Figure 5.

Transformation of [URE3] variants. (A) Conservation of variant characteristics upon transmission of [URE3] variants. The cytoplasm of BY241 strains that were [ure-o] or one of the three different [URE3] variants was transferred to BY251 by cytoduction, and then cytoduced back into BY241. Recipient clones were cured of [URE3] by growth on 1/2 YPD plates containing 3 mM guanidine, and demonstrated reversion to [ure-o] in all cases. Serial 10-fold dilutions were spotted on the indicated media and incubated at 30°C for 3 days (1/2 YPD and SC+Can) or 5 days (SC−Ade). Note the differences in color on 1/2 YPD plates and sensitivity to canavanine between the three [URE3] variants and between the two strains. Data from a parallel experiment performed with BY252 instead of BY251 are provided in Supplementary Figure 5. (B) Infectivity of whole cell extracts from strains BY259 (ure2^fs), BY277 (URE2⁹⁰⁻³⁵⁴), and BY241 with [ure-o] or one of the three [URE3] variants, as determined by transformation into BY241. Protein extracts were transformed at the following concentrations (in mg/ml): ure2^fs, 0.5; URE2⁹⁰⁻³⁵⁴, 0.8; [ure-o], 1.3; [URE3] variant 1, 1.1; [URE3] variant 2, 1.3; [URE3] variant 3, 1.3. A control transformation was performed with 10 μM Ure2p filaments. Control transformations to ensure that the obtained [URE3] clones are indeed transformants and not cells remaining in the extracts are described in Supplementary data. (C) Spectrum of [URE3] variants following successful transformations in panel B. Five randomly chosen clones were streaked on 1/2 YPD plates and incubated at 30°C for 5 days. (D) Seeding experiment with whole cell extracts. Solutions of Ure2p and Ure2p⁹⁰⁻³⁵⁴ were incubated with or without agitation (on a vertical roller at 10 r.p.m.) at 4°C for 12 h with whole cell extracts from BY241 strains that were [ure-o] or contained one of the three [URE3] variants. The Ure2p to extract ratio was roughly 1/150 and corresponds to a Ure2p ratio of about 1/2 × 10⁶. The solutions were sonicated and transformed into BY241 at a concentration of 2 μM. For comparison, soluble Ure2p and Ure2p⁹⁰⁻³⁵⁴ were incubated in the same way and also transformed into BY241. (E) Spectrum of [URE3] variants following transformation of agitated seeding solutions in panel D.