Figure 2
Upstream UUG codons repress the URA6 expression in eIF5G31R mutant and cause DNA damage. (a) Quantification of URA6 protein expression. YP823 strain transformed with pYcplac33-Ura6-3xHA (A1141) or pYcplac33-upUUGlessUra6-3xHA (A1142) constructs along with empty vector pYcplac22 (A823) or pYcplac22-eIF5G31R (A838) construct were grown overnight on synthetic dextrose (SD) plus leucine medium till OD600 ~ 0.8 at 30 °C. Whole cell extract (WCE) was prepared by mechanical cell breaking using glass beads and 40 µg of WCE was subjected to 12% SDS-PAGE and the URA6p was identified by Western blot using an anti-HA antibody and normalized with blot stained by Coomassie brilliant blue (CBB) (left). YP823 strain carrying empty vector pYcplac22 (A823) or pYcplac22-eIF5G31R (A838) constructs were grown overnight on synthetic dextrose (SD) plus leucine medium till OD600 ~ 0.8 at 30 °C. The whole-cell extract (WCE) was treated with TRIzol, followed by ethanol precipitation. The total RNA isolated was subjected to reverse transcription and quantitative PCR using URA6 ORF specific oligonucleotides oPA1101 and oPA1102 (right). The error represents an average deviation. (b) Analysis of URA6-LacZ expression. Yeast strain YP823 containing empty vector pYcplac22 (A823) or pYcplac22-eIF5G31R (A838) constructs were transformed with derivatives of Ura6-LacZ reporter constructs A1074, A1140, A1340, A1341, A1342, and A1343 were grown till OD600 ~ 0.8 in SD plus leucine medium at 30 °C. WCE were prepared, and β-galactosidase activity (nmol of O-nitrophenyl-β-d-galactopyranoside cleaved per min per mg) was measured and plotted. The error represents an average deviation. (c) Growth analysis in the presence of hydroxyurea. High copy (h.c) empty vector (EV) pRS425 (B1378) or high copy pRS425-Ura6 (A1147) constructs were transformed in yeast strain YP828 carrying either EV pYcplac22 (A823) or pYcplac22-eIF5G31R (A838) constructs were grown overnight, serially diluted, and spotted on SD plus uracil or SD plus uracil plus 25 mM hydroxyurea and incubated at 30 °C for 2–3 days. (d) Analysis of DNA fragmentation by TUNEL assay. Yeast strain YP823 carrying either EV pYcplac22 (A823) or pYcplac22-eIF5G31R (A838) constructs were grown till OD600 ~ 0.8. The cells were treated with rTDT and nucleotide mix and imaged using Em λ461 (DAPI) and Em λ529 (FITC) filters. The values are average (n = 300) along with the standard error of the mean (SEM). Statistical differences were determined by the two-tailed Student’s t-test. ns non-significant.
