A high-throughput screening system for genes extending life-span.

We developed a high-throughput functional genomic screening system that allows identification of genes prolonging life-span in the baker's yeast Saccharomyces cerevisiae. The method is based on isolating yeast mother cells with extended number of cell divisions as indicated by the increased number of bud scars on their surface. Fluorescently labelled Wheat Germ Agglutinin was used for specific staining of chitin, a major component of bud scars. Screening of a human HepG2 cDNA expression library in yeast resulted in the isolation of 12 yeast transformants with a potentially prolonged life-span. The transgene in one of the lines was identified as ferritin light chain (FTL) and studied in more detail. Yeast cells containing FTL showed an enhanced iron and H(2)O(2) resistance, a reduced cell death rate and an increased number of cell divisions. Overexpression of FTL in the nematode Caenorhabditis elegans resulted in a life-span increase of 8% confirming our yeast observations in a multicellular organism. Our data demonstrate that this method permits a fast screening of libraries for hunting genes involved in ageing processes.

Nonclassical export pathway: overexpression of NCE102 reduces protein and DNA damage and prolongs lifespan in an SGS1 deficient Saccharomyces cerevisiae.

In this study, we used our recently developed screening method, Bud-Scar-based Screening (BSS), to screen a yeast cDNA expression library in an SGS1 deletion BY4742 yeast strain. One gene involved in a nonclassical export pathway, NCE102, was found to extend the life span of Deltasgs1 yeast. Deletion of NCE102 in a wild type yeast strain increased its sensitivity to oxidative stress upon diethylmaleate (DEM) treatment but did not shorten its lifespan, indicating that this gene is not essential in determining yeast lifespan. Transformation of NCE102 into either Deltance102 or Deltasgs1 strains could rescue its tolerance to DEM stress, indicating that NCE102 is protective during oxidative stress. Moreover, overexpression of NCE102 in Deltasgs1 strain leads to reduced protein damage. However, overexpression of NCE102 in wild type yeast strain BY4742 neither protected against oxidative stress due to DEM nor extended yeast lifespan compared to its parental wild type strain, indicating that nonclassical export is redundant and DNA repair is fully sufficient in the wild type strain. We therefore demonstrate that a nonclassical export pathway functions as an alternative clearance/detoxification pathway to eliminate damaged material, when the basic repair pathway is not sufficient.