Novel Methods for the Identification of Genes that Regulate Chronological Aging

Chronological life-span experiments are conducted using yeast cultures of billions of organisms and are very well suited for the screening of long-lived mutants. In the past we used a transposon mutagenesis technique to generate a pool of yeast carrying mutations that decrease or abolish the expression of the corresponding genes. The mutagenized population was subject to either heat shock or a treatment with the superoxide generator paraquat to select for stress resistance mutants, which were subsequently tested for chronological survival in SDC. Yeast that were viable after 9-15 days were isolated and retested individually to monitor their survival (Fabrizio et al., 2001). The identification of the insertions associated with life-span extension was obtained by sequencing the region adjacent to the transposon, a rather straightforward technique. The approach of preselecting the stress resistant mutants was based on the association between ability to withstand stress and life-span extension, which applies to all model organisms, and allowed us to reduce the representation of non-long lived mutants (false positives) among the day 9-15 survivors.

Recently a more sophisticated genetic tool to study how each individual gene can affect the life span of yeast has become available: the yeast knock-out (YKO) collection. Currently, the collection covers 96% of the yeast open reading frames (ORFs) and is constructed in a way that each deletion represents a complete loss of function of the gene and is uniquely tagged with two 20-nucleotide sequences (UPTAG and DNTAG) (Wach et al., 1994). The abundance of specific deletion mutants in a pool can be measured quantitatively by amplifying the tag sequences and hybridizing them to a high-density oligonucleotide arrays (Affymetrix Tag3) (Giaever et al., 2002). Once completed, the yeast gene-deletion collection will overcome the main limitation of the standard mutagenesis methods used for mutant screening—under-representing the yeast genome. However, with only 4% of the ORFs missing, the YKO collection already represents a remarkable genetic tool for a comprehensive genomic analysis of chronological aging. In a typical experiment, a pool generated using approximately the same number of cells for each deletion strain is (~600) grown in SDC, and viability is measured every 48 hours as described in Survival in SDC: Postdiauxic Phase. Every 48 hours samples of yeast (OD = 0.2-2) are collected and genomic DNA is extracted. 0.2 ^g of DNA is used to amplify the UPTAG and DNTAG molecular bar codes in two







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