Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
2000-07-12
2004-01-06
Paras, Peter (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C435S320100, C435S348000, C435S235100, C536S023500
Reexamination Certificate
active
06673340
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of pest management for agriculture. More particularly the invention relates to the use of certain proteases as pesticidal toxins, particularly for transgenic insecticidal protocols. The invention further comprises insecticidal compositions as well as transgenic techniques including novel expression constructs, vectors, methods for the infection of insects and delivery of toxins, and ultimately for the development of insect resistant transgenic plants.
BACKGROUND OF THE INVENTION
One of the goals of agricultural research is to increase profitability of agriculture while decreasing its environmental impact. Integrated pest management programs need a diversity of control strategies and agents to maximize profits and minimize environmental damage. This need comes at a time when there has been a decrease in the diversity of classical pesticides. Although most experts agree that artificial pest control is needed to maintain our current level of agricultural productivity, over reliance on non selective pesticides has led to resistance, destruction of natural enemies, pest resurgence, and decreased profitability as well as environmental damage.
Synthetic chemical insecticides are effective for controlling pest insects in a wide variety of agricultural, urban, and public health situations. Unfortunately there are significant, often severe, side effects associated with the use of these products. Many pest populations have developed significant resistance to virtually all chemical insecticides, requiring higher and higher rates of usage for continued control. In a number of severe cases, highly resistant pest populations have developed which cannot be controlled by any available product. Chemical insecticides may also have deleterious effects on non-target organisms. Populations of beneficial arthropods, such as predators and parasites, are sometimes more severely affected by chemical applications than the pests themselves. Minor pests, ordinarily held in check by these beneficial organisms, may become serious pests when their natural constraints are removed by the use of chemical insecticides. Thus, new pest problems may be created by attempts to solve established problems.
Chemical insecticides may also have adverse effects on vertebrates. The use of DDT has been banned in the United States, due primarily to the insecticide's great environmental persistence and its resulting tendency to accumulate in the tissues of predatory birds, thereby disrupting their ability to produce viable eggs. The use of carbofuran has been severely restricted due to its avian toxicity, and many species of fish are known to be quite sensitive to a variety of insecticides. A number of insecticides, such as methyl parathion, are also quite toxic to humans and other mammals, and by accident or misuse have caused a number of human poisonings. Clearly, the field of insect control would benefit greatly from the discovery of insecticides with improved selectivity for insects and reduced effects on non-target organisms.
The problems described above, along with other concerns including the possibility that some insecticides may act as human carcinogens, have created a strong demand for the development of safer methods of insect control.
Insect pathogens have been the objects of much study as potential pest control agents. Generally, these pathogens are quite selective for insects and in many cases affect only a few closely related species of insects. A number of insect pathogens have been developed as products, including bacteria (e.g.,
Bacillus thuringiensis
and
Bacillus popiliae
), viruses (e.g., nucleopolyhedroviruses) and protozoa (e.g., the microsporidian
Nosema locustae
). These products occupy only a small fraction of the insecticide market. Although pathogens may ultimately cause a high level of mortality in pest populations, the insects may take weeks to die and continue to feed for much of that time. Thus, an unacceptably high level of crop or commodity damage may be inflicted before control is achieved. Currently, researchers are actively seeking ways to improve the effectiveness of insect pathogens and other biological control tools.
Insecticidal toxins from arthropods have been the objects of increasing interest over the past decade. These materials have proved useful for the detailed study of neural and neuromuscular physiology in insects. They have also been used to enhance the effectiveness of certain insect pathogens. The insecticidal toxin AaIT, from the scorpion
Androctonus australis
, has been employed for both purposes. This toxin belongs to a group of peptides that are lethal to a variety of insects but have no detectable effect in humans. Other toxins in
A. australis
venom are lethal to mammals but have no effect on insects. Understanding the molecular basis of this selectivity may lead to the development of chemical insecticides with reduced effects on mammals and other non-target organisms.
A number of transgenic protocols have been employed to help reduce the environmental impact of non-selective pesticides. A summary of current protocols follows.
One method involves transformation of plants with plant disease resistance genes. Plant defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence (Avr) gene in the pathogen. A plant variety can be transformed with a cloned resistance gene to engineer plants that are resistant to specific pathogen strains. See, for example Jones et al., Science 266: 789 (1994) (cloning of the tomato Cf-9 gene for resistance to
Cladosporium fulvum
); Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistance to
Pseudomonas syringae
pv. tomato encodes a protein kinase); Mindrinos et al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance to
Pseudomonas syringae
).
The most extensively used heterologous gene for insect resistance involves the
Bt
endotoxin. A
Bacillus thuringiensis
protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser et al.,
Gene
48: 109 (1986), who disclose the cloning and nucleotide sequence of a
Bt
Delta-endotoxin gene. Moreover, DNA molecules encoding Detla-endotoxin genes can be purchased from American Type Culture Collection (Rockville, Md.), for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.
Other examples include use of a lectin. See, for example, the disclosure by Van Damme et al.,
Plant Molec. Biol
. 24: 25 (1994), who disclose the nucleotide sequences of several
Clivia miniata
mannose-binding lectin genes, use of a vitamin-binding protein, such as avidin. See PCT application US93/06487 the contents of which are hereby incorporated by reference. (The application teaches the use of avidin and avidin homologues as larvicides against insect pests); and use of an enzyme inhibitor, for example, a protease inhibitor or an amylase inhibitor. See, for example, Abe et al.,
J. Biol. Chem
. 262: 16793 (1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huub et al.,
Plant Molec. Biol
. 21: 985 (1993) (nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I), and Sumitani et al.,
Biosci. Biotech. Biochem
. 57: 1243 (1993) (nucleotide sequence of
Streptomyces nitrosporeus
alpha-amylase inhibitor).
Still other recombinant strategies include use of insect-specific hormones or pheromone such as an ecdysteroid and juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof. See, for example, the disclosure by Hammock et al., Nature 344: 458 (1990), of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone.
Further techniques include an insect-specific peptide or neuropeptide which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of Regan,
J. Biol. Chem
. 269: 9 (1994) (expression cloning yields DNA coding for insect diu
Bonning Bryony C.
Harrison Robert L.
Greenlee Winner and Sullivan P.C.
Iowa State University & Research Foundation, Inc.
Paras Peter
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