Worm Breeder's Gazette 15(1): 37 (October 1, 1997)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
IBG, University of Colorado, Boulder, CO, 80309
Numerous lines of evidence suggest that for beta amyloid peptide (and other amyloidic proteins), the ability to aggregate into insoluble amyloid fibrils is closely linked to pathogenic potential. Transgenic animals containing unc-54/signal peptide/beta 1-42 constructs show putative amyloid deposits, as demonstrated by binding of the specific dye thioflavin S. We have recently succeeded in visualizing green birefringent deposits using Congo Red (the classic characteristic of amyloid deposits) in these animals, further supporting the proposal that these animals are an appropriate model for studying amyloid formation. We have now used this system to investigate beta peptide sequence requirements for amyloid formation by generating transgenic worms expressing 6 different beta peptide variants designed to test models of beta peptide tertiary structure. In summary, we have identified two single amino acid substitutions, L17P and M35C, that completely abrogate the formation of thioflavin S-reactive deposits. Conversely, conservative changes at these positions (L17V and M35L) have no apparent effect on formation of thioflavin S deposits. A proline substitution for A30 significantly reduces, but does not eliminate, thioflavin S deposits. Animals expressing a single chain head-to-tail beta dimer also fail to make thioflavin S deposits. These results support current models of beta peptide folding, but contradict some in vitro studies performed on small sub-peptides derived from the beta peptide sequence. Although the results described above have been consistently observed in multiple independent extrachromosomal and integrated lines, and all lines examined show similar levels of beta peptide by immunohistochemistry, we are developing a quantitative Western blot assay to rigorously compare these lines. Initial experiments with 25 worm samples of an unc-54/signal peptide/ beta 1-42 (non-variant) line indicate that these animals produce an appropriately sized product (confirming signal peptide processing), and that these animals contain as much as 1 ng beta peptide per adult animal.