Figure 6.
A RENT1/HUPF1 chimeric allele functions in translation termination. (A) A RENT1/HUPF1 chimeric allele prevents nonsense suppression in a upf1Δ strain. Strain PLY146 (MATα ura3-52 trp1Δ upf1::URA3 leu2-2 tyr7-1) was transformed with YCplac22 (vector), YCpUPF1 (UPF1), YCpRent1CHI4-2, or YEpRent1CHI4-2, and cells were grown to OD600 = 0.5 in -Trp -Met medium. Dilutions of 1/10, 1/100, and 1/1000 were prepared in -Trp -Met medium and 5 μl of these dilutions was plated simultaneously on -Trp -Met (top plate) or -Trp -Met -Leu -Tyr (bottom plate) media. Cells were monitored for growth at 30°C. (B) A RENT1/HUPF1 chimeric allele does not promote decay of nonsense containing mRNAs. Total RNA was isolated from cells at OD600 = 0.8 from the strains described in A. RNA (40 μg) from strains PLY146 transformed with YCplac22 (vector), YCpUPF1 (UPF1), or YEpRent1CHI4-2 (YEpRENT1CHI4-2)(10) was subjected to Northern blotting analysis and probed with either the LEU2, TYR7, or CYH2 probes.