It contains 3,235 haploid deletion strains covering 65. 8% of the 4,914 protein coding open reading frames based on the annotated genome sequence. As 3,576 genes are nonessen tial, this library represents approximately 90. 5% of the nonessential S. pombe genes. Fission yeast were cultured in YES or EMM medium at 32 C as described concerning before. Screen of deletions sensitive to DNA damage The screen was performed in three rounds. In the first round, deletion strains from the Bioneer library were grown in YES medium till saturation. 20 ul culture from each strain was diluted into 180 ul liquid YES medium contain ing different DNA damage reagents in 96 well microtiter plates. As a control, cells were also diluted into medium without any reagent. Concentrations of reagents were, 7. 5 mM hydroxyurea, 0.
5 mU ml bleomycin, 0. 01% methyl methanesulfonate, 1 uM camptothecin, 15 ug ml thiabendazole and 60 J m2 ultraviolet radiation. After 24 hours of incubation at 32 C, the optical densities Inhibitors,Modulators,Libraries of the cultures were measured at 600 nm and compared to those of the controls. Deletions with A600 that dropped by 5 fold or more upon reagent treatment Inhibitors,Modulators,Libraries were designated as sensitive. Deletion mutants showing sensitivity to at least one reagent were picked to create a sub library. This round of the screen was repeated once. In the second round, strains from the sub library were grown in YES medium overnight, and then inoculated into 1 ml YES medium containing Inhibitors,Modulators,Libraries differ ent reagents at an A600 of 0. 02. After 24 hours of incuba tion at 32 C, A600 was measured and compared to those of no reagent controls.
In the third round, strains Inhibitors,Modulators,Libraries showing sensitivity to at least one DNA damaging agent in the second round were grown in liquid medium to an A600 of 1. 0. Cultures were diluted by five fold for five times, and 2 ul dilutions were spotted onto YES or EMM plates containing DNA damage reagents of indicated concentra tions. The growth of the cells was checked after 3 4 days of incubation at 32 C. If the growth of a mutant on the plate containing certain reagent was 2 spot lesser than that on YES plate, this mutant was designated as sensitive. Gene ontology analysis Gene ontology classifications were performed at with the database filter set as GeneDB S. pombe. Maximum P value was 0. 05 as the threshold for significance assessment, and Inhibitors,Modulators,Libraries minimum number of gene products was 3 in each GO term.
GO analysis was based on the biological process classifications in this study. Flow cytometry 1 2��107 exponentially growing cells were treated with DNA damage reagent for 2 h. For the UV sensitivity assay, cells were exposed to 60 J m2 radiation and then grown for 2 h. Cells were harvested and fixed in 70% cold ethanol at 4 C for 1 h. Cells were resuspended in 0. 5 ml of 50 mM sodium scientific research citrate containing 0. 1 mg ml RNase A and incubated at 37 C for 2 h. Cells were briefly sonicated, and then stained with 4 ug ml propidium iodide at room temperature for 15 min.