The ultimate goal of this research was to perform a domain exchange between a computer identified insect specificity region of the mosquito larvicidal protein Cry IVB and a previously identified domain in a related protein toxin which targets lepidopteran insect larvae. If the insect specificity domain has been correctly identified, an exchange of DNA in this manner transfers the toxicity of one peptide to another by an exchange of the insect specificity domains. New, chimeric peptides may be designed which will target a larger spectrum of insect larvae.In previous research a domain exchange was performed between the two genes carried on plasmid vectors in E. coli and low levels of toxicity to mosquito larvae were observed. Initial efforts of this research attempted to identify these recombinants. However, stability was not achieved by sequential colony screens. Furthermore, a recently published three-dimensional structural model for all the B. thuringiensis crystalline toxins became available and it was quickly determined that the first exchanges excluded most of the f3-sheet domain that is responsible for insect cell receptor binding, the feature that gives the toxins their specificity. Therefore, it was decided that a larger, more inclusive region of Cry IVB DNA must be exchanged between the two toxins.Extensive computer analyses of the Cry IVB sequence and retroactive comparison of these sequences to the three-dimensional model yielded a fragment of DNA that encoded more than 60% of the putative insect specificity domain. Oligonucleotide primers were subsequently designed to flank this region so that the polymerase chain reaction could be employed to amplify the region. Additionally, the primers were engineered to contain terminal restriction endonuclease sites to ease in the exchange of the domain encoding region into Cry IA(c). The region of Cry IVB DNA flanked by the oligonucleotide primers was successfully amplified by the PCR and cloned into the plasmid vector pUC 19 as a reservoir for a future domain exchange. / Department of Biology
| Identifer | oai:union.ndltd.org:BSU/oai:cardinalscholar.bsu.edu:handle/185015 |
| Date | January 1994 |
| Creators | Schmeisser, Glen A. |
| Contributors | Ball State University. Dept. of Biology., Vann, Carolyn N. |
| Source Sets | Ball State University |
| Detected Language | English |
| Format | ii, 87 leaves : ill. ; 28 cm. |
| Source | Virtual Press |
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