Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-05-11
2003-04-01
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S350000, C424S246100
Reexamination Certificate
active
06541448
ABSTRACT:
1.0 BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates generally to the fields of molecular biology. Methods and compositions comprising DNA sequences, and polypeptides derived from
Bacillus thuringiensis
for use in insecticidal formulations and the development of transgenic insect-resistant plants are provided. Novel nucleic acids obtained from
Bacillus thuringiensis
that encode coleopteran-toxic polypeptides are disclosed. Various methods for making and using these nucleic acids, synthetically modified DNA sequences encoding tIC851 polypeptides, and native and synthetic polypeptide compositions are also disclosed. The use of DNA sequences as diagnostic probes and templates for protein synthesis, and the use of polypeptides, fusion proteins, antibodies, and peptide fragments in various insecticidal, immunological, and diagnostic applications are also disclosed, as are methods of making and using nucleic acid sequences in the development of transgenic plant cells comprising the polynucleotides.
1.2 Description of the Related Art
Environmentally-sensitive methods for controlling or eradicating insect infestation are desirable in many instances, in particular when crops of commercial interest are at issue. The most widely used environmentally-sensitive insecticidal formulations developed in recent years have been composed of microbial pesticides derived from the bacterium
Bacillus thuringiensis. B. thuringiensis
is well known in the art, and is characterized morphologically as a Gram-positive bacterium that produces crystal proteins or inclusion bodies which are aggregations of proteins specifically toxic to certain orders and species of insects. Many different strains of
B. thuringiensis
have been shown to produce insecticidal crystal proteins. Compositions including
B. thuringiensis
strains which produce insecticidal proteins have been commercially-available and used as environmentally-acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.
There are several toxin categories established based on primary structure information and the degree of toxin similarities to another. Over the past decade research on the structure and function of
B. thuringiensis
toxins has covered all of the major toxin categories, and while these toxins differ in specific structure and function, general similarities in the structure and function are assumed. Based on the accumulated knowledge of
B. thuringiensis
toxins, a generalized mode of action for
B. thuringiensis
toxins has been created and includes: ingestion by the insect, solubilization in the insect midgut (a combination stomach and small intestine), resistance to digestive enzymes sometimes with partial digestion actually “activating” the toxin, binding to the midgut cells, formation of a pore in the insect cells and the disruption of cellular homeostasis (English and Slatin, 1992).
Many of the &dgr;-endotoxins are related to various degrees by similarities in their amino acid sequences. Historically, the proteins and the genes which encode them were classified based largely upon their spectrum of insecticidal activity. The review by Schnepf et al. (Microbiol. Mol. Biol. Rev. (1998) 62:775-806) discusses the genes and proteins that were identified in
B. thuringiensis
prior to 1998, and sets forth the most recent nomenclature and classification scheme as applied to
B. thuringiensis
insecticidal genes and proteins. Using older nomenclature classification schemes, cry1 genes were deemed to encode lepidopteran-toxic Cry1 proteins, cry2 genes were deemed to encode Cry2 proteins toxic to both lepidopterans and dipterans, cry3 genes were deemed to encode coleopteran-toxic Cry3 proteins, and cry4 genes were deemed to encode dipteran-toxic Cry4 proteins. However, new nomenclature systematically classifies the Cry proteins based upon amino acid sequence homology rather than upon insect target specificities. The classification scheme for many known toxins, not including allelic variations in individual proteins, including dendograms and full
Bacillus thuringiensis
toxin lists is summarized and regularly updated by the
B. thuringiensis
Pesticidal Crystal Protein Nomenclature Committee which will periodically publish a comprshensive list of B.t. toxins which will also be available through the internet as described in Crickmore et al., Microbiol. Mol. Biol. Rev. 62:807-813 (1989).
Most of the nearly 200 Bt crystal protein toxins presently known have some degree of lepidopteran activity associated with them. The large majority of
Bacillus thuringiensis
insecticidal proteins which have been identified do not have coleopteran controlling activity. Therefore, it is particularly important at least for commercial purposes to identify additional coleopteran specific insecticidal proteins.
Cry3 proteins generally display coleopteran activity, however, these generally have limited host range specificity and are not significantly toxic to target pests unless ingested in very high doses. The cloning and expression of the cry3Bb gene has been described (Donovan et al., 1992). This gene codes for a protein of 74 kDa with activity against Coleopteran insects, particularly the Colorado potato beetle (CPB) and the southern corn root worm (SCRW). Improved Cry3Bb proteins have been engineered which display increased toxicity at the same or lower doses than the wild type protein (U.S. Pat. No. 6,023,013; Feb. 8, 2000).
A
B. thuringiensis
strain, PS201T6, was reported to have activity against WCRW (
Diabrotica virgifera virgifera
) (U.S. Pat. No. 5,436,002). This strain also had activity against
Musca domestica, Aedes aegypti,
and
Liriomyza trifoli
. The vip1A gene, which produces a vegetative, soluble, insecticidal protein, has been cloned and sequenced (Intl. Pat. Appl. Pub. No. WO 96/10083, 1996). This gene produces a protein of approximately 80 kDa with activity against both WCRW and Northern Corn Root Worm (NCRW). Another toxin protein with activity against coleopteran insects, including WCRW, is Cry1Ia, an 81-kDa polypeptide, the gene encoding which has been cloned and sequenced (Intl. Pat. Appl. Pub. No. WO 90/13651, 1990).
2.0 SUMMARY OF THE INVENTION
The polypeptide of the present invention and the novel DNA sequences that encode the protein represent a new
B. thuringiensis
crystal protein and gene, and share only insubstantial sequence homology with any previously identified coleopteran inhibitory endotoxins described in the prior art. Similarly, the
B. thuringiensis
strains of the present invention comprise novel gene sequences that express a polypeptide having insecticidal activity against coleopteran insects, the cotton boll weevil (
Anthonomus grandis
Boheman) in particular.
Disclosed and claimed herein is an isolated
Bacillus thuringiensis
&dgr;-endotoxin polypeptide comprising SEQ ID NO:8. The inventors have identified an insecticidally-active polypeptide comprising the 632 amino acid long sequence of SEQ ID NO:8 which displays insecticidal activity against coleopteran insects. For example, the inventors have shown that a &dgr;-endotoxin polypeptide comprising the sequence of SEQ ID NO:8 has insecticidal activity against boll weevil larvae (BWV), but not against western corn rootworm larvae.
The polypeptide of SEQ ID NO:8 is encoded by a nucleic acid segment comprising at least the open reading frame as shown in SEQ ID NO:7 from nucleotide position 28 through nucleotide position 1923. The invention also discloses compositions and insecticidal formulations that comprise such a polypeptide. Such composition may be a cell extract, cell suspension, cell homogenate, cell lysate, cell supernatant, cell filtrate, or cell pellet of a bacteria cell that comprises a polynucleotide that encodes such a polypeptide. Exemplary bacterial cells that produce such a polypeptide include
Bacillus thuringiensis
EG4135 and EG4268, deposited with NRRL respectively on Apr. 28, 2000. The composition as described in detail b
Isaac Barbara
Krieger Elysia K.
Mettus Anne-Marie Light
Moshiri Farhad
Sivasupramanian Sakuntala
Ball Timothy K.
Carlson Karen Cochrane
Hoerner, Jr. Dennis R.
Monsanto Technology LLC
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