U.S. patent application number 11/058727 was filed with the patent office on 2005-11-24 for genes encoding proteins with pesticidal activity.
This patent application is currently assigned to E.I. duPont de Nemours and Company. Invention is credited to Abad, Andre R., Flannagan, Ronald D., Herrmann, Rafael, Kahn, Theodore W., Lu, Albert L., McCutchen, Billy Fred, Presnail, James K., Wong, James F.H., Yu, Cao-Guo.
Application Number | 20050261483 11/058727 |
Document ID | / |
Family ID | 30003210 |
Filed Date | 2005-11-24 |
United States Patent
Application |
20050261483 |
Kind Code |
A1 |
Abad, Andre R. ; et
al. |
November 24, 2005 |
Genes encoding proteins with pesticidal activity
Abstract
The invention provides nucleic acids, and variants and fragments
thereof, obtained from strains of Bacillus thuringiensis encoding
.delta.-endotoxins having pesticidal activity against insect pests.
The invention further provides mutagenized nucleic acids that have
been modified to encode pesticidal polypeptides such as endotoxins
having improved pesticidal activity and/or altered pest
specificity. Particular embodiments of the invention provide
isolated nucleic acids encoding pesticidal proteins that may be
optimized as well as pesticidal compositions, expression cassettes,
and transformed microorganisms and plants comprising a nucleic acid
of the invention. These compositions find use in methods for
controlling pests, especially plant pests.
Inventors: |
Abad, Andre R.; (W. Des
Moines, IA) ; Flannagan, Ronald D.; (Grimes, IA)
; Herrmann, Rafael; (Wilmington, DE) ; Kahn,
Theodore W.; (Durham, NC) ; Lu, Albert L.;
(Newark, DE) ; McCutchen, Billy Fred; (Clive,
IA) ; Presnail, James K.; (Avondale, PA) ;
Wong, James F.H.; (Johnston, IA) ; Yu, Cao-Guo;
(Urbandale, IA) |
Correspondence
Address: |
ALSTON & BIRD LLP
PIONEER HI-BRED INTERNATIONAL, INC.
BANK OF AMERICA PLAZA
101 SOUTH TYRON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
E.I. duPont de Nemours and
Company
Wilmington
DE
|
Family ID: |
30003210 |
Appl. No.: |
11/058727 |
Filed: |
February 15, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11058727 |
Feb 15, 2005 |
|
|
|
10606320 |
Jun 25, 2003 |
|
|
|
60391786 |
Jun 26, 2002 |
|
|
|
60460787 |
Apr 4, 2003 |
|
|
|
Current U.S.
Class: |
530/395 |
Current CPC
Class: |
C12N 15/8286 20130101;
C07K 14/325 20130101; Y02A 40/162 20180101 |
Class at
Publication: |
530/395 |
International
Class: |
C07K 014/195 |
Claims
That which is claimed:
1. An endotoxin comprising a mutation consisting of the alteration
of at least one proteolytic site, whereby the stability of the
endotoxin in an insect gut is increased relative to an endotoxin
lacking said mutation.
2. The endotoxin of claim 1, wherein said mutation consists of an
alteration selected from the group consisting of: a) an addition of
at least one amino acid to at least one proteolytic site; b) a
removal of at least one amino acid from at least one proteolytic
site; c) a replacement of at least one amino acid of at least one
proteolytic site with a different amino acid; and d) a combination
of at least two of (a), (b), and (c).
3. The endotoxin of claim 2, wherein said mutation consists of an
alteration which is the replacement of at least one amino acid of a
proteolytic site with an amino acid selected from the group
consisting of valine and arginine.
4. The endotoxin of claim 1, wherein at least one of said at least
one proteolytic sites is a trypsin site.
5. The endotoxin of claim 1, wherein at least one of said at least
one proteolytic sites is a chymotrypsin site.
6. The endotoxin of claim 1, wherein at least one of said at least
one proteolytic sites is a cathepsin site.
7. The endotoxin of claim 1, further comprising at least one
engineered cathepsin-sensitive proteolytic site, wherein said
endotoxin has improved pesticidal activity relative to an endotoxin
which lacks said at least one cathepsin-sensitive proteolytic site.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 10/606,320, filed Jun. 25, 2003, which claims
the benefit of U.S. Provisional Application No. 60/460,787, filed
Apr. 4, 2003, which claims the benefit of U.S. Provisional
Application No. 60/391,786, filed Jun. 26, 2002, all of which are
herein incorporated by reference in their entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ON COMPACT DISK
[0002] The official copy of the sequence listing is submitted on
compact disk (CD). Two CDs, labeled Copy 1 and Copy 2, containing
an ASCII formatted sequence listing with a file named
287809SEQLIST.TXT, created on Feb. 14, 2005, and having a size of
618 kilobytes are filed concurrently with the specification. The
sequence listing contained on these compact disks is part of the
specification and is herein incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to naturally-occurring and
recombinant nucleic acids that encode polypeptides characterized by
pesticidal activity against insect pests. In some embodiments,
nucleic acids were obtained from Bacillus thuringiensis Cry8-like
genes that encode .delta.-endotoxins characterized by pesticidal
activity against insect pests. Compositions and methods of the
invention utilize the disclosed nucleic acids and their encoded
pesticidal polypeptides to control plant pests.
BACKGROUND OF THE INVENTION
[0004] Insect pests are a major factor in the loss of the world's
agricultural crops. For example, corn rootworm feeding damage or
boll weevil damage can be economically devastating to agricultural
producers. Insect pest-related crop loss from corn rootworm alone
has reached one billion dollars a year.
[0005] Traditionally, the primary methods for impacting insect pest
populations, such as corn rootworm populations, are crop rotation
and the application of broad-spectrum synthetic chemical
pesticides. However, consumers and government regulators alike are
becoming increasingly concerned with the environmental hazards
associated with the production and use of synthetic chemical
pesticides. Because of such concerns, regulators have banned or
limited the use of some of the more hazardous pesticides. Thus,
there is substantial interest in developing alternative
pesticides.
[0006] Biological control of insect pests of agricultural
significance using a microbial agent, such as fungi, bacteria, or
another species of insect affords an environmentally friendly and
commercially attractive alternative. Generally speaking, the use of
biopesticides presents a lower risk of pollution and environmental
hazards, and provides a greater target specificity than is
characteristic of traditional broad-spectrum chemical insecticides.
In addition, biopesticides often cost less to produce and thus
improve economic yield for a wide variety of crops.
[0007] Certain species of microorganisms of the genus Bacillus are
known to possess pesticidal activity against a broad range of
insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera,
and others. Bacillus thuringiensis and Bacillus papilliae are among
the most successful biocontrol agents discovered to date. Insect
pathogenicity has been attributed to strains of: B. larvae, B.
lentimorbus, B. papilliae, B. sphaericus, B. thuringiensis
(Harwook, ed., (1989) Bacillus (Plenum Press), 306) and B. cereus
(WO 96/10083). Pesticidal activity appears to be concentrated in
parasporal crystalline protein inclusions, although pesticidal
proteins have also been isolated from the vegetative growth stage
of Bacillus. Several genes encoding these pesticidal proteins have
been isolated and characterized (see, for example, U.S. Pat. Nos.
5,366,892 and 5,840,868).
[0008] Microbial pesticides, particularly those obtained from
Bacillus strains, have played an important role in agriculture as
alternatives to chemical pest control. Recently, agricultural
scientists have developed crop plants with enhanced insect
resistance by genetically engineering crop plants to produce
pesticidal proteins from Bacillus. For example, corn and cotton
plants genetically engineered to produce pesticidal proteins
isolated from strains of B. thuringiensis, known as
.delta.-endotoxins or Cry toxins (see, e.g., Aronson (2002) Cell
Mol. Life Sci. 59(3): 417-425; Schnepf et al. (1998) Microbiol Mol
Biol Rev. 62(3):775-806) are now widely used in American
agriculture and have provided the farmer with an environmentally
friendly alternative to traditional insect-control methods. In
addition, potatoes genetically engineered to contain pesticidal Cry
toxins have been sold to the American farmer. However, while they
have proven to be very successful commercially, these genetically
engineered, insect-resistant crop plants provide resistance to only
a narrow range of the economically important insect pests. Some
insects, such as Western corn rootworm, have proven to be
recalcitrant.
[0009] Accordingly, efforts have been made to understand the
mechanism of action of Bt toxins and to engineer toxins with
improved properties. It has been shown that insect gut proteases
can affect the impact of Bacillus thuringiensis Cry proteins and
other pesticidal proteins on the insect. Some proteases activate
Cry proteins by processing them from a "protoxin" form into a toxic
form, or "toxin." See, Oppert (1999) Arch. Insect Biochem. Phys.
42: 1-12 and Carroll et al. (1997) J. Invertebrate Pathology 70:
41-49. This activation of the toxin can include the removal of the
N- and C-terminal peptides from the protein and can also include
internal cleavage of the protein. Other proteases can degrade
pesticidal proteins. See Oppert, ibid.; see also U.S. Pat. Nos.
6,057,491 and 6,339,491.
[0010] Research has shown that insect gut proteases include
cathepsins, such as cathepsin B- and L-like proteinases. See, Shiba
et al. (2001) Arch. Biochem. Biophys. 390: 28-34; see also, Purcell
et al. (1992) Insect Biochem. Mol. Biol. 22: 41-47. For example,
cathepsin L-like digestive cysteine proteinases are found in the
larval midgut of Western corn rootworm. See, Koiwa et al. (2000)
FEBS Letters 471: 67-70; see also, Koiwa et al. (2000) Analytical
Biochemistry 282: 153-155. The preferred proteolytic substrate
sites of these proteases have been investigated using synthetic
substrates. See, Alves et al. (2001) Eur. J. Biochem. 268:
1206-1212 and Melo et al. (2001) Anal. Biochem. 293: 71-77.
[0011] Although numerous investigators have attempted to make
mutant pesticidal proteins, including endotoxin proteins, with
improved pesticidal activity, few have succeeded. In fact, the
majority of genetically engineered B. thuringiensis toxins that
have been reported in the literature report endotoxin activity that
is no better than that of the wild-type protein, and in many cases,
the activity is decreased or destroyed altogether. Thus, new
microbial pesticides having altered specificity and/or improved
pesticidal activity are desired for use in pest-management
strategies.
SUMMARY OF THE INVENTION
[0012] Compositions and methods are provided for impacting insect
pests. More specifically, the invention relates to methods of
impacting insect pests utilizing nucleic acids derived from
pesticidal genes to produce transformed microorganisms and plants
that express a pesticidal polypeptide of the invention. The
compositions and methods of the invention find use in agriculture
for controlling pests of many crop plants. Such pests include, but
are not limited to, agriculturally significant pests, such as:
Western corn rootworm, e.g., Diabrotica virgifera virgifera;
Northern corn rootworm, Diabrotica longicornis barberi; Southern
corn rootworm, Diabrotica undecimpunctata howardi; wireworms,
Melanotus spp. and Aeolus spp.; boll weevil, e.g., Anthonomus
grandis; Colorado potato beetle, Leptinotarsa decemlineata; and
alfalfa weevil, Hypera nigrirostris.
[0013] The invention provides nucleic acids and fragments and
variants thereof which encode polypeptides that possess pesticidal
activity against insect pests. The wild-type (e.g., naturally
occurring) nucleotide sequences of the invention obtained from
strains of Bacillus thuringiensis encode Cry8-like
.delta.-endotoxins. The invention further provides fragments and
variants of nucleotide sequences that encode biologically active
(e.g., pesticidal) polypeptides, and the invention thereby also
provides fragments and variants of Cry8-like endotoxins. In some
embodiments, the nucleotide sequences encode polypeptides that are
pesticidal for at least one insect belonging to the order
Coleoptera.
[0014] Other embodiments of the invention provide nucleic acids
encoding truncated versions of a pesticidal protein that are
characterized by pesticidal activity that is either equivalent to
or improved relative to the activity of the corresponding
full-length pesticidal protein. Some of the truncated nucleic acids
of the invention can be referred to as either fragments or
variants. In some embodiments, the nucleic acids of the invention
are truncated at the 3' end or 5' end of a wild-type coding
sequence. In other embodiments, nucleic acids of the invention
comprise a contiguous sequence of nucleotides derived from another
coding sequence of the invention that have been truncated at both
the 5' and 3' ends.
[0015] The invention also pertains to pharmacokinetic studies which
reveal novel mechanisms by which to explore the degradation and/or
stability characteristics of a pesticidal protein utilizing both in
vitro and in vivo conditions. Thus, the invention also provides for
the design and production of mutant nucleotide sequences and their
encoded amino acid sequences that confer additional properties on a
polypeptide encoded by or comprising them. Based on these findings,
multiple pharmacokinetic parameters of the pesticidal protein can
be analyzed to predict, change and produce pesticidal polypeptides
with improved pesticidal characteristics. For example, a
combination of in vitro assays using previously identified,
pest-specific proteases such as L-cathepsins, B-cathepsins,
chymotrypsins, trypsins and the like, with or without known
surrogate proteases, can be utilized to identify potential cleavage
sites within a pesticidal molecule. Furthermore, these data can be
combined with in vivo, insect midgut assays to produce data that
provide a consensus understanding of those areas of the pesticidal
protein which are most likely to be susceptible to proteolytic
degradation and/or instability. In addition, midgut assays
performed at various larval stages will produce data revealing
potential differences in the susceptibility of the pesticidal
protein to proteolytic degradation at different stages of larval
development.
[0016] These data provide for nucleotide sequences that may encode
a previously unknown protease recognition site, which renders a
polypeptide containing it susceptible to digestion by the protease.
These mutations may be placed in the context of a background
sequence, such as a nucleic acid encoding a Bt toxin or other
pesticidal protein, to provide proteins that have been engineered
to have improved and/or altered pesticidal activities. For example,
these mutations may be placed in the context of the pentin-1
protein (see U.S. Pat. Nos. 6,057,491 and 6,339,144, herein
incorporated by reference) to provide proteins with improved and/or
altered pesticidal properties, as demonstrated in Example 21.
[0017] In this manner, the invention provides an array of mutations
that may be used individually or in combination to provide improved
properties to an engineered pesticidal protein. The nucleic acids
of the invention can be used to produce expression cassettes useful
for the production of transformed microorganisms and plants. The
resulting transformants can be used in the preparation of
pesticidal compositions comprising a transformed microorganism, or
for the production and isolation of pesticidal proteins, or for the
production of pest resistant plants. Thus, the invention further
provides pesticidal compositions comprising pesticidal polypeptides
and/or transformed microorganisms as well as methods for producing
and using such compositions. The pesticidal compositions of the
invention find use in agricultural methods for impacting pests.
[0018] The invention further provides isolated pesticidal (e.g.,
insecticidal) polypeptides encoded by either a naturally occurring,
or a modified (e.g., mutagenized or manipulated) nucleic acid of
the invention. In particular examples, pesticidal proteins of the
invention include pesticidal proteins such as pentin-1 like
proteins, full-length .delta.-endotoxin proteins, fragments of
full-length .delta.-endotoxins, and polypeptides that are produced
from mutagenized nucleic acids designed to introduce particular
amino acid sequences into the polypeptides of the invention. In
particular embodiments, the polypeptides of the invention have
enhanced pesticidal activity relative to the activity of the
naturally occurring .delta.-endotoxin or other protein from which
they are derived.
[0019] The nucleic acids of the invention can also be used to
produce transgenic (e.g., transformed) plants that are
characterized by genomes that comprise at least one stably
incorporated nucleotide construct comprising a coding sequence of
the invention operably linked to a promoter that drives expression
of the encoded pesticidal polypeptide. Accordingly, transformed
plant cells, plant tissues, plants, and seeds thereof are also
provided.
[0020] In a particular embodiment, a transformed plant of the
invention can be produced using a nucleic acid that has been
optimized for increased expression in a host plant. For example,
one of the pesticidal polypeptides of the invention can be
back-translated to produce a nucleic acid comprising codons
optimized for expression in a particular host, for example, a crop
plant such as a Zea mays plant. Expression of a coding sequence by
such a transformed plant (e.g., dicot or monocot) will result in
the production of a pesticidal polypeptide and will confer
increased pest resistance to the plant. In some embodiments, the
invention provides transgenic plants expressing pesticidal
polypeptides that find use in methods for impacting various insects
and other pests.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1: Probit Analysis of 1218 Cry8-like (M6) Mutant
against Colorado Potato Beetle (see Example 9). The log
(concentration) of the toxin is graphed on the horizontal axis,
while larval mortality is graphed on the vertical axis. The results
of the probit analysis were: the LC.sub.50 was 0.259 mg/ml; 95%
fiducial limits were 0.171 mg/ml and 0.370 mg/ml. Observed
mortality data points are represented by solid dots, while
predicted mortality is represented by open squares. The 95% upper
and lower limits are indicated by dashed lines.
[0022] FIG. 2: Effect of Wild Type 1218-1 on Colorado Potato Beetle
Larval Mortality. The rate of application of wild type endotoxin in
micrograms per square centimeter is arrayed on the horizontal axis
and the percent mortality is shown on the vertical axis. Two
replicates of the experiment are shown (bars with vertical
stripes=replicate 1; bars with diagonal stripes=replicate 2).
[0023] FIG. 3: Effect of 1218 Cry8-like Mutant K03 on Colorado
Potato Beetle Larval Mortality. The rate of application of wild
type endotoxin in micrograms per square centimeter is arrayed on
the horizontal axis and the percent mortality is shown on the
vertical axis. Two replicates of the experiment are shown (bars
with diagonal stripes=replicate 1; bars with horizontal
stripes=replicate 2).
[0024] FIG. 4: Effect of 1218 Cry8-like Mutant K34 on Colorado
Potato Beetle Larval Mortality. The rate of application of wild
type endotoxin in micrograms per square centimeter is arrayed on
the horizontal axis and the percent mortality is shown on the
vertical axis. Two replicates of the experiment are shown (bars
with diagonal stripes=replicate 1; bars with vertical
stripes=replicate 2).
[0025] FIG. 5: Larval Assays with the Cotton Boll Weevil. This
figure shows results of larval assays with the cotton boll weevil,
as described in Experimental Example 13 and Table 9. Doses are
arrayed on the horizontal axis, while combined larval weight in
milligrams is shown on the vertical axis. K03 mutant data are shown
by vertically-striped bars; M6 mutant data are shown by white bars;
1218-1 (wild type) data are shown by dotted bars; and the buffer
control data are shown by diagonally-striped bars.
[0026] FIG. 6: Probit Analysis of Wild Type 1218-1 against Colorado
Potato Beetle (see Example 6). The log (concentration) of the toxin
is graphed on the horizontal axis, while larval mortality is
graphed on the vertical axis. The results of the probit analysis
were: at probability 0.50, concentration was 1.1098 mg/ml; 95%
fiducial limits were 0.6859 and 2.4485. Observed mortality data
points are represented by solid dots, while predicted mortality is
represented by open squares. The 95% upper and lower limits are
indicated by dashed lines.
[0027] FIG. 7: Probit Analysis of 1218 Cry8-like (K03) Mutant
against Colorado Potato Beetle (see Example 6). The log
(concentration) of the toxin is graphed on the horizontal axis,
while larval mortality is graphed on the vertical axis. The results
of the probit analysis were: at probability 0.50, concentration was
0.00808 mg/ml; 95% fiducial limits were 0.00467 and 0.01184.
Observed mortality data points are represented by solid dots, while
predicted mortality is represented by open squares. The 95% upper
and lower limits are indicated by dashed lines.
[0028] FIG. 8: Distribution Analysis of Coding Regions from Maize
(see Example 14).
[0029] Maize cDNAs with full-length coding regions were analyzed
for GC content and plotted as a function of their GC content (see
top panel, "ORFs"). An EST-based "UniGene" dataset containing
84,085 sequences was also analyzed ("UniGenes," shown in lower
panel).
DETAILED DESCRIPTION OF THE INVENTION
[0030] The invention is drawn to compositions and methods for
impacting insect pests, particularly plant pests. More
specifically, the isolated nucleic acids of the invention, and
fragments and variants thereof, comprise nucleotide sequences that
encode pesticidal polypeptides (e.g., proteins). The disclosed
pesticidal proteins are biologically active (e.g., pesticidal)
against insect pests such as insect pests of the order Coleoptera.
Insect pests of interest include, but are not limited to: western
corn rootworm, e.g., Diabrotica virgifera virgifera; northern corn
rootworm, e.g., Diabrotica longicornis barberi; and southern corn
rootworm, e.g., Diabrotica undecimpunctata howardi. Additional
pests include: wireworms, Melanotus, Eleodes, Conoderus, and Aeolus
spp.; Japanese beetle, Popillia japonica; white grub, Phyllophaga
crinita; corn flea beetle, Chaetocnema pulicaria; sunflower stem
weevil, Cylindrocupturus adspersus; gray sunflower seed weevil,
Smicronyx sordidus; sunflower beetle, Zygogramma exclamationis;
boll weevil, e.g., Anthonomus grandis; alfalfa weevil, Hypera
nigrirostris; crucifer flea beetle, Phyllotreta cruciferae;
Colorado potato beetle, Leptinotarsa decemlineata; striped flea
beetle, Phyllotreta striolata; striped turnip flea beetle,
Phyllotreta nemorum; and rape beetle, Meligethes aeneus.
[0031] The compositions of the invention comprise isolated nucleic
acids, and fragments and variants thereof, that encode pesticidal
polypeptides, expression cassettes comprising nucleotide sequences
of the invention, isolated pesticidal proteins, and pesticidal
compositions. In some embodiments, the invention provides modified
Cry8-like .delta.-endotoxin proteins characterized by improved
insecticidal activity against Coleopterans relative to the
pesticidal activity of the corresponding wild-type protein. In
other embodiments, the invention provides other pesticidal
proteins, such as mutagenized pentin-1 like proteins, characterized
by improved pesticidal activity against Coleopterans relative to
the pesticidal activity of the corresponding wild-type, or
non-mutagenized protein. The invention further provides plants and
microorganisms transformed with these novel nucleic acids, and
methods involving the use of such nucleic acids, pesticidal
compositions, transformed organisms, and products thereof in
impacting insect pests.
[0032] The nucleic acids and nucleotide sequences of the invention
may be used to transform any organism to produce the encoded
pesticidal proteins. Methods are provided that involve the use of
such transformed organisms to impact or control plant pests. The
nucleic acids and nucleotide sequences of the invention may also be
used to transform organelles such as chloroplasts (McBride et al.
(1995) Biotechnology 13:362-365; Kota et al. (1999) Proc. Natl.
Acad Sci. USA 96: 1840-1845).
[0033] The invention further relates to the identification of
fragments and variants of the naturally-occurring coding sequence
that encode biologically active pesticidal proteins. The nucleotide
sequences of the invention find direct use in methods for impacting
pests, particularly insect pests such as pests of the order
Coleoptera. Pests of interest include, for example, the Colorado
potato beetle, western corn rootworm, southern corn rootworm,
northern corn rootworm, Mexican corn rootworm, wireworms, and boll
weevil. Accordingly, the present invention provides new approaches
for impacting insect pests that do not depend on the use of
traditional, synthetic chemical pesticides. The invention involves
the discovery of naturally-occurring, biodegradable pesticides and
the genes that encode them.
[0034] The invention further provides fragments and variants of the
naturally occurring coding sequences that also encode biologically
active (e.g., pesticidal) polypeptides. The nucleic acids of the
invention encompass nucleic acid or nucleotide sequences that have
been optimized for expression by the cells of a particular
organism, for example nucleic acid sequences that have been
back-translated (i.e., reverse translated) using plant-preferred
codons based on the amino acid sequence of a polypeptide having
enhanced pesticidal activity. The invention further provides
mutations which confer improved or altered properties on
polypeptides comprising them. The mutations of the invention may be
utilized with any background sequence so long as the object of the
invention is achieved, i.e., so long as the provided toxin exhibits
altered or improved pesticidal activity.
[0035] In the description that follows, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the invention.
[0036] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogues (e.g., peptide nucleic acids) having the essential
nature of natural nucleotides in that they hybridize to
single-stranded nucleic acids in a manner similar to naturally
occurring nucleotides.
[0037] As used herein, the terms "encoding" or "encoded" when used
in the context of a specified nucleic acid mean that the nucleic
acid comprises the requisite information to direct translation of
the nucleotide sequence into a specified protein. The information
by which a protein is encoded is specified by the use of codons. A
nucleic acid encoding a protein may comprise non-translated
sequences (e.g., introns) within translated regions of the nucleic
acid or may lack such intervening non-translated sequences (e.g.,
as in cDNA).
[0038] As used herein, "full-length sequence" in reference to a
specified polynucleotide or its encoded protein means having the
entire nucleic acid sequence or the entire amino acid sequence of a
native sequence. By "native sequence" is intended an, endogenous
sequence, i.e., a non-engineered sequence found in an organism's
genome. A full-length polynucleotide encodes the full-length,
catalytically active form of the specified protein.
[0039] As used herein, the term "antisense" used in the context of
orientation of a nucleotide sequence refers to a duplex
polynucleotide sequence that is operably linked to a promoter in an
orientation where the antisense strand is transcribed. The
antisense strand is sufficiently complementary to an endogenous
transcription product such that translation of the endogenous
transcription product is often inhibited. Thus, where the term
"antisense" is used in the context of a particular nucleotide
sequence, the term refers to the complementary strand of the
reference transcription product.
[0040] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residues is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0041] The terms "residue" or "amino acid residue" or "amino acid"
are used interchangeably herein to refer to an amino acid that is
incorporated into a protein, polypeptide, or peptide (collectively
"protein"). The amino acid may be a naturally occurring amino acid
and, unless otherwise limited, may encompass known analogues of
natural amino acids that can function in a similar manner as
naturally occurring amino acids.
[0042] Polypeptides of the invention can be produced either from a
nucleic acid disclosed herein, or by the use of standard molecular
biology techniques. For example, a truncated protein of the
invention can be produced by expression of a recombinant nucleic
acid of the invention in an appropriate host cell, or alternatively
by a combination of ex vivo procedures, such as protease digestion
and purification.
[0043] As used herein, the terms "isolated" and "purified" are used
interchangeably to refer to nucleic acids or polypeptides or
biologically active portions thereof that are substantially or
essentially free from components that normally accompany or
interact with the nucleic acid or polypeptide as found in its
naturally occurring environment. Thus, an isolated or purified
nucleic acid or polypeptide is substantially free of other cellular
material or culture medium when produced by recombinant techniques,
or substantially free of chemical precursors or other chemicals
when chemically synthesized.
[0044] An "isolated" nucleic acid is free of sequences (preferably
protein-encoding sequences) that naturally flank the nucleic acid
(i.e., sequences located at the 5' and 3' ends of the nucleic acid)
in the genomic DNA of the organism from which the nucleic acid is
derived. For example, in various embodiments, the isolated nucleic
acids can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5
kb, or 0.1 kb of nucleotide sequences that naturally flank the
nucleic acids in genomic DNA of the cell from which the nucleic
acid is derived.
[0045] As used herein, the term "isolated" or "purified" as it is
used to refer to a polypeptide of the invention means that the
isolated protein is substantially free of cellular material and
includes preparations of protein having less than about 30%, 20%,
10%, or 5% (by dry weight) of contaminating protein. When the
protein of the invention or biologically active portion thereof is
recombinantly produced, preferably culture medium represents less
than about 30%, 20%, 10%, or 5% (by dry weight) of chemical
precursors or non-protein-of-interest chemicals.
[0046] As used herein, the term "impacting insect pests" refers to
effecting changes in insect feeding, growth, and/or behavior at any
stage of development, including but not limited to: killing the
insect; retarding growth; preventing reproductive capability;
antifeedant activity; and the like.
[0047] As used herein, the terms "pesticidal activity" and
"insecticidal activity" are used synonymously to refer to activity
of an organism or a substance (such as, for example, a protein)
that can be measured by but is not limited to pest mortality, pest
weight loss, pest repellency, and other behavioral and physical
changes of a pest after feeding and exposure for an appropriate
length of time. In this manner, pesticidal activity impacts at
least one measurable parameter of pest fitness. For example
"pesticidal proteins" are proteins that display pesticidal activity
by themselves or in combination with other proteins. Endotoxins are
pesticidal proteins. Other examples of pesticidal proteins include,
e.g., pentin-1 (see U.S. Pat. Nos. 6,057,491 and 6,339,144).
[0048] The term "pesticidally effective amount" connotes a quantity
of a substance or organism that has pesticidal activity when
present in the environment of a pest. For each substance or
organism, the pesticidally effective amount is determined
empirically for each pest affected in a specific environment.
Similarly, an "insecticidally effective amount" may be used to
refer to a "pesticidally effective amount" when the pest is an
insect pest.
[0049] As used herein the term "recombinantly engineered" or
"engineered" connotes the utilization of recombinant DNA technology
to introduce (e.g., engineer) a change in the protein structure
based on an understanding of the protein's mechanism of action and
a consideration of the amino acids being introduced, deleted, or
substituted.
[0050] As used herein the term "mutant nucleotide sequence" or
"mutation" or "mutagenized nucleotide sequence" connotes a
nucleotide sequence that has been mutagenized or altered to contain
one or more nucleotide residues (e.g., base pair) that is not
present in the corresponding wild-type or non-mutagenized sequence.
Such mutagenesis or alteration consists of one or more additions,
deletions, or substitutions or replacements of nucleic acid
residues. When mutations are made by adding, removing, or replacing
an amino acid of a proteolytic site, such addition, removal, or
replacement may be within or adjacent to the proteolytic site
motif, so long as the object of the mutation is accomplished (i.e.,
so long as proteolysis at the site is changed).
[0051] A mutant nucleotide sequence can encode a mutant
.delta.-endotoxin showing improved or decreased insecticidal
activity or an amino acid sequence which confers improved or
decreased insecticidal activity on a polypeptide containing it.
Similarly, by "mutant" or "mutation" in the context of a protein is
intended a polypeptide or amino acid sequence which has been
mutagenized or altered to contain one or more amino acid residues
that is not present in the corresponding wild-type or
non-mutagenized sequence. Such mutagenesis or alteration consists
of one or more additions, deletions, or substitutions or
replacements of amino acid residues. A mutant polypeptide shows
improved or decreased insecticidal activity or an amino acid
sequence which confers improved insecticidal activity on a
polypeptide containing it. Thus, by "mutant" or "mutation" may be
intended either or both of the mutant nucleotide sequence and the
encoded amino acids. In some embodiments, the mutant nucleotide
sequences are placed into a sequence background previously known in
the art, such as Cry3A, to confer improved properties on the
encoded polypeptide. Mutants may be used alone or in any compatible
combination with other mutants of the invention or with other
mutants. Where more than one mutation is added to a particular
nucleic acid or protein, the mutations may be added at the same
time or sequentially; if sequentially, mutations may be added in
any suitable order. Thus, a sequence of the invention may be a
mutagenized nucleotide sequence or an optimized nucleotide
sequence, or a sequence of the invention may be both mutagenized
and optimized.
[0052] As used herein the term "improved insecticidal activity" or
"improved pesticidal activity" characterizes a polypeptide or
encoded polypeptide endotoxin of the invention that has enhanced
Coleopteran pesticidal activity relative to the activity of its
corresponding wild-type protein, and/or an endotoxin that is
effective against a broader range of insects, and/or an endotoxin
having specificity for an insect that is not susceptible to the
toxicity of the wild-type protein. A finding of improved or
enhanced pesticidal activity requires a demonstration of an
increase of toxicity of at least 10%, against the insect target,
and more preferably 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,
100%, 200%, or greater increase of toxicity relative to the
insecticidal activity of the wild-type endotoxin determined against
the same insect.
[0053] For example, an improved pesticidal or insecticidal activity
is provided where a wider or narrower range of insects is impacted
by the polypeptide relative to the range of insects that is
affected by a pesticidal protein such as wild-type Bt toxin. A
wider range of impact may be desirable where versatility is
desired, while a narrower range of impact may be desirable where,
for example, beneficial insects might otherwise be impacted by use
or presence of the toxin. While the invention is not bound by any
particular mechanism of action, an improved pesticidal activity may
also be provided by changes in one or more characteristics of a
polypeptide; for example, the stability or longevity of a
polypeptide in an insect gut may be increased relative to the
stability or longevity of a corresponding wild-type or
non-mutagenized protein.
[0054] By "toxin" or "endotoxin" is intended a polypeptide showing
pesticidal activity or insecticidal activity or improved pesticidal
activity or improved insecticidal activity. In some instances,
polypeptide endotoxins of the invention and the nucleotide
sequences encoding them will share a high degree of sequence
identity or similarity to wild-type 1218 Cry8-like sequences. By
"Cry8-like" is intended that the nucleotide or amino acid sequence
shares a high degree of sequence identity or similarity to
previously described sequences categorized as Cry8. Similarly, by
"pentin-1 like" is intended that the nucleotide or amino acid
sequence shares a high degree of sequence identity or similarity to
previously described pentin-1 sequences (see U.S. Pat. Nos.
6,057,491 and 6,339,144). By "Bt" or "Bacillus thuringiensis" toxin
or endotoxin is intended the broader class of toxins found in
various strains of Bacillus thuringiensis, which includes such
toxins as, for example, Cry3A or Cry3B.
[0055] By "proteolytic site" or "cleavage site" is intended an
amino acid sequence which confers sensitivity to a class of
proteases or a particular protease such that a polypeptide
containing the amino acid sequence is digested by the class of
proteases or particular protease. A proteolytic site is said to be
"sensitive" to the protease(s) that recognize that site. It is
recognized that the efficiency of digestion will vary, and that a
decrease in efficiency of digestion can lead to an increase in
stability or longevity of the polypeptide in an insect gut. Thus, a
proteolytic site may confer sensitivity to more than one protease
or class of proteases, but the efficiency of digestion at that site
by various proteases may vary.
[0056] Proteolytic sites include, for example, trypsin sites,
chymotrypsin sites, papain sites, cathepsin sites, and
cathepsin-like sites. Proteolytic sites for particular proteases
often comprise "motifs," or sequence patterns, which are known to
confer sensitivity to a particular protease. Thus, for example,
cathepsin site motifs include FRR, a cathepsin L protease cleavage
site; RR, a trypsin and cathepsin B cleavage site; LKM, a
chymotrypsin site; and FF, a cathepsin D site. A putative
proteolytic site is a sequence that comprises a motif or comprises
a sequence similar to a motif but which has not been shown to be
subject to digestion by the corresponding protease. Units,
prefixes, and symbols may be denoted in their SI accepted form.
Unless otherwise indicated, nucleic acids are written left to right
in 5' to 3' orientation; amino acid sequences are written left to
right in amino to carboxy orientation, respectively. Numeric ranges
are inclusive of the numbers defining the range. Amino acids may be
referred to herein by either their commonly known three letter
symbols or by the one-letter symbols recommended by the IUPAC-IUB
Biochemical Nomenclature Commission. Nucleotides, likewise, may be
referred to by their commonly accepted single-letter codes. The
above-defined terms are more fully defined by reference to the
specification as a whole.
[0057] It is well known that naturally-occurring .delta.-endotoxins
are synthesized by B. thuringiensis sporulating cells as a
proteinaceous crystalline inclusion protoxin. Upon being ingested
by susceptible insect larvae, the microcrystals dissolve in the
midgut, and the protoxin is transformed into a biologically active
moiety by proteases characteristic of digestive enzymes located in
the insect gut. The activated .delta.-endotoxin binds with high
affinity to protein receptors on brush-border membrane vesicles.
The epithelial cells lining the midgut are the primary target of
the endotoxin and are rapidly destroyed as a consequence of
membrane perforation resulting from the formation of gated,
cation-selective channels by the toxin.
[0058] In an effort to better characterize and improve Bt toxins,
strains of the bacterium Bacillus thuringiensis were studied.
Crystal preparations prepared from cultures of the Bacillus
thuringiensis strains were discovered to have pesticidal activity
against Colorado potato beetle, western corn rootworm, and southern
corn rootworm. Crystal proteins were isolated from cultures of the
strains. The isolated crystal proteins were tested for pesticidal
activity in insect feeding assays. The results of the assays
revealed that the isolated crystal proteins possessed Coleopteran
pesticidal activity.
[0059] A comparison of the amino acid sequences of Cry toxins of
different specificities reveals five highly-conserved sequence
blocks. Structurally, the .delta.-endotoxins comprise three
distinct domains which are, from the N- to C-terminus: a cluster of
seven alpha-helices implicated in pore formation (referred to as
"domain 1"), three anti-parallel beta sheets implicated in cell
binding (referred to as "domain 2"), and a beta sandwich (referred
to as "domain 3"). The location and properties of these domains are
known to those of skill in the art. See, for example, Li et al.
(1991) Nature, 305:815-821 and Morse et al. (2001) Structure,
9:409-417.
[0060] An effort was undertaken to identify nucleotide sequences
encoding crystal proteins from the strains, and the wild-type
(i.e., naturally occurring) nucleic acids of the invention were
isolated from the bacterial strains. The nucleotide sequences of
the isolated nucleic acids were demonstrated to encode pesticidal
proteins by transforming Escherichia coli with such nucleotide
sequences. Lysates prepared from the transformed E. coli had
pesticidal activity against corn rootworms, Colorado potato beetles
and cotton boll weevils in feeding assays, demonstrating that the
isolated nucleotide sequences of the invention encode pesticidal
proteins. Depending upon the characteristics of a given lysate
preparation, it was recognized that the demonstration of pesticidal
activity sometimes required trypsin pretreatment to activate the
pesticidal proteins.
[0061] The inventors identified nucleic acid variants and fragments
encoding biologically active pesticidal polypeptides. Some of the
encoded pesticidal proteins require protease digestion (e.g., by
trypsin, chymotrypsin, and the like) for activation, while other
proteins were observed to be biologically active (e.g., pesticidal)
in the absence of activation. In some embodiments, the nucleic acid
encodes a truncated version of the naturally occurring polypeptide
and as such, can be classified either as a variant or a
fragment.
[0062] Further, the inventors determined that the Cry8-like
proteins of the invention were likely to be useful in transgenic
products. Surface plasmon resonance was used to determine the
binding kinetics of the wild-type endotoxin known as 1218-1 to
western corn rootworm midgut brush border membrane vesicles.
Western corn rootworm brush border membrane vesicles were adhered
to a hydrophobic sensor chip and 1218-1 toxin was passed over the
surface at various concentrations while monitoring real time
binding. Five concentrations of toxin were used to generate a
series of binding curves which were analyzed using a standard 1:1
binding model. The analysis generated a KD in the low 10.sup.-9
range. This KD range is consistent with current insecticidal toxins
that have become agricultural transgenic products.
[0063] In addition, nucleic acid sequences were engineered to
encode Cry8-like polypeptides that contain additional mutations
that confer improved or altered pesticidal activity relative to the
pesticidal activity of the naturally occurring polypeptide. Thus,
the nucleotide sequences of these nucleic acids comprise mutations
not found in the wild type sequences.
[0064] The mutant Cry8-like polypeptides of the present invention
were generally prepared by a process that involved the steps of:
obtaining a nucleic acid sequence encoding a Cry8-like polypeptide;
analyzing the structure of the polypeptide to identify particular
"target" sites for mutagenesis of the underlying gene sequence
based on a consideration of the proposed function of the target
domain in the mode of action of the endotoxin; introducing one or
more mutations into the nucleic acid sequence to produce a desired
change in one or more amino acid residues of the encoded
polypeptide sequence; and assaying the polypeptide produced for
pesticidal activity.
[0065] Many of the .delta.-endotoxins are related to various
degrees by similarities in their amino acid sequences and tertiary
structure, and means for obtaining the crystal structures of B.
thuringiensis endotoxins are well known. Exemplary high-resolution
crystal structure solution of both the Cry3A and Cry3B polypeptides
are available in the literature. The inventors of the present
invention used the solved structure of the Cry3A gene (Li et al.
(1991) Nature 353:815-821) to produce a homology model of the
Cry8-like .delta.-endotoxin disclosed herein as SEQ ID NO:2, and
known as Cry8Bb1 (see Genbank Accession No. CAD57542), to gain
insight into the relationship between structure and function of the
endotoxin and to design the recombinantly engineered proteins
disclosed herein. A combined consideration of the published
structural analyses of B. thuringiensis endotoxins and the reported
function associated with particular structures, motifs, and the
like indicates that specific regions of the endotoxin are
correlated with particular functions and discrete steps of the mode
of action of the protein. For example, .delta.-endotoxins isolated
from B. thuringiensis are generally described as comprising three
domains, a seven-helix bundle that is involved in pore formation, a
three-sheet domain that has been implicated in receptor binding,
and a beta-sandwich motif (Li et al. (1991) Nature,
305:815-821).
[0066] The inventors reasoned that the toxicity of Cry8-like
proteins, particularly the toxicity of the Cry8-like protein of the
invention, 1218-1, could be improved by targeting the region
located between alpha helices 3 and 4 of domain 1 of the endotoxin
protein. This theory was premised on a body of knowledge concerning
endotoxins, including: 1) that alpha helices 4 and 5 of domain 1 of
Cry3A 6-endotoxins had been reported to insert into the lipid
bilayer of cells lining the midgut of susceptible insects (Gazit et
al., (1998) PNAS USA 95:12289-12294); 2) the inventors' knowledge
of the location of trypsin and chymotrypsin cleavage cites within
the amino acid sequence of the wild-type protein; 3) the
observation reported herein that the protein encoded by the
wild-type endotoxin 1218-1 (i.e., SEQ ID NO:2) was more active
against certain Coleopterans following in vitro activation by
trypsin or chymotrypsin treatment; and 4) reports that digestion of
toxins from the 3' end resulted in decreased toxicity to insects.
Accordingly, the inventors engineered a series of mutants and
placed them in a variety of background sequences to create novel
polypeptides having enhanced or altered pesticidal activity. These
mutants included, but were not limited to: the addition of at least
one more protease-sensitive site (e.g., Cry8 trypsin cleavage site)
in the region located between helices 3 and 4 of domain 1; the
replacement of the original protease-sensitive site in the
wild-type sequence with a different protease-sensitive site; the
addition of multiple protease-sensitive sites in a particular
location; the addition of amino acid residues near
protease-sensitive site(s) to alter folding of the polypeptide and
thus enhance digestion of the polypeptide at the protease-sensitive
site(s); and adding mutations to protect the polypeptide from
degradative digestion that reduces toxicity, (e.g., making a series
of mutations wherein the wild-type amino acid is replaced by valine
to protect the polypeptide from digestion). Mutations may be used
singly or in any combination to provide polypeptides of the
invention.
[0067] In this manner, the invention provides a variety of
mutations, such as, for example, a mutation that comprises an
additional, or an alternative, protease-sensitive site located in
domain 1 of the polypeptide variant in a region that is located
between alpha-helices 3 and 4 of the encoded polypeptide. A
mutation of the invention which is an additional or alternative
protease-sensitive site may be sensitive to several classes of
proteases such as serine proteases, which include trypsin and
chymotrypsin, or cysteine proteases, such as cathepsin. Thus, a
mutation which is an additional or alternative protease-sensitive
site may be designed so that the site is readily recognized and/or
cleaved by a category of proteases, such as mammalian proteases or
insect proteases. A protease-sensitive site of the invention may
also be designed to be cleaved by a particular class of enzymes or
a particular enzyme known to be produced in an organism, such as,
for example, a cathepsin produced by the alfalfa weevil, Hypera
postica (Wilhite et al., (2000), Insect Biochemistry and Molecular
Biology, 30(12): 1181-1188). Another mutation of the invention is,
for example, a mutation that confers resistance to proteolytic
digestion by chymotrypsin at the C-terminus of the peptide.
[0068] As demonstrated herein, the presence of an additional and/or
alternative protease-sensitive site in the amino acid sequence of
the encoded polypeptide can improve the pesticidal activity and/or
specificity of the polypeptide encoded by the nucleic acids of the
invention. Accordingly, the Cry8-like nucleotide sequences of the
invention can be recombinantly engineered or manipulated to produce
polypeptides having improved or altered insecticidal activity
and/or specificity compared to that of an unmodified wild-type
.delta.-endotoxin. In addition, the mutations disclosed herein may
be placed in or used in conjunction with other nucleotide sequences
to provide improved properties. For example, a protease-sensitive
site that is readily cleaved by insect cathepsin, e.g., a cathepsin
found in the alfalfa weevil or the western corn rootworm (Wilhite
et al. (2000), Insect Biochemistry and Molecular Biology 30(12):
1181-1188; Koiwa et al. (2000), Analytical Biochemistry 282:
153-155; Koiwa et al. (2000), FEBS Letters 471: 67-70), may be
placed in a Cry3A, Cry3B, or Cry8 background sequence to provide
improved toxicity to that sequence. In this manner, the invention
provides toxic polypeptides with improved properties.
[0069] For example, one type of nucleic acid (e.g., mutagenized
Cry8-like nucleotide sequence) disclosed herein provides additional
mutants that comprise additional codons that introduce a second
trypsin-sensitive amino acid sequence (in addition to the naturally
occurring trypsin site) into its encoded polypeptide. An
alternative addition mutant of the invention comprises additional
codons designed to introduce at least one additional different
protease-sensitive site into the polypeptide, for example, a
chymotrypsin-sensitive site located immediately 5' or 3' of the
naturally occurring trypsin site.
[0070] A second alternative type of variant nucleic acid of the
invention provides substitution mutants in which at least one codon
of the nucleic acid that encodes the naturally occurring
protease-sensitive site is destroyed, and alternative codons are
introduced into the variant nucleic acid sequence in order to
introduce a different (e.g., substitute) protease-sensitive site in
its place. In a particular embodiment of this variant
polynucleotide, a replacement mutant is disclosed in which the
naturally-occurring trypsin cleavage site present in the encoded
polypeptide is destroyed and a chymotrypsin cleavage site is
introduced into its place. In another particular embodiment of this
variant polynucleotide, a replacement mutant is disclosed in which
a cathepsin cleavage site is introduced in place of the
naturally-occurring trypsin cleavage site. Another nucleic acid of
the invention provides mutagenized nucleic acids encoding
polypeptides which are resistant to proteolytic digestion by
chymotrypsin. One of skill in the art will recognize that any of
the disclosed mutations can be engineered in any polynucleotide
sequence; accordingly, variants of full-length Cry8-like or Bt
endotoxins, or pentin-1 like proteins, or fragments thereof, can be
modified to contain additional or alternative cleavage sites as
well as to be resistant to proteolytic digestion. In this manner,
the invention provides Cry8-like endotoxins or pentin-1 like
proteins containing mutations that improve pesticidal activity as
well as improved compositions and methods for impacting pests using
pesticidal proteins such as, for example, other Bt toxins.
[0071] The NGSR mutants disclosed herein comprise at least one
additional trypsin-sensitive protease site. These sites may be
provided in a region of the amino acid sequence that encodes domain
1 of the endotoxin polypeptide, for example, between helices 3 and
4. For example, the NGSR.N1218-1 mutant set forth in SEQ ID NO:8
comprises an NGSR sequence introduced between amino acid residues
164 and 165 of the wild-type protein (designated 164-NGSR-165).
This amino acid sequence provides a second trypsin-sensitive
cleavage site in the mutant endotoxin encoded by SEQ ID NO:7. More
specifically, the NGSR sequence (e.g., SEQ ID NO: 10) in
NGSR.N1218-1 duplicates the endogenous trypsin cleavage site that
is present at the target location, thereby introducing a second
protease-sensitive site into the loop region located between alpha
helices 3 and 4 of domain 1. Thus, while the wild-type protein
comprises the sequence NGSR at this location, the amino acid
sequence of SEQ ID NO:8 includes an additional protease-sensitive
site and the amino acid sequence NGSRNGSR (SEQ ID NO: 110).
[0072] The sequence set forth in SEQ ID NO: 22 contains several
mutations, including the "KO mutation" which replaces the NGSR
sequence of the wild-type protein with the sequence FRRGFRRG (SEQ
ID NO: 98). Thus, the FRRGFRRG sequence comprises a duplicated
cathepsin site ((Wilhite et al. (2000) Insect Biochemistry and
Molecular Biology 30(12): 1181-1188; Thie et al. (1990) Insect
Biochemistry 20(3): 313-318; Shiba-Hajime et al. (2001) Archives of
Biochemistry and Biophysics 390(1): 28-34; Melo et al. (2001)
Analytical Biochemistry 293(1): 71-77; Filippova et al. (2000)
Bioorganicheskaya-Khimiya 26(3): 192-196; Gacko et al. (2000)
Bulletin of the Polish Academy of Sciences Biological Sciences
48(1): 11-15; Pimenta et al. (2000) Journal of Protein Chemistry
19(5): 411-418) that is not present in the wild-type 1218-1
polypeptide. Specifically, these additional cathepsin-sensitive
cleavage sites are added to the protein loop region between helix 3
and helix 4 of the protein.
[0073] While the invention is not bound by any particular theory of
operation, it is believed that the presence of a second
protease-sensitive (e.g., trypsin, chymotrypsin, or cathepsin) site
between helices 3 and 4 of these endotoxins facilitates
intramolecular proteolytic cleavage by enhancing the ability of
helices 4 and 5 to separate from the rest of the toxin. The effects
of enhancing the ability of helices 4 and 5 to separate from the
rest of the toxin would be manifest as a more efficient
pore-forming process and hence confer an increase in the pesticidal
or insecticidal activity of the toxin. Indeed, the Cry8-like
mutants described herein show improved toxicity towards several
Coleopteran pests. The data further suggests that the presence of
two or more protease-sensitive sites produces a polypeptide that is
more amenable to activation by the digestive processes of
susceptible insects.
[0074] In this manner, mutations of the invention include mutations
that are directed toward the proteolytic activation of the loop
region between helix 3 and helix 4 in domain I of the Cry8-like
mutants by replacing the wild type loop NGSR with other and/or
additional proteolytic sites, such as chymotrypsin, trypsin, and
cathepsin L and D recognition sites. To further enhance
proteolysis, additional changes may be made to the loop region. For
example, the mutated loop can be engineered to contain pFRRLKMFFa
(SEQ ID NO: 111) where lower-case letters represent the native
sequence and upper-case letters represent the engineered sequence).
More than one recognition site can be added in a particular
location in any combination, and multiple recognition sites can be
added to or removed from the endotoxin. Thus, additional mutations
can comprise three, four, or more recognition sites, for example,
five cathepsin L or D motifs can be added in place of the wild type
NGSR sequence (SEQ ID NO: 10) in the loop region between helices 3
and 4 of domain I.
[0075] Mutations of the invention include mutations that protect
the polypeptide endotoxin from protease degradation, for example by
removing putative proteolytic sites such as putative serine
protease sites and cathepsin recognition sites from different areas
of the endotoxin. Some or all of such putative sites may be removed
or altered so that proteolysis at the location of the original site
is decreased. Changes in proteolysis may be assessed by comparing a
mutant endotoxin with the wild-type endotoxins or by comparing
mutant endotoxins which differ in their amino acid sequence.
Putative proteolytic sites include, but are not limited to, the
following sequences: FRR, a cathepsin L protease cleavage site; RR,
a trypsin and cathepsin B cleavage site; LKM, a chymotrypsin site;
and FF, a cathepsin D site. These sites may be altered by the
addition or deletion of any number and kind of amino acid residues,
so long as the object of the invention is achieved, i.e.,
increasing the pesticidal activity of the pesticidal protein. See,
e.g., Example 21, in which all three N-terminal cleavage sites for
trypsin, chymotrypsin, and papain were mutated simultaneously,
providing pentin-1 like proteins with improved pesticidal
activity.
[0076] Cry8-like mutants K1, K2, K3, K4, K5, K6 and K8 all contain
a mammalian cathepsin-sensitive proteolytic site (Filippova et al.
(2000) Bioorganicheskaya-Khimiya 26(3): 192-196; Gacko et al.
(2000) Bulletin of the Polish Academy of Sciences Biological
Sciences, 48(1): 11-15; Pimenta et al. (2000) Journal of Protein
Chemistry 19(5): 411-418; Melo et al. (2001) Analytical
Biochemistry 293(1): 71-77). The mutants K1, K2, K3, K4, K5, K6 and
K8 set forth in SEQ ID NOs: 39, 41, 43, 45, 47, 49, and 51,
respectively, comprise the "M6 mutation" and the "164-NGSR- 165
mutation" (sequence set forth in SEQ ID NO:33). The proteins
encoded by these nucleic acids are set forth in SEQ ID NOs: 40, 42,
44, 46, 48, 50, and 52, respectively. Similar mutants are set forth
in SEQ ID NOs: 71, 73, 75, 77, 79, 81, and 83, and comprise the "M7
mutation" and the "164-NGSR-165 mutation." The proteins encoded by
these nucleic acids are set forth in SEQ ID NOs: 72, 74, 76, 78,
80, 82, and 84, respectively.
[0077] In each of the K3, K4, K5, and K6 mutants, an additional
mutation was made in which one copy of a cathepsin site with motif
FRSRG was added to the loop between helices 3 and 4 adjacent to
either the N-terminus or the C-terminus of NGSR, a motif that
exists in the 1218 Cry 8-like wild type loop region. While the
invention is not bound by any theory of operation, it is thought
that the addition of this site facilitates toxin activation by
proteolytic cleavage of the loop.
[0078] In some of the mutants, i.e., K1, K4 and K8, a further
mutation was made in which an additional proline was added to the
loop region. This addition may enhance the retention of the loop
structure. For example, in the K4 mutant, a proline was added
immediately after the cathepsin site FRSRG (SEQ ID NO: 95). In the
K8 mutant, serine (S) and leucine (L) amino acid residues were
added just following the c-terminus of the cathepsin motif FRSRG.
This addition is thought to expose the loop to proteases for easier
digestion. Also, an additional proline was also added to the K8
mutant loop region. This addition may enhance the formation of the
loop structure.
[0079] Both the K1 and K2 mutants contain a duplication of the
cathepsin motif FRSRG to form FRSRGFRSRG (SEQ ID NO: 112) in the
mutated loop, thus replacing the wild type NGSR amino acid
residues. The K1 mutant contains an additional proline immediately
after the duplicated FRSRG to favor the retention of the loop
structure.
[0080] The K8 mutant comprises the FRSRG sequence in a particular
relation to several other altered amino acids, so that the K8
mutant comprises the sequence FRSRGSLngsrP (SEQ ID NO: 113), in
which capital letters represent amino acid changes from the native
endotoxin sequence and lower case letters represent the unchanged
native sequence. While the invention is not bound by any particular
mechanism, it is thought that G and S residues favor loop
formation; further, the addition of residues to this loop region is
thought to further favor loop formation and thus enhance the
sensitivity of this site to proteolytic cleavage.
[0081] The "M4, M5, M6, and M7 mutations" comprise changes to
domain 3 of the protein in which valine residues are substituted
for the corresponding amino acid in the wild-type sequence. In each
of the "M4, M5, M6, and M7 mutations," an existing or putative
chymotrypsin-preferred substrate site has been removed and replaced
with a sequence comprising similar amino acids that are not
recognized or preferred by chymotrypsin. Thus, in the sequence
change referred to as the "M4 mutation," the wild-type sequence
"ITTLNLATDSSLALKHNLGED" (SEQ ID NO: 99) is changed to
"ITTLNLATDSSLALKHNVGED" (SEQ ID NO: 100). In the sequence change
referred to as the "M5 mutation," this wild-type sequence is
changed to "ITTLNLATDSSLAVKHNVGED" (SEQ ID NO: 101). In the
sequence change referred to as the "M6 mutation," this wild-type
sequence is changed to "ITTVNLATDSSVAVKHNVGED" (SEQ ID NO: 103). In
the "M7 mutation," this wild-type sequence is changed to
"ITTVNLATDSSVAVKHNLGED" (SEQ ID NO: 102). The "M4, M5, M6, and M7
mutations" are set forth in the 1218-1 background sequence in
combination with the "164-NGSR-165 mutation" in SEQ ID NOs: 26, 30,
34, and 71, respectively.
[0082] By "background sequence" is intended that, but for a
specified change or changes in the amino acid sequence that
correspond to a particular mutation or mutations, the remainder of
the sequence corresponds to another native or engineered or altered
sequence described herein, such as, for example, the sequences set
forth in SEQ ID NOs:2, 4, 6, 8, 12, 14, or 16. Thus, in some
embodiments, multiple mutations are placed into a sequence
background so as to provide the resultant polypeptide with the
attributes of those multiple mutations. For example, the "M6
mutation" comprising four valine substitutions may be combined with
the "K0 mutation" comprising the duplicated cathepsin site sequence
FRRGFRRG (SEQ ID NO: 98) to provide a Cry8-like polypeptide that
resists degradation from the 3' end but is more efficiently cleaved
by cathepsin in the insect gut, thereby increasing the pesticidal
activity of the polypeptide. In this manner, polypeptides and
nucleotides that encode polypeptides are provided that show
improved properties relative to the corresponding wild-type
sequences.
[0083] While the invention is not bound by any theory of operation,
it is believed that alterations of the chymotrypsin site (as in
Cry8-like mutants M4, M5, M6, and M7) interfere with the
degradation of the toxic polypeptides from the C-terminal end,
thereby enhancing the longevity of these polypeptides in the insect
gut.
[0084] The nucleic acid sequences set forth in SEQ ID NOs: 53, 55,
and 57 all encode polypeptides (set forth in SEQ ID NOs: 54, 56,
and 58, respectively) that comprise the "K0 mutation," which is a
duplication of the cathepsin site, FRRG (SEQ ID NO: 97), so that
the wild-type or native amino acid sequence "npngsralr" (SEQ ID NO:
114) is replaced with the sequence "npFRRGFRRGalr" (SEQ ID NO:
116), in which capital letters represent changes from the native
sequence. Each of these sequences also comprises the "M6 mutation,"
in which the wild-type amino acid sequence "ITTLNLATDSSLALKHNLGED"
(SEQ ID NO: 99) is changed to "ITTVNLATDSSVAVKHNVGED" (SEQ ID NO:
103). Each of these sequences further comprises the "C2 mutation,"
which is a change designed to remove the proteolytic site near the
N-terminal of the native endotoxin. In the "C2 mutation," the
native amino acid sequence "dykdylkmsagn" (SEQ ID NO: 104) is
replaced with the sequence "dykdyAVGsagn" (SEQ ID NO: 105).
[0085] The set of mutations found in the nucleic acid sequence of
SEQ ID NO:53 and the amino acid sequence of SEQ ID NO:54 further
comprise the "C3 mutation" in which the amino acid sequence is
changed from the native "innyydrq" (SEQ ID NO: 106) to "innVVdrq"
(SEQ ID NO: 107). This change may reduce salt bridge and
electrostatic hindrances between helices which may promote channel
(pore) formation by the toxin. The sets of changes found in the K34
and K35 mutants further comprise the "C4 mutation," in which the
amino acid sequence is changed from the native "nydtrtypmetka" (SEQ
ID NO: 108) to "nydtItypIetka" (SEQ ID NO: 109). In particular, the
R296I change (i.e., change from R to I at residue 296) is thought
to reduce the polypeptide's susceptibility to proteolytic
attack.
[0086] The invention further provides mutant polypeptides that have
been constructed in various background sequences. Any background
sequence may be used so long as the object of the invention is
achieved, i.e., providing a pesticidal protein with increased or
altered pesticidal activity. Background sequences include Cty8-like
sequences disclosed herein as well as variants and fragments
thereof. Background sequences may also be other Cry or Bt toxin
sequences or other pesticidal polypeptides such as pentin-1, or
pentin-1 like sequences. For example, mutants may be added to a
native 1218-1 background sequence (SEQ ID NOs: 1 and 2) or a
truncated 1218-1 background sequence optimized for expression in
maize (SEQ ID NOs:5 and 6). The mutant endotoxins of the invention
comprise at least one amino acid change or addition relative to the
native or background sequence, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 32, 35, 38, 40, 45, 47, or 50 or more amino acid changes or
additions. Thus, for example, the invention provides nucleotide
sequences encoding mutant endotoxins comprising a second trypsin
cleavage site (e.g., NGSR (SEQ ID NO:10)) introduced into the amino
acid sequence presented in SEQ ID NO:12 (1218-1) or SEQ ID NO: 16
("49PVD"). The "49PVD" fragment was generated by trimming sequence
from both the N-terminus and the C-terminus of the sequence set
forth in SEQ ID NO: 12. More specifically, the N-terminus of the
49PVD polypeptide was trimmed by 47 residues; thus, the polypeptide
starts at aa residue 48(M) of the native polypeptide and the
C-terminus was trimmed by 6 residues up to aa 663(D) of the native
polypeptide. Therefore the 49PVD polypeptide corresponds to the
native 1218-1 polypeptide (SEQ ID NO: 12) from aa residue 48 to aa
663 (see copending application Ser. No. 10/032,717, filed Oct. 23,
2001).
[0087] Thus, for example, SEQ ID NO:22 provides the "K0 mutation"
(ie., FRRGFRRG) as well as the "M6 mutation" in the native 1218-1
background sequence; SEQ ID NO:21 encodes the polypeptide of SEQ ID
NO:22. SEQ ID NO:52 provides the "K8 mutation" (i.e., FRSRGSLngsrP)
as well as the "M6 mutation" in the native 1218-1 background
sequence; SEQ ID NO:52 is encoded by the nucleotide sequence set
forth in SEQ ID NO:51. SEQ ID NO:68 provides the "K0 mutation" in
the native 1218-1 background sequence along with the "M7 mutation,"
in which the wild-type amino acid sequence "ITTLNLATDSSLALKHNLGED"
is changed to "ITTVNLATDSSVAVKHNLGED," a change of 3 Leucines to
Valines (see bolding). SEQ ID NO:68 is encoded by the nucleotide
sequence set forth in SEQ ID NO:67.
[0088] A number of mutant sequences are provided in which the "M7
mutation" is substituted for the "M6 mutation" in a particular
Cry8-like mutant sequence. Thus, SEQ ID NO:68 is the same as SEQ ID
NO:22, except that the "M6 mutation" of SEQ ID NO:21 is replaced
with the "M7 mutation." In the same manner, SEQ ID NOs: 70, 72, 74,
76, 78, 80, 82, 84, 86, 88, 90, 92, and 94 are the same as SEQ ID
NOs: 34, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, and 62, except
that in each sequence the "M6 mutation" is replaced with the "M7
mutation." The amino acid sequences set forth in SEQ ID NOs: 68,
70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, and 94 are encoded
by the nucleotide sequences set forth SEQ ID NOs: 67, 69, 71, 73,
75, 77, 79, 81, 83, 85, 87, 89, 91, and 93, respectively.
[0089] Accordingly, the nucleic acids of the invention comprise
isolated polynucleotides, and variants and fragments thereof, that
encode biologically active (e.g., pesticidal) polypeptide
endotoxins, including but not limited to the nucleotide sequences
set forth in SEQ ID NOs:1, 3, 5, 7, 11, 13, 15, 17, 18, 19, 21, 25,
29, 33, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,
69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, and 93. The
nucleotide sequences disclosed herein further provide background
sequences into which mutations can be introduced, such as the
sequences referred to herein as 1218-1, 1218-2, and 49PVD.
[0090] The polynucleotides of the invention also include any
synthetic or recombinant nucleotide sequence that encodes a
pesticidal polypeptide comprising the amino acid sequences set
forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 26, 30,
34, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,
72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 119, 121, and
123.
[0091] The present invention provides isolated nucleic acids
comprising nucleotide sequences which encode the amino acid
sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20,
22, 26, 30, 34, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,
66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 119,
121, and 123. In particular embodiments, the invention provides
nucleic acids comprising the nucleotide sequences set forth in SEQ
ID NOs:1 (Cry1218-1 CDS) and 3 (Cry1218-2 CDS), the maize-optimized
nucleic acid set forth in SEQ ID NO:5 (mol21 8-1), and the native
genomic sequences set forth in SEQ ID NO: 17 (genomic Cry1218-1)
and SEQ ID NO: 18 (genomic Cry 1218-2). The coding sequence (CDS)
for SEQ ID NO: 17 runs from base pair 731 to 4348. The CDS for SEQ
ID NO: 18 runs from base pair 1254 to 4883. Plasmids comprising
each of these five nucleic acids were deposited on May 5, 2000 and
November 2, 2000 with the Patent Depository of the American Type
Culture Collection (ATCC), Manassas, Va., and assigned Patent
Deposit Nos. PTA-1821 (corresponding to SEQ ID NO:1); PTA-1817
(corresponding to SEQ ID NO:3); PTA-2635 (corresponding to SEQ ID
NO:5); PTA-2634 (comprising SEQ ID NO:17); and PTA-2636 (comprising
SEQ ID NO: 18).
[0092] Patent Deposits PTA-1821 and PTA-1817 comprise a mixture of
2 clones, each of which contains a part of the entire coding
sequence. More specifically, the deposited plasmids encode nucleic
acid molecules cloned into a TA vector (Invitrogen, Carlsbad,
Calif.) that encode two overlapping fragments of the coding
sequence. The full length coding sequence can be produced using an
overlapping PCR strategy. A first PCR reaction should comprise
forward and reverse primers designed to correspond to the 5' and
the 3' ends of the full-length coding sequence. The two DNA bands
generated by the first PCR reaction performed with the
above-identified primer sets should be purified and a second round
of PCR, set for 7 cycles, should be performed utilizing the
purified DNA isolated from the first PCR reaction in the absence of
any primers. The 3' end of the nucleic acid generated by primer set
(a) and the 5' end of the nucleic acid generated by primer set (b)
will overlap and prime the generation of the full-length coding
sequence. A third and final PCR reaction is performed to generate
the full-length coding sequence.
[0093] The above-referenced deposits (e.g., PTA-1 821; PTA- 1817;
PTA-263 5; PTA-2634; and PTA-2636) will be maintained under the
terms of the Budapest Treaty on the International Recognition of
the Deposit of Microorganisms for the Purposes of Patent Procedure.
These deposits were made merely as a convenience for those of skill
in the art and are not an admission that a deposit is required
under 35 U.S.C. .sctn.112.
[0094] Of particular interest are optimized nucleotide sequences
encoding the pesticidal proteins of the invention. As used herein,
the phrase "optimized nucleotide sequences" refers to nucleic acids
that are optimized for expression in a particular organism, for
example a plant. Optimized nucleotide sequences include those
sequences that have been modified such that the GC content of the
nucleotide sequence has been altered. Such a nucleotide sequence
may or may not comprise a coding region. Where the nucleotide
sequence comprises a coding region, the alterations of GC content
may be made in view of other genetic phenomena, such as, for
example, the codon preference of a particular organism or a GC
content trend within a coding region. (See particularly Examples
14, 15, and 16.)
[0095] In some embodiments, where the nucleotide sequence to be
optimized comprises a coding region, the alteration in GC content
does not result in a change in the protein encoded by the
nucleotide sequence. In other embodiments, the alteration in GC
content results in changes to the encoded protein that are
conservative amino acid changes and/or that do not materially alter
the function of the encoded protein. The GC content of an optimized
nucleotide sequence may differ from the first or native nucleotide
sequence by as little as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
or 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,
24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,
37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or
50% or more. Thus, the GC content of an optimized nucleotide
sequence may be 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, or 80% or higher.
[0096] The term "optimized nucleotide sequences" also encompasses
sequences in which the GC content has been altered and, in
addition, other changes have been made to the nucleotide sequence.
Such changes are often made to enhance properties of the sequence,
such as its versatility in genetic engineering (e.g., by adding or
removing restriction enzyme recognition sites) and any other
property which may be desirable for generating a transgenic
organism, such as increased mRNA longevity in the cell. (See
Examples 14, 15, and 16).
[0097] By "derived from" is intended that a sequence is
substantially similar to another sequence. Generally, sequences
derived from a particular nucleotide sequence will have at least
about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence
identity to that particular nucleotide sequence as determined by
sequence alignment programs described elsewhere herein using
default parameters. Sequences derived from a particular nucleotide
sequence may differ from that sequence by as few as 1-15
nucleotides, as few as 1-10, such as 6-10, as few as 5, as few as
4, 3, 2, or even 1 nucleotide. Sequences derived from a particular
nucleotide sequence may also cross-hybridize to that sequence.
[0098] Optimized nucleotide sequences may be prepared for any
organism of interest using methods known in the art. For example,
SEQ ID NO:5 discloses an optimized nucleic acid sequence encoding
the pesticidal protein set forth in SEQ ID NO: 12 (truncated
1218-1). More specifically, the nucleotide sequence of SEQ ID NO:5
comprising maize-preferred codons was prepared by
reverse-translating the amino acid sequence set forth in SEQ ID NO:
12 to comprise maize-preferred codons as described by Murray et al.
(1989) Nucleic Acids Res. 17:477-498. Optimized nucleotide
sequences find use in increasing expression of a pesticidal protein
in a plant, for example monocot plants of the Gramineae (Poaceae)
family such as, for example, a maize or corn plant.
[0099] The invention further provides isolated pesticidal (e.g.,
insecticidal) polypeptides encoded by either a naturally-occurring
or modified (e.g., mutagenized, truncated, and/or optimized)
nucleic acid of the invention. More specifically, the invention
provides polypeptides comprising an amino acid sequence set forth
in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 26, 30, 34, 40,
42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,
76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 119, 121, and 123 and the
polypeptides encoded by nucleic acids described herein, for example
those set forth in SEQ ID NOs: 1, 3, 5, 7, 11, 13, 15, 17, 18, 19,
21, 25, 29, 33, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,
65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 118,
120, and 122 and fragments and variants thereof.
[0100] In particular embodiments, pesticidal proteins of the
invention provide full-length .delta.-endotoxin proteins, fragments
of full-length .delta.-endotoxins, and variant polypeptides that
are produced from mutagenized nucleic acids designed to introduce
particular amino acid sequences into polypeptides of the invention.
In particular embodiments, the amino acid sequences that are
introduced into the polypeptides comprise a sequence that provides
a cleavage site for an enzyme such as a protease.
[0101] Some of the Cry8-like polypeptides of the invention, for
example SEQ ID NOs: 2 and 4, comprise full-length
.delta.-endotoxins. Other polypeptides such as SEQ ID NOs: 6, 12,
and 14 embody fragments of a full-length .delta.-endotoxin. Some of
the polypeptide fragments, variants, and mutations of the invention
have enhanced pesticidal activity relative to the activity of the
naturally occurring .delta.-endotoxin from which they are derived,
particularly in the absence of in vitro activation of the endotoxin
with a protease prior to screening for activity. For example, the
data presented herein in Table 1 of Example 6 indicates that the
NGSR addition mutant, which contains a mutation that was placed in
the background sequence set forth in SEQ ID NO: 12 (truncated
1218-1 endotoxin) and is referred to herein as NGSR.N1218-1 (SEQ ID
NO:8), provides a polypeptide with increased pesticidal activity
against Colorado potato beetle.
[0102] SEQ ID NOs: 6, 12, and 16 provide polypeptides that embody
truncated versions of the 1218-1 polypeptide set forth in SEQ ID
NO:2. SEQ ID NOs: 6 and 12 represent a polypeptide that is
shortened (truncated) at the 3' end of the amino acid sequence set
forth in SEQ ID NO:2. In contrast, the fourth polypeptide variant
set forth in SEQ ID NO: 16 provides a variant that is truncated at
both the 5' and 3' ends of the full-length protein set forth in SEQ
ID NO:2. SEQ ID NO: 14 (1218-2) provides a polypeptide that
embodies a truncated version of the polypeptide set forth in SEQ ID
NO:4. This polypeptide provides a protein that is truncated at the
3' end of the full-length 1218-2 polypeptide set forth in SEQ ID
NO: 4. The mutations of the invention may be placed into any
background sequence, including such truncated polypeptides, so long
as an endotoxin is provided by the polypeptide so produced.
[0103] Thus, one of skill will appreciate that fragments of the
disclosed proteins are also encompassed by the present invention.
By "fragment" is intended a portion of the amino acid sequence of
the exemplary proteins disclosed herein. Fragments of a protein may
retain the pesticidal activity of the full-length protein or they
may have altered or improved pesticidal activity compared to the
full-length protein. Thus, fragments of a protein may range from at
least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,
270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480,
500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740,
760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, or
1000, or up to the full-length sequence of the protein. A
biologically active portion, fragment, or truncated version of a
pesticidal protein can be prepared by isolating a portion of one of
the nucleotide sequences of the invention, expressing the encoded
portion of the pesticidal protein (e.g., by recombinant expression
in vitro), and assessing the activity of the portion of the
pesticidal protein.
[0104] SEQ ID NOs: 8, 20, 22, 26, 30, 34, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,
86, 88, 90, 92, and 94 provide a family of polypeptides that embody
mutants of the biologically active Cry8-like polypeptide endotoxin
set forth in SEQ ID NO:2. For example, SEQ ID NO: 8 provides the
NGSR.N1218-1 mutant, which comprises an additional
trypsin-sensitive cleavage site. Thus, for example, SEQ ID NOs: 26,
30, and 34 provide exemplary mutant polypeptides of the invention.
More specifically, SEQ ID NO:26, in addition to comprising the
"NGSR mutation" (SEQ ID NO: 10) which is an addition of the NGSR
trypsin-sensitive cleavage site, also provides the mutation that is
referred to herein as "M4." SEQ ID NOs: 30 and 34, designated the
"M5 mutant sequence" and the "M6 mutant sequence," respectively,
provide the "NGSR mutation" in addition to the mutations referred
to herein as "M5" and "M6," respectively. The nucleotide sequences
set forth in SEQ ID NOs: 25, 29, and 33 encode the polypeptide
sequences set forth in SEQ ID NOs: 26, 30, and 34, respectively.
SEQ ID NO: 22 provides the mutant referred to herein as K04; the
nucleotide sequence set forth in SEQ ID NO: 21 encodes the
polypeptide sequence set forth in SEQ ID NO: 22. The K04 mutant
sequence comprises the following mutations: the "KO mutation" (in
which native sequence npngsralr is changed to npFRRGFRRGalr) and
the "M6 mutation" (in which native sequence ittlnlatdsslalkhnlged
is changed to ittVnlatdssVaVkhnVged). SEQ ID NO:68 provides the
mutant referred to herein as K03; the nucleotide sequence set forth
in SEQ ID NO:67 encodes the polypeptide sequence set forth in SEQ
ID NO:68. The K03 mutant sequence comprises the following
mutations: the "K0 mutation" (as described above) and the "M7
mutation" (in which native sequence ittlnlatdsslalkhnlged is
changed to ittVnlatdssVaVkhnlged).
[0105] In some instances, mutants disclosed herein were cloned into
the pET expression system, expressed in E. coli, and tested for
pesticidal activity against exemplary insect pests such as southern
corn rootworm (SCRW), western corn rootworm (WCRW), Colorado potato
beetle (CPB, e.g., Leptinotarsa decemlineata), and cotton boll
weevil (e.g., Anthonomus grandis).
[0106] It is to be understood that the polypeptides of the
invention can be produced either by expression of a nucleic acid
disclosed herein, or by the use of standard molecular biology
techniques. For example, a truncated protein of the invention can
be produced by expression of a recombinant nucleic acid of the
invention in an appropriate host cell, or alternatively by a
combination of ex vivo procedures, such as protease digestion and
purification of a purified wild-type protein.
[0107] It is recognized that the pesticidal proteins may be
oligomeric and will vary in molecular weight, number of residues,
component peptides, activity against particular pests, and other
characteristics. However, by the methods set forth herein, proteins
active against a variety of pests may be isolated and
characterized. The pesticidal proteins of the invention can be used
in combination with Bt endotoxins or other insecticidal proteins to
increase insect target range. Furthermore, the use of the
pesticidal proteins of the present invention in combination with Bt
.delta.-endotoxins or other insecticidal principles of a distinct
nature has particular utility for the prevention and/or management
of insect resistance. Other insecticidal principles include, but
are not limited to, protease inhibitors (both serine and cysteine
types), lectins, .alpha.-amylase, and peroxidase.
[0108] Fragments and variants of the nucleotide and amino acid
sequences and the polypeptides encoded thereby are also encompassed
by the present invention. As used herein the term "fragment" refers
to a portion of a nucleotide sequence of a polynucleotide or a
portion of an amino acid sequence of a polypeptide of the
invention. Fragments of a nucleotide sequence may encode protein
fragments that retain the biological activity of the native or
corresponding full-length protein and hence possess pesticidal
activity. Thus, it is acknowledged that some of the polynucleotide
and amino acid sequences of the invention can correctly be referred
to as either fragments or variants. This is particularly true of
truncated sequences that are biologically active.
[0109] It is to be understood that the term "fragment," as it is
used to refer to nucleic acid sequences of the invention, also
encompasses sequences that are useful as hybridization probes. This
class of nucleotide sequences generally do not encode fragment
proteins retaining biological activity. Thus, fragments of a
nucleotide sequence may range from at least about 20 nucleotides,
about 50 nucleotides, about 100 nucleotides, and up to the
full-length nucleotide sequence encoding the proteins of the
invention.
[0110] A fragment of a nucleotide sequence that encodes a
biologically active portion of a pesticidal protein of the
invention will encode at least 15, 25, 30, 50, 100, 200, 300, 400,
500, 600, 700, 800, 900, 1,000, 1,100, or 1,200 contiguous amino
acids, or up to the total number of amino acids present in a
pesticidal polypeptide of the invention (for example, 1,206, 1,210,
and 669 amino acids for SEQ ID NOs:2, 4, and 6, respectively).
Fragments of a nucleotide sequence that are useful as hybridization
probes or PCR primers generally need not encode a biologically
active portion of a pesticidal protein.
[0111] Thus, a fragment of a Cry8-like or pentin-1 like nucleic
acid may encode a biologically active portion of a pesticidal
protein, or it may be a fragment that can be used as a
hybridization probe or PCR primer using methods disclosed below. A
biologically active portion of a pesticidal protein can be prepared
by isolating a portion of one of the nucleotide sequences of the
invention, expressing the encoded portion of the pesticidal protein
(e.g., by recombinant expression in vitro), and assessing the
activity of the encoded portion of the pesticidal protein.
[0112] Nucleic acids that are fragments of a Cry8-like or pentin-1
like nucleotide sequence comprise at least 16, 20, 50, 75, 100,
150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 1,000,
1,200, 1,400, 1,600, 1,800, 2,000, 2,200, 2,400, 2,600, 2,800,
3,000, 3,200, 3,400, or 3,600 nucleotides, or up to the number of
nucleotides present in a Cry8-like or pentin-1 like nucleotide
sequence disclosed herein (for example, 3,621, 3,633, 2,010, 2010,
2022, and 2028 nucleotides for SEQ ID NOs:1, 3, 5, 11, 13, and 39
respectively).
[0113] For example, SEQ ID NOs: 5, 11, and 15 represent fragments
of SEQ ID NO: 1 and SEQ ID NO: 13 represents a fragment of SEQ ID
NO: 3. More specifically, particular embodiments of the nucleic
acids of the invention disclose fragments derived from (e.g.,
produced from) a first nucleic acid of the invention, wherein the
fragment encodes a truncated polypeptide characterized by
pesticidal activity. The truncated polypeptide encoded by the
polynucleotide fragments of the invention are characterized by
pesticidal activity that is either equivalent to, or improved,
relative to the activity of the corresponding full-length
polypeptide encoded by the first nucleic acid from which the
fragment is derived.
[0114] In specific embodiments, some of the nucleic acid fragments
of the invention are truncated at the 3' end of the native or
corresponding full-length coding sequence. For example, SEQ ID NO:
11 represents a fragment of SEQ ID NO: 1 that is truncated at the
3' end. In an alternative embodiment, one of the polynucleotides of
the invention, SEQ ID NO: 15, comprises a nucleic acid sequence
that is truncated at both the 5' and 3' end of the truncated 1218-1
toxin domain encoded by SEQ ID NO:11, respectively.
[0115] By "variants" is intended substantially similar sequences.
For nucleotide sequences, conservative variants include those
sequences that, because of the degeneracy of the genetic code,
encode the amino acid sequence of one of the pesticidal
polypeptides of the invention. Naturally occurring allelic variants
such as these can be identified with the use of well-known
molecular biology techniques, such as, for example, polymerase
chain reaction (PCR) and hybridization techniques as outlined
below.
[0116] Variant nucleotide sequences also include synthetically
derived nucleotide sequences, such as those generated, for example,
by using site-directed mutagenesis but which still encode a
pesticidal protein of the invention, such as a mutant endotoxin.
Generally, variants of a particular nucleotide sequence of the
invention will have at least about 40%, 50%, 60%, 65%, 70%, 75%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or more sequence identity to that particular
nucleotide sequence as determined by sequence alignment programs
described elsewhere herein using default parameters. A variant of a
nucleotide sequence of the invention may differ from that sequence
by as few as 1-15 nucleotides, as few as 1-10, such as 6-10, as few
as 5, as few as 4, 3, 2, or even 1 nucleotide.
[0117] As used herein, the term "variant protein" encompasses
polypeptides that are derived from a native protein by: deletion
(so-called truncation) or addition of one or more amino acids to
the N-terminal and/or C-terminal end of the native protein;
deletion or addition of one or more amino acids at one or more
sites in the native protein; or substitution of one or more amino
acids at one or more sites in the native protein. Accordingly, the
term variant protein encompasses biologically active fragments of a
native protein that comprise a sufficient number of contiguous
amino acid residues to retain the biological activity of the native
protein, i.e., to have pesticidal activity. Such pesticidal
activity may be different or improved relative to the native
protein or it may be unchanged, so long as pesticidal activity is
retained.
[0118] Variant proteins encompassed by the present invention are
biologically active, that is they continue to possess the desired
biological activity of the native protein, that is, pesticidal
activity as described herein. Such variants may result from, for
example, genetic polymorphism or from human manipulation.
Biologically active variants of a native pesticidal protein of the
invention will have at least about 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more
sequence identity to the amino acid sequence for the native protein
as determined by sequence alignment programs described elsewhere
herein using default parameters. A biologically active variant of a
protein of the invention may differ from that protein by as few as
1-15 amino acid residues, as few as 1-10, such as 6-10, as few as
5, as few as 4, 3, 2, or even 1 amino acid residue.
[0119] It is recognized that the nucleic acid sequence of any one
of the polynucleotides of the invention can be altered or
mutagenized to alter (e.g., improve) the biological activity and/or
specificity of its encoded pesticidal polypeptide. For example, SEQ
ID NO: 7 represents a Cry8-like nucleotide sequence that has been
mutagenized to comprise 12 additional nucleotides (SEQ ID NO: 9)
that are not present in the wild-type nucleic acid sequence (SEQ ID
NO: 11). In this manner, the nucleotide sequence inserted into the
coding region of SEQ ID NO: 11 was designed to encode an additional
trypsin cleavage site (referred to herein as the "NGSR mutation")
(SEQ ID NO: 10) in the amino acid sequence of the encoded
polypeptide. In the NGSR mutation, the native sequence "npngsralr"
is replaced with "npNGSRngsralr" (SEQ ID NO: 115).
[0120] More specifically, the amino acid sequence set forth in SEQ
ID NO:10 was introduced between amino acid 164 and 165 of the Cry8
.delta.-endotoxin set forth in SEQ ID NO:12. This particular amino
acid sequence was chosen because it duplicates the endogenous
sequence present in the naturally occurring full-length protein
(SEQ ID NO:2), and creates a second protease-sensitive site. More
specifically, the modification introduces a second trypsin-like
site. It is well known to those of skill in the art that trypsin
cleaves bonds immediately C-terminal to arginine and lysine. As
demonstrated herein the recombinantly engineered protein (SEQ ID
NO:8) encoded by SEQ ID NO:7 is characterized by improved activity
against Coleopterans, for example, against Colorado potato beetle
(see Example 6, Table 1).
[0121] It is recognized that any nucleotide sequence encoding the
amino acid sequences that are proteolytic sites or putative
proteolytic sites (for example, sequences such as NGSR (SEQ ID NO:
10), FRRG (SEQ ID NO: 97), FRR, RR, LKM, FF, or FRSRQ (SEQ ID NO:
117)) can be used and that the exact identity of the codons used to
introduce any of these cleavage sites into a variant polypeptide
may vary depending on the use, i.e., expression in particular plant
species. It is also recognized that any of the disclosed mutations
can be introduced into any polynucleotide sequence of the invention
that comprises the codons for amino acid residues that provide the
native trypsin cleavage site that is targeted for modification.
Accordingly, variants of either full-length pesticidal proteins or
fragments thereof can be modified to contain additional or
alternative cleavage sites, and these embodiments are intended to
be encompassed by the scope of the invention disclosed herein.
[0122] The invention further encompasses a microorganism that is
transformed with at least one nucleic acid of the invention, with
an expression cassette comprising the nucleic acid, or with a
vector comprising the expression cassette. Preferably, the
microorganism is one that multiplies on plants. More preferably,
the microorganism is a root-colonizing bacterium. An embodiment of
the invention relates to an encapsulated pesticidal protein, which
comprises a transformed microorganism comprising at least one
pesticidal protein of the invention.
[0123] The invention provides pesticidal compositions comprising a
transformed organism of the invention. Preferably the transformed
microorganism is present in the pesticidal composition in a
pesticidally effective amount, together with a suitable carrier.
The invention also encompasses pesticidal compositions comprising
an isolated protein of the invention, alone or in combination with
a transformed organism of the invention and/or an encapsulated
pesticidal protein of the invention, in an insecticidally effective
amount, together with a suitable carrier.
[0124] The invention further provides a method of increasing insect
target range by using a pesticidal protein of the invention in
combination with at least one second pesticidal protein that is
different from the pesticidal protein of the invention. Any
pesticidal protein known in the art can be employed in the methods
of the present invention. Such pesticidal proteins include, but are
not limited to, Bt .delta.-endotoxins, protease inhibitors,
lectins, .alpha.-amylases, lipid acyl hydrolases, and
peroxidases.
[0125] The invention also encompasses transformed or transgenic
plants comprising at least one nucleotide sequence of the
invention. Preferably, the plant is stably transformed with a
nucleotide construct comprising at least one nucleotide sequence of
the invention operably linked to a promoter that drives expression
in a plant cell. As used herein, the terms "transformed plant" and
"transgenic plant" refer to a plant that comprises within its
genome a heterologous polynucleotide. Generally, the heterologous
polynucleotide is stably integrated within the genome of a
transgenic or transformed plant such that the polynucleotide is
passed on to successive generations. The heterologous
polynucleotide may be integrated into the genome alone or as part
of a recombinant expression cassette.
[0126] It is to be understood that as used herein the term
"transgenic" includes any cell, cell line, callus, tissue, plant
part, or plant the genotype of which has been altered by the
presence of heterologous nucleic acid including those transgenics
initially so altered as well as those created by sexual crosses or
asexual propagation from the initial transgenic. The term
"transgenic" as used herein does not encompass the alteration of
the genome (chromosomal or extra-chromosomal) by conventional plant
breeding methods or by naturally-occurring events such as random
cross-fertilization, non-recombinant viral infection,
non-recombinant bacterial transformation, non-recombinant
transposition, or spontaneous mutation.
[0127] As used herein, the term "plant" includes reference to whole
plants, plant organs (e.g., leaves, stems, roots, etc.), seeds,
plant cells, and progeny of same. Parts of transgenic plants are to
be understood within the scope of the invention to comprise, for
example, plant cells, protoplasts, tissues, callus, embryos as well
as flowers, ovules, stems, fruits, leaves, roots originating in
transgenic plants or their progeny previously transformed with a
DNA molecule of the invention and therefore consisting at least in
part of transgenic cells, are also an object of the present
invention.
[0128] As used herein, the term "plant cell" includes, without
limitation, seeds suspension cultures, embryos, meristematic
regions, callus tissue, leaves, roots, shoots, gametophytes,
sporophytes, pollen, and microspores. The class of plants that can
be used in the methods of the invention is generally as broad as
the class of higher plants amenable to transformation techniques,
including both monocotyledonous and dicotyledonous plants. Such
plants include, for example, Solanum tuberosum and Zea mays.
[0129] While the invention does not depend on a particular
biological mechanism for increasing the resistance of a plant to a
plant pest, expression of the nucleotide sequences of the invention
in a plant can result in the production of the pesticidal proteins
of the invention and in an increase in the resistance of the plant
to a plant pest. The plants of the invention find use in
agriculture in methods for impacting insect pests. Certain
embodiments of the invention provide transformed crop plants, such
as, for example, maize plants, which find use in methods for
impacting insect pests of the plant, such as, for example, western,
northern, southern and Mexican corn rootworms. Other embodiments of
the invention provide transformed potato plants, which find use in
methods for impacting the Colorado potato beetle, transformed
cotton plants, which find use in methods for impacting the cotton
boll weevil, and transformed turf grasses, which find use in
methods for impacting the bluegrass billbug, Sphenophorous
parvulus.
[0130] One of skill in the art will readily acknowledge that
advances in the field of molecular biology such as site-specific
and random mutagenesis, polymerase chain reaction methodologies,
and protein engineering techniques provide an extensive collection
of tools and protocols suitable for use to alter or engineer both
the amino acid sequence and underlying genetic sequences of
proteins of agricultural interest. Thus, the pesticidal proteins of
the invention may be altered in various ways including amino acid
substitutions, deletions, truncations, and insertions. Methods for
such manipulations are generally known in the art. For example,
amino acid sequence variants of the pesticidal proteins can be
prepared by introducing mutations into a synthetic nucleic acid
(e.g., DNA molecule). Methods for mutagenesis and nucleic acid
alterations are well known in the art. For example, designed
changes can be introduced using an oligonucleotide-mediated
site-directed mutagenesis technique. See, for example, Kunkel
(1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987)
Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker
and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan
Publishing Company, New York), and the references cited
therein.
[0131] The mutagenized nucleotide sequences of the invention may be
modified so as to change about 1, 2, 3, 4, 5, 6, 8, 10, 12 or more
of the amino acids present in the primary sequence of the encoded
polypeptide. Alternatively even more changes from the native
sequence may be introduced such that the encoded protein may have
at least about 1% or 2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, or even about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or
20%,21%,22%,23%,24%, or 25%,30%,35%, or 40% or more of the codons
altered, or otherwise modified compared to the corresponding
wild-type protein. In the same manner, the encoded protein may have
at least about 1% or 2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, or even about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%,
21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or more additional
codons compared to the corresponding wild-type protein. It should
be understood that the mutagenized nucleotide sequences of the
present invention are intended to encompass biologically
functional, equivalent peptides which have pesticidal activity,
such as an improved pesticidal activity as determined by
antifeedant properties against boll weevil larvae. Such sequences
may arise as a consequence of codon redundancy and functional
equivalency that are known to occur naturally within nucleic acid
sequences and the proteins thus encoded.
[0132] One of skill in the art would recognize that amino acid
additions and/or substitutions are generally based on the relative
similarity of the amino acid side-chain substituents, for example,
their hydrophobicity, charge, size, and the like. Exemplary
substitutions that 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.
[0133] Guidance as to appropriate amino acid substitutions that do
not affect biological activity of the protein of interest may be
found in the model of Dayhoff et al. (1978) Atlas of Protein
Sequence and Structure (Natl. Biomed. Res. Found., Washington,
D.C.), herein incorporated by reference. Conservative
substitutions, such as exchanging one amino acid with another
having similar properties, may be made.
[0134] Thus, the genes and nucleotide sequences of the invention
include both the naturally occurring sequences as well as mutant
forms. Likewise, the proteins of the invention encompass both
naturally occurring proteins as well as variations (e.g., truncated
polypeptides) and modified (e.g., mutant) forms thereof. Such
variants will continue to possess the desired pesticidal activity.
Obviously, the mutations that will be made in the DNA encoding the
variant must not place the sequence out of reading frame and
preferably will not create complementary regions that could produce
secondary mRNA structure. See, EP Patent Application Publication
No. 75,444.
[0135] The deletions, insertions, and substitutions of the protein
sequences encompassed herein are not expected to produce radical
changes in the characteristics of the protein. However, when it is
difficult to predict the exact effect of the substitution,
deletion, or insertion in advance of doing so, one skilled in the
art will appreciate that the effect will be evaluated by routine
screening assays, such as insect-feeding assays. See, for example,
Marrone et al. (1985) J. Econ. Entomol. 78:290-293 and Czapla and
Lang (1990) J. Econ. Entomol. 83:2480-2485, herein incorporated by
reference.
[0136] Variant nucleotide sequences and proteins also encompass
sequences and proteins derived from a mutagenic and recombinogenic
procedure such as DNA shuffling. With such a procedure, one or more
different coding sequences can be manipulated to create a new
pesticidal protein possessing the desired properties. In this
manner, libraries of recombinant polynucleotides are generated from
a population of related sequence polynucleotides comprising
sequence regions that have substantial sequence identity and can be
homologously recombined in vitro or in vivo. For example, using
this approach, full-length coding sequences, sequence motifs
encoding a domain of interest, or any fragment of a nucleotide
sequences of the invention may be shuffled between the nucleotide
sequences encoding the pesticidal proteins of the invention and
corresponding portions of other nucleotide sequences known to
encode pesticidal proteins to obtain a new gene coding for a
protein with an improved property of interest.
[0137] Properties of interest include, but are not limited to,
pesticidal activity per unit of pesticidal protein, protein
stability, and toxicity to non-target species particularly humans,
livestock, and plants and microbes that express the pesticidal
polypeptides of the invention. The invention is not bound by a
particular shuffling strategy, only that at least one nucleotide
sequence of the invention, or part thereof, is involved in such a
shuffling strategy. Shuffling may involve only nucleotide sequences
disclosed herein or may additionally involve shuffling of any other
nucleotide sequences known in the art including, but not limited
to, GenBank Accession Nos. U04364, U04365, and U04366. Strategies
for DNA shuffling are known in the art. See, for example, Stemmer
(1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994)
Nature 370:389-391; Crameri et al. (1997) Nature Biotech.
15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et
al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al.
(1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and
5,837,458.
[0138] The nucleotide sequences of the invention can also be used
to isolate corresponding sequences from other organisms,
particularly other bacteria, and more particularly other Bacillus
strains. In this manner, methods such as PCR, hybridization, and
the like can be used to identify such sequences based on their
sequence homology to the sequences set forth herein. Sequences
isolated based on their sequence identity to the entire Cry8-like
sequences set forth herein or to fragments thereof are encompassed
by the present invention. Such sequences include sequences that are
orthologs of the disclosed sequences. By "orthologs" is intended
genes derived from a common ancestral gene and which are found in
different species as a result of speciation. Genes found in
different species are considered orthologs when their nucleotide
sequences and/or their encoded protein sequences share substantial
identity as defined elsewhere herein. Functions of orthologs are
often highly conserved among species.
[0139] In a PCR approach, oligonucleotide primers can be designed
for use in PCR reactions to amplify corresponding DNA sequences
from cDNA or genomic DNA extracted from any organism of interest.
Methods for designing PCR primers and PCR cloning are generally
known in the art and are disclosed in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor
Laboratory Press, Plainview, N.Y.). See also Innis et al., eds.
(1990) PCR Protocols: A Guide to Methods and Applications (Academic
Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies
(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR
Methods Manual (Academic Press, New York). Known methods of PCR
include, but are not limited to, methods using paired primers,
nested primers, single specific primers, degenerate primers,
gene-specific primers, vector-specific primers,
partially-mismatched primers, and the like.
[0140] In hybridization techniques, all or part of a known
nucleotide sequence is used as a probe that selectively hybridizes
to other corresponding nucleotide sequences present in a population
of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or
cDNA libraries) from a chosen organism. The hybridization probes
may be genomic DNA fragments, cDNA fragments, RNA fragments, or
other oligonucleotides, and may be labeled with a detectable group
such as .sup.32P, or any other detectable marker. Thus, for
example, probes for hybridization can be made by labeling synthetic
oligonucleotides based on the nucleotide sequences of the
invention. Methods for preparation of probes for hybridization and
for construction of cDNA and genomic libraries are generally known
in the art and are disclosed in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, N.Y.).
[0141] For example, an entire Cry8-like sequence disclosed herein,
or one or more portions thereof, may be used as a probe capable of
specifically hybridizing to corresponding Cry8-like sequences and
messenger RNAs. To achieve specific hybridization under a variety
of conditions, such probes include sequences that are unique among
Cry8-like sequences and are preferably at least about 10
nucleotides in length, and most preferably at least about 20
nucleotides in length. Such probes may be used to amplify
corresponding Cry8-like sequences from a chosen organism by PCR.
This technique may be used to isolate additional coding sequences
from a desired organism or as a diagnostic assay to determine the
presence of coding sequences in a an organism. Hybridization
techniques include hybridization screening of plated DNA libraries
(either plaques or colonies; see, for example, Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring
Harbor Laboratory Press, Plainview, N.Y.).
[0142] Hybridization of such sequences may be carried out under
stringent conditions. By "stringent conditions" or "stringent
hybridization conditions" is intended conditions under which a
probe will hybridize to its target sequence to a detectably greater
degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will
be different in different circumstances. By controlling the
stringency of the hybridization and/or washing conditions, target
sequences that are 100% complementary to the probe can be
identified (homologous probing). Alternatively, stringency
conditions can be adjusted to allow some mismatching in sequences
so that lower degrees of similarity are detected (heterologous
probing). Generally, a probe is less than about 1000 nucleotides in
length, preferably less than 500 nucleotides in length.
[0143] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37.degree.
C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M
NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary
moderate stringency conditions include hybridization in 40 to 45%
formamide, 1.0 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high
stringency conditions include hybridization in 50% formamide, 1 M
NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to
65.degree. C. The duration of hybridization is generally less than
about 24 hours, usually about 4 to about 12 hours.
[0144] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the
T.sub.m can be approximated from the equation of Meinkoth and Wahl
(1984) Anal. Biochem. 138:267-284: T.sub.m=81.5.degree. C.+16.6
(log M)+0.41 (%GC)-0.61 (% form)-500/L; where M is the molarity of
monovalent cations, %GC is the percentage of guanosine and cytosine
nucleotides in the DNA, % form is the percentage of formamide in
the hybridization solution, and L is the length of the hybrid in
base pairs. The T.sub.m is the temperature (under defined ionic
strength and pH) at which 50% of a complementary target sequence
hybridizes to a perfectly matched probe. T.sub.m is reduced by
about 1.degree. C. for each 1% of mismatching; thus, T.sub.m,
hybridization, and/or wash conditions can be adjusted to hybridize
to sequences of the desired identity. For example, if sequences
with .gtoreq.90% identity are sought, the T.sub.m can be decreased
10.degree. C. Generally, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point (T.sub.m)
for the specific sequence and its complement at a defined ionic
strength and pH. However, severely stringent conditions can utilize
a hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than
the thermal melting point (T.sub.m); moderately stringent
conditions can utilize a hybridization and/or wash at 6, 7, 8, 9,
or 10.degree. C. lower than the thermal melting point (T.sub.m);
low stringency conditions can utilize a hybridization and/or wash
at 11, 12, 13, 14, 15, or 20.degree. C. lower than the thermal
melting point (T.sub.m). Using the equation, hybridization and wash
compositions, and desired T.sub.m, those of ordinary skill will
understand that variations in the stringency of hybridization
and/or wash solutions are inherently described. If the desired
degree of mismatching results in a T.sub.m of less than 45.degree.
C. (aqueous solution) or 32.degree. C. (formamide solution), it is
preferred to increase the SSC concentration so that a higher
temperature can be used. An extensive guide to the hybridization of
nucleic acids is found in Tijssen (1993) Laboratory Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel et al.,
eds. (1995) Current Protocols in Molecular Biology, Chapter 2
(Greene Publishing and Wiley-Interscience, New York). See Sambrook
et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold
Spring Harbor Laboratory Press, Plainview, N.Y.). Thus, for
example, isolated sequences that encode a Cry8-like protein of the
invention and hybridize under stringent conditions to the Cry8-like
sequences disclosed herein, or to fragments thereof, are
encompassed by the present invention.
[0145] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence," (b) "comparison window," (c) "sequence
identity," (d) "percentage of sequence identity," and (e)
"substantial identity."
[0146] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence,
or the complete cDNA or gene sequence.
[0147] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0148] Methods of alignment of sequences for comparison are well
known in the art. Thus, the determination of percent identity
between any two sequences can be accomplished using a mathematical
algorithm. Non-limiting examples of such mathematical algorithms
are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the
local homology algorithm of Smith et al. (1981) Adv. Appl. Math.
2:482; the homology alignment algorithm of Needleman and Wunsch
(1970) J. Mol. Biol. 48:443-453; the search-for-similarity-method
of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448;
the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci.
USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl.
Acad. Sci. USA 90:5873-5877.
[0149] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0); the ALIGN
PLUS program (Version 3.0, copyright 1997); and GAP, BESTFIT,
BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package
of Genetics Computer Group, Version 10 (available from Accelrys,
9685 Scranton Road, San Diego, Calif., 92121, USA). The scoring
matrix used in Version 10 of the Wisconsin Genetics Software
Package is BLOSUM62 (see Henikoff and Henikoff(1989) Proc. Natl.
Acad. Sci. USA 89:10915).
[0150] Alignments using these programs can be performed using the
default parameters. The CLUSTAL program is well described by
Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989)
CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res.
16:10881-90; Huang et al. (1992) CABIOS 8:155-65; and Pearson et
al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN and the ALIGN
PLUS programs are based on the algorithm of Myers and Miller (1988)
supra. A PAM120 weight residue table, a gap length penalty of 12,
and a gap penalty of 4 can be used with the ALIGN program when
comparing amino acid sequences. The BLAST programs of Altschul et
al (1990) J. Mol. Biol. 215:403 are based on the algorithm of
Karlin and Altschul (1990) supra. BLAST nucleotide searches can be
performed with the BLASTN program, score=100, word length=12, to
obtain nucleotide sequences homologous to a nucleotide sequence
encoding a protein of the invention. BLAST protein searches can be
performed with the BLASTX program, score=50, wordlength=3, to
obtain amino acid sequences homologous to a protein or polypeptide
of the invention. To obtain gapped alignments for comparison
purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described
in Altschul et al. (1997) Nucleic Acids Res. 25:3389.
Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an
iterated search that detects distant relationships between
molecules. See Altschul et al. (1997) supra. When utilizing BLAST,
Gapped BLAST, or PSI-BLAST, the default parameters of the
respective programs (e.g., BLASTN for nucleotide sequences, BLASTX
for proteins) can be used. On the world wide web see
ncbi.hlm.nih.gov. Alignment may also be performed manually by
inspection.
[0151] Unless otherwise stated, nucleotide and amino acid sequence
identity/similarity values provided herein refer to the value
obtained using GAP with default parameters, or any equivalent
program. By "equivalent program" is intended any sequence
comparison program that, for any two sequences in question,
generates an alignment having identical nucleotide or amino acid
residue matches and an identical percent sequence identity when
compared to the corresponding alignment generated by the preferred
program.
[0152] GAP uses the algorithm of Needleman and Wunsch (1970) J.
Mol. Biol. 48:443-453, to find the alignment of two complete
sequences that maximizes the number of matches and minimizes the
number of gaps. GAP considers all possible alignments and gap
positions and creates the alignment with the largest number of
matched bases and the fewest gaps. It allows for the provision of a
gap creation penalty and a gap extension penalty in units of
matched bases. GAP must make a profit of gap creation penalty
number of matches for each gap it inserts. If a gap extension
penalty greater than zero is chosen, GAP must, in addition, make a
profit for each gap inserted of the length of the gap times the gap
extension penalty. Default gap creation penalty values and gap
extension penalty values in Version 10 of the Wisconsin Genetics
Software Package for protein sequences are 8 and 2, respectively.
For nucleotide sequences, the default gap creation penalty is 50
while the default gap extension penalty is 3. The gap creation and
gap extension penalties can be expressed as an integer selected
from the group of integers consisting of from 0 to 200. Thus, for
example, the gap creation and gap extension penalties can each be
0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65 or greater.
[0153] For purposes of the present invention, comparison of
nucleotide or protein sequences for determination of percent
sequence identity to the Cry8-like sequences disclosed herein is
preferably made using the GAP program in the Wisconsin Genetics
Software Package (Version 10 or later) or any equivalent program.
For GAP analyses of nucleotide sequences, a GAP Weight of 50 and a
Length of 3 was used.
[0154] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity." Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0155] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0156] (e)(i) The term "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70% sequence identity, preferably at least 80%, more
preferably at least 90%, and most preferably at least 95%, compared
to a reference sequence using one of the alignment programs
described using standard parameters. One of skill in the art will
recognize that these values can be appropriately adjusted to
determine corresponding identity of proteins encoded by two
nucleotide sequences by taking into account codon degeneracy, amino
acid similarity, reading frame positioning, and the like.
Substantial identity of amino acid sequences for these purposes
normally means sequence identity of at least 60%, more preferably
at least 70%, 80%, 90%, and most preferably at least 95%.
[0157] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. However, stringent conditions encompass
temperatures in the range of about 1.degree. C. to about 20.degree.
C., depending upon the desired degree of stringency as otherwise
qualified herein. Nucleic acids that do not hybridize to each other
under stringent conditions are still substantially identical if the
polypeptides they encode are substantially identical. This may
occur, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code. One
indication that two nucleic acid sequences are substantially
identical is when the polypeptide encoded by the first nucleic acid
is immunologically cross reactive with the polypeptide encoded by
the second nucleic acid.
[0158] (e)(ii) The term "substantial identity" in the context of a
peptide indicates that a peptide comprises a sequence with at least
70% sequence identity to a reference sequence, preferably 80%, more
preferably 85%, most preferably at least 90% or 95% sequence
identity to the reference sequence over a specified comparison
window. Preferably, optimal alignment is conducted using the
homology alignment algorithm of Needleman and Wunsch (1970) J. Mol.
Biol. 48:443-453. An indication that two peptide sequences are
substantially identical is that one peptide is immunologically
reactive with antibodies raised against the second peptide. Thus, a
peptide is substantially identical to a second peptide, for
example, where the two peptides differ only by a conservative
substitution. Peptides that are "substantially similar" share
sequences as noted above except that residue positions that are not
identical may differ by conservative amino acid changes.
[0159] The use of the term "nucleotide constructs" herein is not
intended to limit the present invention to nucleotide constructs
comprising DNA. Those of ordinary skill in the art will recognize
that nucleotide constructs, particularly polynucleotides and
oligonucleotides composed of ribonucleotides and combinations of
ribonucleotides and deoxyribonucleotides, may also be employed in
the methods disclosed herein. The nucleotide constructs, nucleic
acids, and nucleotide sequences of the invention additionally
encompass all complementary forms of such constructs, molecules,
and sequences. Further, the nucleotide constructs, nucleotide
molecules, and nucleotide sequences of the present invention
encompass all nucleotide constructs, molecules, and sequences which
can be employed in the methods of the present invention for
transforming plants including, but not limited to, those comprised
of deoxyribonucleotides, ribonucleotides, and combinations thereof.
Such deoxyribonucleotides and ribonucleotides include both
naturally occurring molecules and synthetic analogues. The
nucleotide constructs, nucleic acids, and nucleotide sequences of
the invention also encompass all forms of nucleotide constructs
including, but not limited to, single-stranded forms,
double-stranded forms, hairpins, stem-and-loop structures, and the
like.
[0160] A further embodiment of the invention relates to a
transformed organism such as an organism selected from the group
consisting of plant and insect cells, bacteria, yeast,
baculoviruses, protozoa, nematodes, and algae. The transformed
organism comprises: a DNA molecule of the invention, an expression
cassette comprising the said DNA molecule, or a vector comprising
the said expression cassette, preferably stably incorporated into
the genome of the transformed organism.
[0161] The sequences of the invention are provided in expression
cassettes for expression in the organism of interest. The cassette
will include 5' and 3' regulatory sequences operably linked to a
sequence of the invention. By "operably linked" is intended a
functional linkage between a promoter and a second sequence,
wherein the promoter sequence initiates and mediates transcription
of the DNA sequence corresponding to the second sequence.
Generally, operably linked means that the nucleic acid sequences
being linked are contiguous and, where necessary to join two
protein coding regions, contiguous and in the same reading frame.
The cassette may additionally contain at least one additional gene
to be cotransformed into the organism. Alternatively, the
additional gene(s) can be provided on multiple expression
cassettes.
[0162] Such an expression cassette is provided with a plurality of
restriction sites for insertion of the sequence to be under the
transcriptional regulation of the regulatory regions. The
expression cassette may additionally contain selectable marker
genes.
[0163] The expression cassette will include in the 5' to 3'
direction of transcription: a transcriptional and translational
initiation region, a DNA sequence of the invention, and a
transcriptional and translational termination region functional in
the organism serving as a host. The transcriptional initiation
region (i.e., the promoter) may be native or analogous or foreign
or heterologous to the host organism. Additionally, the promoter
may be the natural sequence or alternatively a synthetic sequence.
By "foreign" is intended that the transcriptional initiation region
is not found in the native organism into which the transcriptional
initiation region is introduced. As used herein, a chimeric gene
comprises a coding sequence operably linked to a transcription
initiation region that is heterologous to the coding sequence.
Where the promoter is a native or natural sequence, the expression
of the operably linked sequence is altered from the wild-type
expression, which results in an alteration in phenotype.
[0164] The termination region may be native with the
transcriptional initiation region, may be native with the operably
linked DNA sequence of interest, or may be derived from another
source. Convenient termination regions are available from the
Ti-plasmid of A. tumefaciens, such as the octopine synthase and
nopaline synthase termination regions. See also Guerineau et al.
(1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell
64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et
al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene
91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903;
and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
[0165] Where appropriate, a nucleic acid may be optimized for
increased expression in the host organism. Thus, where the host
organism is a plant, a sequence may be optimized using
plant-preferred codons for improved expression. See, for example,
Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion
of host-preferred codon usage. For example, although nucleic acid
sequences of the present invention may be expressed in both
monocotyledonous and dicotyledonous plant species, sequences can be
modified to account for the specific codon preferences and GC
content preferences of monocotyledons or dicotyledons as these
preferences have been shown to differ (Murray et al. (1989) Nucleic
Acids Res. 17:477-498). Thus, the maize-preferred codon for a
particular amino acid may be derived from known gene sequences from
maize. Maize codon usage for 28 genes from maize plants are listed
in Table 4 of Murray et al., supra. Methods are available in the
art for synthesizing plant-preferred genes. See, for example, U.S.
Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989)
Nucleic Acids Res. 17:477-498, herein incorporated by
reference.
[0166] In addition to altering codons of a sequence in accordance
with an organism's codon preference, optimization of a sequence can
include modification of the GC content of the sequence. Gene GC
content is a common metric of gene structure. GC content can vary
greatly within and between genes, and between genes of the same or
different organisms. The reasons for this variation are not
definitively known, but may include factors such as chromosome
organization and function, methylation pressure, presence of
repetitive DNA, adaptations for gene expression, and
codon-anticodon coadapted biases. Most organisms have gene
populations that display a fairly normal GC content distribution,
but some warm-blooded vertebrates as well as cereal plants,
including maize, have a curious bimodal distribution of GC content
(e.g. Campbell and Gowri (1990), supra; Bernardi (1995) Annual
Review of Genetics 29:445-475; Carels and Bernardi (2000) Genetics
154:1819-1825). The biological significance of this bimodality
remains unknown, but observations concerning GC content
distributions and bimodal tendencies are mounting, especially with
the completion of genome sequencing, for example, in humans and in
rice (International Human Genome Sequencing Consortium (2001)
Nature 409:860-921; Yu et al. (2002) Science 296:79-91; Wong et al.
(2002) Genome Research 12:851-856).
[0167] Maize and other cereals have distinctly bimodal gene GC
content distributions not observed in other taxonomic groups such
as dicot plants, animals, fungi, bacteria, and archaea. Using the
largest maize gene dataset to date, we explored differences in mRNA
structure and expression between the high and low GC modes. The
bimodality phenomenon is observed in nuclear-encoded genes. In
maize, the two modes occur at approximately 51 % and 67% GC content
(which may be referred to as "low (GC) mode" and "high (GC) mode.")
Most maize genes are "low mode" and have GC content at the lower
level of approximately 51%. Most GC content variation is found in
the coding region, particularly in the third codon position. GC
content in the third codon position can reach 100%, and in high GC
mode genes, C can predominate over G by a ratio of 1:3.
[0168] Analysis of GC content also reveals patterns within genes,
particularly within the coding region (also called the "ORF," or
Open Reading Frame). For example, if GC content is evaluated along
the coding region of a gene, maize genes have a generally negative
GC gradient (i.e., GC content decreases toward the 3' end of the
coding region). However, this gradient pattern is not present in
most high GC mode genes and about half of the low GC mode genes.
Further, the coding regions of the remaining low GC mode genes
(i.e., the other half) shows a reversal of the marked negative GC
gradient into a positive gradient towards the end of the coding
region.
[0169] Another GC content pattern observed in maize is that high GC
mode genes are richer in GC-rich codon amino acids, and this
variation also occurs in a gradient along the length of the coding
sequence. For example, in high GC mode genes, the amino acid bias
for alanine is greatest near the beginning of the coding sequence.
While gene expression varies widely, we have determined that the
overall average expression of high and low GC mode genes is similar
as revealed by both EST and Lynx MPSS mRNA profiling (see Brenner
et al. (2000) Nature Biotechnology 18: 630-634; Brenner et al.
(2000) PNAS 97: 1665-1670 for information on Lynx MPSS; see Simmons
et al., Maize Coop Newsletter 2002, on the world wide web at
Agron.Missouri.edu/mnl/77 /10simmons.html for comment on high and
low GC mode gene expression). However, high GC mode genes were
observed to show higher tissue-preferred expression, especially in
vegetative and non-kernel reproductive tissues, while low GC mode
genes showed higher expression levels in endosperm, pericarp and R1
kernel tissues.
[0170] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other
well-characterized sequences that may be deleterious to gene
expression. Also, as described herein, particularly in Examples 14,
15, and 16, the GC content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. By "host cell"
is meant a cell that contains a vector and supports the replication
and/or expression of the expression vector. A host organism is an
organism that contains a host cell. Host cells may be prokaryotic
cells such as E. coli, or eukaryotic cells such as yeast, insect,
amphibian, or mammalian cells. Preferably, host cells are
monocotyledonous or dicotyledonous plant cells. A particularly
preferred monocotyledonous host cell is a maize host cell. When
possible, the sequence is modified to avoid predicted hairpin
secondary mRNA structures.
[0171] The expression cassettes may additionally contain 5' leader
sequences in the expression cassette construct. Such leader
sequences can act to enhance translation. Translation leaders are
known in the art and include: picornavirus leaders, for example,
EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein
et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6126-6130); potyvirus
leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et
al. (1995) Gene 165(2): 233-238), MDMV leader (Maize Dwarf Mosaic
Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain
binding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94);
untranslated leader from the coat protein mRNA of alfalfa mosaic
virus (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625);
tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in
Molecular Biology of RNA, ed. Cech (Liss, N.Y.), pp. 237-256); and
maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)
Virology 81: 382-385). See also, Della-Cioppa et al. (1987) Plant
Physiol. 84: 965-968. Other methods known to enhance translation
can also be utilized, for example, introns and the like.
[0172] In preparing the expression cassette, the various DNA
fragments may be manipulated so as to provide for the DNA sequences
in the proper orientation and, as appropriate, in the proper
reading frame. Toward this end, adapters or linkers may be employed
to join the DNA fragments or other manipulations may be involved to
provide for convenient restriction sites, removal of superfluous
DNA, removal of restriction sites, or the like. For this purpose,
in vitro mutagenesis, primer repair, restriction, annealing,
resubstitutions, e.g., transitions and transversions, may be
involved.
[0173] A number of promoters can be used in the practice of the
invention. The promoters can be selected based on the desired
outcome. The nucleic acids can be combined with constitutive,
tissue-preferred, inducible, or other promoters for expression in
the host organism. Suitable constitutive promoters for use in a
plant host cell include, for example, the core promoter of the
Rsyn7 promoter and other constitutive promoters disclosed in WO
99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter
(Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et
al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al.
(1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992)
Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl.
Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730);
ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other
constitutive promoters include, for example, those discussed in
U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;
5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
[0174] Depending on the desired outcome, it may be beneficial to
express the gene from an inducible promoter. Of particular interest
for regulating the expression of the nucleotide sequences of the
present invention in plants are wound-inducible promoters. Such
wound-inducible promoters, may respond to damage caused by insect
feeding, and include potato proteinase inhibitor (pin II) gene
(Ryan (1990) Ann. Rev. Phytopath. 28: 425-449; Duan et al. (1996)
Nature Biotechnology 14: 494-498); wun1 and wun2, U.S. Pat. No.
5,428,148; win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet.
215: 200-208); systemin (McGurl et al. (1992) Science 225:
1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22:
783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73-76); MPI gene
(Corderok et al. (1994) Plant J. 6(2): 141-150); and the like,
herein incorporated by reference.
[0175] Additionally, pathogen-inducible promoters may be employed
in the methods and nucleotide constructs of the present invention.
Such pathogen-inducible promoters include those from
pathogenesis-related proteins (PR proteins), which are induced
following infection by a pathogen; e.g., PR proteins, SAR proteins,
beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et
al. (1983) Neth. J Plant Pathol. 89: 245-254; Uknes et al. (1992)
Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol.
4:111-116. See also WO 99/43819, herein incorporated by
reference.
[0176] Of interest are promoters that are expressed locally at or
near the site of pathogen infection. See, for example, Marineau et
al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989)
Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al.
(1986) Proc. Natl. Acad Sci. USA 83:2427-2430; Somsisch et al.
(1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad
Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J.
10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA
91:2507-2511;Warner et al. (1993) Plant J. 3:191-201; Siebertz
etal. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386
(nematode-inducible); and the references cited therein. Of
particular interest is the inducible promoter for the maize PRms
gene, whose expression is induced by the pathogen Fusarium
moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol.
Plant Path. 41:189-200).
[0177] Chemical-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Depending upon the objective, the
promoter may be a chemical-inducible promoter, where application of
the chemical induces gene expression, or a chemical-repressible
promoter, where application of the chemical represses gene
expression. Chemical-inducible promoters are known in the art and
include, but are not limited to, the maize In2-2 promoter, which is
activated by benzenesulfonamide herbicide safeners, the maize GST
promoter, which is activated by hydrophobic electrophilic compounds
that are used as pre-emergent herbicides, and the tobacco PR-la
promoter, which is activated by salicylic acid. Other
chemical-regulated promoters of interest include steroid-responsive
promoters (see, for example, the glucocorticoid-inducible promoter
in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425
and McNellis et al. (1998) Plant J. 14(2):247-257) and
tetracycline-inducible and tetracycline-repressible promoters (see,
for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and
U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by
reference.
[0178] Tissue-preferred promoters can be utilized to target
enhanced pesticidal protein expression within a particular plant
tissue. Tissue-preferred promoters include those discussed in
Yamamoto et al. (1997) Plant J. 12(2)255-265; Kawamata et al.
(1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol.
Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res.
6(2):157-168; Rinehart et al. (1996) Plant Physiol.
112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.
112(2):525-535; Canevascini et al. (1996) Plant Physiol.
112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.
35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196;
Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et
al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and
Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters
can be modified, if necessary, for weak expression.
[0179] Leaf-specific promoters are known in the art. See, for
example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al.
(1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka
et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
[0180] Root-specific promoters are known and can be selected from
those available from the literature or isolated de novo from
various compatible species. See, for example, Hire et al. (1992)
Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine
synthetase gene); Keller and Baumgartner (1991) Plant Cell
3(10):1051-1061 (root-specific control element in the GRP 1.8 gene
of French bean); Sanger et al. (1990) Plant Mol. Biol.
14(3):433-443 (root-specific promoter of the mannopine synthase
(MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991)
Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic
glutamine synthetase (GS), which is expressed in roots and root
nodules of soybean). See also Bogusz et al. (1990) Plant Cell
2(7):633-641, where two root-specific promoters isolated from
hemoglobin genes from the nitrogen-fixing nonlegume Parasponia
andersonii and the related non-nitrogen-fixing nonlegume Trema
tomentosa are described. The promoters of these genes were linked
to a .beta.-glucuronidase reporter gene and introduced into both
the nonlegume Nicotiana tabacum and the legume Lotus corniculatus,
and in both instances root-specific promoter activity was
preserved. Leach and Aoyagi (1991) describe their analysis of the
promoters of the highly expressed rolC and rolD root-inducing genes
of Agrobacterium rhizogenes (see Plant Science (Limerick)
79(1):69-76). They concluded that enhancer and tissue-preferred DNA
determinants are dissociated in those promoters. Teeri et al.
(1989) used gene fusion to lacZ to show that the Agrobacterium
T-DNA gene encoding octopine synthase is especially active in the
epidermis of the root tip and that the TR2' gene is root specific
in the intact plant and stimulated by wounding in leaf tissue, an
especially desirable combination of characteristics for use with an
insecticidal or larvicidal gene (see EMBO J 8(2):343-350). The TR1'
gene fused to nptII (neomycin phosphotransferase II) showed similar
characteristics. Additional root-preferred promoters include the
VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol.
29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol.
Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386;
5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.
[0181] "Seed-preferred" promoters include both "seed-specific"
promoters (those promoters active during seed development such as
promoters of seed storage proteins) as well as "seed-germinating"
promoters (those promoters active during seed germination). See
Thompson et al. (1989) BioEssays 10:108, herein incorporated by
reference. Such seed-preferred promoters include, but are not
limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa
zein); and milps (myo-inositol-1-phosphate synthase); (see WO 00/1
1177, herein incorporated by reference). Gamma-zein is a preferred
endosperm-specific promoter. Glob-1 is a preferred embryo-specific
promoter. For dicots, seed-specific promoters include, but are not
limited to, bean .beta.-phaseolin, napin, .beta.-conglycinin,
soybean lectin, cruciferin, and the like. For monocots,
seed-specific promoters include, but are not limited to, maize 15
kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1,
shrunken 2, globulin 1, etc. See also WO 00/12733, where
seed-preferred promoters from end1 and end2 genes are disclosed;
herein incorporated by reference. A promoter that has "preferred"
expression in a particular tissue is expressed in that tissue to a
greater degree than in at least one other plant tissue. Some
tissue-preferred promoters show expression almost exclusively in
the particular tissue.
[0182] Where low level expression is desired, weak promoters will
be used. Generally, by "weak promoter" is intended a promoter that
drives expression of a coding sequence at a low level. By low level
is intended at levels of about 1/1000 transcripts to about
1/100,000 transcripts to about 1/500,000 transcripts.
Alternatively, it is recognized that the term "weak promoters" also
encompasses promoters that are expressed in only a few cells and
not in others to give a total low level of expression. Where a
promoter is expressed at unacceptably high levels, portions of the
promoter sequence can be deleted or modified to decrease expression
levels.
[0183] Such weak constitutive promoters include, for example the
core promoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No.
6,072,050), the core 35S CaMV promoter, and the like. Other
constitutive promoters include, for example, U.S. Pat. Nos.
5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;
5,268,463; 5,608,142; and 6,177,611; herein incorporated by
reference.
[0184] Generally, the expression cassette will comprise a
selectable marker gene for the selection of transformed cells.
Selectable marker genes are utilized for the selection of
transformed cells or tissues. Marker genes include genes encoding
antibiotic resistance, such as those encoding neomycin
phosphotransferase II (NEO) and hygromycin phosphotransferase
(HPT), as well as genes conferring resistance to herbicidal
compounds, such as glufosinate ammonium, bromoxynil,
imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See
generally, Yarranton (1992) Curr. Opin. Biotech. 3: 506-511;
Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:
6314-6318; Yao et al. (1992) Cell 71: 63-72; Reznikoff (1992) Mol.
Microbiol. 6: 2419-2422; Barkley et al. (1980) in The Operon, pp.
177-220; Hu et al. (1987) Cell 48: 555-566; Brown et al. (1987)
Cell 49: 603-612; Figge et al. (1988) Cell 52: 713-722; Deuschle et
al. (1989) Proc. Natl. Acad. Sci. USA 86: 5400-5404; Fuerst et al.
(1989) Proc. Natl. Acad. Sci. USA 86: 2549-2553; Deuschle et al.
(1990) Science 248: 480-483; Gossen (1993) Ph.D. Thesis, University
of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:
1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10: 3343-3356;
Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89: 3952-3956;
Baim et al. (1991) Proc. Natl. Acad Sci. USA 88: 5072-5076;
Wyborski et al. (1991) Nucleic Acids Res. 19: 4647-4653;
Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162;
Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:
1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104;
Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.
(1992) Proc. Natl. Acad. Sci. USA 89: 5547-5551; Oliva et al.
(1992) Antimicrob. Agents Chemother. 36: 913-919; Hlavka et al.
(1985) Handbook of Experimental Pharmacology, Vol. 78
(Springer-Verlag, Berlin); Gill et al. (1988) Nature 334: 721-724.
Such disclosures are herein incorporated by reference.
[0185] The above list of selectable marker genes is not meant to be
limiting. Any selectable marker gene can be used in the present
invention.
[0186] Transformation protocols as well as protocols for
introducing nucleotide sequences into plants may vary depending on
the type of plant or plant cell, i.e., monocot or dicot, targeted
for transformation. Suitable methods of introducing nucleotide
sequences into plant cells and subsequent insertion into the plant
genome include microinjection (Crossway et al. (1986) Biotechniques
4: 320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83: 5602-5606, Agrobacterium-mediated transformation
(Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al., U.S. Pat.
No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO
J. 3: 2717-2722), and ballistic particle acceleration (see, for
example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al.,
U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No. 5,886,244;
Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995) "Direct
DNA Transfer into Intact Plant Cells via Microprojectile
Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental
Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and
McCabe et al. (1988) Biotechnology 6: 923-926); and Lecl
transformation (WO 00/28058). For potato transformation see Tu et
al. (1998) Plant Molecular Biology 37: 829-838 and Chong et al.
(2000) Transgenic Research 9: 71-78. Additional transformation
procedures can be found in Weissinger et al. (1988) Ann. Rev.
Genet. 22: 421-477; Sanford et al. (1987) Particulate Science and
Technology 5: 27-37 (onion); Christou et al. (1988) Plant Physiol.
87: 671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:
923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev.
Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl.
Genet. 96: 319-324 (soybean); Datta et al. (1990) Biotechnology 8:
736-740 (rice); Klein et al. (1988) Proc. Natl. Acad Sci. USA 85:
4305-4309 (maize); Klein et al. (1988) Biotechnology 6: 559-563
(maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat.
Nos. 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol.
91: 440-444 (maize); Fromm et al. (1990) Biotechnology 8: 833-839
(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:
763-764; Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier
et al. (1987) Proc. Natl. Acad. Sci. USA 84: 5345-5349 (Liliaceae);
De Wet et al. (1985) in The Experimental Manipulation of Ovule
Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen);
Kaeppler et al. (1990) Plant Cell Reports 9: 415-418 and Kaeppler
et al. (1992) Theor. Appl. Genet. 84: 560-566 (whisker-mediated
transformation); D'Halluin et al. (1992) Plant Cell 4: 1495-1505
(electroporation); Li et al. (1993) Plant Cell Reports 12: 250-255
and Christou and Ford (1995) Annals of Botany 75: 407-413 (rice);
Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (maize via
Agrobacterium tumefaciens); all of which are herein incorporated by
reference.
[0187] The cells that have been transformed may be grown into
plants in accordance with conventional ways. See, for example,
McCormick et al. (1986) Plant Cell Reports 5: 81-84. These plants
may then be grown, and either pollinated with the same transformed
strain or different strains, and the resulting hybrid having
constitutive or inducible expression of the desired phenotypic
characteristic identified. Two or more generations may be grown to
ensure that expression of the desired phenotypic characteristic is
stably maintained and inherited and then seeds harvested to ensure
expression of the desired phenotypic characteristic has been
achieved.
[0188] The nucleotide sequences of the invention may be provided to
the plant by contacting the plant with a virus or viral nucleic
acids. Generally, such methods involve incorporating the nucleotide
construct of interest within a viral DNA or RNA molecule. It is
recognized that the recombinant proteins of the invention may be
initially synthesized as part of a viral polyprotein, which later
may be processed by proteolysis in vivo or in vitro to produce the
desired pesticidal protein. It is also recognized that such a viral
polyprotein, comprising at least a portion of the amino acid
sequence of a pesticidal protein of the invention, may have the
desired pesticidal activity. Such viral polyproteins and the
nucleotide sequences that encode for them are encompassed by the
present invention. Methods for providing plants with nucleotide
constructs and producing the encoded proteins in the plants, which
involve viral DNA or RNA molecules are known in the art. See, for
example, U.S. Pat. Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367;
and 5,316,931; herein incorporated by reference.
[0189] The invention further relates to plant propagating material
of a transformed plant of the invention including, but not limited
to, seeds, tubers, corms, bulbs, leaves, and cuttings of roots and
shoots.
[0190] The present invention may be used for transformation of any
plant species, including, but not limited to, monocots and dicots.
Examples of plants of interest include, but are not limited to,
corn (Zea mays), Brassica spp. (e.g., canola (B. napus), B. rapa,
B. juncea), particularly those Brassica species useful as sources
of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye
(Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare),
millet (e.g, pearl millet (Pennisetum glaucum), proso millet
(Panicum miliaceum), foxtail millet (Setaria italica), finger
millet (Eleusine coracana)), sunflower (Helianthus annuus),
safflower (Carthamus tinctorius), wheat (Triticum aestivum),
soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum
tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium
barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus),
cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos
nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.),
cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa
spp.), avocado (Persea americana), fig (Ficus casica), guava
(Psidium guajava), mango (Mangifera indica), olive (Olea europaea),
papaya (Carica papaya), cashew (Anacardium occidentale), macadamia
(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets
(Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,
vegetables, ornamentals, and conifers.
[0191] Vegetables include tomatoes (Lycopersicon esculentum),
lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris),
lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members
of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include
azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),
hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa
spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),
carnation (Dianthus caryophyllus), poinsettia (Euphorbia
pulcherrima), and chrysanthemum. Conifers that may be employed in
practicing the present invention include, for example, pines such
as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),
ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta),
and Monterey pine (Pinus radiata); Douglas fir (Pseudotsuga
menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea
glauca); redwood (Sequoia sempervirens); true firs such as silver
fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars
such as Western red cedar (Thuja plicata) and Alaska yellow cedar
(Chamaecyparis nootkatensis). Plants of the present invention
include crop plants (for example, corn, alfalfa, sunflower,
Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,
millet, tobacco, etc.), as well as turf grasses.
[0192] Turfgrasses include, but are not limited to: annual
bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canada
bluegrass (Poa compressa); Chewings fescue (Festuca rubra);
colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis
palustris); crested wheatgrass (Agropyron desertorum); fairway
wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia);
Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis
glomerata); perennial ryegrass (Lolium perenne); red fescue
(Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa
trivialis); sheep fescue (Festuca ovina); smooth bromegrass (Bromus
inermis); tall fescue (Festuca arundinacea); timothy (Phleum
pratense); velvet bentgrass (Agrostis canina); weeping alkaligrass
(Puccinellia distans); western wheatgrass (Agropyron smithii);
Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrum
secundatum); zoysia grass (Zoysia spp.); Bahia grass (Paspalum
notatum); carpet grass (Axonopus affinis); centipede grass
(Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum);
seashore paspalum (Paspalum vaginatum); blue gramma (Bouteloua
gracilis); buffalo grass (Buchloe dactyloids); sideoats gramma
(Bouteloua curtipendula).
[0193] Plants of interest include grain plants that provide seeds
of interest, oil-seed plants, and leguminous plants. Seeds of
interest include grain seeds, such as corn, wheat, barley, rice,
sorghum, rye, millet, etc. Oil-seed plants include cotton, soybean,
safflower, sunflower, Brassica, maize, alfalfa, palm, coconut,
flax, castor, olive etc. Leguminous plants include beans and peas.
Beans include guar, locust bean, fenugreek, soybean, garden beans,
cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
[0194] Compositions of the invention find use in protecting plants,
seeds, and plant products in a variety of ways. For example, the
compositions can be used in a method that involves placing an
effective amount of the pesticidal composition in the environment
of the pest by a procedure selected from the group consisting of
spraying, dusting, broadcasting, or seed coating.
[0195] Before plant propagation material (fruit, tuber, bulb, corm,
grains, seed), but especially seed, is sold as a commercial
product, it is customarily treated with a protectant coating
comprising herbicides, insecticides, fungicides, bactericides,
nematicides, molluscicides, or mixtures of several of these
preparations, if desired together with further carriers,
surfactants, or application-promoting adjuvants customarily
employed in the art of formulation to provide protection against
damage caused by bacterial, fungal, or animal pests. In order to
treat the seed, the protectant coating may be applied to the seeds
either by impregnating the tubers or grains with a liquid
formulation or by coating them with a combined wet or dry
formulation. In addition, in special cases, other methods of
application to plants are possible, e.g., treatment directed at the
buds or the fruit.
[0196] The plant seed of the invention comprising a DNA molecule
comprising a nucleotide sequence encoding a pesticidal protein of
the invention may be treated with a seed protectant coating
comprising a seed treatment compound, such as, for example, captan,
carboxin, thiram, methalaxyl, pirimiphos-methyl, and others that
are commonly used in seed treatment. In one embodiment within the
scope of the invention, a seed protectant coating comprising a
pesticidal composition of the invention is used alone or in
combination with one of the seed protectant coatings customarily
used in seed treatment.
[0197] It is recognized that the genes encoding the pesticidal
proteins can be used to transform insect pathogenic organisms. Such
organisms include Baculoviruses, fungi, protozoa, bacteria, and
nematodes.
[0198] A gene encoding a pesticidal protein of the invention may be
introduced via a suitable vector into a microbial host, and said
host applied to the environment, or to plants or animals. The term
"introduced" in the context of inserting a nucleic acid into a
cell, means "transfection" or "transformation" or "transduction"
and includes reference to the incorporation of a nucleic acid into
a eukaryotic or prokaryotic cell where the nucleic acid may be
incorporated into the genome of the cell (e.g., chromosome,
plasmid, plastid, or mitochondrial DNA), converted into an
autonomous replicon, or transiently expressed (e.g., transfected
mRNA).
[0199] Microorganism hosts that are known to occupy the
"phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or
rhizoplana) of one or more crops of interest may be selected. These
microorganisms are selected so as to be capable of successfully
competing in the particular environment with the wild-type
microorganisms, provide for stable maintenance and expression of
the gene expressing the pesticidal protein, and desirably, provide
for improved protection of the pesticide from environmental
degradation and inactivation.
[0200] Such microorganisms include bacteria, algae, and fungi. Of
particular interest are microorganisms such as bacteria, e.g.,
Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas,
Streptomyces, Rhizobium, Rhodopseudomonas, Methylius,
Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter,
Azotobacter, Leuconostoc, and Alcaligenes, fungi, particularly
yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,
Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular
interest are such phytosphere bacterial species as Pseudomonas
syringae, Pseudomonasfluorescens, Serratia marcescens, Acetobacter
xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas
campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter
xyli and Azotobacter vinlandir 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 rosues, S.
odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of
particular interest are the pigmented microorganisms.
[0201] A number of ways are available for introducing a gene
expressing the pesticidal protein into the microorganism host under
conditions that allow for stable maintenance and expression of the
gene. For example, expression cassettes can be constructed which
include the nucleotide constructs of interest operably linked with
the transcriptional and translational regulatory signals for
expression of the nucleotide constructs, and a nucleotide sequence
homologous with a sequence in the host organism, whereby
integration will occur, and/or a replication system that is
functional in the host, whereby integration or stable maintenance
will occur.
[0202] Transcriptional and translational regulatory signals
include, but are not limited to, promoters, transcriptional
initiation start sites, operators, activators, enhancers, other
regulatory elements, ribosomal binding sites, an initiation codon,
termination signals, and the like. See, for example, U.S. Pat. Nos.
5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992)
Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Davis et
al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor
Laboratory Press), Cold Spring Harbor, N.Y.; and the references
cited therein.
[0203] Suitable host cells, where the pesticidal protein-containing
cells will be treated to prolong the activity of the pesticidal
proteins in the cell when the treated cell is applied to the
environment of the target pest(s), may include either prokaryotes
or eukaryotes, normally being limited to those cells that do not
produce substances toxic to higher organisms, such as mammals.
However, organisms that produce substances toxic to higher
organisms could be used, where the toxin is unstable or the level
of application sufficiently low as to avoid any possibility of
toxicity to a mammalian host. As hosts, of particular interest will
be the prokaryotes and the lower eukaryotes, such as fungi.
Illustrative prokaryotes, both Gram-negative and gram-positive,
include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella,
Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as
Rhizobium; Spirillaceae, such as photobacterium, Zymomonas,
Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum;
Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and
Acetobacter; Azotobacteraceae 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.
[0204] Characteristics of particular interest in selecting a host
cell for purposes of pesticidal protein production include ease of
introducing the pesticidal protein gene into the host, availability
of expression systems, efficiency of expression, stability of the
protein in the host, and the presence of auxiliary genetic
capabilities. Characteristics of interest for use as a pesticide
microcapsule include protective qualities for the pesticide, such
as thick cell walls, pigmentation, and intracellular packaging or
formation of inclusion bodies; leaf affinity; lack of mammalian
toxicity; attractiveness to pests for ingestion; ease of killing
and fixing without damage to the toxin; and the like. Other
considerations include ease of formulation and handling, economics,
storage stability, and the like.
[0205] Host organisms of particular interest include yeast, such as
Rhodotorula spp., Aureobasidium spp., Saccharomyces spp., and
Sporobolomyces spp., phylloplane organisms such as Pseudomonas
spp., Erwinia spp., and Flavobacterium spp., and other such
organisms, including Pseudomonas aeruginosa, Pseudomonas
fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis,
Escherichia coli, Bacillus subtilis, and the like.
[0206] Genes encoding the pesticidal proteins of the invention can
be introduced into microorganisms that multiply on plants
(epiphytes) to deliver pesticidal proteins to potential target
pests. Epiphytes, for example, can be gram-positive or
gram-negative bacteria.
[0207] Root-colonizing bacteria, for example, can be isolated from
the plant of interest by methods known in the art. Specifically, a
Bacillus cereus strain that colonizes roots can be isolated from
roots of a plant (see, for example, Handelsman et al. (1991) Appl.
Environ. Microbiol. 56:713-718). Genes encoding the pesticidal
proteins of the invention can be introduced into a root-colonizing
Bacillus cereus by standard methods known in the art.
[0208] Genes encoding pesticidal proteins can be introduced, for
example, into the root-colonizing Bacillus by means of
electrotransformation. Specifically, genes encoding the pesticidal
proteins can be cloned into a shuttle vector, for example, pHT3101
(Lerecius et al. (1989) FEMS Microbiol. Letts. 60: 211-218. The
shuttle vector pHT3101 containing the coding sequence for the
particular pesticidal protein gene can, for example, be transformed
into the root-colonizing Bacillus by means of electroporation
(Lerecius et al. (1989) FEMS Microbiol. Letts. 60: 211-218).
[0209] Expression systems can be designed so that pesticidal
proteins are secreted outside the cytoplasm of gram-negative
bacteria, E. coli, for example. Advantages of having pesticidal
proteins secreted are: (1) avoidance of potential cytotoxic effects
of the pesticidal protein expressed; and (2) improvement in the
efficiency of purification of the pesticidal protein, including,
but not limited to, increased efficiency in the recovery and
purification of the protein per volume cell broth and decreased
time and/or costs of recovery and purification per unit
protein.
[0210] Pesticidal proteins can be made to be secreted in E. coli,
for example, by fusing an appropriate E. coli signal peptide to the
amino-terminal end of the pesticidal protein. Signal peptides
recognized by E. coli can be found in proteins already known to be
secreted in E. coli, for example the OmpA protein (Ghrayeb et al.
(1984) EMBO J, 3:2437-2442). OmpA is a major protein of the E. coli
outer membrane, and thus its signal peptide is thought to be
efficient in the translocation process. Also, the OmpA signal
peptide does not need to be modified before processing as may be
the case for other signal peptides, for example lipoprotein signal
peptide (Duffaud et al. (1987) Meth. Enzymol. 153:492).
[0211] Pesticidal proteins of the invention can be fermented in a
bacterial host and the resulting bacteria processed and used as a
microbial spray in the same manner that Bacillus thuringiensis
strains have been used as insecticidal sprays. In the case of a
pesticidal protein(s) that is secreted from Bacillus, the secretion
signal is removed or mutated using procedures known in the art.
Such mutations and/or deletions prevent secretion of the pesticidal
protein(s) into the growth medium during the fermentation process.
The pesticidal proteins are retained within the cell, and the cells
are then processed to yield the encapsulated pesticidal proteins.
Any suitable microorganism can be used for this purpose.
Pseudomonas has been used to express Bacillus thuringiensis
endotoxins as encapsulated proteins and the resulting cells
processed and sprayed as an insecticide (Gaertner et al. (1993),
in: Advanced Engineered Pesticides, ed. Kim).
[0212] Alternatively, the pesticidal proteins are produced by
introducing a heterologous gene into a cellular host. Expression of
the heterologous gene results, directly or indirectly, in the
intracellular production and maintenance of the pesticide. These
cells are then treated under conditions that prolong the activity
of the toxin produced in the cell when the cell is applied to the
environment of target pest(s). The resulting product retains the
toxicity of the toxin. These naturally encapsulated pesticidal
proteins may then be formulated in accordance with conventional
techniques for application to the environment hosting a target
pest, e.g., soil, water, and foliage of plants. See, for example
EPA 0192319, and the references cited therein.
[0213] In the present invention, a transformed microorganism (which
includes whole organisms, cells, spore(s), pesticidal protein(s),
pesticidal component(s), pest-impacting component(s), mutant(s),
preferably living or dead cells and cell components, including
mixtures of living and dead cells and cell components, and
including broken cells and cell components) or an isolated
pesticidal protein can be formulated with an acceptable carrier
into a pesticidal composition(s) that is, for example, a
suspension, a solution, an emulsion, a dusting powder, a
dispersible granule, a wettable powder, and an emulsifiable
concentrate, an aerosol, an impregnated granule, an adjuvant, a
coatable paste, and also encapsulations in, for example, polymer
substances.
[0214] Such compositions disclosed above may be obtained by the
addition of a surface-active agent, an inert carrier, a
preservative, a humectant, a feeding stimulant, an attractant, an
encapsulating agent, a binder, an emulsifier, a dye, a UV
protectant, a buffer, a flow agent or fertilizers, micronutrient
donors, or other preparations that influence plant growth. One or
more agrochemicals including, but not limited to, herbicides,
insecticides, fungicides, bactericides, nematicides, molluscicides,
acaracides, plant growth regulators, harvest aids, and fertilizers,
can be combined with carriers, surfactants or adjuvants customarily
employed in the art of formulation or other components to
facilitate product handling and application for particular target
pests. Suitable carriers and adjuvants can be solid or liquid and
correspond to the substances ordinarily employed in formulation
technology, e.g., natural or regenerated mineral substances,
solvents, dispersants, wetting agents, tackifiers, binders, or
fertilizers. The active ingredients of the present invention are
normally applied in the form of compositions and can be applied to
the crop area, plant, or seed to be treated. For example, the
compositions of the present invention may be applied to grain in
preparation for or during storage in a grain bin or silo, etc. The
compositions of the present invention may be applied simultaneously
or in succession with other compounds. Methods of applying an
active ingredient of the present invention or an agrochemical
composition of the present invention that contains at least one of
the pesticidal proteins produced by the bacterial strains of the
present invention include, but are not limited to, foliar
application, seed coating, and soil application. The number of
applications and the rate of application depend on the intensity of
infestation by the corresponding pest.
[0215] Suitable surface-active agents include, but are not limited
to, anionic compounds such as a carboxylate of, for example, a
metal; carboxylate of a long chain fatty acid; an
N-acylsarcosinate; mono or di-esters of phosphoric acid with fatty
alcohol ethoxylates or salts of such esters; fatty alcohol sulfates
such as sodium dodecyl sulfate, sodium octadecyl sulfate or sodium
cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated
alkylphenol sulfates; lignin sulfonates; petroleum sulfonates;
alkyl aryl sulfonates such as alkyl-benzene sulfonates or lower
alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;
salts of sulfonated naphthalene-formaldehyde condensates; salts of
sulfonated phenol-formaldehyde condensates; more complex sulfonates
such as the amide sulfonates, e.g., the sulfonated condensation
product of oleic acid and N-methyl taurine; or the dialkyl
sulfosuccinates, e.g., the sodium sulfonate or dioctyl succinate.
Non-ionic agents include condensation products of fatty acid
esters, fatty alcohols, fatty acid amides or fatty-alkyl- or
alkenyl-substituted phenols with ethylene oxide, fatty esters of
polyhydric alcohol ethers, e.g., sorbitan fatty acid esters,
condensation products of such esters with ethylene oxide, e.g.,
polyoxyethylene sorbitar fatty acid esters, block copolymers of
ethylene oxide and propylene oxide, acetylenic glycols such as
2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic
glycols. Examples of a cationic surface-active agent include, for
instance, an aliphatic mono-, di-, or polyamine such as an acetate,
naphthenate or oleate; or oxygen-containing amine such as an amine
oxide of polyoxyethylene alkylarnine; an amide-linked amine
prepared by the condensation of a carboxylic acid with a di- or
polyamine; or a quaternary ammonium salt.
[0216] Examples of inert materials include but are not limited to
inorganic minerals such as kaolin, phyllosilicates, carbonates,
sulfates, phosphates, or botanical materials such as cork, powdered
corncobs, peanut hulls, rice hulls, and walnut shells.
[0217] The compositions of the present invention can be in a
suitable form for direct application or as a concentrate of primary
composition that requires dilution with a suitable quantity of
water or other diluant before application. The pesticidal
concentration will vary depending upon the nature of the particular
formulation, specifically, whether it is a concentrate or to be
used directly. The composition contains 1 to 98% of a solid or
liquid inert carrier, and 0 to 50%, preferably 0.1 to 50% of a
surfactant. These compositions will be administered at the labeled
rate for the commercial product, preferably about 0.01 lb-5.0 lb.
per acre when in dry form and at about 0.01 pts.-10 pts. per acre
when in liquid form.
[0218] In a further embodiment, the compositions, as well as the
transformed microorganisms and pesticidal proteins, of the
invention can be treated prior to formulation to prolong the
pesticidal activity when applied to the environment of a target
pest as long as the pretreatment is not deleterious to the
activity. Such treatment can be by chemical and/or physical means
as long as the treatment does not deleteriously affect the
properties of the composition(s). Examples of chemical reagents
include but are not limited to halogenating agents; aldehydes such
a formaldehyde and glutaraldehyde; anti-infectives, such as
zephiran chloride; alcohols, such as isopropanol and ethanol; and
histological fixatives, such as Bouin's fixative and Helly's
fixative (see, for example, Humason (1967) Animal Tissue Techniques
(W.H. Freeman and Co.).
[0219] In other embodiments of the invention, it may be
advantageous to treat the polypeptides with a protease, for example
trypsin, to activate the protein prior to application of a
pesticidal protein composition of the invention to the environment
of the target pest. Methods for the activation of protoxin by a
serine protease are well known in the art. See, for example,
Cooksey (1968) Biochem. J. 6:445-454 and Carroll and Ellar (1989)
Biochem. J. 261:99-105, the teachings of which are herein
incorporated by reference. For example, a suitable activation
protocol includes, but is not limited to, combining a polypeptide
to be activated, for example a purified 1218-1 polypeptide, and
trypsin at a 1/100 weight ratio of 1218-1 protein/trypsin in 2 l nM
NaHCO3, pH 8 and digesting the sample at 36.degree. C. for 3
hours.
[0220] The compositions (including the transformed microorganisms
and pesticidal proteins of the invention) can be applied to the
environment of an insect pest by, for example, spraying, atomizing,
dusting, scattering, coating or pouring, introducing into or on the
soil, introducing into irrigation water, by seed treatment or
general application or dusting at the time when the pest has begun
to appear or before the appearance of pests as a protective
measure. For example, the pesticidal protein and/or transformed
microorganisms of the invention may be mixed with grain to protect
the grain during storage. It is generally important to obtain good
control of pests in the early stages of plant growth, as this is
the time when the plant can be most severely damaged. The
compositions of the invention can conveniently contain another
insecticide if this is thought necessary. In an embodiment of the
invention, the composition is applied directly to the soil, at a
time of planting, in granular form of a composition of a carrier
and dead cells of a Bacillus strain or transformed microorganism of
the invention. Another embodiment is a granular form of a
composition comprising an agrochemical such as, for example, a
herbicide, an insecticide, a fertilizer, an inert carrier, and dead
cells of a Bacillus strain or transformed microorganism of the
invention.
[0221] The embodiments of the present invention may be effective
against a variety of pests. For purposes of the present invention,
pests include, but are not limited to, insects, fungi, bacteria,
nematodes, acarids, protozoan pathogens, animal-parasitic liver
flukes, and the like. Pests of particular interest are insect
pests, particularly insect pests that cause significant damage to
agricultural plants. By "insect pests" is intended insects and
other similar pests such as, for example, those of the order Acari
including, but not limited to, mites and ticks. Insect pests of the
present invention include, but are not limited to, insects of the
order Lepidoptera, e.g. Achoroia grisella, Acleris gloverana,
Acleris variana, Adoxophyes orana, Agrotis ipsilon, Alabama
argillacea, Alsophila pometaria, Amyelois transitella, Anagasta
kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea
pernyi, Anticarsia gemmatalis, Archips sp., Argyrotaenia sp.,
Athetis mindara, Bombyx mori, Bucculatrix thurberiella, Cadra
cautella, Choristoneura sp., Cochylls hospes, Colias eurytheme,
Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana
integerrima, Dendrolimus sibericus, Desmiafeneralis, Diaphania
hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea
saccharalis, Ennomos subsignaria, Eoreuma loftini, Esphestia
elutella, Erannis tilaria, Estigmene acrea, Eulia salubricola,
Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis
chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita
molesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa
zea, Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum,
Hyphantia cunea, Keiferia lycopersicella, Lambdina fiscellaria
fiscellaria, Lambdina fiscellaria lugubrosa, Leucoma salicis,
Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macalla
thyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra
configurata, Manduca quinquemaculata, Manduca sexta, Maruca
testulalis, Melanchra picta, Operophtera brumata, Orgyia sp.,
Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes,
Pectinophora gossypiella, Phryganidia californica, Phyllonorycter
blancardella, Pieris napi, Pieris rapae, Plathypena scabra,
Platynota flouendana, Platynota stultana, Platyptilia
carduidactyla, Plodia interpunctella, Plutella xylostella, Pontia
protodice, Pseudaletia unipuncta, Pseudoplasia includens, Sabulodes
aegrotata, Schizura concinna, Sitotroga cerealella, Spilonta
ocellana, Spodoptera sp., Thaurnstopoea pityocampa, Tinsola
bisselliella, Trichoplusia hi, Udea rubigalis, Xylomyges curiails,
and Yponomeutapadella.
[0222] Also, the embodiments of the present invention may be
effective against insect pests including insects selected from the
orders Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera,
Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera,
Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera,
especially Diabrotica virgifera and Lepidoptera. Insect pests of
the invention for the major crops include, but are not limited to:
Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon,
black cutworm; Helicoverpa zea, corn earworm; Spodoptera
frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn
borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea
saccharalis, surgarcane borer; western corn rootworm, e.g.,
Diabrotica virgifera virgifera; northern corn rootworm, e.g.,
Diabrotica longicornis barberi; southern corn rootworm, e.g.,
Diabrotica undecimpunctata howardi; Melanotus spp., wireworms;
Cyclocephala borealis, northern masked chafer (white grub);
Cyclocephala immaculata, southern masked chafer (white grub);
Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn
flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum
maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid;
Blissus leucopterus leucopterus, chinch bug; Melanoplus
femurrubrum, redlegged grasshopper; Melanoplus sanguinipes,
migratory grasshopper; Hylemya platura, seedcorn maggot; Agromyza
parvicornis, corn blotch leafminer; Anaphothrips obscrurus, grass
thrips; Solenopsis milesta, thief ant; Tetranychus urticae, two
spotted spider mite; Sorghum: Chilo partellus, sorghum borer;
Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn
earworm; Elasmopalpus lignosellus, leser cornstalk borer; Feltia
subterranea, granulate cutworm; Phyllophaga crinita, white grub;
Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema melanopus,
cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle;
Sphenophorus maidis, maize billbug; Rhopalosiphum maidis; corn leaf
aphid; Sipha flava, yellow sugarcane aphid; chinch bug, e.g.,
Blissus leucopterus leucopterus; Contarinia sorghicola, sorghum
midge; Tetranychus cinnabarinus, carmine spider mite; Tetranychus
urticae, two-spotted spider mite; Wheat: Pseudaletia unipunctata,
army worm; Spodoptera frugiperda, fall armyworm; Elasmopalpus
lignosellus, lesser cornstalk borer; Agrotis orthogonia, pale
western cutworm; Elasmopalpus lignosellus, lesser cornstalk borer;
Oulema melanopus, cereal leaf beetle; Hypera punctata, clover leaf
weevil; southern corn rootworm, e.g., Diabrotica undecimpunctata
howardi; Russian wheat aphid; Schizaphis graminum, greenbug;
Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum,
redlegged grasshopper; Melanoplus differentialis, differential
grasshopper; Melanoplus sanguinipes, migratory grasshopper;
Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat
midge; Meromyza americana, wheat stem maggot; Hylemya coarctata,
wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus
cinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite;
Sunflower: Cylindrocupturus adspersus, sunflower stem weevil;
Smicronyx fulus, red sunflower seed weevil; Smicronyx sordidus,
gray sunflower seed weevil; Suleima helianthana, sunflower bud
moth; Homoeosoma electellum, sunflower moth; Zygogramma
exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle;
Neolasioptera murtfeldtiana, sunflower seed midge; Cotton:
Heliothis virescens, tobacco budworm; Helicoverpa zea, cotton
bollworm; Spodoptera exigua, beet armyworm; Pectinophora
gossypiella, pink bollworm; boll weevil, e.g., Anthonomus grandis;
Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton
fleahopper; Trialeurodes abutilonea, bandedwinged whitefly; Lygus
lineolaris, tarnished plant bug; Melanoplus femurrubrum, redlegged
grasshopper; Melanoplus differentialis, differential grasshopper;
Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips;
Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae,
two-spotted spider mite; Rice: Diatraea saccharalis, sugarcane
borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn
earworm; Colaspis brunnea, grape colaspis; Lissorhoptrus
oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil;
Nephotettix nigropictus, rice leafhoper; chinch bug, e.g., Blissus
leucopterus leucopterus; Acrosternum hilare, green stink bug;
Soybean: Pseudoplusia includens, soybean looper; Anticarsia
gemmatalis, velvetbean caterpillar; Plathypena scabra, green
cloverworm; Ostrinia nubilalis, European corn borer; Agrotis
ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis
virescens, tobacco budworm; Helicoverpa zea, cotton bollworm;
Epilachna varivestis, Mexican bean beetle; Myzus persicae, green
peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare,
green stink bug; Melanoplus femurrubrum, redlegged grasshopper;
Melanoplus differentialis, differential grasshopper; Hylemya
platura, seedcorn maggot; Sericothrips variabilis, soybean thrips;
Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry
spider mite; Tetranychus urticae, two-spotted spider mite; Barley:
Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black
cutworm; Schizaphis graminum, greenbug; chinch bug, e.g., Blissus
leucopterus leucopterus; Acrosternum hilare, green stink bug;
Euschistus servus, brown stink bug; Jylemya platura, seedcorn
maggot; Mayetiola destructor, Hessian fly; Petrobia latens, brown
wheat mite; Oil Seed Rape: Vrevicoryne brassicae, cabbage aphid;
Phyllotreta cruciferae, crucifer flea beetle; Phyllotreta
striolata, striped flea beetle; Phyllotreta nemorum, striped turnip
flea beetle; Meligethes aeneus, rapeseed beetle; and the pollen
beetles Meligethes rufimanus, Meligethes nigrescens, Meligethes
canadianus, and Meligethes viridescens; Potato: Leptinotarsa
decemlineata, Colorado potato beetle.
[0223] Furthermore, embodiments of the present invention may be
effective against Hemiptera such as Lygus hesperus, Lygus
lineolaris, Lyguspratensis, Lygus rugulipennis Popp, Lygus
pabulinus, Calocoris norvegicus, Orthops compestris, Plesiocoris
rugicollis, Cyrtopeltis modestus, Cyrtopeltis notatus, Spanagonicus
albofasciatus, Diaphnocoris chlorinonis, Labopidicola allii,
Pseudatomoscelis seriatus, Adelphocoris rapidus, Poecilocapsus
lineatus, Blissus leucopterus, Nysius ericae, Nysiusraphanus,
Euschistus servus, Nezara viridula, Eurygaster, Coreidae,
Pyrrhocoridae, Tinidae, Blostomatidae, Reduviidae, and Cimicidae.
Pests of interest also include Araecerus fasciculatus, coffee bean
weevil; Acanthoscelides obtectus, bean weevil; Bruchus rufimanus,
broadbean weevil; Bruchus pisorum, pea weevil; Zabrotes
subfasciatus, Mexican bean weevil; Diabrotica balteata, banded
cucumber beetle; Cerotoma trifurcata, bean leaf beetle; Diabrotica
virgifera, Mexican corn rootworm; Epitrix cucumeris, potato flea
beetle; Chaetocnema confinis, sweet potato flea beetle; Hypera
postica, alfalfa weevil; Anthonomus quadrigibbus, apple curculio;
Sternechus paludatus, bean stalk weevil; Hypera brunnipennis,
Egyptian alfalfa weevil; Sitophilus granaries, granary weevil;
Craponius inaequalis, grape curculio; Sitophilus zeamais, maize
weevil; Conotrachelus nenuphar, plum curculio; Euscepes
postfaciatus, West Indian sweet potato weevil; Maladera castanea,
Asiatic garden beetle; Rhizotrogus majalis, European chafer;
Macrodactylus subspinosus, rose chafer; Tribolium confusum,
confused flour beetle; Tenebrio obscurus, dark mealworm; Tribolium
castaneum, red flour beetle; Tenebrio molitor, yellow mealworm.
[0224] Nematodes include plant-parasitic nematodes such as
root-knot, cyst, and lesion nematodes, including Heterodera and
Globodera spp. such as Globodera rostochiensis and Globodera
pailida (potato cyst nematodes); Heterodera glycines (soybean cyst
nematode); Heterodera schachtii (beet cyst nematode); and
Heterodera avenae (cereal cyst nematode).
[0225] The preferred developmental stage for testing for pesticidal
activity is larvae or immature forms of these above mentioned
insect pests. The insects may be reared in total darkness at from
about 20.degree. C. to about 30.degree. C. and from about 30% to
about 70% relative humidity. Bioassays may be performed as
described in Czapla and Lang (1990) J. Econ. Entomol. 83(6):
2480-2485. Methods of rearing insect larvae and performing
bioassays are well known to one of ordinary skill in the art.
[0226] A wide variety of bioassay techniques is known to one
skilled in the art. General procedures include addition of the
experimental compound or organism to the diet source in an enclosed
container. Pesticidal activity can be measured by, but is not
limited to, changes in mortality, weight loss, attraction,
repellency and other behavioral and physical changes after feeding
and exposure for an appropriate length of time. Bioassays described
herein can be used with any feeding insect pest in the larval or
adult stage.
[0227] The following examples are presented by way of illustration,
not by way of limitation.
Experimental
EXAMPLE 1
Bioassay for Testing the Pesticidal Activity of B. thuringiensis
Strains Against Western Corn Rootworm and Southern Corn
Rootworm
[0228] Insect diets for Colorado potato beetle (CPB), southern corn
rootworm (SCRW), and western corn rootworm (WCRW) larvae are known
in the art. See, for example, Rose and McCabe (1973) J. Econ.
Entomology 66:393, herein incorporated by reference. The insect
diet is prepared and poured into a CD International bioassay tray.
Generally 1.5 ml of diet is dispensed into each cell with an
additional 150 .mu.l of sample preparation applied to the diet
surface.
[0229] Bacterial colonies from an original plate of transformants
expressing the pesticidal proteins of interest are spotted on
replica plates and inoculated in 5 ml 2.times.YT broth with 500
.mu.l/1000 ml kanamycin antibiotic. The tubes are grown overnight.
If no growth is present, the tubes are incubated for an additional
24 hours. Following incubation, the tubes are centrifuged at 3500
rpm for 5-8 minutes. The supernatant is discarded and the pellet
resuspended in 1000 .mu.l PBS. The sample is then transferred to
1.5 ml Eppendorf tubes and incubated on ice until the temperature
is 3 to 4.degree. C., followed by sonication for 12-15 seconds.
[0230] Microbial culture broths (150 .mu.l) or other samples (150
.mu.l) are overlaid onto artificial diets. The trays are allowed to
dry. Rootworm larvae are dispensed into the wells of the bioassay
tray. Lids are placed on the bioassay trays and the samples are
incubated for 4-7 days at a temperature of 26.degree. C. The
bioassays are then scored by counting "live" versus "dead" larvae.
Mortality is calculated as percentage of dead larvae out of the
total larvae tested.
EXAMPLE 2
Pesticidal Activity of B. thuringiensis Strain 1218 Lysates
[0231] Samples prepared from cultures of B. thuringiensis strains
1218 were tested for the presence of pesticidal activity against
CPB, WCRW, and SCRW as described in Example 1. As a control, the
diet was treated with phosphate-buffered saline (PBS).
[0232] To prepare each sample, an individual colony of a strain
growing on an LB plate was selected and used to inoculate a flask
containing 50 ml of TB medium. The flask was incubated overnight at
28.degree. C. and 250 rpm. Following the incubation, the culture in
the flask was transferred to a tube, and the tube was centrifuged
at 4300.times.g for 15 minutes. The supernatant was discarded and
the pellet resuspended in 50 ml of sporulation medium. The tube was
centrifuged again at 4300.times.g for 15 minutes. The second
supernatant was discarded, and the second pellet resuspended in 50
ml of sporulation medium. The resuspended culture solution was
transferred to a flask, and the flask was then incubated for 48
hours at 28.degree. C. and 250 rpm. Following this incubation, the
culture in the flask was transferred to a tube, and the tube was
centrifuged at 4300.times.g for 15 minutes. The supernatant was
discarded, and the pellet was resuspended in 10 ml of 1.times.M9
medium. The sample was then transferred to a 1.5 ml microfuge tube,
incubated on ice until the temperature was about 3 to 4.degree. C.,
and then sonicated for 12-15 seconds. For bioassays, 150 .mu.l of a
sonicated sample was used.
[0233] Sporulation medium comprises 200 ml of 5.times.M9 salts
solution, 5 ml of salts solution, 5 ml of CaCl.sub.2 solution, and
dH.sub.2O to a final volume of 1 liter. The solution of 5.times.M9
salts comprises: 64 g Na.sub.2HPO.sub.4.7H.sub.2O; 15 g
KH.sub.2PO.sub.4; 2.5 g NaCl; 5 g NH.sub.4Cl; and dH.sub.2O to a
final volume of 1 liter. Salts solution comprises: 2.46 g
MgSO.sub.4.7H.sub.2O; 0.04 g MnSO.sub.4.H.sub.2O; 0.28 g
ZnSO.sub.4.7H.sub.2O; 0.40 g FeSO.sub.4.7H.sub.2O; and dH.sub.2O to
a final volume of 1 liter. CaCl.sub.2 solution comprises 3.66 g
CaCl.sub.2.2H.sub.20 and dH.sub.2O to a final volume of 100 ml.
[0234] Samples were tested with and without heating to determine
whether the component(s) responsible for the pesticidal activity is
heat stable. For the heat treatment, the samples were boiled for 15
minutes prior to use in the bioassay. Unheated samples prepared
from strain 1218 exhibited pesticidal activity against southern
corn rootworm, with lesser pesticidal activity against western corn
rootworm. The samples prepared from strain 1218 lysates caused
moderate stunting in the southern corn rootworm larvae. Following
heating, the samples had greatly reduced pesticidal activity
against both species of rootworms.
[0235] The reduction in pesticidal activity following heating
indicated that the one or more components of the sample from strain
1218 that is responsible for the pesticidal activity is heat
labile. Such a reduction is consistent with one or more of the
components being a protein.
EXAMPLE 3
Pesticidal Activity of Crystal Proteins Isolated from B.
thuringiensis Strain 1218
[0236] Using samples of sporulated cultures of B. thuringiensis
strain 1218 prepared as described in Example 2, crystal proteins
were isolated and then trypsin-treated using methods known in the
art. Briefly, after purification (zonal gradient centrifugation,
Renografin-76), the purified crystals were dissolved in alkaline
buffer (50 mM Na.sub.2CO.sub.3, 10 mM dithiothreitol, pH 10). Prior
to use in the assays, the dissolved crystal proteins were
concentrated by filtration with Centriprep.RTM. (Millipore Corp.)
centrifugal filter units with a MW cutoff of 10,000.
[0237] It is recognized that under some experimental conditions, it
may be advantageous to treat the Cry8-like polypeptides with a
protease, for example trypsin, to activate the protein prior to
determining the pesticidal activity of a particular sample. Methods
for the activation of protoxin by a serine protease are well known
in the art. See, for example, Cooksey (1968) Biochem J. 6:445-454
and Carroll and Ellar (1989) Biochem J. 261:99-105; herein
incorporated by reference. Isolated crystal proteins were screened
for pesticidal activity against western corn rootworm larvae as
described in Example 1. Both a new crystal protein preparation and
a previously made preparation ("old preparation") from strain 1218
possessed pesticidal activity against western corn rootworms.
Dissolved crystal proteins were stored at -80.degree. C. for 20
days before use in the assays.
[0238] A skilled artisan will acknowledge that there are numerous
indicators of pesticidal activity and that variables such as number
of dead insects, or average weight of treated insects can be
monitored. For example, pesticidal activity can be conveniently
expressed as percent (%) mortality, which is the percentage of dead
rootworm larvae out of the total number of larvae.
EXAMPLE 4
Nucleotide Sequences Isolated from B. thuringiensis Strain 1218
[0239] An effort was undertaken to isolate the nucleotide sequences
that encode the crystal proteins from B. thuringiensis strain 1218.
Two nucleotide sequences were isolated from 1218 that have
nucleotide sequence and amino acid sequence homology to Cry8Bal
(GenBank Accession No. U04365). The two Cry8-like coding sequences
isolated from strain 1218 have been designated Cry1218-1 (SEQ ID
NO:1), also known as Cry8Bb1, see Genbank Accession No. AX543924
and Cry1218-2 (SEQ ID NO:3), also known as Cry8Bc1, see Genbank
Accession No. AX543926. SEQ ID NO:17 and SEQ ID NO:18 provide the
nucleic acid sequences of native genomic clones of Cry1218-1 and
Cry1218-2, respectively.
[0240] To determine if the proteins encoded by variant or mutant
polynucleotides of the invention encode proteins with pesticidal
activity, each of the nucleic acid sequences was expressed in
Escherichia coli. For example, to determine if the 1218-1 or 1218-2
polynucleotide sequences provided herein encode polypeptides with
pesticidal activity, truncated nucleotide sequences were prepared.
SEQ ID NO: 11 corresponds to nucleotides 1 through 2007 of the
nucleotide sequence of Cry1218-1 (SEQ ID NO: 1). SEQ ID NO: 13
corresponds to nucleotides 1 through 2019 of the nucleotide
sequence of Cry1218-2 (SEQ ID NO:3).
[0241] SEQ ID NOs:11 and 13 encode truncated Cry8-like polypeptides
having the amino acid sequences set forth in SEQ ID NO:12 and 14,
respectively. Each of the truncated nucleotide sequences (SEQ ID
NOs:11 and 13) was separately cloned into a pET28a expression
vector and then used to transform E. coli. Transformed colonies
were selected and grown in liquid culture as described in Example
1. The expressed, N-terminal-His-tagged, truncated Cry8-like
proteins were isolated from E. coli lysates by affinity
chromatography using a nickel affinity column. The column fractions
with the protein of interest were dialyzed extensively against 10
mM Tris-HCl (pH 8.5) and then concentrated using Centriprep.RTM.
(Millipore Corp.) centrifugal filter units with a MW cutoff of
10,000 according to the manufacturer's directions. The concentrated
Cry8-like protein samples were tested for the presence of
pesticidal activity against western corn rootworm as described in
Example 1.
[0242] Bioassays evaluating the pesticidal activity of recombinant
Cry8-like proteins purified from E. coli-expressed preparations
were conducted as described in Example 1 with the aqueous protein
samples overlaid on the surface of the rootworm diet. The
pesticidal activity of wild-type (e.g., native) and mutant
endotoxin were assessed against southern corn rootworms. As
expected, it was observed that the pesticidal activity decreased as
the concentration of the truncated Cry8-like proteins applied to
the diet decreased.
[0243] Pesticidal activity was also assessed by incorporating the
pesticidal proteins into the rootworm diet, as opposed to the
method described above, which involved incorporating a
protein-containing solution into the diet mixture. For example,
sample diets comprising 1000, 500, 400, 300, 200, or 100 ppm of a
pesticidal polypeptide incorporated into the diet were
assessed.
EXAMPLE 5
Preparation of a Plant-Preferred Nucleotide Sequence Encoding a
Pesticidal Protein
[0244] Because codon usage is different between plants and
bacteria, the expression in a plant of a protein encoded by
nucleotide sequence of bacterial origin can be limited due to
translational inefficiency in the plant. It is known in the art
that expression can be increased in a plant by altering the coding
sequence of the protein to contain plant-preferred codons. For
optimal expression of a protein in a plant, a synthetic nucleotide
sequence may be prepared using the amino acid sequence of the
protein and back-translating the sequence using plant-preferred
codons.
[0245] Using such an approach, a portion of the amino acid sequence
of the protein encoded by Cry1218-1 (SEQ ID NO:2) was
back-translated (i.e., reverse translated) using maize-preferred
codons. The resulting plant-preferred nucleotide sequence is set
forth in SEQ ID NO:5. The nucleotide sequence set forth in SEQ ID
NO:5 encodes a polypeptide (SEQ ID NO:6) that comprises the first
669 amino acids of the amino acid sequence set forth in SEQ ID
NO:2. Thus, SEQ ID NOs:6 and 12 encode polypeptides comprising the
same amino acid sequence and SEQ ID NO:11 provides a second
polynucleotide that encodes the amino acid sequences set forth in
SEQ ID NO:6.
EXAMPLE 6
Bioassay for Testing the Pesticidal Activity of Mutant Cry8-Like
Polypeptides against Colorado Potato Beetle (Leptinotarsa
decemlineata)
[0246] Protocol
[0247] Briefly, bioassay parameters were as follows: Bio-Serv diet
(catalog number F9800B, from: BIOSERV, Entomology Division, One 8h
Street, Suite 1, Frenchtown, N.J. 08825) was dispensed in a 96 well
microtiter plate (catalog number 353918, Becton Dickinson, Franklin
Lakes, N.J. 07417-1886) having a surface area of 0.33 cm.sup.2.
Cry8-like samples (1218-1 and K03) were applied topically to the
diet surface. The amino acid sequence of the 1218-1 endotoxin is
set forth in SEQ ID NO:2, while the amino acid sequence of the K03
mutant endotoxin is set forth in SEQ ID NO:68. Enough sample
material was supplied to provide for 8 observations/sample. After
the sample dried, 1 Colorado potato beetle (CPB) neonate was added
to each well. Therefore, there was a total of 8 larvae/sample. A
Mylar.RTM. lid (Clear Lam Packaging, Inc., 1950 Pratt Blvd., Elk
Grove Village, Ill. 60007-5993) was affixed to each tray. Bioassay
trays were placed in an incubator at 25.degree. C.
[0248] The test was scored for mortality on the 7.sup.th day
following live infesting. The resulting mortality data was analyzed
by a probit model (SAS/STAT Users Guide Version 8 Chapter 54,
1999). The probit analysis of wild type 1218-1 and Cry8-like mutant
K03 is shown in FIG. 6 and FIG. 7 respectively.
[0249] Results
[0250] Sample labeled "I and R" in Table 1 was a control sample
consisting of 10 mM carbonate buffer at pH 10. All of the cry 8
like mutant protein samples, 1218-1 (A-H) and K03 (J-Q) were
solubilized in 10 mM carbonate buffer at pH 10. Bioassays of 1218-1
and K03 indicated that both protein samples were efficacious
against CPB. Cry8-like mutant K03 was found to be more potent than
the parent 1218-1 endotoxin. The LC.sub.50 for Cry8-like mutant K03
was much lower when compared to the wild type 1218-1 protein (Table
2.) Thus, based on diet surface area, it requires about 137 times
less protein to achieve a LC.sub.50 using Cry8-like mutant K03
versus 1218-1 (0.61 .mu.g/cm.sup.2 for K03 versus 84 .mu.g/cm.sup.2
for 1218-1). Based on probit analysis and LC.sub.50 determination
(Table 2), Cry8-like mutant K03 shows significantly better
bioactivity against CPB than 1218-1 wild type.
1TABLE 1 Pesticidal Activity of a 1218 Cry8-like (K03) Mutant and
Wild Type 1218-1 against Colorado Potato Beetle Protein Mortality
Mortality Code Samples (mg/ml) Rep 1 Rep 2 A 1218-1 0.5 *100% 100%
B 1218-1 0.25 75% 100% C 1218-1 0.125 50% 100% D 1218-1 0.0625 25%
63% E 1218-1 0.03125 25% 25% F 1218-1 0.0156 38% 25% G 1218-1
0.0078 13% 38% H 1218-1 0.0039 13% 0% I buffer 13% 13% J K03 0.5
100% 100% K K03 0.25 100% 100% L K03 0.125 100% 100% M K03 0.0625
100% 100% N K03 0.03125 88% 63% O K03 0.0156 75% 75% P K03 0.0078
38% 38% Q K03 0.0039 38% 38% R buffer 25% 13% *Percent mortality
was calculated from 8 observations per concentration.
[0251]
2TABLE 2 LC.sub.50 Determination of a 1218 Cry8-like (K03) Mutant
and Wild Type 1218-1 against Colorado Potato Beetle Sample
LC.sub.50 (mg/ml) 95% Fiducial Limits 1218-1 1.1098 0.6859-2.4485
K03 0.00808 0.00467-0.01184
EXAMPLE 7
Bioassay for Testing the Pesticidal Activity of Mutant Cry8-Like
Polypeptides against Southern Corn Rootworm and Western Corn
Rootworm
[0252] Protocol
[0253] The assay parameters described above in Example 6 are
modified to allow for the evaluation of the pesticidal activity of
additional mutant polypeptides against western corn rootworm (WCRW)
and southern corn rootworm (SCRW). Briefly, Bio-Serv diet (catalog
number F9800B, from: BIOSERV, Entomology Division, One 8.sup.th
Street, Suite 1, Frenchtown, N.J. 08825) is dispensed in 128-well
CD International bioassay trays (catalog number BIO-BA-128 from CD
International, Pitman, N.J. 08071).
[0254] Endotoxin samples are applied topically to the diet. Enough
sample material is supplied to provide for replicate observations
per sample. The trays are allowed to dry. Rootworm larvae are
dispensed into the wells of the bioassay trays. Lids are placed on
the bioassay trays and the samples are incubated for 4-7 days at a
temperature of 26.degree. C.
[0255] For the evaluation of pesticidal activity against SCRW,
insects are exposed to a solution comprising either buffer (50 mM
carbonate buffer (pH 10)) or a solution of mutant polypeptide at
selected doses, for example, 36 or 3.6 .mu.g/cm.sup.2.
[0256] For the evaluation of pesticidal activity against WCRW,
insects are exposed to a solution comprising either buffer (50 mM
carbonate buffer (pH 10)) or to a limited number of mutant
polypeptides at a particular dose, e.g., 88 .mu.g/cm.sup.2.
[0257] The bioassays are then scored by counting "live" versus
"dead" larvae. Mortality is calculated as percentage of dead larvae
out of the total larvae tested.
EXAMPLE 8
Construction and Evaluation of Mutant Sequences
[0258] An experiment was conducted to create and evaluate
particular examples of mutant polynucleotide sequences and their
encoded mutant proteins. The NGSR1218-1 polynucleotide sequence was
cloned into the pET28a-c(+) vector (Novagen Corporation) as a
BamHI-XhoI fragment. This construct (pET28/NGSR1218-1) was then
used as the starting material for further genetic modification.
[0259] A multistep PCR procedure was employed to generate the
mutants. Mutagenesis primers were first used in combination with
two primers designed from the pET 28 vector as pET forward primer
(SEQ ID NO:37) and pET reverse primer (SEQ ID NO:38). The
mutagenesis primers used to create the M4 mutant were the M4
forward primer (SEQ ID NO: 27) and the M4 reverse primer (SEQ ID
NO: 28); the mutagenesis primers used to create the M5 mutant were
the M5 forward primer (SEQ ID NO: 31) and the M5 reverse primer
(SEQ ID NO: 32); and the mutagenesis primers used to create the K04
mutant were the K04 forward primer (SEQ ID NO: 23) and the K04
reverse primer (SEQ ID NO: 24). Thus, the amino acid sequence of
the M4 mutant endotoxin is set forth in SEQ ID NO:26; the amino
acid sequence of the M5 mutant endotoxin is set forth in SEQ ID
NO:30; and the amino acid sequence of the K04 mutant endotoxin is
set forth in SEQ ID NO:22.
[0260] After a first round of PCR, the samples were loaded into a
1% agarose gel, and the expected bands were excised and purified
using the Qiaquick gel extraction kit (Qiagen). To generate the
mutant polynucleotide, a second round of PCR was performed for 7
cycles without primers. This procedure generated the mutant
polynucleotide via overlapping of the homologous mutated region.
Subsequently, the flanking pET 28 primers (forward and reverse)
were added to generate the mutated polynucleotide sequence.
[0261] These modified polynucleotide fragments were then used to
replace the corresponding fragment in the pET28/NGSR1218-1 plasmid
using standard cloning procedures so that the mutated portions of
the polynucleotide were substituted for the corresponding portions
of the original polynucleotide. The pET28-based plasmids were used
to express the encoded proteins in E. coli.
[0262] BL21 Star.TM. (DE3) cells (Invitrogen) were used as the E.
coli host for protein production from the pET28-derived plasmids.
The pET28 plasmid provides a "tag," which is a short polypeptide
linked to the 3' end of polypeptides generated from the plasmid.
This tag provides a mechanism by which the protein can be purified
from solution. To produce the protein, the bacterial cultures were
grown to a density of approximately OD.sub.600 1.0 at 37.degree. C.
Cultures were then induced with 200 .mu.g/ml IPTG and incubated
overnight at 16.degree. C. The culture cells were then collected
and lysed to produce lysate containing the tagged fusion protein of
interest. The fusion proteins were purified using the Novagen His
tag purification kit. Purified protein concentrations were
determined using the BCA protein assay (Pierce).
[0263] Mutant proteins were used in a bioassay procedure to
evaluate the effect of the mutant polypeptides on pests of
interest. Specifically, an experiment was conducted to compare the
effects of wild type (native) and mutant polypeptides on WCRW. The
rootworms were cultured in bioassay trays. Insect diet was
dispensed into each well of the bioassay tray. Test protein samples
or control samples were applied topically to the diet. Samples were
dried down in a laminar flow hood. Test protein samples were used
in the bioassays as described in Table 3 to determine what
concentration of protein to use in tests to compare the original
protein to the mutant proteins.
3TABLE 3 Test protein samples used in bioassays. Western Corn
Rootworm Assays: Sample Stock Sample Concentration Concentration
(mg/ml) on Diet (.mu.g/cm.sup.2) 2.5 225 1.25 112.5 0.625 56.25
0.3125 28.13 0.1563 14.06 0.0781 7.03
[0264]
4 Colorado Potato Beetle Assays: Sample Sample Concentration
Concentration in stock(mg/ml) on diet (.mu.g/cm.sup.2) 0.500 38
0.250 19 0.125 9.5 0.0625 4.7 0.03125 2.4 0.0156 1.2 0.0078 0.6
0.0039 0.3 Buffer 0
[0265] Four observations were made per concentration of test
protein.
[0266] Mortality and stunting were evaluated at 5 and 7 days post
western corn rootworm infestation. The term "stunting" (or
"stunted") means the WCRW larva is severely retarded in growth and
turns pale yellow to brown in coloration, in contrast to normal
larvae of the same stage or instar, which are large, round and
creamy white in color.
[0267] Another assay format referred to as the "128-well bioassay
tray protocol" was also used to evaluate the mutant proteins.
Again, insect diet was added to each well of the bioassay tray.
Either test protein sample or control sample was applied topically
to the diet. After the samples had thoroughly dried, wells were
infested with 10 larvae per well. The wells were then covered with
a sealable lid and the trays were incubated at 27.degree. C. in the
dark. Mortality and stunting were evaluated at 5 and 7 days after
infestation, and surviving larvae were weighed (Table 4).
[0268] Similar tests were conducted for the Colorado potato beetle
(CPB). CPB neonates were infested at a rate of one per well; the
test was scored after 6 days and percent mortality for each rate
was calculated. Results (shown in FIGS. 2-4) indicate that CPB
larvae are much more susceptible to mutant endotoxins K03 and K34
relative to the wild type endotoxin (1218-1). Further, survivors
that fed on diets treated with K03 and K34 endotoxin were severely
stunted as compared to buffer controls, while CPB survivors from
the 1218-1 test were relatively large.
5TABLE 4 Initial Results of WCRW Bioassays 5-day 7-day 5-day 7-day
% Samples [PROTEIN] SCORE MORTALITY WCRW Test # 1 1 Buffer 6/40
6/40 15 15 2 1218 132 .mu.g/cm.sup.2 4/40 4/40 10 10 3 NGSR 132
.mu.g/cm.sup.2 22/40 23/40 55 57 4 M6 132 .mu.g/cm.sup.2 38/40
40/40 95 100 WCRW Test # 2 1 Buffer 4/40 5/40 10 12 2 1218 132
.mu.g/cm.sup.2 7/40 7/40 17 17 3 NGSR 132 .mu.g/cm.sup.2 24/40
26/40 62 65 4 M6 132 .mu.g/cm.sup.2 31/40 35/40 78 88
EXAMPLE 9
LC.sub.50 Determination of Cry8 Like Mutants
[0269] A bioassay experiment was conducted to determine the
LC.sub.50 of a Cry8-like mutant M6 for western corn rootworm (WCRW)
neonates. These bioassays were conducted essentially as set forth
in Example 8. Five observations were made per treatment level
(Table 5). Three WCRW neonates were added to each well for a total
of 15 larvae/dose. Percent mortality was scored after 5 days of
incubation at 27.degree. C. PROBIT analysis (SAS/STAT Users Guide
Version 8 Chapter 54, 1999) was used to calculate the lethal
concentration of sample at which 50% of the larvae died (i.e., the
LC.sub.50).
[0270] The summary of the dose-mortality response of WCRW neonates
for this experiment is shown in Table 6. Probit analysis was
performed and the result indicated that the LC.sub.50 of the
Cry8-like mutant M6 protein was 26 .mu.g/cm.sup.2, with 95%
fiducial limits at 17.1 and 37.0.
6TABLE 5 M6 Protein Samples Used in LC.sub.50 Bioassays Sample
Stock Sample Concentration Concentration (mg/ml) on Diet
(.mu.g/cm.sup.2) 2.44 244 1.22 122 0.610 61 0.305 30.5 0.153 15.3
0.076 7.6 0.038 3.8
[0271]
7TABLE 6 Percent Mortality of WCRW Larvae at Various Concentrations
of M6 Protein Protein Concentration on Diet Surface
(.mu.g/cm.sup.2) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6
244 100 100 100 93 80 80 122 47 93 40 53 100 53 61 83 79 67 47 73
57 30.5 53 79 40 13 67 21 15.3 27 40 33 33 73 8 7.6 53 27 53 20 81
14 3.8 ND ND 0 27 75 25 0 (buffer) 7 7 0 7 20 0 (ND = no data)
[0272] Probit analysis of the above data indicated that the
LC.sub.50 of the M6 protein corresponded to a concentration of 26
.mu.g/cm.sup.2, with 95% fiducial limits at 17.1 and 37.0. A graph
of the larval mortality rate as a function of the log of the
concentration of M6 protein is shown in FIG. 1.
EXAMPLE 10
Transformation of Maize by Particle Bombardment and Regeneration of
Transgenic Plants
[0273] Immature maize embryos from greenhouse donor plants are
bombarded with a DNA molecule containing the plant-optimized
Cry1218-1 nucleotide sequence (SEQ ID NO:5) operably linked to a
ubiquitin promoter and the selectable marker gene PAT (Wohlleben et
al. (1988) Gene 70:25-37), which confers resistance to the
herbicide Bialaphos. Alternatively, the selectable marker gene is
provided on a separate DNA molecule. Transformation is performed as
follows. Media recipes follow below.
[0274] Preparation of Target Tissue
[0275] The ears are husked and surface sterilized in 30% Clorox.TM.
bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two
times with sterile water. The immature embryos are excised and
placed embryo axis side down (scutellum side up), 25 embryos per
plate, on 560Y medium for 4 hours and then aligned within the
2.5-cm target zone in preparation for bombardment.
[0276] Preparation of DNA
[0277] A plasmid vector comprising a plant-optimized Cry8-like
nucleotide sequence (e.g., Cry1218-1, SEQ ID NO:5) operably linked
to a ubiquitin promoter is made. For example, a suitable
transformation vector comprises a UBIL promoter from Zea mays, a 5'
UTR from UBI1 and a UBI1 intron, in combination with a PinII
terminator. The vector additionally contains a PAT selectable
marker gene driven by a CAMV35S promoter and includes a CAMV35S
terminator. Optionally, the selectable marker can reside on a
separate plasmid. A DNA molecule comprising a Cry8-like nucleotide
sequence as well as a PAT selectable marker is precipitated onto
1.1 .mu.m (average diameter) tungsten pellets using a CaCl.sub.2
precipitation procedure as follows:
[0278] 100 .mu.l prepared tungsten particles in water
[0279] 10 .mu.l (1 .mu.g) DNA in Tris EDTA buffer (1 .mu.g total
DNA)
[0280] 100 .mu.l 2.5 M CaCl.sub.2
[0281] 10 .mu.l 0.1 M spermidine
[0282] Each reagent is added sequentially to a tungsten particle
suspension, while maintained on the multitube vortexer. The final
mixture is sonicated briefly and allowed to incubate under constant
vortexing for 10 minutes. After the precipitation period, the tubes
are centrifuged briefly, liquid removed, washed with 500 ml 100%
ethanol, and centrifuged for 30 seconds. Again the liquid is
removed, and 105 .mu.l 100% ethanol is added to the final tungsten
particle pellet. For particle gun bombardment, the tungsten/DNA
particles are briefly sonicated and 10 .mu.l spotted onto the
center of each macrocarrier and allowed to dry about 2 minutes
before bombardment.
[0283] Particle Gun Treatment
[0284] The sample plates are bombarded at level #4 in particle gun
#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI,
with a total of ten aliquots taken from each tube of prepared
particles/DNA.
[0285] Subsequent Treatment
[0286] Following bombardment, the embryos are kept on 560Y medium
for 2 days, then transferred to 560R selection medium containing 3
mg/liter Bialaphos, and subcultured every 2 weeks. After
approximately 10 weeks of selection, selection-resistant callus
clones are transferred to 288J medium to initiate plant
regeneration. Following somatic embryo maturation (2-4 weeks),
well-developed somatic embryos are transferred to medium for
germination and transferred to the lighted culture room.
Approximately 7-10 days later, developing plantlets are transferred
to 272V hormone-free medium in tubes for 7-10 days until plantlets
are well established. Plants are then transferred to inserts in
flats (equivalent to 2.5" pot) containing potting soil and grown
for 1 week in a growth chamber, subsequently grown an additional
1-2 weeks in the greenhouse, then transferred to classic 600 pots
(1.6 gallon) and grown to maturity. Plants are monitored and scored
for expression of the Cry1218-1 protein by assays known in the art,
such as, for example, immunoassays and western blotting with an
antibody that binds to the Cry1218-1 protein.
[0287] Bombardment and Culture Media
[0288] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts
(SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix
(1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose,
1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with dl
H.sub.2O following adjustment to pH 5.8 with KOH); 2.0 g/l
Gelrite.TM. (added after bringing to volume with dl H.sub.2O); and
8.5 mg/l silver nitrate (added after sterilizing the medium and
cooling to room temperature). Selection medium (560R) comprises 4.0
g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix
(1000.times. SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose,
and 2.0 mg/l 2,4-D (brought to volume with dl H.sub.2O following
adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite.TM. (added after
bringing to volume with dl H.sub.2O); and 0.85 mg/l silver nitrate
and 3.0 mg/l Bialaphos (both added after sterilizing the medium and
cooling to room temperature).
[0289] Plant regeneration medium (288J) comprises 4.3 g/l MS salts
(GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g
nicotinic acid, 0.02 g/l thiamine HCl, 0.10 g/l pyridoxine HCl, and
0.40 g/l Glycine brought to volume with polished D-I H.sub.2O)
(Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l
myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1
mM abscisic acid (brought to volume with polished dl H.sub.2O after
adjusting to pH 5.6); 3.0 g/l Gelrite.TM. (added after bringing to
volume with dl H.sub.2O); and 1.0 mg/l indoleacetic acid and 3.0
mg/l Bialaphos (added after sterilizing the medium and cooling to
60.degree. C.). Hormone-free medium (272V) comprises 4.3 g/l MS
salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100
g/l nicotinic acid, 0.02 g/l thiamine HCl, 0.10 g/l pyridoxine HCl,
and 0.40 g/l Glycine brought to volume with polished dl H.sub.2O),
0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with
polished dl H.sub.2O after adjusting pH to 5.6); and 6 g/l
Bacto-agar (added after bringing to volume with polished dl
H.sub.2O), sterilized and cooled to 60.degree. C.
EXAMPLE 11
Agrobacterium-Mediated Transformation of Maize and Regeneration of
Transgenic Plants
[0290] For Agrobacterium-mediated transformation of maize with a
plant-optimized Cry1218-1 nucleotide sequence (SEQ ID NO: 5),
preferably the method of Zhao is employed (U.S. Pat. No. 5,981,840,
and PCT patent publication WO98/32326; the contents of which are
hereby incorporated by reference). Briefly, immature embryos are
isolated from maize and the embryos contacted with a suspension of
Agrobacterium under conditions whereby the bacteria are capable of
transferring the plant-optimized Cry1218-1 nucleotide sequence (SEQ
ID NO:5) to at least one cell of at least one of the immature
embryos (step 1: the infection step). In this step the immature
embryos are preferably immersed in an Agrobacterium suspension for
the initiation of inoculation. The embryos are co-cultured for a
time with the Agrobacterium (step 2: the co-cultivation step).
Preferably the immature embryos are cultured on solid medium
following the infection step. Following this co-cultivation period
an optional "resting" step is contemplated. In this resting step,
the embryos are incubated in the presence of at least one
antibiotic known to inhibit the growth of Agrobacterium without the
addition of a selective agent for plant transformants (step 3:
resting step). Preferably the immature embryos are cultured on
solid medium with antibiotic, but without a selecting agent, for
elimination of Agrobacterium and for a resting phase for the
infected cells. Next, inoculated embryos are cultured on medium
containing a selective agent and growing transformed callus is
recovered (step 4: the selection step). Preferably, the immature
embryos are cultured on solid medium with a selective agent
resulting in the selective growth of transformed cells. The callus
is then regenerated into plants (step 5: the regeneration step),
and preferably calli grown on selective medium are cultured on
solid medium to regenerate the plants.
EXAMPLE 12
Dose-Response Bioassay for Mutant Endotoxins against the Boll
Weevil, Anthonomus grandis
[0291] Treatments:
[0292] Four endotoxins were tested by diet incorporation for
activity against the boll weevil, Anthonomus grandis, obtained from
USDA APHIS PPQ MPPC Insect Production; Moore Air Base, Bldg. S-6414
Mission, Tex.: wild type (1218-1); K03 mutant endotoxin; M6 mutant
endotoxin; and K40 mutant endotoxin. Controls included buffer alone
and untreated diet.
[0293] Method:
[0294] Five 24-well plates were set up for each treatment, and 200
ml Bioserv boll weevil diet (#F9247B) was prepared according to
manufacturer's specifications. The diet was held in a 40.degree. C.
water bath.
[0295] A serial dilution of the endotoxin samples was prepared in
microfuge tubes using sample aliquots of 3 mg, 1.5 mg, 0.75 mg,
0.37 mg, 0.19 mg. 5 ml of diet was removed from the water bath and
placed in a scintillation vial. A protein sample was then added to
the diet and mixed thoroughly. After mixing with 5 ml of diet the
resulting concentrations were 600, 300, 150, 75, and 37 .mu.g/ml
diet (these rates were chosen to correspond to topical rates of
100, 50, 25, 12.5, and 6.25 .mu.g/cm.sup.2.) 150 microliters of
diet was added to four wells of each of the five 24-well plates.
Each plate had the following configuration:
8TABLE 7 Configuration of Test Plates 600 300 150 75 37 Blank 600
300 150 75 37 Blank 600 300 150 75 37 Blank 600 300 150 75 37
Blank
[0296] Controls included a single plate of buffer treatment, which
was produced with all 24 wells receiving 500 microliters of buffer.
Another control plate was produced with no addition to the diet.
The M6 mutant endotoxin amino acid sequence is set forth in SEQ ID
NO:70; the K03 mutant endotoxin amino acid sequence is set forth in
SEQ ID NO:68; and the K40 mutant endotoxin amino acid sequence is
set forth in SEQ ID NO:94.
[0297] Results:
[0298] One week after boll weevil infestation, boll weevil larvae
were recovered from the diet plugs of all 5 plates containing the
same Cry8-like mutant and combined. The diet pills were carefully
dissected under 4.times. magnification in order to recover all
larvae.
9TABLE 8 Results of Bioassay on Boll Weevil Larvae Buffer
Concentration Protein (500 ul/ (ug/ml diet) 1218-1 M6 K03 K40 well)
600 5ss 4s 0 3ss 4 + 1s 300 3ss 6s 0 1ss 5 + 1ss 150 2s 7s 3s 3ss 3
+ 1ss 75 2 9 3s 3ss 2 + 4s 38 3 11 2s 3ss 4 + 1s (s = stunted; ss =
severely stunted).
EXAMPLE 13
Second Dose-Response Bioassay for Mutant Endotoxins against the
Boll Weevil, Anthonomus grandis
[0299] An examination of the effect of wild type endotoxin (1218-1)
and two endotoxin mutant proteins (M6 and K03) on total biomass
using a high and low dose of toxin shows that the mutants have
enhanced pesticidal activity relative to the wild type endotoxin.
Results are shown in Table 8.
[0300] Bioassays were conducted as described in Example 12, with
the following modifications. Three replicate plates were produced
for each sample with four observations per dose per plate.
[0301] Results were scored at 96 hours post-emergence, when larvae
were recovered from the diet, counted, and weighed. All larvae from
a particular treatment plate were weighed together this number was
divided by the number of individuals to give an average weight.
10TABLE 9 Effect of Endotoxins on Cotton Boll Weevil Larval Weight
Larval weight Larval weight (mg) on 600 .mu.g/ml (mg) on 19
.mu.g/ml Endotoxin diet diet 1218-1 9.00 42.23 K03 0.00 14.70 M6
4.07 30.60 Buffer 79.10 84.40 (control)
[0302] (These results are also shown graphically in FIG. 5).
[0303] Thus, at the highest endotoxin dose of 600 .mu.g per ml of
diet, 1218-1 and M6 treatments show a very significant reduction in
biomass of 88.6% and 94.9%, respectively. These data represent an
8.80 and 19.4 fold increase in activity for 1218 and M6,
respectively, when compared to buffer control. Treatment with K03
protein yielded no survivors at the 600 .mu.g treatment in any of
the replicates.
[0304] In comparison, at the lowest dose of 19 .mu.g per ml of
diet, the data indicate a 50.0%, 63.7%, and 82.6% reduction in
biomass for 1218, M6 and K03, respectively, when compared to the
buffer control. Thus, at a dose that is over 30 fold lower, the K03
mutation at 19 .mu.g per ml of diet exhibits nearly equivalent
activity (82.6% reduction in biomass) when compared to wild type
endotoxin (1218) at 600 .mu.g per ml of diet (88.6% reduction in
biomass). Furthermore, at a dose of 19 .mu.g per ml of diet, K03
endotoxin shows activity that is 2.08 and 2.87 fold better activity
than the M6 and wild type (1218-1) endotoxins, respectively.
[0305] Explanation of Results:
[0306] The data indicate a clear reduction in weight for all
polypeptide samples when compared to the buffer control.
Additionally, all mutant endotoxins reduced larval growth below the
growth seen for the native or wild type (1218-1) endotoxin. The
mutants K03, K35, and K40 produced results of few or no larvae
recovered at the highest doses and a high degree of stunting at
lower doses. The K40 mutant protein produced an approximately
5-fold reduction in weight gain at the highest doses when compared
to wild type endotoxin. When compared to the buffer control, the
K40 mutant produced reductions ranging from 46 fold at the highest
dose to 5 fold at the lowest dose based on comparison of average
larval weights at those doses. Similarly, results for the K03
mutant showed effects ranging from complete mortality at the
highest dose to 200-fold weight reduction at the next dose and
5-fold weight reduction at the lowest dose. The K35 mutant showed a
pattern similar to that of the K03 mutant.
EXAMPLE 14
Bioassay for Testing the Pesticidal Activity of Mutant Cry8-Like
K03 Polypeptide Against Corn Flea Beetle (Chaetocnema
pulicaria)
[0307] A bioassay experiment was conducted to determine if corn
flea beetles (Chaetocnema pulicaria) are susceptible to the mutant
K03 endotoxin (SEQ ID NO:68). Since corn leaf beetles feed
predominately on the upper layer of leaf cells, a known amount of
toxin may be applied to the leaf surface or leaves may be coated
with toxin by dipping. Insects are then allowed to feed on toxin
treated leaves and after a prescribed time period, percent
mortality can be calculated.
[0308] For this assay, corn flea beetles were field collected and
presented with leaf discs that were dipped in either a K03 or
buffer solution. Leaf discs were evaluated in a 128-well CD
International bioassay tray (catalog number BIO-BA-128 from CD
International, Pitman, N.J. 08071) in which each well was first
filled with 1 ml molten agar solution. Once the agar solidified, a
1.5 cm filter paper (VWR, catalog number 28309-989) was placed on
top of the agar plug and wetted with 25 .mu.l of sterile water.
Next, leaf discs (1 cm diameter) were punched from whorl leaves
(collected from V8 stage corn plants) and dipped in either in a K03
(1 mg/ml) solution or a 20 mM sodium carbonate (pH 10.5) buffer
solution. Both solutions contained 0.01% Tween 20 to aid in the
dispersal of sample over the entire leaf surface. Once the wetted
dipped leaf discs dried, they were placed on top of the filter
paper in the bioassay tray so that 1 disc was present per well in
the 128 well bioassay tray. Each well was then infested with one
corn flea beetle and covered with sealable lids supplied by CD
International, Pitman, N.J. 08071. The assay was scored after 5
days and percent mortality was calculated.
[0309] Examination of leaf discs after 5 days showed moderate
levels of feeding damage as noted by the presence of thin brown
stripes on both K03 and buffer treated leaves. It was observed that
a greater number of corn flea beetles died after they fed on leaf
discs treated with K03 as compared to those that fed on buffer
treated leaf discs (see Table 10).
11TABLE 10 Corn flea beetle bioassay results. Treatment Mortality
(%) Buffer 14/32 = 44 K03 23/31 = 74
EXAMPLE 15
Modification of GC Content to Create Optimized Nucleotide
Sequences
[0310] Analysis of Coding Regions from Various Organisms
[0311] A dataset containing 1831 maize cDNAs with full-length
coding regions were plotted versus GC content of the coding
sequence (FIG. 8, "ORFs" shown in upper panel). The plot showed a
bimodal distribution with the majority of sequences (about 2/3) in
the low GC mode peaked at about 51% GC and about a third in the
high GC mode peaked at about 67% GC.
[0312] While this is the largest set of maize full-length cDNAs so
analyzed to date, based on a total gene count estimate of 50,000,
this dataset may only represent about 3.6% of the transcriptome.
Consequently, an EST-based UniGene assembly sequence dataset
believed to represent most maize genes and containing 84,085
sequences was also analyzed (FIG. 8, "UniGenes" shown in lower
panel). As used herein, a Unigene represents a consensus sequence
of assembled Est's. The Unigene dataset results from an application
of the CAP3 assembly algorithm (see Huang and Madan (1999) Genome
Research 9:868-877). The analysis of this dataset confirmed the
earlier full-length cDNA results by showing a bimodal distribution
with a similar proportion of high and low GC genes. The bimodal
distribution for the UniGene dataset was centered at 45% and 64%
GC, slightly lower than for the smaller full-length cDNA dataset,
probably due to the inclusion of remaining untrimmed AT-rich 3'-UTR
non-coding sequences.
[0313] The GC analysis was performed for other plants. A
corresponding survey of coding regions (i.e., cDNA "ORFs," or Open
Reading Frames) revealed very similar bimodal distributions for
rice and wheat (2,400 rice sequences and 800 wheat sequences were
analyzed). In contrast, analysis of Arabidopsis (25,700 sequences),
Solanaceae ssp. (2,000 sequences), and soybean (G. max, 400 cDNAs,
or 49,300 UniGene assemblies), all revealed single mode
distributions with peaks between 42-44% GC content.
[0314] In an examination of other organisms, a survey of cDNA ORFs
from warm-blooded mammals all revealed broad GC content
distributions with suggested bimodality. In this analysis, 19,200
sequences were analyzed from human, 12,000 from mouse (M.
musculus), 900 from cattle (B. taurus), and 1,100 from chicken (G.
gallus). An examination of organisms from other major eukaryotic
groups showed unimodal distributions with peaks ranging from
38%-56% GC content for C. elegans (16,000 sequences analyzed), D.
melanogaster (14,800 sequences), and S. cereviseae (6,300
sequences). Unimodal distributions were also found for sequences
from three eubacteria (E. coli, 4,200 sequences; B. subtilus, 4,000
sequences; Synechocystis sp. 3,200 sequences) and four Archaea (T.
maritima, 1,800 sequences; T. jannaschii, 1,800 sequences; A.
fulgidus, 2,400 sequences; H. halobium, 2,600 sequences (with very
high overall GC content).
[0315] Thus, a broad survey of GC content distribution showed that,
in contrast to most organisms, monocot cereals have a clearly
bimodal GC content distribution. Warm-blooded vertebrates also
showed a bimodal tendency, but this was less pronounced than in
monocots.
[0316] mRNA Profiling
[0317] To examine the relationship between gene expression and GC
content, mRNA expression of high (centered at approximately 67% GC
content) and low (centered at approximately 51% GC content) GC mode
maize genes was investigated using both EST distribution analysis
(over 400,000 ESTs) and Lynx MPSS technology (63.4 million 17-mer
tags) (see Brenner et al. (2000) Nature Biotechnology 18:630-634,
Brenner et al. (2000) PNAS 97:1665-1670 for information on Lynx
MPSS). The data showed that while gene expression varied widely
within high and low GC modes, when the average expression levels
among 12 key distinct tissue categories were considered, the
overall average expression level of high and low GC mode genes in
maize was similar.
EXAMPLE 16
Method of Optimizing GC Content of Genes
[0318] In light of the findings about GC content described above,
it was of interest to develop computerized methods to modify coding
sequences of any gene from any source organism into a structure
compatible with that preferred by maize and other cereals. As
discussed above, other major cereals such as wheat and rice show
similar bimodal distributions to maize, and the high GC preferred
codons are the same. Consequently, the methods for sequence
optimization described below would be useful not only for enhanced
gene expression in maize but also in all the cereals. These methods
allow coding sequences from various organisms to be optimized for
expression in cereals and in this manner provide for improved
transgenic plants, for example, a crop plant such as maize. Two
exemplary optimization methods are presented below. However, it is
recognized that one of skill in the art would be able to optimize a
sequence using a variety of procedures and still create a sequence
of the invention.
[0319] Method 1: Dialed-In GC Content
[0320] This method allows selection and generation of an altered
nucleotide sequence containing a specified percentage of GC content
(within 0.5%). This method employs proportional codon usage
frequencies and takes into account the tendency of coding regions
to have a gradient of GC content from 5' to 3' end. The
proportional codon usage frequencies are arrayed in weighted tables
to implement the method.
[0321] Step 1. Determine Whether the Selected GC Content is
Theoretically Feasible.
[0322] First, the theoretical highest and lowest GC content are
calculated for the sequence of interest. In this step, codon
substitutions are made in the original sequence to generate altered
sequences with the highest and lowest possible GC content that
still encode the same polypeptide as the original sequence. The
original sequence may of course be a coding sequence or predicted
polypeptide from any source.
[0323] Where there are two codons that are equally GC-poor, the
codons are substituted in proportion according to the low GC mode
proportional codon tables (see Table 11, GC-Richest and Poorest
Proportional Codon Table, Proportional Codon Frequency Columns (on
left)). For example, the GC-poor codons corresponding to alanine
include both GCT and GCA. From the low GC mode proportional codon
table, the relative frequencies of GCA and GCT are 30.4% and 36.5%,
respectively. Thus, in proportion with their relative frequencies,
for low GC mode substitution, the GCA substitution frequency should
be 30.4/(36.5+30.4)=45.4% and the GCT substitution frequence should
be 36.5/(36.5+30.4)=55.6%. These percentages have been calculated
and are presented in Table 11, Proportional Extreme GC Columns/
Lowest GC (on right). Thus, for low GC mode, GCA should be
substituted for 45.4% of the alanine codons and GCT for 55.6% of
the alanine codons.
[0324] Similarly, for determining the highest possible GC content,
substitution frequencies are presented in Table 11, Proportional
Extreme GC Columns/Highest GC. Thus, for alanine, the high GC
content codons are GCC and GCG, which are found at frequencies of
47.2% and 38.7% overall, respectively. Thus, in high GC mode, the
GCC codon is substituted for 54.9% of alanine codons
[47.2/(47.2+38.7)=54.9%] and the GCT codon is substituted for 45.1%
of alanine codons [38.7/47.2+38.7)=45.1%].
[0325] In this manner, two new altered nucleotide sequences are
created, one with the lowest possible GC content and the other with
the highest possible GC content, according to the proportional
codon usage of Table 11. These altered nucleotide sequences still
encode the same polypeptide as the original nucleotide sequence. In
a computer program written to implement this algorithm, if the
desired GC content is at or outside these high and low GC content
values, the program can output the altered nucleotide sequence for
the higest and lowest GC content. One characteristic of this method
is that in the altered sequence, the codons for any given amino
acid may not be uniformly distributed and there could be block
stretches of the same codon for a particular amino acid.
12TABLE 11 GC-Richest and Poorest Proportional Codon Table
Proportional Codon Frequency Proportional Extreme GC Amino acid
Codon General High GC Low GC Highest GC Lowest GC GCA 19.88% 5.96%
30.38% 45.43% Ala GCC 32.00% 47.20% 20.61% 54.93% GCG 22.83% 38.72%
12.51% 45.07% GCT 25.29% 8.13% 36.49% 54.56% AGA 16.20% 3.57%
24.18% 100.00% AGG 25.71% 22.04% 26.57% Arg CGA 7.82% 3.43% 10.24%
CGC 23.11% 40.18% 13.28% 61.20% CGG 15.94% 25.47% 11.56% 38.80% CGT
11.22% 5.31% 14.17% Asn AAC 60.68% 92.55% 46.57% 100.00% AAT 39.32%
7.45% 53.43% 100.00% Asp GAC 55.30% 90.32% 37.75% 100.00% GAT
44.70% 9.68% 62.25% 100.00% Cys TGC 67.97% 92.08% 54.31% 100.00%
TGT 32.03% 7.92% 45.69% 100.00% Gln CAA 34.97% 9.41% 47.49% 100.00%
CAG 65.03% 90.59% 52.51% 100.00% Glu GAA 34.46% 9.55% 46.37%
100.00% GAG 65.54% 90.45% 53.63% 100.00% GGA 20.26% 7.62% 28.39%
48.83% Gly GGC 37.85% 62.57% 23.22% 72.82% GGG 20.48% 23.35% 18.65%
27.18% GGT 21.41% 6.45% 29.74% 51.16% His CAC 56.40% 87.35% 40.16%
100.00% CAT 43.60% 12.65% 59.84% 100.00% ATA 19.32% 4.90% 24.91%
37.25% Ile ATC 48.33% 88.53% 33.13% 100.00% ATT 32.34% 6.57% 41.96%
62.75% CTA 8.04% 2.73% 10.82% CTC 25.61% 44.16% 15.63% 50.06% Leu
CTG 27.10% 44.05% 19.29% 49.94% CTT 18.24% 4.61% 24.48% TTA 6.63%
0.54% 10.18% 100.00% TTG 14.37% 3.91% 19.59% Lys AAA 28.98% 7.57%
39.06% 100.00% AAG 71.02% 92.43% 60.94% 100.00% Met ATG 100.00%
100.00% 100.00% 100.00% 100.00% Phe TTC 64.74% 94.80% 50.08%
100.00% TTT 35.26% 5.20% 49.92% 100.00% CCA 26.66% 10.21% 36.80%
51.94% Pro CCC 22.07% 31.91% 15.40% 40.09% CCG 25.74% 47.67% 13.76%
59.90% CCT 25.53% 10.21% 34.05% 48.05% TAA 30.64% 24.89% 33.00%
100.00% STOP TAG 34.95% 38.33% 33.00% 51.03% TGA 34.41% 36.78%
34.00% 48.97% AGC 21.90% 32.94% 16.65% 37.50% AGT 10.93% 2.56%
15.26% 25.34% Ser TCA 15.95% 4.23% 21.75% 36.12% TCC 20.60% 31.87%
14.46% 36.29% TCG 13.22% 23.02% 8.68% 26.21% TCT 17.40% 5.38%
23.20% 38.53% ACA 23.81% 5.61% 34.03% 51.40% Thr ACC 31.88% 46.40%
22.29% 52.75% ACG 20.74% 41.57% 11.50% 47.25% ACT 23.57% 6.42%
32.18% 48.60% Trp TGG 100.00% 100.00% 100.00% 100.00% 100.00% Tyr
TAC 63.47% 94.76% 47.77% 100.00% TAT 36.53% 5.24% 52.23% 100.00%
GTA 9.86% 2.37% 14.58% 28.73% Val GTC 29.82% 42.63% 21.73% 45.93%
GTG 35.25% 50.19% 27.52% 54.07% GTT 25.07% 4.81% 36.17% 71.27%
[0326] Step 2. If the Desired GC Content is Between the Highest and
Lowest Possible GC Percentage for the Original Sequence, the
Sequence may be Altered Accordingly.
[0327] The altered sequence from step 1 is selected which has GC
content closest to the desired GC content. This sequence is then
further altered according to the codon usage tables so that the GC
content is increased or decreased to the desired level. As an
initial step in changing GC content, changing only the third codon
positions should be considered. (However, for arginine codons,
there could theoretically be changes in the first two codon
positions when substituting the preferred low or high GC codon--see
Table 12 below). If the GC content needs to be increased, changes
may be made from the N-terminal or 5'-end to the C-terminal or
3'-end so as to preserve and even enhance the negative GC gradient
in the coding region. Similarly, if the GC content needs to be
decreased, changes may be made from the C-terminal or 3'-end to the
N-terminal or 5'-end so as to preserve and even enhance the
negative GC gradient. Not all amino acid codons will be substituted
because some rare codons may be avoided. Among the amino acids and
their codons available to change in method 1 are the following:
13TABLE 12 Codon Substitutions to Increase or Decrease GC Content
AA To Decrease GC To Increase GC Ala GCT GCC Arg AGA CGC Asn AAT
AAC Asp GAT GAC Gly GGT GGC His CAT CAC Ile ATT ATC Leu CTT CTC Pro
CCA CCG Ser TCT AGC Thr ACA ACC Val GTT GTC
[0328] Results Output
[0329] Where a computer program implements the method, the output
can include a nucleotide sequence which is the altered sequence
according to the method(s) above. This sequence is then translated
into a predicted polypeptide which is compared with the polypeptide
encoded or predicted to be encoded by the original nucleotide
sequence to ensure that, where desired, the polypeptide sequence
has not been changed by the alterations in the GC content of the
nucleotide sequence.
[0330] Method 2for Optimizing Genes:
[0331] Step 1. The first step is the same as described for method 1
except that the appropriate codons are substituted in an
alternating pattern, with any excess of one applied to the
beginning (i.e., oriented toward the N-terminal), and codons ending
in G or C are applied first where possible. As in method 1, two
altered sequences are generated that represent the highest and
lowest possible GC content for a sequence that (if desired) still
encodes the same polypeptide as the original sequence. If the
desired GC content is at or outside these theoretical highest and
lowest GC content values, the sequence closest to the desired level
of GC content is chosen for further alteration.
[0332] Step 2. If the Desired GC Content is Between the Highest and
Lowest Possible GC Percentage for the Original Sequence, the
Sequence may be Altered Accordingly.
[0333] The study of the 1831 maize ORFs described in Example 15
revealed patterns in the GC content and codon content of maize
genes. The coding regions of maize genes were shown to have an
overall GC content of 54.5%, with an overall GC content in the
third codon position of 63%. The GC content of the third position
varies as a function of relative position in the coding region.
Thus, for the first 180 nucleotides (first 60 codons, or roughly
first sixth of coding region), the third codon position GC content
is 70%. For the second 180 nucleotides (second 60 codons, or
roughly second sixth of coding region), the third codon position GC
content is 65%. For the remainder of the coding region, the third
codon position GC content is about 60%. Thus, in approximately the
first 60 codons, the third codon position GC content is 11% higher
than the overall GC content; in approximately the second 60 codons,
it is 3% higher, and in the remainder of the coding region it is
4.8% lower than the overall GC content.
[0334] A scatter plot of the third codon position GC content
(designated "ORF3GC") versus the overall GC content (designated
"ORFGC") was used to determine the best fitting line to this data
using the least squares method. The resulting equation gives the
general relationship between ORF3GC and ORFGC for maize genes, as
follows: ORF3GC=2.03*ORFGC-47.2. Changes made to the third codon
position will generally have an effect on the ORFGC content in a
manner according to this equation.
[0335] However, the plot of ORF3GC versus ORFGC is actually
slightly curved at the ends, especially at the high-end GC levels,
where the slope decreases. This decrease in slope is probably the
result of amino acid composition biases as well as saturation of GC
content in codons that may vary in third position GC content. Thus,
unless the above equation is modified, it will generally
underestimate the correct ORF3GC value in relation to ORFGC. This
is especially true where the overall GC percentage of a sequence is
intermediate, a situation in which GC content alteration is
particularly likely to be desirable. A computer program was
designed and implemented to perform the above methods. After using
this program (method 2, also known as "10.2") to apply the methods
in equation form and using the above original linear equation,
empirical observations permitted correction of the original
equation to one that resulted in better correlation of ORF3GC with
ORFGC. The resulting modified equation is ORF3GC =2.06*ORFGC-44.2.
Thus, changing ORF3GC will be expected to generally cause a
concomitant change in the ORFGC.
[0336] Given the other information above regarding the tendency
towards a negative ORF ORF3GC content gradient, the following
equation can be developed.
[0337] Let L=length of protein in amino acids or codons
[0338] Let B=Base ORF3GC% level to which, for example 11% will be
added in first ORF section
[0339] Let ORF3GC=Overall ORF3GC% of the ORF
[0340] Let ORFGC=Overall ORFGC% of the ORF
[0341] Line equation=ORF3GC=2.06*ORFGC-44.2
[0342] So:
Number 3GC nts=Number 3GC nts in first ORF section+Number 3GCnts in
second ORF section+Number 3GC nts in remainder of the ORF
[0343] Which equals:
L*(ORF3GC/100)=60*(B+11)/100+60*(B+3)/100+(L-120)(B-4.8)/100
[0344] Substitute with line equation:
L*(2.06*ORFGC-44.2)/100=60*(B+11)/100+60*(B+3)/100+(L-120)(B-4.8)/100
[0345] Simplify:
2.06*L*ORFGC-44.2*L=60B+660+60B+180+LB-4.8*L-120B+576
2.06*L*ORFGC-44.2*L=1416+LB-4.8*L
2.06*L*ORFGC-39.4*L=1416+LB
[0346] Example Solve:
[0347] Let Length=300
[0348] Let ORFGC=60
[0349] Then:
2.06*300*60-39.4*300=1416+300B
37080-11820=1416+300B
23844=300 B
B=79.48 or 79.48% ORF3GC as the base
[0350] Therefore the ORF3GC target in the first section will be
90.48, in the second section 82.48, and in the last section
approximately 74.68. The ORF3GC target in the last section will be
affected by protein length due to limitation of the first two
sections to 60 codons each, leaving the remainder of the ORF to the
last section. Thus, the number of codons in the last section will
vary depending upon the length of the protein. As the described
methods are applied to proteins of various lengths, the amount of
GC adjustments that are performed in the last section will then be
affected by the length of this section.
[0351] Step 3. Creation of a Template ORF
[0352] For the process a "template ORF" or coding sequence is
created based on the general maize codon table so that the normal
relative proportion of codons is preserved (rounded off to the
nearest whole integer). Codons having a G or C in the third
position are generally concentrated at the N-terminal or 5' end.
Also, codons are distributed such that excess codons are
substituted into the 5' or N-terminal of the coding region,
followed by an alteration of the codons so as to disperse their
location in the protein.
14TABLE 13 General Maize Codon Table (1831 seqs) Codon Amino acid
Codon Freq Ala GCA 19.88% GCC 32.00% GCG 22.83% GCT 25.29% Arg AGA
16.20% AGG 25.71% CGA 7.82% CGC 23.11% CGG 15.94% CGT 11.22% Asn
AAC 60.68% AAT 39.32% Asp GAC 55.30% GAT 44.70% Cys TGC 67.97% TGT
32.03% Gln CAA 34.97% CAG 65.03% Glu GAA 34.46% GAG 65.54% Gly GGA
20.26% GGC 37.85% GGG 20.48% GGT 21.41% His CAC 56.40% CAT 43.60%
Ile ATA 19.32% ATC 48.33% ATT 32.34% Leu CTA 8.04% CTC 25.61% CTG
27.10% CTT 18.24% TTA 6.63% TTG 14.37% Lys AAA 28.98% AAG 71.02%
Met ATG 100.00% Phe TTC 64.74% TTT 35.26% Pro CCA 26.66% CCC 22.07%
CCG 25.74% CCT 25.53% STOP TAA 30.64% TAG 34.95% TGA 34.41% Ser AGC
21.90% AGT 10.93% TCA 15.95% TCC 20.60% TCG 13.22% TCT 17.40% Thr
ACA 23.81% ACC 31.88% ACG 20.74% ACT 23.57% Trp TGG 100.00% Tyr TAC
63.47% TAT 36.53% Val GTA 9.86% GTC 29.82% GTG 35.25% GTT
25.07%
[0353] This template ORF is then used to adjust the original coding
sequence to conform to the GC gradient according to the principles
outlined above. In this process, the linear equation discussed
above is used to calculate the base ORF3GC. In addition, the OFR3GC
content is adjusted in view of the increased GC content in the
first and second 60-codon regions of the ORF, as discussed above.
Thus, the ORF3GC content is adjusted by dividing the template ORF
into the three sections: the first 60 codons, the second 60 codons,
and the rest of the ORF. For each section, the ORFGC and ORF3GC are
determined and compared and alterations made to the original
sequence accordingly. Thus, for example, the first 60-codon ORF
section is evaluated to determine whether the ORF3GC needs to be
raised or lowered. (Often the ORF3GC will need to be raised to be
in compliance with the negative GC gradient along the coding
sequence). If the ORF3GC needs to be raised, then codon
substitutions are made according to Table 11 beginning at the
N-terminal end of the section. Similarly, if the ORF3GC needs to be
lowered, corresponding substitutions are made to lower the GC
content according to Table 11 and beginning at the 3' end or
C-terminal region as described in more detail above. Codons which
have a G or C in the third position are used in relative
proportions as they occur naturally (as shown in Table 11,
Proportional Extreme GC Columns/Highest GC or Lowest GC, as
appropriate). In this manner, alterations are made in this section
until the desired level of ORF3GC is reached. If the desired level
cannot be reached without changing the encoded polypeptide, then
changes may be made to bring the GC content as close as possible to
the desired level or alternatively amino acid changes can be
considered which would allow alteration of the GC content of the
nucleotide sequence but which would not significantly affect the
function of the encoded polypeptide. One of skill in the art is
familiar with the genetic code and would be able to make such
sequences and perform functional tests to determine whether
function had been so affected by the sequence change as to render
the change undesirable.
[0354] This process is then applied to the second section of 60
codons in the same manner and then to the remainder of the coding
region. Again, if the ORF3GC needs to be lowered, which will often
be the case in the remainder of the coding region, it is done so
starting from the C-terminus and moving in an N-terminal direction.
Once the sequences of these three sections have been altered as
described, the sections are combined to create a second template
ORF and the ORFGC and ORF3GC of this sequence are determined.
Because changes in this example were made to the ORF3GC rather than
the ORFGC, the ORFGC may need to be adjusted to the desired level.
If the difference between the second template ORFGC and the desired
ORFGC is less than 1 nucleotide equivalent, the sequence need not
be changed. However, if the difference is more than one nucleotide
equivalent, then the number of needed changes is determined
according to the following equation:
Percent ORFGC difference=Desired ORFGC-Template ORFGC
100*N/L=100*(G+C)d/L-100*(G+C).sub.t/L
N=(G+C)d-(G+C).sub.t
[0355] A positive number indicates the number of G or C to be
added; a negative number indicates the number of G or C to be
subtracted. Additional changes are made in the same manner as
described above for adjusting the GC content of the entire coding
region. In this manner, an altered nucleotide sequence is obtained
having the desired GC content and conforming to other known
properties of the coding regions of the desired host organism, as
particularly exemplified herein for maize. It will be apparent from
the methodologies described herein that any host organism could be
studied for GC content patterns and a corresponding pattern of
substitution designed and implemented for making suitable GC
content alterations in a sequence of interest.
[0356] Further Adjustments to Sequences
[0357] Additional changes may be made to an altered sequence to
optimize its expression and conformity to the maize gene structural
norm. For example, it may be desirable to make changes to the Kozak
context, which is thought to be involved in the optimization of
translation efficiency through proper docking of the ribosomal
complex. The Kozak context ("ATGGc") occurs around the start codon.
Thus, the second amino acid usually begins with a codon that starts
with "G", especially "GC", which corresponds to the amino acid
alanine. If, on the other hand, the codon following the ATG start
codon does not begin with a G, then changing that G generally
results in a change in the corresponding amino acid (except for
arginine). Such a change may not be desirable if it is important
that the sequence continue to encode exactly the same polypeptide
sequence, but if this first portion of the protein is a transit
peptide or is otherwise cleaved from the final mature protein, such
changes may have no effect on the final polypeptide product. Other
adjustments can also be made to the coding region, such as the
removal of potential RNA processing sites or degradation sequences,
removal of premature polyadenylation sequences, and the removal of
intron splice or donor sites. Possible intron splice-donor sites
may be identified by publicly available computer programs such as
GeneSeqer (see Usuka et al. (2000) Bioinformatics 16:203-211).
[0358] Further changes can be made to add or subtract restriction
enzyme sites or, for example, to disrupt regions of strong
palindromic tendency which might result in mRNA hairpin loop
formation. As one of skill in the art will appreciate, such changes
are made with consideration of whether the encoded amino acid is
also changed. Where possible, sequence changes that substitute
frequently used codons should be chosen over changes that
substitute less frequently used codons. Example 17: Optimization of
the Mutant Cry8-like K04 Nucleotide Sequence The original K04
mutant nucleotide sequence (set forth in SEQ ID NO:21) was modified
for optimal GC content. This modified sequence is set forth in SEQ
ID NO: 63 and encodes the original K04 mutant protein (set forth in
SEQ ID NO:22), as demonstrated by the translation of SEQ ID NO:63
set forth in SEQ ID NO:64. Additional changes were then made to
improve expression. These changes to improve expression of this
sequence included the removal of potential intron splice-donor
sites (i.e., GT--AG), the modification of potential premature
polyadenylation sites, removal of a potential RNA degradation
signal, and modification of restriction sites to facilitate cloning
without appreciably altering the codon usage of the reconditioned
sequence. These changes are shown in Table 14. The sequence
containing these additional changes is known as "1218-1K054B" and
is set forth in SEQ ID NO:65 and, as demonstrated by the
translation of SEQ ID NO:65 set forth in SEQ ID NO:66, SEQ ID NO:65
encodes the original K04 mutant protein as set forth in SEQ ID NO:
22.
15TABLE 14 Changes made to K04 sequence in addition to optimization
of GC content. Purpose Position Change Removal of potential intron
76, 78 AGG to CGC, preserving Arg splice-donor sites 1098 AGG to
AGA, preserving Arg 1500 GGT to GGC, preserving Gly 1839 GGT to
GGC, preserving Gly 1935 GGT to GGC, preserving Gly Removal of
potential polyA 1506 ACA to ACT, preserving Thr sites 1563 ACA to
ACT, preserving Thr 1926 CAT to CAC, preserving His Removal of
potential RNA 1566 ATT to ATC, preserving Ile degradation signal
(ATTTA) Modification of restriction 111 CTG to CTC, preserving Leu
and sites removing a PstI site 268 GTG to GTT, preserving Val and
removing an ApaI site 417 CTG to CTC, preserving Leu and creating
an XhoI site 567 CCA to CCT, preserving Pro and removing a HindIII
site 615 GCC to GCT, preserving Ala and removing an NcoI site 1641
GGT to GGC, preserving Gly and creating an ApaI site 1941 GAT to
GAC, preserving Asp and removing a BamHI site Change to preferred
codon 1980 AGA to AGG, preserving Arg and utilizing the preferred
AGG Arg codon
EXAMPLE 18
Bioassay for Testing the Pestcidal Activity of Mutant Cry8-Like K04
Polypeptide Against Western Corn Rootworm and Southern Corn
Rootworm
[0359] A bioassay experiment was conducted to determine the
efficacy of Cry8-like mutant K04 polypeptide against western corn
rootworm (WCRW) and southern corn rootworm (SCRW) larvae. These
bioassays were conducted essentially as set forth in Example 8
except that individual wells were infested with eggs instead of
neonates. Approximately 25 eggs were added to each bioassay well
with a total of 7 observations at each dose level. The majority of
eggs hatched within 24 hours. Percent mortality was scored after 5
days of incubation at 27.degree. C.
[0360] The summary of the mortality data shown in Table 15
indicates that the Cry8-like mutant K04 killed over half of the
WCRW larvae with moribund (dying or near death) survivors. The
results shown in Table 16 reveal that SCRW is much more susceptible
to the Cry8-like mutant K04. It was observed that 80 % of the SCRW
larvae died within 72 hours after feeding on 50 .mu.g/cm.sup.2
Cry8-like mutant K04 protein (data not shown) and by day 5, all
SCRW were dead (see Table 16).
16TABLE 15 Bioassay results of WCRW fed K04. Sample Conc. On Diet
Sample Surface (.mu.g/cm.sup.2) Mortality (%) K04 50 37/60 = 62*
Buffer 4/80 = 5 *Moribund survivors.
[0361]
17TABLE 16 Bioassay results of SCRW fed K04. Sample Conc. On Diet
Sample Surface (.mu.g/cm.sup.2) Mortality (%) K04 100 39/39 = 100
K04 50 53/53 = 100 Buffer 0/41 = 0
EXAMPLE 19
In Vivo Study of 1218-1 Protein Degradation by Western Corn
Rootworm (WCRW) Gut Proteases
[0362] An in vivo investigation of the degradation pattern of the
1218-1 truncated protein molecule produced by Western corn rootworm
gut proteases was undertaken in order to identify proteolytic sites
that may cause degradation and loss of insecticidal activity of the
1218-1 protein molecule. The truncated 1218-1 protein used for this
experiment (SEQ ID NO: 12) was generated from a pET-28a expression
vector (Novagen, San Diego, Calif.). The expressed protein was
His-Tag purified and thrombin treated according to the
manufacturer's protocol. A small T7 tag was retained with the
1218-1 protein sample. An additional 19 amino acid residues
(1868.01 Da) before the first Methionine of the 1218-1 truncated
protein were retained after thrombin treatment.
[0363] Protocol
[0364] Actively feeding, mid to late 3.sup.rd instar WCRW larvae
were starved on agar plates overnight. Starved larva were fed with
a 0.5 mg/ml 1218-1 protein solution that contained blue food
coloring and sucrose, or were fed with solution alone (a control
preparation containing sucrose and food coloring). Larvae which
imbibed a sufficient quantity of the test or control solution
(which stained the food bolus) were allowed to sit at ambient
temperatures for 1 hour. After 1 hour, larvae were placed on ice
for dissection.
[0365] Midguts were carefully removed under cold carbonate buffer
fortified with a protease inhibitor cocktail (Complete.TM. Protease
Inhibitor Cocktail fortified with 5 mM EDTA; Roche Diagnostics,
Mannheim, Germany). After the fat body and trachea were removed,
each midgut was rinsed with several drops of the same buffer.
Midguts were then retrieved from the buffer and excess buffer was
removed with a paper towel. The middle region of the midgut was
then cut with a razor blade and 5 .mu.l buffer was added to the
spilled lumenal contents. Therefore, one midgut equivalent was
equal to a 5 .mu.l aliquot of the retrieved gut/buffer
solution.
[0366] Western analysis was performed to identify the 1218-1 sample
and its degraded fragments from the gut lumenal contents.
WesternBreeze.TM. Chemiluminescent Immunodetection Kit from
Invitrogen (Carlsbad, Calif.) was used according to the
manufacturer's protocol for the analysis and visualization of
1218-1 samples.
[0367] Results
[0368] The majority of the 1218-1 protein fed to Western corn
rootworm larvae is processed into a single predominant band of less
than 62 kDa, as observed on a 10 minute exposure of the Western
blot. Numerous smaller and distinct immunoreactive bands were
observed in a 30 minute exposure of the Western blot which were
different from the immuno(cross)-reactive protein moieties present
in the control preparation. The immunoreactive bands in the control
preparation were used to discriminate the background from the true
1218-1 degraded protein fragments shown on the blot. These results
indicate that in the Western corn rootworm, the 1218-1 protein is
first processed into a protein of approximately 62 kDa, and then is
further degraded by gut proteases into small protein fragments. The
Western analysis following the in vivo digestion of the 1218-1
protein allowed for the identification of proteolytic sites and
provided for a modification of these sites in order to produce a
more efficacious insecticidal protein. 1
[0369] An in vitro investigation of the degradation pattern of the
1218-1 truncated protein molecule by proteolytic enzymes was
undertaken in order to identify proteolytic sites in the molecule
that may be available for modification. The truncated 1218-1
protein used for this experiment (SEQ ID NO: 12) was generated from
a pET-28a expression vector (Novagen, San Diego, Calif.). The
expressed protein was His-Tag purified according to the
manufacturer's protocol. Both the His-Tag and a small T7 tag were
retained with the 1218-1 protein sample.
[0370] Western analysis was performed according to the
manufacturer's protocol (Western Breeze.TM. Chemiluminescent
Immunodetection Kit; Invitrogen, Carlsbad, Calif.) in order to
identify the 1218-1 protein sample and the protein fragments
resulting from the proteolytic digestion. For each test digest, 3
.mu.g of 1218-1 protein and 0.03 .mu.g of enzyme were used. The
following enzymes were utilized for this analysis: chymotrypsin,
trypsin and papain. The digested 1218-1 samples, as well as an
undigested 1218-1 sample, were run out on a gel and blotted.
[0371] Results
[0372] Micrographs were developed and protein bands were removed
from the gel and submitted for N-terminal sequencing. The
sequencing results revealed cleavage sites generated from the
proteolytic digestion. Residue positions indicated below are
relative to the first Methionine of the 1218-1 protein sample, not
the Methionine of the His-Tag.
[0373] N-terminus sequencing of the approximately 70 kDa band in
the chymotrypsin treated sample indicated cleavage of the 1218-1
protein at the carboxyl side of Methionine at position 48. Thus
chymotrypsin removed the first 48 amino acid residues at the
N-terminus of the 1218-1 protein. N-terminus sequencing of the
approximately 57 kDa band in the trypsin treated sample indicated
cleavage of the 1218-1 sample at the carboxyl side of Arginine at
position 164. In addition, N-terminus sequencing of the
approximately 70 kDa band indicated that the 1218-1 protein sample
was cleaved by trypsin at the carboxyl side of Lysine at position
47.
[0374] At least 9 major bands were observed from the papain digest
of the 1218-1 protein sample. When these digested fragments were
isolated and sent for N-terminus sequencing, results from the
sequence analysis indicated that 7 of these major bands all
contained the same N-terminal sequence at position 49. Thus, these
results indicate that there were multiple cleavages of the 1218-1
protein molecule by papain and that these proteolytic sites occur
in the C-terminus of the molecule.
EXAMPLE 21: Mutation of Proteolytic Sites in a Modified Pentin-1
Protein Proteolytic Digestion of a Modified Pentin-1 Protein
[0375] Pentin-1 protein was modified by the removal of the putative
signal sequence and the addition at the N-terminus of the 4
following amino acids; MADV (SEQ ID NO: 124) (see U.S. Pat. No.
6,057,491 and 6,339,144, herein incorporated by reference). These 4
amino acids were added in order to enhance the production of the
modified pentin-1 protein in a host cell.
[0376] Modified pentin-1 protein (Mod P-1) was produced using the
pET30 protein expression system following the manufacturer's
protocol (Novagen, Madison, Wis.). The purified, modified pentin-1
protein, at a concentration of 1 mg/ml, was subjected to
proteolysis by trypsin, chymotrypsin and papain (digestions
occurring at 1/50 w/w). After electrophoresis and blotting of the
digested protein samples, select digestion fragments of modified
pentin-1 were cut from the trypsin, chymotrypsin, and papain lanes
on the blot and sent for N-terminal sequencing. Results from the
sequencing indicated that trypsin, chymotrypsin, and papain all
cleaved the modified pentin-1 protein at the N-terminus. Those
cleavage sites are designated by capital letters in the following
set of contiguous amino acids from the N-terminus of the modified
pentin-1 protein: madvaFstQaKaskd (SEQ ID NO: 125). More
specifically, chymotrypsin cleaved after 6-F, papain cleaved after
9-Q, and trypsin cleaved after 11-K.
[0377] Site-Directed Mutagenesis of Modified Pentin-1
[0378] Mutagenesis of the modified pentin-1 sequence to remove
proteolytic cleavage sites was initiated in an effort to increase
pentin-1 toxicity against the Western corn rootworm, WCRW. Due to
the close proximity of the three N-terminal cleavage sites
associated with trypsin, chymotrypsin, and papain, all three
N-terminal cleavage sites were mutated simultaneously. Mutations
were introduced using the GeneTailor.TM. Site-Directed Mutagenesis
System following the manufacturer's protocol (Invitrogen, Carlsbad,
Calif.). The first 30 amino acids of the modified pentin-1 protein
(Mod P-1) as well as the first 30 amino acids of the modified
pentin-1 mutant sequences named NEZ1, NEZ2, and NEZ3 are shown in
the alignment below. Those amino acids that were changed in the
mutants are shown in bold.
18 Mod P-1: MADVAFSTQAKASKDGNLVTVLAIDGGGIR (SEQ ID NO: 126) NEZ 1:
MADVAGSTGAGASKDGNLVTVLAIDGGGIR (SEQ ID NO: 127) NEZ 2:
MADVAGSTGAHASKDGNLVTVLAIDGGGIR (SEQ ID NO: 128) NEZ 3:
MADVAGSTHAHASKDGNLVTVLAIDG- GGIR (SEQ ID NO: 129)
[0379] Primers Used to Create the Mutant Sequences NEZ1, NEZ2 and
NEZ3:
[0380] The reverse primer (SEQ ID NO: 130):
19 GCCACATCAGCCATGGCCTTGTCGTCGTCG
[0381] The mutation forward primer for mutant NEZ1 (SEQ ID NO:
131):
20 GACAAGGCCatggctgatgtggcaggctccacaggtgcgggagcttctaa
agatggaaac
[0382] The mutation forward primer for mutant NEZ2 (SEQ ID NO:
132):
21 GACAAGGCCatggctgatgtggcaggctccacaggtgcgcatgcttctaaagatggaaac
[0383] The mutation forward primer for mutant NEZ3 (SEQ ID NO:
133):
22 GACAAGGCCatggctgatgtggcaggctccacacacgcgcatgcttctaaagatggaaac
[0384] The following sequence represents the 5' end of the modified
pentin-1 expression sequence as it exists in the bacterial host
cell and indicates the start of the modified pentin-1 coding
sequence (coding region in small letters):
23 CGACGACGACAAGGCCatggctgatgtggc. (SEQ ID NO: 134)
[0385] Expression and Digestion of Mutants
[0386] After the mutations were confirmed by DNA sequencing, the
mutant genes were placed into pET30 vectors and expressed, and the
corresponding mutant proteins were purified. The NEZ3 mutant
protein was subsequently subjected to proteolytic digestion using
the enzymes chymotrypsin, trypsin, and papain and utilizing the
protocol described above. This mutant protein was not digested by
any of the enzymes used.
[0387] Insect Bioassay
[0388] Modified pentin-1 protein and the modified pentin-1 mutants,
NEZ1 and NEZ3, were used in WCRW insect bioassays essentially as
described in Example 1. More specifically, 3 neonate larvae were
placed into each well (20 wells per sample), each sample contained
protein at a concentration of 1 mg/ml, the test sample volume
topically applied to each well was 501.mu.l, and larval mortality
was scored at 5 days post infestation.
[0389] The results shown below in Table 17 for a first experiment
indicate that the pentin-1 mutant named NEZ3 inhibits the growth of
WCRW larvae more than the modified pentin-1 protein (Mod P-1). The
results shown below in Table 18 for a second experiment indicate
that the modified pentin-1 mutants NEZ1 and NEZ3 inhibit the growth
of WCRW larvae more than modified pentin-1 (Mod P-1).
24TABLE 17 WCRW Bioassay of Modified Pentin-1 (Mod P-1) and its
Mutant NEZ3 Sample Mortality(%) Comment Replicate 1: NEZ3 29/59 =
49% Moderate-severe stunting Mod P-1 26/60 = 43% Moderate stunting
Replicate 2: NEZ3 34/54 = 62% Moderate-severe stunting Mod P-1
33/51 = 65% Moderate stunting
[0390]
25TABLE 18 WCRW Bioassay of Modified Pentin-1 (Mod P-1) and its
Mutants NEZ1 and NEZ3 Average Sample Concentration Larval Weight
(.mu.g) Mod P-1 1 .mu.g/.mu.l 154 Mod P-1 0.67 .mu.g/.mu.l 115 Mod
P-1 0.33 .mu.g/.mu.l 137 NEZ1 1 .mu.g/.mu.l 109 NEZ1 0.67
.mu.g/.mu.l 116 NEZ1 0.33 .mu.g/.mu.l 121 NEZ3 1 .mu.g/.mu.l 130
NEZ3 0.67 .mu.g/.mu.l 122 NEZ3 0.33 .mu.g/.mu.l 110 Buffer 19 395
Diet 18 347
[0391] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0392] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the embodiments.
Sequence CWU 0
0
* * * * *