Use of a genome-wide approach to identify new genes that control resistance of Saccharomyces cerevisiae to ionizing radiation.
We have used the recently completed set of all homozygous diploid deletion mutants in budding yeast, S. cerevisiae, to screen for new mutants conferring sensitivity to ionizing radiation. In each strain a different open reading frame (ORF) has been replaced with a cassette containing unique 20-mer sequences that allow the relative abundance of each strain in a pool to be determined by hybridization to a high-density oligonucleotide array. Putative radiation-sensitive mutants were identified as having a reduced abundance in the pool of 4,627 individual deletion strains after irradiation. Of the top 33 strains most sensitive to radiation in this assay, 14 contained genes known to be involved in DNA repair. Eight of the remaining deletion mutants were studied. Only one, which deleted for the ORF YDR014W (which we name RAD61), conferred reproducible radiation sensitivity in both the haploid and diploid deletions and had no problem with spore viability when the haploid was backcrossed to wild-type. The rest showed only marginal sensitivity as haploids, and many had problems with spore viability when backcrossed, suggesting the presence of gross aneuploidy or polyploidy in strains initially presumed haploid. Our results emphasize that secondary mutations or deviations from euploidy can be a problem in screening this resource for sensitivity to ionizing radiation.
Game
John C
JC
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Birrell
Geoff W
GW
Brown
James A
JA
Shibata
Toru
T
Baccari
Clelia
C
Chu
Angela M
AM
Williamson
Marsha S
MS
Brown
J Martin
JM
D015153:Blotting, Western; D002470:Cell Survival; D002588:Cesium Radioisotopes; D003433:Crosses, Genetic; D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D004307:Dose-Response Relationship, Radiation; D016681:Genome, Fungal; D005838:Genotype; D006238:Haploidy; D006720:Homozygote; D009154:Mutation; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016366:Open Reading Frames; D011003:Ploidies; D016133:Polymerase Chain Reaction; D011836:Radiation Tolerance / Q000235:genetics*; D011839:Radiation, Ionizing; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology*; D012542:Scattering, Radiation; D013997:Time Factors
D002588:Cesium Radioisotopes; C476199:RAD61 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA