A NEW METHOD FOR MANIPULATING TRANSGENES - ENGINEERING HEAT TOLERANCEIN A COMPLEX, MULTICELLULAR ORGANISM

Background Heat-shock proteins (hsps) are thought to protect cells aga inst stresses, especially due to elevated temperatures. But while gene tic manipulation of hsp gene expression can protect microorganisms and cultured metazoan cells against lethal stress, this has so far not be en demonstrated in multicellular organisms. Testing whether expression of an hsp transgene contributes to increased stress tolerance is comp licated by a general problem of transgene analysis: if the transgene c annot be targeted to a precise site in the genome, newly observed phen otypes may be due to either the action of the transgene or mutations c aused by the transgene insertion. Results: To study the relationship b etween heat tolerance and hsp expression in Drosophila melanogaster, w e have developed a novel method for transgene analysis, based upon the site-specific FLP recombinase. The method employs site-specific siste r chromatid exchange to create an allelic series of transgene insertio ns that share the same integration site, but differ in transgene copy number. Phenotypic differences between members of this series can be c onfidently attributed to the transgenes. Using such an allelic series and a novel thermotolerance assay for Drosophila embryos, we investiga ted the role of the 70kD heat-shock protein, Hsp70, in thermotolerance . At early embryonic stages, Hsp70 accumulation was rate-limiting for thermotolerance, and elevated Hsp70 expression increased survival at e xtreme temperatures. Conclusion: Our results provide an improved metho d for analyzing transgenes and demonstrate that, in Drosophila, Hsp70 is a critical thermotolerance factor. They shaw, moreover, that manipu lating the expression of a single hsp can be sufficient to improve the stress tolerance of a complex multicellular organism.