodotorula rubra,
R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C.
diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.
cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces
veronae, and Aureobasidium pollulans.
[0138] Characteristics of particular interest in selecting a host
cell for purposes of production include ease of introducing a
selected genetic construct into the host, availability of
expression systems, efficiency of expression, stability of the
polynucleotide in the host, and the presence of auxiliary genetic
capabilities. Other considerations include ease of formulation and
handling, economics, storage stability, and the like.
[0139] 4.11 Polynucleotide Sequences
[0140] DNA compositions encoding the insecticidally-active
polypeptides of the present invention are particularly preferred
for delivery to recipient plant cells, and ultimately in the
production of insect-resistant transgenic plants. For example, DNA
segments in the form of vectors and plasmids, or linear DNA
fragments, in some instances containing only the DNA element to be
expressed in the plant cell, and the like, may be employed.
[0141] 4.12 Methods for Preparing Mutagenized Polynucleotide
Sequences
[0142] In certain circumstances, it may be desirable to modify or
alter one or more nucleotides in one or more of the polynucleotide
sequences disclosed herein for the purpose of altering or changing
the insecticidal activity or insecticidal specificity of the
encoded polypeptide. In general, the means and methods for
mutagenizing a DNA sequences are well-known to those of skill in
the art. Modifications to such sequences may be made by random, or
site-specific mutagenesis procedures. The polynucleotides may be
modified by the addition, deletion, or substitution of one or more
nucleotides from the sequence encoding the insecticidally-active
polypeptide.
[0143] Mutagenesis may be performed in accordance with any of the
techniques known in the art such as and not limited to synthesizing
an oligonucleotide having one or more mutations within the sequence
of a particular region. In particular, site-specific mutagenesis is
a technique useful in the preparation of mutants, through specific
mutagenesis of the underlying DNA. The technique further provides a
ready ability to prepare and test sequence variants, for example,
incorporating one or more of the foregoing considerations, by
introducing one or more nucleotide sequence changes into the DNA.
Site-specific mutagenesis allows the production of mutants through
the use of specific oligonucleotide sequences which encode the DNA
sequence of the desired mutation, as well as a sufficient number of
adjacent nucleotides, to provide a primer sequence of sufficient
size and sequence complexity to form a stable duplex on both sides
of the deletion junction being traversed. Typically, a primer of
about 17 to about 75 nucleotides or more in length is preferred,
with about 10 to about 25 or more residues on both sides of the
junction of the sequence being altered.
[0144] In general, the technique of site-specific mutagenesis is
well known in the art, as exemplified by various publications. As
will be appreciated, the technique typically employs a phage vector
which exists in both a single stranded and double stranded form.
Typical vectors useful in site-directed mutagenesis include vectors
such as the M13 phage. These phage are readily commercially
available and their use is generally well known to those skilled in
the art. Double stranded plasmids are also routinely employed in
site directed mutagenesis which eliminates the step of transferring
the gene of interest from a plasmid to a phage.
[0145] The preparation of sequence variants of the selected
.delta.-endotoxin-encoding DNA segments using site-directed
mutagenesis is provided as a means of producing potentially useful
species and is not meant to be limiting as there are other ways in
which sequence variants of DNA sequences may be obtained. For
example, recombinant vectors encoding the desired sequence may be
treated with mutagenic agents, such as hydroxylamine, to obtain
sequence variants.
[0146] As used herein, the term "oligonucleotide directed
mutagenesis procedure" refers to template-dependent processes and
vector-mediated propagation which result in an increase in the
concentration of a specific nucleic acid molecule relative to its
initial concentration, or in an increase in the concentration of a
detectable signal, such as amplification. As used herein, the term
"oligonucleotide directed mutagenesis procedure" also is intended
to refer to a process that involves the template-dependent
extension of a primer molecule. The term template-dependent process
refers to nucleic acid synthesis of an RNA or a DNA molecule
wherein the sequence of the newly synthesized strand of nucleic
acid is dictated by the well-known rules of complementary base
pairing (Watson, 1987). Typically, vector mediated methodologies
involve the introduction of the nucleic acid fragment into a DNA or
RNA vector, the clonal amplification of the vector, and the
recovery of the amplified nucleic acid fragment. Examples of such
methodologies are provided by U.S. Pat. No. 4,237,224.
[0147] A number of template dependent processes are available to
amplify the target sequences of interest present in a sample. One
of the best known amplification methods is the polymerase chain
reaction (PCR.TM.) which is described in detail in U.S. Pat. Nos.
4,683,195, 4,683,202 and 4,800,159. Briefly, in PCR.TM., two primer
sequences are prepared which are complementary to regions on
opposite complementary strands of the target sequence. An excess of
deoxynucleoside triphosphates are added to a reaction mixture along
with a DNA polymerase (e.g., Taq polymerase). If the target
sequence is present in a sample, the primers will bind to the
target and the polymerase will cause the primers to be extended
along the target sequence by adding on nucleotides. By raising and
lowering the temperature of the reaction mixture, the extended
primers will dissociate from the target to form reaction products,
excess primers will bind to the target and to the reaction products
and the process is repeated. Preferably a reverse transcriptase
PCR.TM. amplification procedure may be performed in order to
quantify the amount of mRNA amplified. Polymerase chain reaction
methodologies are well known in the art.
[0148] Another method for amplification is the ligase chain
reaction (referred to as LCR), disclosed in Eur. Pat. Appl. Publ.
No. 320,308. In LCR, two complementary probe pairs are prepared,
and in the presence of the target sequence, each pair will bind to
opposite complementary strands of the target such that they abut.
In the presence of a ligase, the two probe pairs will link to form
a single unit. By temperature cycling, as in PCR.TM., bound ligated
units dissociate from the target and then serve as "target
sequences" for ligation of excess probe pairs. U.S. Pat. No.
4,883,750, incorporated herein by reference in its entirety,
describes an alternative method of amplification similar to LCR for
binding probe pairs to a target sequence.
[0149] An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification of
target molecules that contain nucleotide
5'-[.alpha.-thioltriphosphates in one strand of a restriction site
(Walker et al., 1992, incorporated herein by reference in its
entirety), may also be useful in the amplification of nucleic acids
in the present invention.
[0150] 4.13 Post-Transcriptional Events Affecting Expression of
Transgenes in Plants
[0151] In many instances, the level of transcription of a
particular transgene in a given host cell is not always indicative
of the amount of protein being produced in the transformed host
cell. This is often due to post-transcriptional processes, such as
splicing, polyadenylation, appropriate translation initiation, and
RNA stability, that affect the ability of a transcript to produce
protein. Such factors may also affect the stability and amount of
mRNA produced from the given transgene. As such, it is often
desirable to alter the post-translational events through particular
molecular biology techniques. The inventors contemplate that in
certain instances it may be desirable to alter the transcription
and/or expression of the polypeptide-encoding nucleic acid
constructs of the present invention to increase, decrease, or
otherwise regulate or control these constructs in particular host
cells and/or transgenic plants.
[0152] 4.13.1 Efficient Initiation of Protein Translation
[0153] The 5'-untranslated leader (5'-UTL) sequence of eukaryotic
mRNA plays a major role in translational efficiency. Many early
chimeric transgenes using a viral promoter used an arbitrary length
of viral sequence after the transcription initiation site and fused
this to the AUG of the coding region. More recently studies have
shown that the 5'-UTL sequence and the sequences directly
surrounding the AUG can have a large effect in translational
efficiency in host cells and particularly certain plant species and
that this effect can be different depending on the particular cells
or tissues in which the message is expressed.
[0154] In most eukaryotic mRNAs, the point of translational
initiation occurs at the AUG codon closest to the 5' cap of the
transcript. Comparison of plant mRNA sequences and site directed
mutagenesis experiments have demonstrated the existence of a
consensus sequence surrounding the initiation codon in plants,
5'-UAAACAAUGGCU-3' (SEQ ID NO: 4) (Joshi, 1987; Lutcke et al.,
1987). However, consensus sequences will be apparent amongst
individual plant species. For example, a compilation of sequences
surrounding the initiation codon from 85 maize genes yields a
consensus of 5'-(C/G)AUGGCG-3' (Luehrsen et al., 1994). In tobacco
protoplasts, transgenes encoding .beta.-glucuronidase (GUS) and
bacterial chitinase showed a 4-fold and an 8-fold increase in
expression, respectively, when the native sequences of these genes
were changed to encode 5'-ACCAUGG-3' (Gallie et al., 1987b; Jones
et al., 1988). Interestingly, B. thuringiensis has chosen to
utilize an alternative initiation codon for the native gene
encoding tIC851. The inventors find, as described below, that this
codon, although not generally known to encode for other than
leucine, is believed to code for methionine in the first position
of the tIC851 polypeptide toxin as judged by N-terminal amino acid
sequence analysis of the purified toxin. Therefore, for efficiency
inplanta, it is intended that the more frequently utilized ATG
initiation codon will be used instead.
[0155] When producing chimeric transgenes (i.e. transgenes
comprising DNA segments from different sources operably linked
together), often the 5'-UTL of plant viruses are used. The alfalfa
mosaic virus (AMV) coat protein and brome mosaic virus (BMV) coat
protein 5'-UTLs have been shown to enhance mRNA translation 8-fold
in electroporated tobacco protoplasts (Gallie et al., 1987a;
1987b). A 67-nucleotide derivative (.OMEGA.) of the 5'-UTL of
tobacco mosaic virus RNA (TMV) fused to the chloramphenicol
acetyltransferase (CAT) gene and GUS gene has been shown to enhance
translation of reporter genes in vitro (Gallie et al., 1987a;
1987b; Sleat et al., 1987; Sleat et al., 1988). Electroporation of
tobacco mesophyll protoplasts with transcripts containing the TMV
leader fused to reporter genes CAT, GUS, and LUC produced a 33-,
21-, and 36-fold level of enhancement, respectively (Gallie et al.,
1987a; 1987b; Gallie et al., 1991). Also in tobacco, an 83-nt
5'-UTL of potato virus X RNA was shown to enhance expression of the
neomycin phosphotransferese II (NptIII) 4-fold (Poogin and
Skryabin, 1992).
[0156] The effect of a 5'-UTL may be different depending on the
plant, particularly between dicots and monocots. The TMV 5'-UTL has
been shown to be more effective in tobacco protoplasts (Gallie et
al., 1989) than in maize protoplasts (Gallie and Young, 1994).
Also, the 5'-UTLs from TMV-.OMEGA. (Gallie et al., 1988), AMV-coat
(Gehrke et al., 1983; Jobling and Gehrke, 1987), TMV-coat (Goelet
et al., 1982), and BMV-coat (French et al., 1986) worked poorly in
maize and inhibited expression of a luciferase gene in maize
relative to its native leader (Koziel et al., 1996). However, the
5'-UTLs from the cauliflower mosaic virus (CaMV) 35S transcript and
the maize genes glutelin (Boronat et al., 1986), PEP-carboxylase
(Hudspeth and Grula, 1989) and ribulose biphosphate carboxylase
showed a considerable increase in expression of the luciferase gene
in maize relative to its native leader (Koziel et al., 1996).
[0157] These 5'-UTLs had different effects in tobacco. In contrast
to maize, the TMV .OMEGA. 5'-UTL and the AMV coat protein 5'-UTL
enhanced expression in tobacco, whereas the glutelin, maize
PEP-carboxylase and maize ribulose-1,5-bisphosphate carboxylase
5'-UTLs did not show enhancement relative to the native luciferase
5'-UTL (Koziel et al., 1996). Only the CaMV 35S 5'-UTL enhanced
luciferase expression in both maize and tobacco (Koziel et al.,
1996). Furthermore, the TMV and BMV coat protein 5'-UTLs were
inhibitory in both maize and tobacco protoplasts (Koziel et al.,
1996).
[0158] 4.13.2 Use of Introns to Increase Expression
[0159] Including one or more introns in the transcribed portion of
a gene has been found to increase heterologous gene expression in a
variety of plant systems (Callis et al., 1987; Maas et al., 1991;
Mascerenhas et al., 1990; McElroy et al., 1990; Vasil et al.,
1989), although not all introns produce a stimulatory effect and
the degree of stimulation varies. The enhancing effect of introns
appears to be more apparent in monocots than in dicots. Tanaka et
al., (1990) has shown that use of the catalase intron 1 isolated
from castor beans increases gene expression in rice. Likewise, the
first intron of the alcohol dehydrogenase 1 (Adh1) has been shown
to increase expression of a genomic clone of Adh1 comprising the
endogenous promoter in transformed maize cells (Callis et al.,
1987; Dennis et al., 1984). Other introns that are also able to
increase expression of transgenes which contain them include the
introns 2 and 6 of Adh1 (Luehrsen and Walbot, 1991), the catalase
intron (Tanaka et al., 1990), intron 1 of the maize bronze 1 gene
(Callis et al., 1987), the maize sucrose synthase intron 1 (Vasil
et al., 1989), intron 3 of the rice actin gene (Luehrsen and
Walbot, 1991), rice actin intron 1 (McElroy et al., 1990), and the
maize ubiquitin exon 1 (Christensen et al., 1992).
[0160] Generally, to achieve optimal expression, the selected
intron(s) should be present in the 5' transcriptional unit in the
correct orientation with respect to the splice junction sequences
(Callis et al., 1987; Maas et al., 1991; Mascerenhas et al., 1990;
Oard et al., 1989; Tanaka et al., 1990; Vasil et al., 1989). Intron
9 of Adh1 has been shown to increase expression of a heterologous
gene when placed 3' (or downstream of) the gene of interest (Callis
et al., 1987).
[0161] 4.13.3 Use of Synthetic Genes to Increase Expression of
Heterologous Genes in Plants
[0162] When introducing a prokaryotic gene into a eukaryotic host,
or when expressing a eukaryotic gene in a non-native host, the
sequence of the gene must often be altered or modified to allow
efficient translation of the transcript(s) derived form the gene.
Significant experience in using synthetic genes to increase
expression of a desired protein has been achieved in the expression
of Bacillus thuringiensis in plants. Native B. thuringiensis genes
are often expressed only at low levels in dicots and sometimes not
at all in many species of monocots (Koziel et al., 1996). Codon
usage in the native genes is considerably different from that found
in typical plant genes, which have a higher G+C content. Strategies
to increase expression of these genes in plants generally alter the
overall G+C content of the genes. For example, synthetic B.
thuringiensis crystal-protein encoding genes have resulted in
significant improvements in expression of these endotoxins in
various crops including cotton (Perlak et al., 1990; Wilson et al.,
1992), tomato (Perlak et al., 1991), potato (Perlak et al., 1993),
rice (Cheng et al., 1998), and maize (Koziel et al., 1993).
[0163] In a similar fashion the inventors contemplate that the
genetic constructs of the present invention, because they contain
one or more genes of bacterial origin, may in certain circumstances
be altered to increase the expression of these prokaryotic-derived
genes in particular eukaryotic host cells and/or transgenic plants
which comprise such constructs. Using molecular biology techniques
which are well-known to those of skill in the art, one may alter
the coding or non coding sequences of the particular
tIC851-encoding gene sequences to optimize or facilitate its
expression in transformed plant cells at levels suitable for
preventing or reducing insect infestation or attack in such
transgenic plants.
[0164] 4.13.4 Use of Promotors in Expression Vectors
[0165] The expression of a gene which exists in double-stranded DNA
form involves transcription of messenger RNA (mRNA) from the coding
strand of the DNA by an RNA polymerase enzyme, and the subsequent
processing of the mRNA primary transcript inside the nucleus.
Transcription of DNA into mRNA is regulated by a region of DNA
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. The
particular promoter selected should be capable of causing
sufficient expression of the coding sequence to result in the
production of an effective insecticidal amount of the B.
thuringiensis protein.
[0166] A promoter is selected for its ability to direct the
transformed plant cell's or transgenic plant's transcriptional
activity to the coding region, to ensure sufficient expression of
the enzyme coding sequence to result in the production of
insecticidal amounts of the B. thuringiensis protein. Structural
genes can be driven by a variety of promoters in plant tissues.
Promoters can be near-constitutive (i.e. they drive transcription
of the transgene in all tissue), such as the CaMV35S promoter, or
tissue-specific or developmentally specific promoters affecting
dicots or monocots. Where the promoter is a near-constitutive
promoter such as CaMV35S or FMV35S, increases in polypeptide
expression are found in a variety of transformed plant tissues and
most plant organs (e.g., callus, leaf, seed and root). Enhanced or
duplicate versions of the CaMV35S and FMV35S promoters are
particularly useful in the practice of this invention (Kay et al.,
1987; Rogers, U.S. Pat. No. 5,378,619).
[0167] Those skilled in the art will recognize that there are a
number of promoters which are active in plant cells, and 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 A. 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), the rice Act1 promoter 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., McElroy et al., 1990, U.S. Pat. No. 5,463,175).
[0168] In addition, it may also be preferred to bring about
expression of the B. thuringiensis .delta.-endotoxin in specific
tissues of the plant by using plant integrating vectors containing
a tissue-specific promoter. Specific target tissues may include the
leaf, stem, root, tuber, seed, fruit, etc., and the promoter chosen
should have the desired tissue and developmental specificity.
Therefore, promoter function should be optimized by selecting a
promoter with the desired tissue expression capabilities and
approximate promoter strength and selecting a transformant which
produces the desired insecticidal activity in the target tissues.
This selection approach from the pool of transformants is routinely
employed in expression of heterologous structural genes in plants
since there is variation between transformants containing the same
heterologous gene due to the site of gene insertion within the
plant genome (commonly referred to as "position effect"). In
addition to promoters which are known to cause transcription
(constitutive or tissue-specific) of DNA in plant cells, other
promoters may be identified for use in the current invention by
screening a plant cDNA library for genes which are selectively or
preferably expressed in the target tissues and then determine the
promoter regions.
[0169] An exemplary tissue-specific promoter is the lectin
promoter, which is specific for seed tissue. The lectin protein in
soybean seeds is encoded by a single gene (Le1) that is only
expressed during seed maturation and accounts for about 2 to about
5% of total seed mRNA. The lectin gene and seed-specific promoter
have been fully characterized and used to direct seed specific
expression in transgenic tobacco plants (Vodkin et al., 1983;
Lindstrom et al., 1990). An expression vector containing a coding
region that encodes a polypeptide of interest can be engineered to
be under control of the lectin promoter and that vector may be
introduced into plants using, for example, a protoplast
transformation method (Dhir et al., 1991). The expression of the
polypeptide would then be directed specifically to the seeds of the
transgenic plant.
[0170] 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.
[0171] Other 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 (McBride and Summerfelt,
1989), Ti plasmid nopaline synthase (Langridge et al., 1989),
petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine
rich protein 1 (Keller et al., 1989), CaMV 35s transcript (Odell et
al., 1985) and Potato patatin (Wenzler et al., 1989). Preferred
promoters are the cauliflower mosaic virus (CaMV 35S) promoter and
the S-E9 small subunit RuBP carboxylase promoter.
[0172] The promoters used in the DNA constructs 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.
Examples of such enhancer sequences have been reported by Kay et
al. (1987). Chloroplast or plastid specific promoters are known in
the art (Daniell et al., U.S. Pat. No. 5,693,507; herein
incorporated by reference), for example promoters obtainable from
chloroplast genes, such as the psbA gene from spinach or pea, the
rbcL and atpB promoter region from maize, and rRNA promoters. Any
chloroplast or plastid operable promoter is within the scope of the
present invention.
[0173] 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
RNAs, from suitable eukaryotic 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. As
shown below, a plant gene leader sequence which is useful in the
present invention is the petunia heat shock protein 70 (hsp70)
leader (Winter et al., 1988).
[0174] An exemplary embodiment of the invention involves the
plastid targeting or plastid localization of the B. thuringiensis
amino acid sequence. Plastid targeting sequences have been isolated
from numerous nuclear encoded plant genes and have been shown to
direct importation of cytoplasmically synthesized proteins into
plastids (reviewed in Keegstra and Olsen, 1989). A variety of
plastid targeting sequences, well known in the art, including but
not limited to ADPGPP, EPSP synthase, or ssRUBISCO, may be utilized
in practicing this invention. In alternative embodiments preferred,
plastidic targeting sequences (peptide and nucleic acid) for
monocotyledonous crops may consist of a genomic coding fragment
containing an intron sequence as well as a duplicated proteolytic
cleavage site in the encoded plastidic targeting sequences.
[0175] Tables 5-7 list promoters which are illustrative of those
known in the art, but which are not meant to be limiting.
5TABLE 5 PLANT PROMOTERS Promoter Reference Viral Figwort Mosaic
Virus (FMV) U.S. Pat. No. 5,378,619 Cauliflower Mosaic Virus (CaMV)
U.S. Pat. No. 5,530,196 U.S. Pat. No. 5,097,025 U.S. Pat. No.
5,110,732 Plant Elongation Factor U.S. Pat. No. 5,177,011 Tomato
Polygalacturonase U.S. Pat. No. 5,442,052 Arabidopsis Histone H4
U.S. Pat. No. 5,491,288 Phaseolin U.S. Pat. No. 5,504,200 Group 2
U.S. Pat. No. 5,608,144 Ubiquitin U.S. Pat. No. 5,614,399 P119 U.S.
Pat. No. 5,633,440 .alpha.-amylase U.S. Pat. No. 5,712,112 Viral
enhancer/Plant promoter CaMV 35Senhancer/mannopine U.S. Pat. No.
5,106,739 synthase promoter
[0176]
6TABLE 6 TISSUE SPECIFIC PLANT PROMOTERS Tissue Specific Promoter
Tissue(s) Reference Blec epidermis U.S. Pat. No. 5,646,333 malate
synthase seeds; seedlings U.S. Pat. No. 5,689,040 isocitrate lyase
seeds; seedlings U.S. Pat. No. 5,689,040 patatin tuber U.S. Pat.
No. 5,436,393 ZRP2 root U.S. Pat. No. 5,633,363 ZRP2(2.0) root U.S.
Pat. No. 5,633,363 ZRP2(1.0) root U.S. Pat. No. 5,633,363 RB7 root
U.S. Pat. No. 5,459,252 root U.S. Pat. No. 5,401,836 fruit U.S.
Pat. No. 4,943,674 meristem U.S. Pat. No. 5,589,583 guard cell U.S.
Pat. No. 5,538,879 stamen U.S. Pat. No. 5,589,610 SodA1 pollen;
middle layer; stomium Van Camp et al., 1996 of anthers SodA2
vasular bundles; stomata; Van Camp et al., 1996 axillary buds;
pericycle; stomium; pollen CHS15 flowers; root tips Faktor et al.,
1996 Psam-1 phloem tissue; cortex; Vander et al., 1996 root tips
ACT11 elongating tissues and organs; Huang et al., 1997 pollen;
ovules zmGBS pollen; endosperm Russell and Fromm, 1997 zmZ27
endosperm Russell and Fromm, 1997 osAGP endosperm Russell and
Fromm, 1997 osGT1 endosperm Russell and Fromm, 1997 RolC phloem
tissue; bundle sheath; Graham et al., 1997 vascular parenchyma Sh
phloem tissue Graham et al., 1997 CMd endosperm Grosset et al.,
1997 Bnm1 pollen Treacy et al., 1997 rice tungro bacilliform virus
phloem Yin et al., 1997a; 1997b S2-RNase pollen Ficker et al., 1998
LeB4 seeds Baumlein et al., 1991 gf-2.8 seeds; seedlings Berna and
Bernier, 1997
[0177] The ability to express genes in a tissue specific manner in
plants has led to the production of male and female sterile plants.
Generally, the production of male sterile plants involves the use
of anther-specific promoters operably linked to heterologous genes
that disrupt pollen formation (U.S. Pat. Nos. 5,689,051; 5,689,049;
5,659,124). U.S. Pat. No. 5,633,441 discloses a method of producing
plants with female genetic sterility. The method comprises the use
of style-cell, stigma-cell, or style- and stigma-cell specific
promoters that express polypeptides that, when produced in the
cells of the plant, kills or significantly disturbs the metabolism,
functioning or development of the cells.
7TABLE 7 INDUCIBLE PLANT PROMOTERS Promoter Reference heat shock
promoter U.S. Pat. No. 5,447,858 Em U.S. Pat. No. 5,139,954 Adh1
Kyozoka et al., 1991 HMG2 U.S. Pat. No. 5,689,056 cinnamyl alcohol
dehydrogenase U.S. Pat. No. 5,633,439 asparagine synthase U.S. Pat.
No. 5,595,896 GST-II-27 U.S. Pat. No. 5,589,614
[0178] 4.13.5 Chloroplast Sequestering and Targeting
[0179] Another approach for increasing expression of A+T rich genes
in plants has been demonstrated in tobacco chloroplast
transformation. High levels of expression of an unmodified Bacillus
thuringiensis crystal protein-encoding genes in tobacco has been
reported by McBride et al., (1995).
[0180] Additionally, methods of targeting proteins to the
chloroplast have been developed. This technique, utilizing the pea
chloroplast transit peptide, has been used to target the enzymes of
the polyhydroxybutyrate synthesis pathway to the chloroplast
(Nawrath et al., 1994). Also, this technique negated the necessity
of modification of the coding region other than to add an
appropriate targeting sequence.
[0181] U.S. Pat. No. 5,576,198 discloses compositions and methods
useful for genetic engineering of plant cells to provide a method
of controlling the timing or tissue pattern of expression of
foreign DNA sequences inserted into the plant plastid genome.
Constructs include those for nuclear transformation which provide
for expression of a viral single subunit RNA polymerase in plant
tissues, and targeting of the expressed polymerase protein into
plant cell plastids. Also included are plastid expression
constructs comprising a viral gene promoter region which is
specific to the RNA polymerase expressed from the nuclear
expression constructs described above and a heterologous gene of
interest to be expressed in the transformed plastid cells. 4.13.6
Effects of 3' Regions on Transgene Expression
[0182] The 3'-end regions of transgenes have been found to have a
large effect on transgene expression in plants (Ingelbrecht et al.,
1989). In this study, different 3' ends were operably linked to the
neomycin phosphotransferase II (NptII) reporter gene and expressed
in transgenic tobacco. The different 3' ends used were obtained
from the octopine synthase gene, the 2S seed protein from
Arabidopsis, the small subunit of rbcS from Arabidopsis, extension
form carrot, and chalcone synthase from Antirrhinum. In stable
tobacco transformants, there was about a 60-fold difference between
the best-expressing construct (small subunit rbcS 3' end) and the
lowest expressing construct (shalcone synthase 3' end).
[0183] 4.14 Antibody Compositions and Methods of Making
[0184] In particular embodiments, the inventors contemplate the use
of antibodies, either monoclonal 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). The methods for generating monoclonal
antibodies (mAbs) generally begin along the same lines as those for
preparing polyclonal antibodies. mAbs may be readily prepared
through use of well-known techniques, such as those exemplified in
U.S. Pat. No. 4,196,265. Antibody use is well known in the art and
can be used for purification, immunoprecipitation, ELISA and
western blot for resolving the presence of molecules having
identifiable epitopes. Those skilled in the art would not encounter
undue experimentation in using antibodies and such methods to
idolate, identify, and characterize genes and proteins expressed
from such genes as contemplated herein. Immuno-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.
[0185] 4.15 Biological Functional Equivalents
[0186] Modification and changes may be made in the structure of the
peptides of the present invention and DNA sequences 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 8.
8TABLE 8 Amino Acids Codon Abbreviations.sup.1 Codons Alanine Ala A
GCA GCC GCG GCU Arginine Arg R AGA AGG CGA CGC CGG CGU Asparagine
Asn N AAC AAU Aspartic acid Asp D GAC GAU Cysteine Cys C UGC UGU
Glutamic acid Glu E GAA GAG Glutamine Gln Q CAA CAG Glycine Gly G
GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUG
AUU Leucine Leu L UUA UUG CUA CUC CUG CUU Lysine Lys K AAA AAG
Methionine Met M AUG UUG* Phenylalanine Phe F UUC UUU Proline Pro P
CCA CCC CCG CCU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr
T ACA ACC ACG ACU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
Valine Val V GUA GUC GUG GUU *the codon UUG is also utilized as an
initiation codon as a part of the tIC851 coding sequence
.sup.1three letter code and corresponding single letter code
abbreviations
[0187] 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.
[0188] 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.
[0189] 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).
[0190] 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.
[0191] 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.
[0192] 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).
[0193] 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 .+-.5 are even more particularly preferred.
[0194] 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.
5.0 EXAMPLES
[0195] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
5.1 Example 1
[0196] Bacillus thuringiensis Stains with Sequences Related to
CryET70
[0197] We previously identified a B. thuringiensis strain
expressing a protein which we designated CryET70. The CryET70
protein had effective coleopteran specific bioactivity when
provided in bioassay feeding studies to western corn rootworm
larvae, but not against southern corn rootworm larvae. We were
interested in identifying additional B. thuringiensis strains which
contained DNA encoding CryET70 and closely related genes. Colony
blot hybridization experiments were completed as indicated below,
using a probe prepared from cryET70 DNA. Wild-type B. thuringiensis
strains were patched onto LB plates and incubated at 30.degree. C.
for four hours. A Nytran.RTM. Maximum-Strength Plus (Schleicher and
Schuell, Keene, N.H.) circular (82 mm) membrane filter was then
placed on the plates and the plates and filters were incubated at
25.degree. C. overnight. The filters, which contained an exact
replica of the patches, were then placed on fresh LB plates, and
the filters and the original plates were incubated at 30.degree. C.
for 4 hr to allow for growth of the colonies. To release the DNA
from the B. thuringiensis cells onto the nitrocellulose filter, the
filters were placed, colony-side up, on Whatman 3 MM Chromatography
paper (Whatman International LTD., Maidstone, England) soaked with
0.5 N NaOH, 1.5 M NaCl for 15 min. The filters were then
neutralized by placing the filters, colony-side up, on Whatman
paper soaked with 1 M NH.sub.4-acetate, 0.02 M NaOH for 10 min. The
filters were then rinsed in 3.times.SSC, 0.1% SDS, air dried, and
baked for one hr at 80.degree. C. in a vacuum oven to prepare them
for hybridization.
9 Oligonucleotide primers were designed based on the cryET70
sequence (SEQ ID NO:1): AM34: 5'-GACATGATTTTACTTTTAGAGC-3' (SEQ ID
NO:3) AM43: 5'-CATCACTTTCCCCATAGC-3' (SEQ ID NO:4)
[0198] A PCR.TM. with primers AM 34 and AM 43 was used to amplify a
cryET70 fragment from pEG1648 DNA. This PCR.TM. product was labeled
with [.alpha..sup.32P]dATP using the Prime-a-Gene.RTM. kit (Promega
Corporation, Madison, Wis.) to generate a cryET70-specific probe.
Hybridizations were performed overnight with the hybridization
temperature at 63.degree. C. Filters were washed in 1.times.SSC,
0.1% SDS at 63.degree. C. Hybridizing colonies were detected by
autoradiography using Kodak X-OMAT AR X-ray film. The results
indicated that several B. thuringiensis strains in our collection
contained DNA sequences which hybridized to cryET70 sequences under
specified conditions. The strains identified by colony blot
hybridization are listed in Table 9.
5.2 Example 2
[0199] Production of Antibody to CryET70
[0200] CryET70 specific polyclonal antibody was prepared so that
proteins containing CryET70-related epitopes could be identified
using immunological methods. Recombinant B. thuringiensis strain
EG11839 containing plasmid pEG1648 expressing CryET70 was grown in
C2 medium for four days at 25.degree. C. The resulting spores and
crystals were washed in 2.5.times. volume H.sub.2O and resuspended
at 1/20 the original volume in 0.005% Triton X-100.RTM.. The
spore-crystal suspension was then loaded on a sucrose step gradient
consisting of 79%, 72% and 55% sucrose. The gradient was spun
overnight in a Beckman SW28 at 18,000 RPM. CryET70 crystals banded
between the 79% and the 72% sucrose layers. CryET70 crystals were
washed several times in H.sub.2O and resuspended in 0.005% Triton
X-100.RTM.. The purified crystals were then solubilized in 50 mM
sodium carbonate (pH 10), 5 mM DTT, and any contaminating
vegetative cells or spores were removed by centrifugation. The
supernatant was neutralized with boric acid to pH8.4, and the
solubilized crystals were sent to Rockland Laboratories
(Gilbertsville, Pa.) for antibody production in rabbits according
to standard procedures. The rabbits received two intradermal
injections on days zero and seven with 50% CryET70 protein in
sterile phosphate buffered saline, 50% complete Freund's adjuvant.
Two additional boosts were given subcutaneously on days 14 and 28
before a test bleed on day 38. Two hundred fifty .mu.g of CryET70
were used per rabbit for the initial injection, and 125 .mu.g of
CryET70 were used per rabbit for the subsequent boosts. On day 56
the rabbits were boosted again, as before, prior to a production
bleed on day 71. The final boost was with 160 .mu.g CryET70 on day
80, followed by a termination bleed on day 90.
5.3 Example 3
[0201] Southern and Western Blot Analyses
[0202] Strains identified in Example 1 as containing sequences
related to cryET70 were examined further by Southern and Western
blot analyses.
[0203] Total DNA was prepared from the strains by the following
procedure. Vegetative cells were resuspended in a lysis buffer
containing 50 mM glucose, 25 mM Tris-HCl (pH8.0), 10 mM EDTA, and 4
mg/ml lysozyme. The suspension was incubated at 37.degree. C. for
one hr. Following incubation, SDS was added to 1%. The suspension
was then extracted with an equal volume of
phenol:chloroform:isoamyl alcohol (50:48:2). 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 with a glass rod, washed with 70% ethanol, and
resuspended in dH.sub.2O.
[0204] Total DNA was digested with EcoRI and separated on a 0.8%
agarose gel in TAE buffer (40 mM Tris-acetate, 2 mM Na.sub.2EDTA,
pH 8). The DNA was blotted onto an Immobilon-NC nitrocellulose
filter (Millipore Corp., Bedford, Mass.) according to the method of
Southern (1975). DNA was fixed to the filter by baking at
80.degree. C. in a vacuum oven.
[0205] The blot was then hybridized with the cryET70 probe
described in Example 1. The filters were exposed to the labeled
probe diluted in 3.times.SSC, 0.1% SDS, 10.times. Denhardt's
reagent (0.2% bovine serum albumin (BSA), 0.2%
polyvinylpyrrolidone, 0.2% Ficoll.RTM.), 0.2 mg/mi heparin and
incubated overnight at 60.degree. C. Following the incubation, the
filters were washed in three changes of 3.times.SSC, 0.1% SDS at
60.degree. C. The filters were blotted dry and exposed to Kodak
X-OMAT AR X-ray film (Eastman Kodak Company, Rochester, N.Y.)
overnight at -70.degree. C. with an intensifying screen (Fisher
Biotech, Pittsburgh, Pa.). Strains containing hybridizing DNA
fragments are listed in Table 9.
[0206] For the Western blot analysis, B. thuringiensis strains were
grown in C2 medium (Donovan et al., 1988) at 25.degree. C. for four
days until sporulation and cell lysis had occurred. The resulting
spores and crystals were harvested by centrifugation, washed in
approximately 2.5 times the original volume with H.sub.2O, and
resuspended in 0.005% Triton X-100.RTM. at one-tenth the original
volume. Proteins from 10-fold concentrated cultures of the strains
were run on a 10% SDS-polyacrylamide gel (Owl Separation Systems,
Woburn, Mass.). Twenty .mu.l of culture was added to 10 .mu.l of
3.times. Laemmli buffer and heated at 100.degree. C. for five
minutes. Fifteen .mu.l were loaded per lane. Following
electrophoresis, the gel was blotted to nitrocellulose following
standard Western blotting procedures (Towbin et al., 1979). The
filter was blocked with TBSN (10 mM Tris, pH 7.8, 0.9% NaCl, 0.1%
globulin-free BSA, 0.03% NaN.sub.3) +2% BSA. The filter was then
washed with TBSN twice and then incubated with anti-CryET70 rabbit
antiserum diluted 1/1,000 in TBSN. The filter was then washed in
TBSN and incubated with alkaline phosphatase conjugated sheep
anti-rabbit IgG (1/1,000 dilution in TBSN). After washing in TBSN,
proteins antigenically related to CryET70 were detected with
ImmunoPure.RTM. NBT/BCIP Substrate Kit (Pierce, Rockford, Ill.). B.
thuringiensis strains producing proteins antigenically related to
CryET70 as judged by Western blot analysis are indicated in Table
9.
5.4 Example 4
[0207] Bioassay Evaluation of B. thuringiensis Strains
[0208] Insect bioassays were used to characterize B. thuringiensis
strains having activity directed against western corn rootworm
larvae. B. thuringiensis strains were grown in C2 medium (Donovan
et al., 1988) at 25.degree. C. for four days at which time
sporulation and lysis had occurred. The resulting spores and
crystals were harvested by centrifugation, washed in approximately
2.5 times the original volume with water, and resuspended in 0.005%
Triton X-100.RTM. at one-tenth the original culture volume. The
spore-crystal suspensions were used directly in bioassay.
[0209] Insecticidal activity against WCRW larvae was determined via
a surface contamination assay on an artificial diet (20 g agar, 50
g wheat germ, 39 g sucrose, 32 g casein, 14 g fiber, 9 g Wesson
salts mix, 1 g methyl paraben, 0.5 g sorbic acid, 0.06 g
cholesterol, 9 g Vaderzant's vitamin mix, 0.5 ml linseed oil, 2.5
ml phosphoric/propionic acid per liter) in a plastic feeding cup
(175 mm.sup.2 surface). All bioassays were performed using 128-well
trays containing approximately 1 ml of diet per well with
perforated mylar sheet covers (C-D International Inc., Pitman,
N.J.). Thirty-two larvae (one per well) were tested per bioassay
screen at 50 ul of a spore-crystal suspension per well of diet. The
results of the bioassay screen are shown in Table 9.
10TABLE 9 SUMMARY OF SOUTHERN, WESTERN, AND BIOASSAY ANALYSES
Strains Southern blot Western blot % Control WCRW EG2929 + + 26
EG3218 +/- - 30 EG3221 +/- - 63 EG3303 +/- - 15 EG3304 +/- 0 EG3707
+ - 45 EG3803 - 0 EG3953 + 100 EG3966 + - 7 EG4113 - - 40 EG4135 +
+ 45 EG4150 - - 64 EG4268 - + 46 EG4375 - 100 EG4447 +/- 0 EG4448 +
- 100 EG4503 +/- - 56 EG4541 +/- - 72 EG4580 + + 33 EG4640 - - 95
EG4737 - - 72 EG4741 + - 73 EG5233 - - 52 EG5366 + - 69 EG5370 - -
16 EG5422 - 8
5.5 Example 5
[0210] Analysis of Wild-Type B. thuringiensis Strains
[0211] The CryET70 peptide sequence has previously been shown to
share significant amino acid sequence identity with Cry22Aa. Based
on the known nucleotide and amino acid sequences of CryET70 and
Cry22Aa, thermal amplification primers were designed for sequences
similar or identical to those of the CryET70 and Cry22Aa coding
sequences.
11TABLE 10 Thermal Amplification Oligonucleotide Sequence Alignment
in cry22Aa and cryET70 Corresponding Position of Oligo in: Sequence
(5'-3') & cry22Aa cryET70 Oligo.sup.a Corresponding SEQ ID NO
(SEQ ID NO:9) (SEQ ID NO:1) 2270-1 GCATTTCATAGAGGATCAAT SEQ. ID
NO:5 262-281 350-369 2270-2 ATTGATCCTCTATGAAATGC SEQ ID NO:11
281-262 369-350 2270-3 GTTTCCCAAATGGATATCC SEQ ID NO:12 428-446
516-534 2270-4 GGATATCCATTTGGGAAAC SEQ ID NO:13 446-428 534-516
2270-5 ATCTAATAACCTACATCAGA SEQ ID NO:14 726-745 814-833 2270-6
TCTGATGTAGGTTATTAGAT SEQ ID N0:15 745-726 833-814 2270-7
TATGGGGAAAGTGATGAAAA SEQ ID NO:16 973-992 1061-1080 2270-8
TTTTCATCACTTTCCCCATA SEQ ID NO:6 992-973 1080-1061 2270-9
ATGTTGAATTAGAAATAG SEQ ID NO:17 1280-1297 1368-1385 2270-10
CTATTTCTAATTCAACAT SEQ ID NO:18 1297-1280 1385-1358 2270-11
AAGTCCTTGTTCTAGGAGAA SEQ ID NO:19 1481-1500 1569-1588 2270-12
TTCTCCTAGAACAAGGACTT SEQ ID NO:20 1500-1481 1588-1569 2270-13
TATGTATTCTATGATTCTAG SEQ ID NO:21 1840-1859 1928-1947 2270-14
CTAGAATCATAGAATACATA SEQ ID NO:22 1859-1840 1947-1928 a: odd
numbered oligonucleotides represent sequences identical to the
indicated position for each gene (SEQ ID NO), and even numbered
oligonucleotides represent sequences complementary to the indicated
position for each gene (SEQ ID NO).
[0212] Even numbered oligonucleotides were paired with odd numbered
oligonucleotides in various combinations in thermal amplification
reactions in order to confirm the expected size of fragments from
amplification of sequences from both cryET70 and cry22Aa. DNA
obtained from strains EG4135 and EG4268 was also used in separate
thermal reactions with all primer pairs. While all pairs produced
amplification fragments from both cryET70 and cry22Aa, the only
oligonucleotide primer pair which produced a product from DNA of
strains EG4135 and EG4268 was the 2270-1 and 2270-8 primer pair
(SEQ ID NO: 5 & SEQ ID NO: 6 respectively).
[0213] Amplification reactions were performed using `Taq-Beads`
(Pharmacia Biotech), a Stratagene Robocycler.TM., and the following
cycling regimen: 94 C for 30 seconds, 45 C for 45 seconds, and 72 C
for 1 minute for 30 cycles. Thermocycling was preceded by a 5
minute incubation at 94 C, followed by a 5 minute incubation at 72
C. The amplification products produced from strains EG4135 and
EG4268 were cloned as blunt-end fragments into the SmaI site of
pBluescript KSII(+) and sequenced. The sequences of the DNA inserts
indicated the presence of an open reading frame (ORF) which
displayed approximately 65% sequence identity to the corresponding
region from either CryET70 or Cry22Aa.
5.6 Example 6
[0214] Sequence Analysis of the Full-Length Gene
[0215] Genomic DNA libraries from strains EG4135 and EG4268 were
constructed in the Lambda Zap.RTM. II vector (Stratagene; La Jolla,
Calif.) and used to isolate recombinant clones containing the
entire ORF identified in Example 5. The ORF encodes a protein of
632 amino acids, designated tIC851. The nucleotide sequence
encompassing the tIC851 gene (SEQ ID NO: 7) is shown below:
12 AAATATTTTT AAAGGGGGAT ACGTAATTTG AATTCTAAAT CTATCATCGA
AAAAGGGGTA 60 CAAGAGAATC AATATATTGA TATTCGTAAC ATATGTAGCA
TTAATGGTTC TGCTAAATTT 120 GATCCTAATA CTAACATTAC AACCTTAACA
GAAGCTATCA ATTCTCAAGC AGGAGCGATT 180 GCTGGAAAAA CTGCCCTAGA
TATGAGACGT GATTTTACTC TCGTAGCAGA TATATACCTA 240 GGGTCTAAAA
GTAGTGGAGC TGATGGTATT GCTATAGCGT TTCATAGAGG ATCAATTGGT 300
TTTATCGGTA CCATGGGTGG AGGCTTAGGG ATTCTAGGAG CACCAAACGG GATAGGATTT
360 GAAATAGATA CGTATTGGAA AGCAACTTCA GATGAAACAG GCGATTCATT
TGGACATGGT 420 CAAATGAATG GAGCACATGC GGGATTTGTA AGTACAAATC
GAAATGCAAG CTATTTAACA 480 GCCTTAGCTC CTATGCAAAA AATACCTGCA
CCTAATAATA AATGGCGGGT TCTAACTATC 540 AATTGGGATG CGCGTAACAA
CAAACTAACA GCACGGCTTC AAGAGAAAAG TAATGATGCT 600 TCTACTAGCA
CTCCTAGTCC AAGATATCAA ACATGGGAAC TATTAAATCC TGCGTTTGAT 660
TTAAATCAGA AATATACTTT TATTATCGGC TCAGCTACAG GGGCTGCTAA TAACAAGCAT
720 CAGATTGGAG TTACTTTGTT TGAAGCATAC TTTACAAAAC CAACTATAGA
GGCAAATCCT 780 GTTGATATTG AACTAGGCAC AGCGTTTGAT CCATTAAACC
ATGAGCCAAT TGGACTCAAA 840 GCAACAGATG AAGTAGATGG AGATATAACA
AAGGACATTA CGGTAGAATT TAATGACATA 900 GATACCTCCA AACCAGGTGC
ATACCGTGTA ACATATAAAG TAGTAAATAG TTATGGAGAA 960 AGTGATGAGA
AAACAATAGA AGTCGTAGTA TACACGAAAC CAACTATAAC TGCACATGAT 1020
ATTACGATTA AGAAAGACTT AGCATTTGAT CCATTAAACT ATGAACCAAT TGGACTCAAA
1080 GCAACCGATC CAATTGATGG AGATATAACA GATAAAATCG CTGTAAAATT
TAATAATGTC 1140 GATACCTCTA AACCGGGTAA ATACCATGTA ACATATAAAG
TGATAAATAG TTATGAAAAA 1200 ATTGATGAAA AAACAATAGA GGTCACAGTA
TATACGAAAC CATCTATAGT GGCACATGAT 1260 GTTGAGATTA AAAAAGATAC
GGCATTTGAT CCGTTAAACT ATGAACCAAT TGGGCTCAAA 1320 GCAACCGATC
CAATTGATGG AGATATAACA GATAAAATTA CGGTAGAATC TAATGATGTT 1380
GATACCTCTA AACCAGGTGC ATATAGTGTG AAATATAAAG TAGTAAATAA TTATGAAGAA
1440 AGTGACGAAA AAACAATTGC CGTTACAGTA CCTGTTATAG ATGATGGGTG
GGAGAATGGC 1500 GATCCGACAG GATGGAAATT CTTCTCTGGT GAAACCATTA
CTCTAGAAGA TGATGAAGAG 1560 CATGCTCTTA ATGGTAAATG GGTATTTTAT
GCTGATAAAC ATGTAGCAAT ATACAAACAA 1620 GTAGAGTTGA AGAATAATAT
CCCTTATCAA ATTACACTAT ATGTTAAACC AGAAGATGAA 1680 GGAACTGTGG
CACACCATAT TGTTAAAGTA TCTTTCAAAT CTGATTCTGC TGGTCCAGAA 1740
AGTGAAGAAG TTATAAATGA AAGATTAATT GATGCAGAAC AGATACAAAA AGGATACAGA
1800 AAGTTAACAA GTATTCCATT TACACCAACA ACCATTGTTC CCAACAAAAA
ACCAGTGATA 1860 ATTGTTGAAA ACTTTTTACC AGGATGGATA GGTGGAGTTA
GAATAATTGT AGAGCCTACA 1920 AAGTAAGAAT TATAAACTAG CTTTTAATAA
ATATATTTAA AAAAT 1965
[0216] The tIC851 ORF initiation codon is TTG beginning at
nucleotide 28 of the sequence shown above. The deduced amino acid
sequence (SEQ ID NO. 8) of the tIC851 protein is shown below, as
translated from the ORF described above:
13 MNSKSIIEKG VQENQYIDIR NICSINGSAK FDPNTNITTL TEAINSQAGA
IAGKTALDMR 60 RDFTLVADIY LGSKSSGADG IAIAFHRGSI GFIGTMGGGL
GILGAPNGIG FEIDTYWKAT 120 SDETGDSFGH GQMNGAHAGF VSTNRNASYL
TALAPMQKIP APNNKWRVLT INWDARNNKL 180 TARLQEKSND ASTSTPSPRY
QTWELLNPAF DLNQKYTFII GSATGAANNK HQIGVTLFEA 240 YFTKPTIEAN
PVDIELGTAF DPLNHEPIGL KATDEVDGDI TKDITVEFND IDTSKPGAYR 300
VTYKVVNSYG ESDEKTIEVV VYTKPTITAH DITIKKDLAF DPLNYEPIGL KATDPIDGDI
360 TDKIAVKFNN VDTSKPGKYH VTYKVINSYE KIDEKTIEVT VYTKPSIVAH
DVEIKKDTAF 420 DPLNYEPIGL KATDPIDGDI TDKITVESND VDTSKPGAYS
VKYKVVNNYE ESDEKTIAVT 480 VPVIDDGWEN GDPTGWKFFS GETITLEDDE
EHALNGKWVF YADKHVAIYK QVELKNNIPY 540 QITVYVKPED EGTVAHHIVK
VSFKSDSAGP ESEEVINERL IDAEQIQKGY RKLTSIPFTP 600 TTIVPNKKPV
IIVENFLPGW IGGVRIIVEP TK 632
[0217] The predicted molecular weight for this protein is 69,398
Daltons.
[0218] The amino acid sequences of tIC851, CryET70, and Cry22Aa
were aligned as shown below using the CLUSTAL alignment program
(PC/GENE.RTM.). The tIC851 protein shares approximately 56% amino
acid sequence identity with CryET70 and approximately 57% amino
acid sequence identity with Cry22Aa. According to current Bacillus
thuringiensis crystal protein nomenclature rules, the tIC851
protein should be assigned to a new secondary class of Cry
proteins.
[0219] For the three way alignment, the K-tuple value was set at 1,
the gap penalty value was set at 5, the window size was set at 10,
the filtering level was set at 2.5, the open gap cost was set at
10, and the unit gap cost was set at 10. An "*" indicates that a
position in the alignment is perfectly conserved, and a `.`
indicates that a position is well conserved.
14 Cry22Aa MKEQNLNKYDEITVQAASDYIDIRPIFQTNGSATFNSNTNITTLTQAINS 50
ET70 MKDSISKGYDEITVQA-SDYIDIRSIFQTNGSATFNSTTNITTLTQATNS 49 tIC851
MN---SKSIIEKGVQE-NQYIDIRNICSINGSAKFDPNTNITTLTEAINS 46 *. . * **.
..***** * .****.*...*******.*.** Cry22Aa
QAGAIAGKTALDMRHDFTFRADIFLGTKSNGADGIAIAFHRGSIGFVGTK 100 ET70
QAGAIAGKTALDMRHDFTFRADIFLGTKSNGADGIAIAFHRGSIGFVGEK 99 tIC851
QAGAIAGKTALDMRRDFTLVADIYLGSKSSGADGIAIAFHRGSIGFIGTM 96
**************.***. ***.**.**.****************.*.. Cry22Aa
GGGLGILGAPKGIGFELDTYANAPEDEVGDSFGHGAMKGSFPSFPNGYPH 150 ET70
GGGLGILGALKGIGFELDTYANAPQDEQGDSFGHGAMRGLFPGFPNGYPH 149 tIC851
GGGLGILGAPNGIGFEIDTYWKATSDETGDSFGHGQMNG---------AH 137 *********
.*****.*** .*. ** *******.*.* .* Cry22Aa
AGFVSTDKNSRWLSALAQMQRIAAPNGRWRRLEIRWDARNKELTANLQDL 200 ET70
AGFVSTDKNRGWLSALAQMQRIAAPNGRWRRLAIHWDARNKKLTANLEDL 199 tIC851
AGFVSTNRNASYLTALAPMQKIPAPNNKWRVLTINWDARNNKLTARLQE- 186 ******..*
.*.***.**.*.***..** *.*.*****..***.*.. Cry22Aa
TFNDITVGEKPRTPRTATWRLVNPAFELDQKYTFVIGSATGASNNLHQIG 250 ET70
TFNDSTVLVKPRTPRYARWELSNPAFELDQKYTFVIGSATGASNNLHQIG 249 tIC851
--KSNDASTSTPSPRYQTWELLNPAFDLNQKYTFIIGSATGAANNKHQIG 234 .. .. ....**
. * * ****.*.*****.*******.** **** Cry22Aa
IIEFDAYFTKPTIEANNVNVPVGATFNPKTYPGINLRATDEIDGDLTSKI 300 ET70
IIEFDAYFTKPTIEANNVSVPVGATFNPKTYPGINLRATDEIDGDLTSEI 299 tIC851
VTLFEAYFTKPTIEANPVDIELGTAFDPLNHEPIGLKATDEVDGDITKDI 284 ..
*.*********** *.. .*..*.* .. *.*.****.***.*..* Cry22Aa
IVKANNVNTSKTGVYYVTYYVENSYGESDEKTIEVTVFSNPTIIASDVEI 350 ET70
IVTDNNVNTSKSGVYNVTYYVKNSYGESDEKTIEVTVFSNPTIIASDVEI 349 tIC851
TVEFNDIDTSKPGAYRVTYKVVNSYGESDEKTIEVVVYTKPTITAHDITI 334 .*.
*...***.*.* *** * *************.*...***.* *..* Cry22Aa
EKGESFNPLTDSRVGLSAQDSLGNDITQNVKVKSSNVDTSKPGEYEVVFE 400 ET70
EKGESFNPLTDSRVRLSAQDSLGNDITSKVKVKSSNVDTSKPGEYDVVFE 399 tIC851
KKDLAFDPL----------------------------------NYE---- 346 .*. .*.**
.*. Cry22Aa VTDSFGGKAEKDFKVTVLGQPSIEANNVELEIDDSLDPLTDAKVGLRAKD 450
ET70 VTDNFGGKAEKEIKVTVLGQPSIEANDVELEIGDLFNPLTDSQVGLRAKD 449 tIC851
------------------------------------------PIGLKATD 354 .**.*.*
Cry22Aa SLGNDITKDIKVKFNNVDTSNSGKYEVIFEVTDRFGKKAEKSIEVLVLGE 500 ET70
SLGKDITNDVKVKSSNVDTSKPGEYEVVFEVTDRFGKKAEKSIKVLVLGE 499 tIC851
PIDGDITDKIAVKFNNVDTSKPGKYHVTYKVINSYEKIDEKTIEVTVYTK 404 ... ***...
** .*****..*.*.*...*.....* .**.*.* * .. Cry22Aa
PSIEANDVEVNKGETFEPLTDSRVGLRAKDSLGNDITKDVKIKSSNVDTS 550 ET70
PSIEANNVEIEKDERFDPLTDSRVGLRAKDSLGKDITNDVKVKSSNVDTS 549 tIC851
PSIVAHDVEIKKDTAFDPLNYEPIGLKATDPIDGDITDKITVESNDVDTS 454 ***
*..**..*.. *.**. ...**.*.*... ***......*..**** Cry22Aa
KPGEYEVVFEVTDRFGKYVEKTIGVIVPVIDDEWEDGNVNGWKFYAGQDI 600 ET70
KPGEYEVVFEVTDRFGKYVKKLIVVIVPVIDDEWEDGNVNGWKFYAGQDI 599 tIC851
KPGAYSVKYKVVNNYEESDEKTIAVTVPVIDDGWENGDPTGWKFFSGETI 504 ***.*.*
..*...... .* * *.******.**.*. .****..*..* Cry22Aa
KLLKDPDKAYKGDYVFYDSRHVAISKTIPLTDLQINTNYEITVYAKAES- 649 ET70
TLLKDPEKAYKGEYVFYDSRHAAISKTIPVTDLQVGGNYEITVYVKAES- 648 tIC851
TLEDDEEHALNGKWVFYADKHVAIYKQV---ELKNNIPYQITVYVKPEDE 551 .* .* ..*
.*..***...*.** * . .*. . *.****.*.*. Cry22Aa
---GDHHLKVTYKKDPAGPEEPPVFNRLISTGTLVEKDYRELKGT-FRVT 695 ET70
---GDHHLKVTYKKDPKGPEEPPVFNRLISTGKLVEKDYRELKGT-FRVT 694 tIC851
GTVAHHIVKVSFKSDSAGPESEEVINERLIDAEQIQKGYRKLTSIPFTPT 601 ..*
.**..*.*. ***. *.* . ... ..*.**.*... * * Cry22Aa
EL--NKAPLIIVENFGAGYIGGIRIV--KIS 722 ET70 EL--NQAPLIIVENFGAGYIGGIRI-
V--KIS 721 tIC851 TIVPNKKPVIIVENFLPGWIGGVRIIVEPTK 632 .. *.
*.****** .*.***.**. ..
5.7 Example 7
[0220] Expression of the tIC851 Protein in B. thuringiensis and
Bioassay Evaluation
[0221] The coding region for tIC851 was cloned into the B.
thuringiensis shuttle vector pEG597 (Baum et al., 1990) together
with about 0.6 kb of flanking native DNA both up and down stream of
the ORF, giving rise to the recombinant plasmids pIC17501 and
pIC17502. These plasmids contain a gene which confers
chloramphenicol resistance on a B. thuringiensis host cell. Plasmid
pMON56207, containing the cryET70 coding sequence, confers
erythromycin resistance to a B. thuringiensis host. These plasmids
were introduced into the Cry-B. thuringiensis strain EG10650 by
electroporation. Recombinants harboring the correct plasmids were
selected for growth on starch agar medium supplemented with the
appropriate antibiotic.
[0222] Recombinants were grown in C2 medium for 72-96 hours, at
which time the cultures were sporulated and the cells lysed.
Plasmids pIC17501 and plC17502, differing only with respect to the
orientation of the tIC851 gene insert, directed the production of a
protein with an apparent molecular mass of approximately 75 kDa, as
judged by SDS polyacrylamide gel electrophoresis. EG10650
recombinants harboring the cloning vector pEG597 did not produce a
crystal protein. Plasmid pMON56207 directed the production of
CryET70, with an apparent molecular mass of approximately 80
kDa.
[0223] tIC851 was tested against boll weevil larvae and western
corn rootworm (WCRW) larvae in an insect feeding bioassay and shown
not to have activity against WCRW, but surprisingly good activity
against boll weevil. Based on the similarity of tIC851 to CryET70
and Cry22Aa, these two proteins were also tested against boll
weevil. A dose-response study on the susceptibility of the boll
weevil to these B. thuringiensis toxins was performed by diet
incorporation (Stone et al. 1991). A series of 3 to 8
concentrations prepared by serial dilution was used in each
instance. First instar larvae were manually infested onto the diet.
Mortality and weight measurements were recorded 10 days after
infestation. Larvae that were dead or were still at the neonate
stage were considered dead in tabulating larval responses to the
individual proteins. Concentration-mortality regressions were
estimated assuming the probit model (SAS software 1995). Weight
records were used to calculate effective concentrations using the
non-linear regression model (SAS 1995).
[0224] Surprisingly, Cry22Aa was also found to have significant
toxicity to boll weevil larvae comparable to that of CryET70, as
indicated in Table 11. This is the first report that Cry22Aa and
CryET70 have activity against this target insect pest.
15TABLE 11 Cotton boll weevil Bioassay Protein LC.sub.50
(.mu.g/well) EC.sub.50 (.mu.g/well) CryET70 3.12 (1.95-5.00) 1.92
.+-. 0.37 Cry22Aa 0.72 (0.022-1.70) 0.36 .+-. 0.18
[0225] The toxin encoded by the tIC851 gene has interesting
similarities as well as differences when compared with the toxins
encoded by the CryET70 and Cry22Aa genes. Both CryET70 and Cry22Aa
have within their primary sequence four repeating regions of
approximately 80 amino acids each, aligned in a head-to-tail
fashion. The sequence of tIC851 shows that the tIC851 protein has
only three of the four `repeat domains` found in CryET70 and
Cry22Aa. This accounts for most of the approximately 90 amino acids
by which the tIC851 coding sequence is shorter than that of either
CryET70 or Cry22Aa. Despite this difference in structure, tIC851
has significant activity on boll weevil larvae. The novel modular
structure of these three Bt toxins should be of value in
semi-rational engineering of variants, which could have increased
potency or spectrum of activity.
5.8 Example 8
[0226] Transgenic Plants Expressing tIC851
[0227] One or more transgenes, each containing a structural coding
sequence of the present invention can be inserted into the genome
of a plant by any suitable method such as those detailed herein.
Suitable plant transformation vectors include those derived from a
Ti plasmid of Agrobacterium tumefaciens, as well as those
disclosed, e.g., by Herrera-Estrella (1983), Bevan (1983), Klee
(1985) and Eur. Pat. Appl. Publ. No. EP0120516. In addition to
plant transformation vectors derived from the Ti or root-inducing
(Ri) plasmids of Agrobacterium, alternative methods can be used to
insert the DNA constructs of this invention into plant cells. Such
methods may involve, for example, the use of liposomes,
electroporation, chemicals that increase free DNA uptake, free DNA
delivery via microprojectile bombardment, and transformation using
viruses or pollen (Fromm et al., 1986; Armstrong et al., 1990;
Fromm et al., 1990). For efficient expression of the
polynucleotides disclosed herein in transgenic plants, the selected
sequence region encoding the insecticidal polypeptide must have a
suitable sequence composition (Diehn et al., 1996).
[0228] Expression of the tIC851 protein from within a plant
expression vector is then confirmed in plant protoplasts by
electroporation of the vector into protoplasts followed by protein
blot and ELISA analysis. This vector can be introduced into the
genomic DNA of plant embryos such as cotton by particle gun
bombardment followed by paromomycin selection to obtain cotton
plants expressing the cry gene essentially as described in U.S.
Pat. No. 5,424,412. For example, the plant transformation and
expression vector can be introduced via co-bombardment with a
hygromycin resistance conferring plasmid into transformation
susceptible cotton tissue, followed by hygromycin selection, and
regeneration. Transgenic cotton lines expressing the tIC851 protein
can then identified by ELISA analysis. Progeny seed from these
events can then subsequently be tested for protection from
susceptible insect feeding.
[0229] The B. thuringiensis polypeptides described herein are
primarily localized to the cytoplasm of the plant cell, and this
cytoplasmic localization results in plants that are insecticidally
effective. However, in certain embodiments, it may be advantageous
to direct the B. thuringiensis polypeptide 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.
[0230] 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.
[0231] In addition to tIC851 expression in plants as described
herein, it is specifically intended that Cry22Aa and CryET70 be
used alone or in combination with each other or in combinations
along with tIC851 in plants to protect plants from boll weevil
infestation and in particular combinations to prevent the onset of
resistance of boll weevils to any of the proteins when used
alone.
[0232] 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.
[0233] 6.0 References
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References