A new high resolution screening method for study of phenotype stress responses of Saccharomyces cerevisae mutants.
A high resolution high throughput screening method has been developed for stress response phenotyping of the global Saccharomyces cerevisiae knock out mutant collection. Stress causing agent is added at three concentrations to individual mutant cultures growing in early exponentially phase in 384-well microplates, and the dynamic effect of stress agent exposure is measured by following subsequent growth profiles of individual mutants with a resolution of three optical density measurements per hour. Software was written for calculation of sensitivity coefficients and efficient visual inspection of the growth and inhibition curves. Three DNA damage response causing agents were chosen to explore the feasibility of the new screening method: methyl methanesulphonate, 5-fluorouracil and cisplatin. They were tested in three biological replicas on a 1400 mutant large sub-library of the homozygote diploid S. cerevisiae gene knock out collection. The sub-library consisted of only mutants with a human ortholog to the inactivated gene. Almost 400 mutants were found more sensitive to one or more of the agents. Forty-nine mutants were sensitive to all three agents. One of the mutants, ERK5, sensitive to all three agents was chosen for follow-up human cell experiments to verify that such yeast screens can be used as hypothesis generator for human cell studies. Similar to yeast, HeLa cells became more sensitive against all three DNA damaging agents when co-treated with the ERK5 inhibitor BIX21088, thus supporting the result from the yeast phenotype screen.
SINTEF Materials and Chemistry, Department of Biotechnology, Trondheim, Norway. Havard.Sletta@sintef.no
Håvard
H
Sletta
Geir
G
Klinkenberg
Asgeir
A
Winnberg
Hans Fredrik Nyvold
HF
Kvitvang
Marianne Bakke
MB
Nilsen
Hans Einar
HE
Krokan
Marit
M
Otterlei
Per
P
Bruheim
D002945:Cisplatin / Q000633:toxicity; D004249:DNA Damage; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005472:Fluorouracil / Q000633:toxicity; D055786:Gene Knockout Techniques; D006367:HeLa Cells; D006801:Humans; D008741:Methyl Methanesulfonate / Q000633:toxicity; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D013312:Stress, Physiological
D008741:Methyl Methanesulfonate; D002945:Cisplatin; D005472:Fluorouracil