Figure 2.

The Ure2 protein induces [URE3] generation. (A) One base was deleted in codon 44 of URE2, or inserted in codon 80, or both. This changed the reading frame for most of the prion domain. The insertion at codon 80 makes it a UAA codon. Deleting a base from codon 44 produces a fusion peptide that terminates … IISRIPter after residue 80, with reduced mRNA levels (compare lanes 1 and 7 with 5 in C). (B) Induction of [URE3] was tested by introducing each plasmid into strain 3469 (= MATa/MATα ura2/ura2 kar1/kar1 leu2/leu2 trp1/+ +/his⁻ [ure-o]). Mixed transformant clones were spotted on SGal + Ura plates. After 3–5 days cells were suspended in water, cell number was measured, and dilutions were plated on SD + USA. After 5 days at 30°C clones were counted. Ten or more clones from each sample were tested for curing by growth on SD + Ura + 5 mM guanidine. Complementation of ure2Δ was carried out by introducing each plasmid into strain 3718 (= MATα his3 leu2 ura2 ure2::URA3), growing transformants on SGal + His + Ura and then replicaplating to SGal + His + USA. Values shown are USA⁺ clones per 10⁶ cells plated. To the right are shown diagramatically the protein sequences expressed in the various constructs. The solid line represents Ure2p sequences and the dashed line the normally out-of-frame sequences that are expressed because of the frameshift mutations. Vector = p554, Ure2p = p646, Ure2p 1–65 = p680, Ure2p Δ3–65 = p682, Ure2p −1at44 = average of three plasmids containing the −1 frameshift at codon 44: E2+1, E2+7, E2+15; Ure2p+1at80 = +1 frameshift at codon 80: E2+3; Ure2p−1at44&+1at80 = average of two plasmids with both frameshifts: E+7–3, E+7–1. (C) Northern blot hypbridization of URE2 mRNA. Lane 1, −FS1D (−1 frameshift at residue 44). Lane 2, E2+3 (+1 at 80). Lane 3, E2+7–3 (both frameshifts). Lane 4, p554 (vector). Lane 5, p644 (wild type). Lane 6, E2+7–1 (both frameshifts). Lane 7, E2+1 (−1 frameshift at residue 44). Lane 8, E2+3 (+1 frameshift at residue 80).