PIL1 Involvement In Cyr1 Mutant Life Span Extension

ObjectiveMutations that decrease the activities of the Akt/PKB,Tor, and Ras pathways extend the lifespan of several model organisms, suggesting that the underlying mechanisms of longevity regulation are conserved in many eukaryotic organisms.Our previous data showed that PIL1 is involved in sch9 mutant life span extension, but was not reproducible. We decided to focus on the Ras/cAMP/PKA pathway and investigate any P1L1 involvement in ras/PKA life span extension.We hypothesize that:PIL1 is involved in the stabilization of cyrl mutant actin cytoskeleton dynamics and life span extension.MethodsYeast strains, plasmids and media. All strains used in these studies are DBY746 background. Other mutated strains are described in Table 1. The stains were grown with shaking at 30℃in Longo's Aging medium[5]with 2% glucose as carbon sources. The fresh colonies were inoculated into 2ml of Aging medium and get SONC (saturated overnight culture). Dilute SONC into 25ml of aging medium in 125 flasks, let initial OD=0.1, grew the cells at 30℃with shaking (220rpm). After 3 days, the cells reached the stationary phase and stopped dividing at this time and still survived. We treated it as day 1. The plasmids Ycplac22-PIL1 was constructed by PCR amplification and cloning of PIL1 ORF with its own promoter into the BamHl-Sall sites of yeast pRS315 vector.Chronological life span assay according Longo's methods[3]Hydrogen peroxide stress resistance assays.1 OD cells are taken from cultures and was treated by 135mM H2O2 at RT for lhr and do 10 fold serial dilutions, spread onto YPD plates (1% yeast extract,2% peptone,1.5%agar and 2%glucose) and incubate at 30℃for 2-3 days. Caspase activity using Flow Cytometry.CaspACE-FITC-VAD-FMK in situ marker (Promega G746A. Part#9PIG746) is a FITC-conjugate in situ marker for apoptosis. which binds to activated caspase. Staining the cells of final concentration 5-10μM in the dark for 20 min. after washing with PBS 3 times and being ready for Flow cytometry (excitation 488nm, emission 452-450nm).ResultsThe deletion of pill influence cyrl::mTn remarkably:the life span decurtate. The oxidative stress resistance decrease, and the caspase activity upgrade, it acts as WT. But the similar phenomenon is not surveyed in ras2 mutant。ConclusionPIL1 is involved in cyrl::mTn longevity and increased oxidative stress resistance, but has little effect in ras2A longevity or stress resistance...