Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Václav Vopálenský; Michal Sýkora; Tomáš Mašek; Martin Pospíšek

Tested strains (50 ml) were cultured in an appropriate medium at 24°C with shaking. When the A 600 reached 0.9, cells were harvested and washed three times with the same medium preheated to 24°C. One-half of the culture (25 ml) was mixed with 25 ml of fresh medium preheated to 24°C and then cultivated at 24°C for more than 20 hours. The second half of the tested culture (25 ml) was mixed with 25 ml of fresh medium preheated to 52°C and then cultivated at 37°C for more than 20 hours. Aliquots (2 ml) were taken at 0, 3, 6 and 12 hours. Samples were harvested, sterilized using 0.2 μm filters and assayed for the presence of the killer toxin activity in the culture medium by an agar well diffusion assay using S. cerevisiae S6/1 as a sensitive strain.