Please click image to view Figure 1.

This assay uses a specific yeast strain (SH1274) which has markers allowing measurement of the frequency of meiotic non-disjunction at Chromosome II. The basis for the assay is described in Figure 1.

1. Cells are grown to stationary phase in 10 ml YPA cultures and then harvested, washed two times, and transferred to 6 ml of sporulation medium (1% KOAc + amino acids). This culture is split into 3 culture tubes, each containing 2 ml of the culture. The culture tubes are placed at 30°C on a rotating wheel.

2. After two hours in sporulation medium, benomyl is added to the medium. 100 ml aliquots of benomyl (10 mg / ml in DMSO) are stored at –20°C. For each culture, additional DMSO is added to bring the final DMSO concentration to 1%. Cultures are replaced on the rotating wheel at 30°C.

3. After 72 hours in sporulation medium, 1 ml aliquots of each culture are harvested, washed twice in water, and then resuspended in 1 ml of water. Cells are examined in the microscope for sporulation. Cell concentration is determined using a hemacytometer. Cells are diluted serially in water to generate a 10^-2 dilution and a 10^-4 dilution.

4. 50 ml of the 10^-4 dilution are plated on complete medium (COM plates) and complete medium lacking arginine and containing canavanine (CAN plates). 5ml of the undiluted cells are plated on complete medium lacking arginine, histidine and lysine and containing canavanine (HLCAN plates).

Please click image to view Figure 2.

5. The frequency of cells that form colonies on COM plates indicates the viability of the culture. The frequency of cells forming colonies on CAN approximates the fraction of cells in the culture that have undergone meiosis and sporulation. The frequency of cells that can grow into colonies on HLCAN plates is proportional to the frequency of Chr. II disomes in the culture. Frequency of Chr. II disomes divided by the viability yields the frequency of Chr. II disomes / cfu.