The long-term objective of the project is to understand the mechanisms by which the toxins of the bacterium, Bacillus thuringiensis Subsp. israelensis (BTI) exert their toxic effect. The toxins from this bacterium are increasingly being used for insect control. However, in spite of this increasing use there is little information on how the toxins exert their precise mode of action. In this renewal our principal focus will be to understand the structure and function relationships of the cytolytic 25KD protein toxin of BTI. While this toxin has been sequenced, the relationship of its structure to its mode of action is not known. Such studies will not only enable us to further understand the molecular basis of toxicity of this toxin both on mammalian and insect systems, but will also enable us to further understand cell membranes, both in mammals and insects. The specific objectives of this proposal are therefore to: I. Evaluate the structure an function relationships of the 25KD BTI toxin. Within this objective we will characterize the mechanism of action of the 25KD toxin, evaluate the interaction of the toxin on cell membranes, both mammalian and insect derived, and characterize the structural features of the 25KD toxin which cause the toxin to exert its toxin effect. II. Determine the similarities/differences in the action of toxins derived from Bacillus thuringiensis subsp. morrisoni (BTM) with those derived from BTI. III. Identify and characterize the nematocidal factor in BTI. Characterization of the structure-function relationships will be achieved by a variety of approaches including electrophsiological and biochemical techniques using purified toxins, antibodies, cell culture and liposomes. Purification of toxins from both BTI and BTM will be achieved by gel-filtration and ion-exchange chromatography, and toxin activity assay against insect and mammalian cels. The nematocidal factor from BTI will be purified by high-pressure liquid chromatography. Bioassays for this factor will be performed with nematode eggs.
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