Multiple-stress analysis for isolation of Drosophila longevity genes

Abstract

Long-lived organisms tend to be more resistant to various forms of environmental stress. An example is the Drosophila longevity mutant, methuselah, which has enhanced resistance to heat, oxidants, and starvation. To identify genes regulated by these three stresses, we made a cDNA library for each by subtraction of "unstressed" from "stressed" cDNA and used DNA hybridization to identify genes that are regulated by all three. This screen indeed identified 13 genes, some already known to be involved in longevity, plus candidate genes. Two of these, hsp26 and hsp27, were chosen to test for their effects on lifespan by generating transgenic lines and by using the upstream activating sequence/GAL4 system. Overexpression of either hsp26 or hsp27 extended the mean lifespan by 30%, and the flies also displayed increased stress resistance. The results demonstrate that multiple-stress screening can be used to identify new longevity genes.

Fig. 1.

The screen for multiple-stress-responsive genes. (A) The strategy. Three subtracted cDNA libraries were prepared, one for each stress. The paraquat stress-subtracted cDNA library was used to make duplicate microarray membranes. Each microarray membrane was probed separately with one of the other two stress-subtracted cDNA libraries. Triple-stress up-regulated genes were identified by comparison of the two membranes. (B) Triple-stress-responsive candidate genes were identified by DNA sequencing and blast search. (C) Confirmation of up-regulation of three heat-shock genes by Northern blot. Ten micrograms of total RNA from either control or stressed flies was run on the gel and the blots hybridized with ³²P-labeled clones as indicated. 16S rRNA was used as an internal loading control.

Fig. 2.

Generation of hsp26 and hsp27 transgenic flies. (A) The transgenic constructs. Full-length hsp26 and hsp27 cDNA were subcloned into expression vector pINDY6, driven by the yeast USA enhancer linked with an hsp70 minimal promoter, to generate pINDY6-hsp26 and pINDY6-hsp27. Each construct was used to make transgenic flies, as described in Materials and Methods. Two different insertion lines, one on the second chromosome and the other on the third chromosome, were obtained for each transgene. These are named UAS-hsp26.II, UAS-hsp26.III, UAS-hsp27.II, and UAS-hsp27.III. The primer set HDW54 and HDW55 detects the hsp26 transcripts, generating a 600-bp DNA fragment. The primer set HDW54 and HDW57 amplifies the hsp27 transcript, generating a 500-bp DNA fragment. (B) RT-PCR to detect expression of hsp26 and hsp27 in the transgenic flies. Five micrograms of total RNA was used in the RT-PCR reaction, as described in Materials and Methods. Equal amounts (10 μl) of each PCR product were applied to 1.5% agarose gel for electrophoresis.

Fig. 3.

Lifespan extension by overexpression of hsp26 and hsp27 in transgenic flies. Each experiment was repeated four times with ≈75 flies per trial. (A) UAS-hsp26.II. (B) UAS-hsp26.III. (C) UAS-hsp27.II. (D) UAS-hsp27.III. Each of the four transgenic strains was tested with and without the hs-GAL4 driver, as compared with the driver alone.

Fig. 4.

Increased stress resistance in transgenic flies overexpressing hsp26 or hsp27. Three- to 5-day-old adult male transgenic flies overexpressing hsp26 or hsp27, driven by hs-GAL4, as compared with controls without the driver, were tested with the three separate stresses, as described in Materials and Methods. The measure of stress resistance is mean survival time under paraquat (A), heat (B), or starvation (C).