A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Summary

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null mutant.

Abstract

Aging is the time dependent deterioration of an organism’s normal biological processes that increases the probability of death. Many genetic factors contribute to alterations in the normal aging process. These factors intersect in complex ways, as evidenced by the wealth of documented links identified and conserved in many organisms. Most of these studies focus on loss-of-function, null mutants that allow for rapid screening of many genes simultaneously. There is much less work that focuses on characterizing the role that overexpression of a gene in this process. In the present work, we present a straightforward methodology to identify and clone genes in the budding yeast, Saccharomyces cerevisiae, for study in suppression of the short-lived chronological lifespan phenotype seen in many genetic backgrounds. This protocol is designed to be accessible to researchers from a wide variety of backgrounds and at various academic stages. The SIR2 gene, which codes for a histone deacetylase, was selected for cloning in the pRS315 vector, as there have been conflicting reports on its effect on the chronological lifespan. SIR2 also plays a role in autophagy, which results when disrupted via the deletion of several genes, including the transcription factor ATG1. As a proof of principle, we clone the SIR2 gene to perform a suppressor screen on the shortened lifespan phenotype characteristic of the autophagy deficient atg1Δ mutant and compare it to an otherwise isogenic, wild type genetic background.

Introduction

Aging is the time-dependent loss of integrity in myriad biological processes that ultimately increases the probability of organismal death. Aging is nearly inevitable for all species. On a cellular level there are several well characterized hallmarks that are associated with aging, including: genomic instability, epigenetic alterations, loss-of-proteostasis, mitochondrial dysfunction, deregulated nutrient sensing, cellular senescence, and telomere attrition^1,2. In single celled organisms, such as yeasts, this leads to a reduction in replicative potential and chronological life span³

Protocol

1. Identify potential genetic interactions for screening

1. Identify the genetic background(s) for characterization, that results in an abnormally shorted chronological life span (CLS) in Saccharomyces cerevisiae using the Saccharomyces Genome Database (the SGD, https://www.yeastgenome.org^29,30), which compiles known phenotypic information for this organism.
   1. Select the Function tab from the options on the top of the webpage.
   2. Select Phenotype followed by selecting Browse all Phenotypes.
   3. From the

Results

As there are conflicting reports on the role of SIR2 during aging, we chose this gene for study as a potential suppressor of the atg1Δ mutant’s shorten CLS phenotype²⁶. The role of SIR2 is somewhat controversial, with conflicting reports on its role in extending CLS, however it has been clearly linked to increased CLS in at least one yeast background, with a role in both autophagy and mitophagy^22,31,

Discussion

Unravelling the genetics of aging is a difficult challenge, with many opportunities for further study that can potentially yield significant insights into the complex interactions that exist. There are many methods that allow for the rapid generation of loss-of-function mutants for the study of null strains of yeast^45,46. This method presents a straightforward approach to identify and clone genes onto the pRS315 vector for overexpression suppressor studies. One a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fungal/Bacterial DNA kit	Zymo Research	D6005
HindIIIHF enzyme	New England Biolabs	R3104S
Phusion High-Fidelity DNA Polymerase	New England Biolabs	M0530S
Plasmid miniprep kit	Qiagen	12123
SacII enzyme	New England Biolabs	R0157S
Salmon sperm DNA	Thermofisher	AM9680
T4 DNA ligase	New England Biolabs	M0202S