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







so so 4

Cyr1 (cAMP)

Pkh1 (homolog of PDK1)

Sch9 (homolog of PKA Akt/PKB) J




(Receptors) (G-proteins)


Blood Pressure Health

Blood Pressure Health

Your heart pumps blood throughout your body using a network of tubing called arteries and capillaries which return the blood back to your heart via your veins. Blood pressure is the force of the blood pushing against the walls of your arteries as your heart beats.Learn more...

Get My Free Ebook

Post a comment