A high-throughput method to measure the sensitivity of yeast cells to genotoxic agents in liquid cultures.
The sensitivity of yeast Saccharomyces cerevisiae to DNA damaging agents is better represented when cells are grown in liquid media than on solid plates. However, systematic assessment of several strains that are grown in different conditions is a cumbersome undertaking. We report an assay to determine cell growth based on automatic measurements of optical densities of very small (100 microl) liquid cell cultures. Furthermore, an algorithm was elaborated to analyze large data files obtained from the cell growth curves, which are described by the growth rate--that starts at zero and accelerates to the maximal rate (mu(m))--and by the lag time (lambda). Cell dilution spot test for colony formation on solid media and the growth curve assay were used in parallel to analyze the phenotypes of cells after treatments with three different classes of DNA damaging agents (methyl methanesulfonate, bleomycin, and ultraviolet light). In these experiments the survival of the WT (wild type) and a number of DNA repair-deficient strains were compared. The results show that only the cell growth curve assay could uncover subtle phenotypes when WT cells, or mutant strains that are only weakly affected in DNA repair proficiency, were treated with low doses of cytotoxic compounds. The growth curve assay was also applied to establish whether histone acetyltransferases and deacetylases affect the resistance of yeast cells to UV irradiation. Out of 20 strains tested the sir2delta and rpd3delta cells were found to be more resistant than the WT, while gcn5delta and spt10delta cells were found to be more sensitive. This new protocol is sensitive, provides quantifiable data, offers increased screening capability and speed compared to the colony formation test.
Toussaint
Martin
M
Département de Microbiologie et d'Infectiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Que. J1H 5N4, Canada.
Levasseur
Geneviève
G
Gervais-Bird
Julien
J
Wellinger
Raymund J
RJ
Elela
Sherif Abou
SA
Conconi
Antonio
A
D001761:Bleomycin / Q000494:pharmacology; D003470:Culture Media / Q000494:pharmacology*; D051548:Histone Acetyltransferases / Q000378:metabolism; D006655:Histone Deacetylases / Q000378:metabolism; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009153:Mutagens / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects* / Q000235:genetics* / Q000528:radiation effects; D013997:Time Factors; D014466:Ultraviolet Rays
D003470:Culture Media; D009153:Mutagens; D001761:Bleomycin; D008741:Methyl Methanesulfonate; D051548:Histone Acetyltransferases; D006655:Histone Deacetylases