Expression of hsp22 and hsp70 transgenes is partially predictive of drosophila survival under normal and stress conditions

Abstract

Drosophila Hsp70 is a highly conserved molecular chaperone with numerous cytoplasmic targets. Hsp22 is an alpha-crystallin-related chaperone (small hsp) that localizes to the mitochondrial matrix. The hsp70 and hsp22 genes are induced in response to acute heat and oxidative stress and are also upregulated during normal aging. Here the hsp22 promoter (-314 to +10) and the hsp70 promoter (-194 to +10) were used to drive expression of the fluorescent reporter proteins green fluorescent protein (GFP) and Discosoma sp. red fluorescent protein (DsRED) in transgenic flies. Multiple transgenic lines were analyzed under normal culture conditions and under oxidative stress and heat stress conditions that significantly shorten life span. Flies were individually housed, and GFP (or DsRED) was quantified at young-age time points using the fluorescence stereomicroscope and image analysis software. Expression of the hsp reporters in young flies was partially predictive of remaining life span: Young flies with high expression tended to die sooner under both control and stress conditions.

Figure 1.

Transgenic hsp22 and hsp70 reporters. (A), Construct maps. The vector region between the P element inverted repeats (P) is diagrammed at the top, and the hsp gene sequences are diagrammed below. Solid boxes (I) indicate the insulator elements. (B) Image capture assay. Representative images are presented for male flies of the hsp22-GFP and hsp70-GFP transgenic strains under normal aging conditions (young = 4-d old; old = 70-d old) and heat stress (3 d at 34°C) and oxidative stress conditions (4 d in oxygen stress), as indicated. For each fly, the GFP fluorescence image is presented to the left and an overlay of the GFP and visible images is presented to the right. The young fly pictures are from line hsp22-GFP(3)1/TM3 Sb e and hsp70-GFP(3)2/TM3 Sb e. All other pictures are from lines hsp22-GFP(3)1 MI1/TM3 Sb e and hsp70-GFP(3)2 MI4/TM3 Sb e.

Figure 2.

Survival curves for transgenic fly cohorts. (A) Survival curves for flies under normal aging conditions. Time points for fluorescence assay were 10 d (time point 1), 20 d (time point 2), and 30 d (time point 3), respectively. (B) Survival curves for flies under oxygen stress conditions. Time points for fluorescence assay were 2 d (time point 1), 4 d (time point 2), and 6 d (time point 3), respectively. (C) Survival curves for flies under heat stress conditions. Time points for fluorescence assay were 2 d (time point 1), 4 d (time point 2), and 6 d (time point 3), respectively. Median life spans for each cohort are summarized in Table 1.

Figure 2.

Survival curves for transgenic fly cohorts. (A) Survival curves for flies under normal aging conditions. Time points for fluorescence assay were 10 d (time point 1), 20 d (time point 2), and 30 d (time point 3), respectively. (B) Survival curves for flies under oxygen stress conditions. Time points for fluorescence assay were 2 d (time point 1), 4 d (time point 2), and 6 d (time point 3), respectively. (C) Survival curves for flies under heat stress conditions. Time points for fluorescence assay were 2 d (time point 1), 4 d (time point 2), and 6 d (time point 3), respectively. Median life spans for each cohort are summarized in Table 1.

Figure 3.

GFP levels of flies with different life spans under normal aging conditions. Image capture was performed in triplicate for each fly at time point 1 (TP1 = Day 10), time point 2 (TP2 = Day 20), and time point 3 (TP3 = Day 30). (A) Scatter plot, line hsp22-GFP (3)1/TM3 Sb e, data for time point 3. (B–D) The data for the flies were divided into three equal groups based on their life spans. (B) hsp22-GFP(3)1MI1/TM3 Sb e. (C) hsp70-GFP(3)2MI4/TM3 Sb e. (D) PEPCK-GFP.

Figure 4.

GFP level of flies with different life span ranges under oxygen stress. Image capture was performed in triplicate for each fly at time point 1 (TP1 = Day 2), time point 2 (TP2 = Day 4), and time point 3 (TP3 = Day 6). (A) Scatter plot, line hsp22-GFP (3)1/TM3 Sb e, data for time point 1. (B–D) The data for the flies were divided into three equal groups based on their life spans. (B) hsp22-GFP(3)/TM3 Sb e. (C) hsp70-GFP(3)1MI2/TM3 Sb e. (D) PEPCK-GFP.