High copy plasmids that enhance induction of [<i>PSI</i>⁺] also cure pre-existing [<i>PSI</i>⁺] and [<i>URE3</i>].

<p>(A) Overexpressed Q/N-rich proteins cause loss of [<i>PSI</i>⁺]. Weak (w) [<i>PSI</i>⁺][<i>PIN</i>⁺] (L1758) was transformed with plasmids encoding the indicated Q/N-rich proteins or fragments (<i>PIN4C</i> and <i>CYC8C</i>), or with the empty vector pHR81. Representative images of transformants plated on YPD following amplification of plasmids on SD-Leu are shown (upper). The efficiency of curing (lower) was determined as the percentage of red colonies indicative of [<i>psi</i>⁻] among ∼1100 colonies. (B) Induction of [<i>PSI</i>⁺] in a <i>rnq1Δ</i>::<i>HIS3</i> [<i>psi</i>⁻][<i>pin</i>⁻] 74-D694 strain (L3125) carrying the same plasmids as in (A). [<i>PSI</i>⁺] was induced by overexpression of <i>SUP35NM-GFP</i> from p<i>CUP1</i>-<i>SUP35NM::GFP-TRP1</i> in 50 µM Cu²⁺ following library plasmid amplification on SD-Leu. Shown is growth on SD-Ade, which indicates the presence of [<i>PSI</i>⁺]: spots are representative of three repeated experiments. The Ade⁺ colonies were verified to be [<i>PSI</i>⁺] by visualization of Sup35NM-GFP dots. (C) Overexpressed Q/N-rich domains cause [<i>URE3</i>] curing. The [<i>URE3</i>] derivative of 74D-694 [<i>PIN</i>⁺][<i>psi</i>⁻] expressing the GFP tagged endogenous Sup35 (L3154), was transformed with high copy plasmids encoding the Q/N-rich proteins or protein fragments, or with the empty vector pHR81. The percentage of cured [<i>ure-o</i>] cells among ∼1000 colonies was determined using the color assay described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003236#s4" target="_blank"><i>Materials and Methods</i></a>. Error bars show standard error of the mean.