A centromere-based plasmid carrying URA3 and KAR4 with an internal 3xHA tag under the control of its native promoter (pMR2654) was mutagenized with hydroxylamine (see Table S2). The plasmid (10 μg) was incubated with 500 μl of 7% hydroxylamine solution for 20 hours at 37° (Rose, Winston et al. 1990 ). The reaction was stopped by adding 10 μl 5M NaCl and 50 μl 1mg/ml BSA, and the DNA was ethanol-precipitated and resuspended in TE. The mutagenized DNA was transformed into the yeast strain MY 10128 (see Table S1). Individual transformants were patched onto SC-URA media, 50 to a plate, with a positive and negative control (MY 11596 and 11597). The patches were replica-printed onto YEPD with a lawn of MATα MY11297 and allowed to mate at 30° C for 3 hours (limited mating) before replica-printing on SC-LEU-LYS media to detect mating defects. The mating plates were returned to the 30° C incubator overnight, then replica printed onto SC-LEU-LYS-URA media the next day to select for diploids from all patches (overnight mating). Diploids from the overnight mating were replica printed onto Complete Sporulation media and incubated at room temperature for one day, then at 30° for three days. The sporulation plates were finally replica printed on SC-HIS-ARG media with added canavanine to detect successful entry into meiosis. The limited mating plates were compared to the meiosis plates to detect potential separation of function mutants. Potential hits were repatched (nine to a plate) from the original transformation plate and rescreened. When transformants displayed a consistent phenotype, the plasmid was extracted using a yeast mini-prep kit (Zymo Research, Irvine, CA) and electroporated into bacteria. Bacterial colonies were grown up and the plasmids were extracted using a kit (Qiagen, Germantown, MD). The samples were sent for sequencing with KAR4-specific primers. After sequencing, the plasmids were retransformed into MY 10128 and assayed a final time for both mating and meiotic defects. To further address the severity of the meiotic defect, cells were sporulated as described above and spotted in 10-fold serial dilutions on the SC-HIS-ARG media. Alleles that supported wild type levels (formed colonies at the highest dilution in the series) of recombination were scored as “++++”. Alleles that only formed colonies on the 10−2 serial dilution were scored as “+++” and so on.