Chronological aging of budding yeast cells results in a reduction in subsequent replicative life span through unknown mechanisms. (CLS) is defined as the length of time that a quiescent yeast cell can retain the capacity to re-enter mitotic growth upon appropriate nutritional cues (Fabrizio and Longo, 2003). The determinants of chronological and replicative longevity in yeast appear to be both overlapping and distinct (Longo et al., 2012). Among those features that are shared between the two aging models is a robust lifespan extension in response to dietary restriction (DR), accomplished by reducing the glucose concentration of the culture medium from 2% to 0.5% or lower (Jiang et al., 2000; Kaeberlein et al., 2004; Lin et al., 2000; Murakami et al., 2008; Smith et al., 2007). Several interventions that decrease signaling through the nutrient-responsive target of rapamycin (TOR) pathway also extend both CLS and RLS (Laun et al., 2006), including deletion of genes encoding the mechanistic target of rapamycin (mTOR) homolog Tor1 or the ribosomal S6 kinase homolog Sch9, as well as treating yeast cells with the mTOR complex 1 inhibitor rapamycin (Fabrizio et al., 2004b; Fabrizio et al., 2001; Kaeberlein et al., 2005; Powers et al., 2006). Each of these interventions has also been shown to extend lifespan in nematodes, fruit flies, and mice, demonstrating that WYE-687 both yeast aging paradigms share conservation with aging in evolutionarily divergent multicellular organisms (Longo et al., 2012). Although both types of yeast aging are strongly influenced by glucose availability and nutrient signaling, it remains unclear to what extent aging is caused by similar downstream molecular events in each system. Both chronological and replicative aging are associated with increased accumulation of mitochondrial damage and oxidatively damaged/aggregated proteins (Kaeberlein, 2010). Although these correlated molecular factors are very likely to play a causal role in determining both CLS and RLS, this has been difficult to convincingly establish. Instead, some causes of aging in WYE-687 each system appear to be private for each type of yeast aging: genomic instability within the rDNA limiting RLS, and cell death arising from acidification of the culture medium limiting CLS. Genomic instability within the rDNA array during replicative aging can be observed through an age-associated accumulation of extrachromosomal rDNA circles within the mother cell (Sinclair and Guarente, 1997). DNA episomes induce a similar life shortening stress (Falcon and Aris, 2003). This instability can be suppressed by overexpression of the histone deacetylase Sir2 or deletion of the replication fork block protein Fob1, which both extend RLS (Defossez et al., 1999; Kaeberlein et al., 1999). Deletion of SIR2, on the other hand, increases rDNA instability and dramatically shortens RLS, but does not shorten CLS, and actually extends CLS under certain conditions (Fabrizio et al., WYE-687 2005; Kaeberlein et al., 1999; Smith et al., 2007; Wu et al., 2011). These observations have led to the idea that rDNA instability is specific to yeast replicative aging and does not influence chronological aging (Steinkraus et al., 2008). Acidification of the culture medium is a limiting factor for CLS when performed by quantifying survival of yeast cells aged in expired synthetic complete (SC) medium, conditions employed in the majority of published CLS studies (Longo et al., 2012). Buffering the culture medium to pH 6.0, addition of NaOH, or a shift to water after acid production during aging is sufficient to dramatically increase CLS (Burtner et al., 2009b; Burtner et al., Rabbit polyclonal to ZFP2. 2011; Fabrizio et al., 2004a; Fabrizio et al., 2005; Murakami et al., 2011). Since RLS analysis is performed by isolation and microdissection of individual mother cells on the surface of an agar-based rich growth medium, medium acidification does not occur during this assay and buffering the medium does not extend RLS (our unpublished data). In order to better understand the shared molecular features of replicative and.