FIG. 5.
[PSI+] maintenance at low Hsp104 levels. (A) A representative tetratype tetrad obtained from dissection of GT84 (HSP104+/hsp104Δ) transformed with the [PMOD-HSP104] plasmid. WT, wild type. (B) Levels of the Hsp104 protein in the spore clones shown in panel A as well as those in the Ade+ papillae obtained from the hsp104Δ [PMOD-HSP104] spore clone. Protein amounts, determined by densitometry (as described in Materials and Methods), are relative to wild-type Hsp104 levels (100%). (C) Plasmid copy number analysis. DNA was isolated from the following three types of colonies, originated from the Ade+ papillae of the hsp104Δ [PMOD-HSP104] spore clone: (i) colonies retaining the persistent Ade+ phenotype (light pink); (ii) red-pink sectored Ade+ colonies; and (iii) Ade− colonies (dark red), originated from the same hsp104Δ [PMOD-HSP104] (Ade+ papillae) spore clone. Southern blot hybridization was performed on the HSP104 probe that identifies both the vestige sequence of the disrupted chromosomal copy of HSP104 (4.5-kb band, used as a loading control) and the plasmid-borne [PMOD-HSP104] (1.3-kb band). Higher intensity of suppression (lighter color) correlates with higher copy number.