All experiments were conducted with the diploid strain PG311 (Lee et al. 2009 (link)). The relevant genotype of PG311 is MATα::NATR/MATa URA3 /ura3 -1 ade2 -1/ade2 -1 TRP1 /trp1 -1 HIS3 /his3 -11,15 GAL2 /gal2 SUP4 -o/can1 -100 V9229/V9229::HYG V261553/V261553::LEU2 . This diploid was generated by crossing the haploid strains PSL2 and PSL5, which are isogenic with strains W303a and YJM789, respectively, except for alterations introduced by transformation (Lee et al. 2009 (link)). From this point on, we will refer to the haploid parents of PG311 as W303a and YJM789. In general, PG311 has the SNPs predicted from the haploid parents. The disruption of MATα in PG311 allows synchronization of this diploid by α-factor. Although diploids that lack MATα do not sporulate under normal conditions, such strains can be sporulated on plates containing 5 mM nicotinamide (J. Rine, personal communication). For experiments in which we analyzed meiotic products of PG311, the stains were pregrown on YPD plates with 5 mM of nicotinamide and then transferred to sporulation plates containing 5 mM nicotinamide. Plates were incubated at 25° for 2–4 days before tetrad dissection.
Standard media were used (Guthrie and Fink 1991 ) unless noted. To detect spontaneous crossovers, we first isolated single colonies of PG311 grown on rich growth medium (YPD) at 30° for 2 days. Individual colonies were suspended in 400 μl of dH2O, and 100 μl of this mixture was plated on canavanine-containing plates (SD-arg + 120 μg/ml canavanine). The plates were incubated 4 days at room temperature, followed by incubation for 16 hr at 4°; the 4° incubation allows better visualization of the red sectors. We purified cells from the red and white sectors for subsequent analysis.
In the experiments in which cells were irradiated, we synchronized cells in G1 using α-factor (Lee and Petes 2010 (link)). The synchronized cells were treated with either γ-radiation in a Shepherd Mark 1 137Cs irradiator at 100 Gy or with UV (254 nm) derived from a TL-2000 Ultraviolet Translinker at a dosage of 10 or 15 J/m2. Following radiation, the cells were plated either on nonselective plates (SD-arg) or plates that lacked arginine and contained 120 μg/ml canavanine. The subsequent growth of the cells and the analysis of sectors were done as described above for the spontaneous selection with the exception that sectored colonies for the UV-treated samples were isolated from nonselective plates grown at 30° instead of room temperature.
Standard media were used (Guthrie and Fink 1991 ) unless noted. To detect spontaneous crossovers, we first isolated single colonies of PG311 grown on rich growth medium (YPD) at 30° for 2 days. Individual colonies were suspended in 400 μl of dH2O, and 100 μl of this mixture was plated on canavanine-containing plates (SD-arg + 120 μg/ml canavanine). The plates were incubated 4 days at room temperature, followed by incubation for 16 hr at 4°; the 4° incubation allows better visualization of the red sectors. We purified cells from the red and white sectors for subsequent analysis.
In the experiments in which cells were irradiated, we synchronized cells in G1 using α-factor (Lee and Petes 2010 (link)). The synchronized cells were treated with either γ-radiation in a Shepherd Mark 1 137Cs irradiator at 100 Gy or with UV (254 nm) derived from a TL-2000 Ultraviolet Translinker at a dosage of 10 or 15 J/m2. Following radiation, the cells were plated either on nonselective plates (SD-arg) or plates that lacked arginine and contained 120 μg/ml canavanine. The subsequent growth of the cells and the analysis of sectors were done as described above for the spontaneous selection with the exception that sectored colonies for the UV-treated samples were isolated from nonselective plates grown at 30° instead of room temperature.
