Figure 5

Double mutants sup45 upf1Δ are characterized by defects of NMD. A. Representative hybridization signals specific to precursor and mature forms of CYH2. Total RNA was isolated from strain 3v-D1658 (sup45Δ upf1Δ pRS315/SUP45) and its derivates (sup45Δ upf1Δ pRS315/sup45-n) transformed with pRS316 and pRS316/UPF1 plasmids, designated as (UPF1 -) and (UPF1 +), respectively. The Northern blots were hybridized with radiolabeled CYH2 probe. The CYH2 precursor/mature ratio in wild-type strain was set as 1.0. B. The fold increase in CYH2 precursor/mature mRNA accumulation measured in the same strains as in panel A are represented relative to such in wild-type strain. C. The same transformants as in panel A were tested by plating 10⁰, 10^-1and 10^-2 serial dilutions of overnight cultures (left to right) on synthetic complete plates lacking adenine and incubated 5 days at 25°C. The same serially diluted cultures were also spotted on plates lacking leucine and uracil (-L -U) to estimate the total number of cells analyzed. D. Norhern blots prepared with total RNA from the same transformants as in panel A were hybridized with radiolabeled probes, detecting ade1-14, SUP45 and scR1 mRNA (scR1 was used as a control). eRF1 and eRF3 protein levels in the same transformants were analyzed by western blot. Tubulin was used as a loading control. WT – wild type, 102 – sup45-102 (nonsense), 107 – sup45-107 (nonsense).