ena-STUDY-University of Latvia Faculty of Medicine-26-09-2020-19:12:29:617-197
Adenine auxotrophic strains are widely used, but their response to purine starvation is not studied in detail. We report effects harnessed by purine  or nitrogen starvation in purine synthesis ade8 knockout in CEN.PK2-1D strain.
We performed genome wide transcriptomic response analyses on CEN.PK2-1D strain growing in full meda and media with adenine omitted. ade8 knockout was cultivated in SD media  with added 80 mg/L tryptophan, 100 mg/L uracil, 480 mg/L leucine, 100 mg/L histidine and 100 mg/L adenine . To ensure, that yeast culture is in it's exponential growth phase, we prepared cultures by reinoculating overnight cultures (grown from a single colony) into fresh media, where at least 6 doublings occured and OD600 0.5-1 corresponding to 1-2*10^7 cells/mL is reached. To ensure specific media dependent conditions, cultures in exponential growth phase (OD 0.5-1) were washed with distilled water twice and resuspended at OD 0.5 in full SD media (SD+) or SD media adenine omitted (SD ade-) or SD addenine omitted +guanine (gua+) added.  All cultures were prepared in biological triplicates, incubated on a rotary shaker, +30°C, 180 rpm. Total yeast RNA after 4 hour cultivation in aforementioned medias were isolated with RiboPure™ RNA Purification Kit, yeast.
We performed genome wide transcriptomic response analyses on CEN.PK2-1D strain growing in full meda and media with adenine omitted. ade8 knockout was cultivated in SD media  with added 80 mg/L tryptophan, 100 mg/L uracil, 480 mg/L leucine, 100 mg/L histidine and 100 mg/L adenine . To ensure, that yeast culture is in it's exponential growth phase, we prepared cultures by reinoculating overnight cultures (grown from a single colony) into fresh media, where at least 6 doublings occured and OD600 0.5-1 corresponding to 1-2*10^7 cells/mL is reached. To ensure specific media dependent conditions, cultures in exponential growth phase (OD 0.5-1) were washed with distilled water twice and resuspended at OD 0.5 in full SD media (SD+) or SD media adenine omitted (SD ade-) or SD addenine omitted +guanine (gua+) added.  All cultures were prepared in biological triplicates, incubated on a rotary shaker, +30°C, 180 rpm. Total yeast RNA after 4 hour cultivation in aforementioned medias were isolated with RiboPure™ RNA Purification Kit, yeast.