Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-10-23
2001-04-17
Nelson, Amy J. (Department: 1638)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S252300, C435S320100, C435S449000, C435S468000, C536S023710
Reexamination Certificate
active
06218188
ABSTRACT:
BACKGROUND OF THE INVENTION
Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by insect pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.
Chemical pesticides have provided an effective method of pest control; however, the public has become concerned about the amount of residual chemicals which might be found in food, ground water, and the environment. Therefore, synthetic chemical pesticides are being increasingly scrutinized, and correctly so, for their potential toxic environmental consequences. Synthetic chemical pesticides can poison the soil and underlying aquifers, pollute surface waters as a result of runoff, and destroy non-target life forms. Synthetic chemical control agents have the further disadvantage of presenting public safety hazards when they are applied in areas where pets, farm animals, or children may come into contact with them. They may also provide health hazards to applicants, especially if the proper application techniques are not followed. Regulatory agencies around the world are restricting and/or banning the uses of many pesticides and particularly the synthetic chemical pesticides which are persistent in the environment and enter the food chain. Examples of widely used synthetic chemical pesticides include the organochlorines, e.g., DDT, mirex, kepone, lindane, aldrin, chlordane, aldicarb, and dieldrin; the organophosphates, e.g., chlorpyrifos, parathion, malathion, and diazinon; and carbamates. Stringent new restrictions on the use of pesticides and the elimination of some effective pesticides from the market place could limit economical and effective options for controlling costly pests.
Because of the problems associated with the use of synthetic chemical pesticides, there exists a clear need to limit the use of these agents and a need to identify alternative control agents. The replacement of synthetic chemical pesticides, or combination of these agents with biological pesticides, could reduce the levels of toxic chemicals in the environment.
A biological pesticidal agent that is enjoying increasing popularity is the soil microbe
Bacillus thuringiensis
(B.t.) The soil microbe
Bacillus thuringiensis
(B.t.) is a Gram-positive, spore-forming bacterium. Most strains of
B.t
. do not exhibit pesticidal activity. Some
B.t
. strains produce, and can be characterized by, parasporal crystalline protein inclusions. These “&dgr;-endotoxins,” which typically have specific pesticidal activity, are different from exotoxins, which have a non-specific host range. These inclusions often appear microscopicallyas distinctively shaped crystals. The proteins can be highly toxic to pests and are specific in their toxic activity.
Preparations of the spores and crystals of
B. thuringiensis
subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example,
B. thuringiensis
var. kurstaki HD-1 produces a crystalline &dgr;-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
The cloning and expression of a
B.t
. crystal protein gene in
Escherichia coli
was described in the published literature more than 15 years ago (Schnepf, H. E., H. R. Whiteley [1981
] Proc. Natl. Acad. Sci. USA
78:2893-2897.). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expression of
B.t
. crystal protein in
E. coli
. Recombinant DNA-based
B.t
. products have been produced and approved for use.
Commercial use of
B.t
. pesticides was originally restricted to a narrow range of lepidopteran(caterpillar)pests. More recently, however, investigators have discovered
B.t
. pesticides with specificities for a much broader range of pests. For example, other species of
B.t
., namely israelensis and morrisoni (a.k.a. tenebrionis, a.k.a.
B.t
. M-7), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F. H. [1989] “Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms,” in
Controlled Delivery of Crop Protection Agents
, R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255).
New subspecies of
B.t
. have now been identified, and genes responsible for active &dgr;-endotoxin proteins have been isolated and sequenced (Höfte, H., H. R. Whiteley [1989
] Microbiological Reviews
52(2):242-255). Höfte and Whiteley classified
B.t
. crystal protein genes into four major classes. The classes were cryI (Lepidoptera-specific), cryII (Lepidoptera- and Diptera-specific), cryIII (Coleoptera-specific), and cryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. S., J. Payne, L. Kim [1992
] Bio/Technology
10:271-275). For example, the designations CryV and CryVI have been proposed for two new groups of nematode-active toxins.
Many
Bacillus thuringiensis
&dgr;-endotoxin crystal protein molecules are composed of two functional segments. For these proteins, the protease-resistant core toxin is the first segment and corresponds to about the first half of the protein molecule. The three-dimensional structure of a core segment of a CryIIIA
B.t
. &dgr;-endotoxin is known, and it was proposed that all related toxins have that same overall structure (Li, J., J. Carroll, D. J. Ellar [1991
] Nature
353:815-821). The second half of the molecule is often referred to as the “protoxin segment.” The protoxin segment is believed to participate in toxin crystal formation (Arvidson,H., P. E. Dunn, S. Strand, A. I. Aronson [1989
] Molecular Microbiology
3:1533-1534; Choma, C. T., W. K. Surewicz, P. R. Carey, M. Pozsgay, T. Raynor, H. Kaplan [1990
] Eur. J Biochem
. 189:523-527). The fill 130 kDa toxin molecule is typically processed to the resistant core segment by proteases in the insect gut. The protoxin segment may thus convey a partial insect specificity for the toxin by limiting the accessibility of the core to the insect by reducing the protease processing of the toxin molecule (Haider, M. Z., B. H. Knowles, D. J. Ellar [1986
] Eur. J Biochem
. 156:531-540) or by reducing toxin solubility (Aronson, A. I., E. S. Han, W. McGaughey, D. Johnson [1991
] Appl. Environ. Microbiol
. 57:981-986).
The 1989 nomenclature and classification scheme of Höfte and Whiteley was based on both the deduced amino acid sequence and the host range of the toxin. That system was adapted to cover 14 different types of toxin genes which were divided into five major classes. The number of sequenced
Bacillus thuringiensis
crystal protein genes currently stands at more than 50. A revised nomenclature scheme has been proposed which is based solely on amino acid identity (Crickmoreet et al. [1996] Society for Invertebrate Pathology, 29th Annual Meeting, IlIrd International Colloquium on
Bacillus thuringiensis
, University of Cordoba, Cordoba, Spain, Sep. 1-6, 1996, abstract). The mnemonic “cry” has been retained for all of the toxin genes except cytA and cytB, which remain a separate class. Roman numerals have been exchanged for Arabic numerals in the primary rank, and the parentheses in the tertiary rank have been removed. Many of the original names have been retained, although a number have been reclassified.
With the use of genetic engineering techniques, new approaches for delivering
B.t
. toxins to agricultural environments are under development, including the use of plants genetically engineered with
B.t
. toxin genes for insect resistance and the use of stabilized, microbial cells as delivery vehicles of
B.t
. toxins (Gaertner, F. H., L. Kim [1988
] TIBTECH
6:S4-S7). Thus, isolated
B.t
. endotoxin genes are becoming commercially valuable.
Various improvements have been achieved by modifying
B.t
. toxins and/or their genes. For example, U.S. Pat. Nos. 5,380,831 and 5,567,862 r
Cardineau Guy A.
Narva Kenneth E.
Stelman Steven J.
Mycogen Corporation
Nelson Amy J.
Saliwanchik Lloyd & Saliwanchik
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