FIG 2.

Mutant Ure2 proteins propagate [URE3] but maintain it only under selective conditions. (A) Representative meiotic progeny of [URE3] diploids heterozygous for ure2Δ and the mutant Ure2, as indicated on the left (see the text). The dissection plates (1/2YPD) were replica plated to YPAD containing G418, which selects for ure2Δ cells, −ade medium, which selects for cells lacking Ure2 or propagating [URE3], and medium lacking uracil (−ura), which selects for cells with plasmid-borne URE2. All Ure2 point mutants (G418 sensitive) that are Ura^– (i.e., lacking wild-type Ure2) fail to grow on −ade plates, showing that they lose [URE3]. Point mutants that retain the plasmid encoding URE2 dominantly inhibit [URE3] as indicated by pink color and weakened Ade⁺ phenotype. (B) After continued incubation, rare Ade⁺ colonies of Ure2 mutants arose on the −ade plates. Cells from these colonies were recovered and streaked onto similar [URE3]-selective plates (−ade, left) and from these onto nonselective 1/2YPD indicator medium (right). Both plates were incubated at 30°C for 2 days. (C) [URE3] was induced de novo in cells of the indicated Ure2 mutants by galactose-induced overexpression of Ure2(1-64) followed by spreading of 10⁶ cells on −ade medium (left, sectors labeled +). The same numbers of identically treated cells carrying an empty vector were plated on alternate sectors (labeled –). The frequency of Ade⁺ colonies reflects the efficiency of [URE3] induction. This plate was replica plated onto 1/2YPD (center) and onto a similar −ade plate that selects for [URE3] (right). Rare colonies on −ade plates from uninduced cultures are uncharacterized Ade⁺ revertants.