The structure of the DNA-protein complex in the insecticidal crystals produced by Bacillus thuringiensis var. kurstaki HD-73 was investigated. The role of DNA in the activation mechanism of the crystals was determined by monitoring the fate of the DNA and protein during the activation process with gut juice enzymes from spruce budworm, Choristeneura fumiferana. The results demonstrate that the DNA is co-processed with the protein, giving rise to novel DNA-protein complexes as intermediates. The role of the DNA appears to be mainly structural, providing a framework for the sequestering of the protein into the crystal. Based on the results obtained, a structural model for the arrangement of the DNA-protein complex is proposed. An activation model is also proposed, which accounts for the processing of the DNA and protein components of the crystals. The model offers explanations for the unusual mechanism of proteolytic activation of the Bt crystal protein. The sunlight inactivation (photo-inactivation) of Bt crystals was studied by UV-irradiation studies. The results of these studies suggest that the photo-inactivation is due to the formation of covalent linkages between the DNA and crystal protein. UV irradiation at 302 nm for 8 hours was sufficient to cause the total loss of biological activity of the crystals. Incorporation of water-soluble antioxidants into crystal preparations inhibits the photo-inactivation reaction. Ascorbic acid was found to be an effective antioxidant and inhibited the photo-inactivation of Bt crystals. In the presence of ascorbic acid, the toxicity of the irradiated crystals did not decrease significantly under the same conditions and the retention of toxicity is estimated to be approximately one order of magnitude longer than unprotected crystals. The inactivation of crystals was inducible in the presence of a free-radical generator, suggesting that a free-radical mechanism is responsible for the photo-inactivation of the crystals. A highly sensitive method has been developed for the determination of the C-terminal sequence of proteins. The C-terminal sequence of chymotrypsin was determined using this methodology. C-terminal peptides from ribonuclease and from the crystal protein from Bacillus thuringiensis var. kurstaki HD-73 were also isolated using this procedure.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/8513 |
| Date | January 1999 |
| Creators | Clairmont, François. |
| Contributors | Kaplan, Harvey, |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 203 p. |
Page generated in 0.0131 seconds