Screening of Ade⁺ colonies for killer [PSI⁺]. Isolated Ade⁺ colonies were restreaked three times on −Ade −Ura + 10 μg/mL doxycycline before being stamped on FOA. Circled colonies have killer [PSI⁺]. The fractions of isolates with mild, sick, and killer [PSI⁺] are shown below.

Killer [PSI⁺] strains are sick because of Sup35p deficiency. Cells carrying killer [PSI⁺], a nontoxic [PSI⁺], and a [psi⁻] control were streaked on ½YPD with (A) or without (B) doxycycline (10 μg/mL).

Toxicity and instability of [PSI⁺] isolates. (A) Sick and killer [PSI⁺] candidates were streaked on ½YPD and on FOA plates. After 2 d of growth on ½YPD, strains were restreaked on FOA plates. (B–D) Individual sick [PSI⁺] and nontoxic [PSI⁺] colonies were streaked on ½YPD from FOA. (Lower) Single colonies restreaked on ½YPD are indicated.

Multiple Sup35NM-GFP foci for killer [PSI⁺] and single foci for benign [PSI⁺]. (A) Newly induced 74-D694 [PSI⁺] colonies that could not grow on FOA plates (killer [PSI⁺]) were transformed with the pSupNM-GFP plasmid directly from −Ade −Ura + doxycycline plates. Transformants were selected only for the presence of pSupNM-GFP and were highly heterogeneous by color. White and sectoring transformants appeared as a result of the loss of the Sup35C plasmid, and their [PSI⁺] has become benign. Red transformants still carry the Sup35C-expressing plasmid and contained [PSI⁺] (probably killer [PSI⁺]), as was shown by fluorescent microscopy analysis. (B and C) Red transformants from A of two independent killer [PSI⁺] variants contain [PSI⁺] and show cells with multiple Sup35NM-GFP foci. (D) Typical white transformants from A and benign 74-D694 [PSI⁺] isolates show cells with mostly single Sup35NM-GFP foci. Six benign [PSI⁺] and four killer [PSI⁺] isolates were transformed as described in A. Transformants of both [PSI⁺] types that tend to retain the Sup35C plasmid were compared, and about 2,000 individual cells containing [PSI⁺] were analyzed for the presence of multiple Sup35NM-GFP foci per cell by fluorescent microscopy analysis. About 50% of putative killer [PSI⁺] cells had multiple foci, compared with only about 10% of benign [PSI⁺] cells under these conditions.

Spontaneously formed [URE3] clones with dramatically slowed growth and mitotic instability. BY241 (A), BY241 Δure2::TRP1 (B), BY241 individual spontaneously formed [URE3] colonies (C–F), and second streaks of small BY241 [URE3] colonies (G and H) from E and F. All strains were grown on ½YPD for 3 d before the pictures were taken.