To confirm this, we have examined other conditions known to provoke apoptosis in yeast, i.e. acetic acid, ageing and hyperosmotic shock ( Figure 1E -H). For treatment with acetic acid, cells were grown in SC complete medium (pH 3) to exponential phase and exposed to 175 mM acetic acid for up to 200 min (29,30). Hybridizations with probes against 25S (probe 007, position +40, lanes 1-6; probe W234, position +344, lanes 7-12; probe W236, position +600, lanes 13-18; probe W238, position +843, lanes 19-24; probe W239, position +2168, lanes 25-30 and probe W240, position +3323, lanes 31-36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3 0 product of the major cleavage, 5 0 product is marked with one asterisk. (B-C) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5 0 end of the mature 25S. DNA sequencing on a PCR product encompassing the 5 0 end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1-4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/. (D-E) 3 0 ends of cleaved-off products for the major cleavage at positions +610-611 were mapped by 3 0 RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated Hyperosmotic shock was achieved by growth of exponential cells in SC complete media supplemented with 60% (wt/wt) glucose or 30% (wt/wt) sorbitol (31) . And finally, chronological ageing was performed by constant growth of yeast cultures in SC complete medium for 2-16 days (32) . This analysis revealed that all apoptotic stimuli tested resulted in the 25S and 5.8 rRNA fragmentation with the degradation pattern specific for each condition (shown in Figure 1E -G for 25S in all apoptotic conditions and in Figure 1H for 5.8S during ageing). The accumulating intermediates generated by some factors were comparable (see for example, cleavages mediated by 60% glucose and 30% sorbitol, acetic acid and H 2 O 2 , Figure 1E and F); however, the general outcome of each treatment indicated differences in the course of events during each response.