], were recently described (6, 7). Thus, it is evident that protein inheritance is a widespread phenomenon, at least in lower eukaryotes.The discovery of prions in yeast occurred in different ways. Some (i.e., [PSI + ] and [URE3]) were long known as genetic determinants of mysterious nature until their prion nature was proposed (8). The others were revealed by purposeful screening of potentially prionogenic proteins and corresponding determinants. The prion-like determinant [ISP + ], described in our earlier work (9), belongs to the first group, because it was detected as a nonchromosomal antisuppressor in strains containing specific sup35 nonsense suppressor mutations and the nonsense mutations his7-1 (UAA) and lys2-87 (UGA (Fig. 1A). The Sup + phenotype cosegregated with Ura + in tetrads of the diploid that was obtained by crossing the sfp1Δ and [ISP + ] strains (Fig. 1B). These findings indicate either that [ISP + ] is a prion form of Sfp1 or that the change in phenotype was caused by an independent manifestation of the SFP1-null allele.To distinguish between these two possibilities, the sfp1Δ strain was transformed with the centromeric vector pRS315-SFP1. Introduction of the wild-type SFP1 allele did not change the phenotype of the sfp1Δ strain [i.e., the absolute majority (556 of 559) of transformants has retained the Sup + phenotype]. This fact suggests that the change of phenotype in the sfp1Δ strain was caused by [ISP + ] loss rather than phenotypic effects of the SFP1 deletion; otherwise, restoration of the Sup − phenotype would be observed. Notably, this loss was irreversible, because we have not observed a single example of Sup -clones appearing in the mitotic progeny of sfp1Δ strains in contrast to [isp -] strains obtained by GuHCl treatment, which produced Sup -clones at a high frequency (9). These results confirmed that SFP1 could be considered as a likely host gene for [ISP + ].

Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was proposed by Vitaly V. Kushnirov in the Michael D. Ter-Avanesyan's laboratory as a method to compare sizes of amyloid aggregates. Currently, this method is widely used for amyloid investigation, but mostly as a qualitative approach. In this work, we assessed the possibilities and limitations of the quantitative analysis of amyloid aggregate size distribution using SDD-AGE results. For this purpose, we used aggregates of two well-characterized yeast amyloid-forming proteins, Sup35 and Rnq1, and developed a protocol to standardize image analysis and process the result. A detailed investigation of factors that may affect the results of SDD-AGE revealed that both the cell lysis method and electrophoresis conditions can substantially affect the estimation of aggregate size. Despite this, quantitative analysis of SDD-AGE results is possible when one needs to estimate and compare the size of aggregates on the same gel, or even in different experiments, if the experimental conditions are tightly controlled and additional standards are used.

No abstract