Yeast prion variants are distinguishable based on intensity of the prion phenotype, stability or instability of prion propagation, sensitivity of prion stability to overproduction or deficiency of several chaperones and other cellular components and ability to overcome barriers to transmission between species 
– or even within species, the last documented here for transmission across the barriers found in wild strains of S. cerevisiae
. Yeast prion amyloids are all folded parallel in-register β-sheet structures 
, but within this architectural restraint, different prion variant structures are proposed to vary in the extent of the β-sheet structure (how much of the N and M domains are in β-sheet), the locations of the folds in the sheets and the association of protofilaments to form fibers.
We find that separation of prion variants based on sensitivity to intra-species barriers cuts across separation based on ‘strong’ vs ‘weak’ assessment of strength of prion phenotype. The four transmission variant types derived from the [PSI+
] in strain 779-6A were all strong [PSI+
], like the parent prion. Interestingly, the prions in wild strains were all weak [PSI+
], presumed to arise independently and thus not part of the same ‘prion cloud’, but fell into the same four transmission variant types. Likewise, two similarly ‘weak’ [PSI+
] variants showed different transmission across a barrier set up by deletions in the prion domain 
. These results show that prion variant uniformity is not demonstrated by showing uniformity of a single property (for example, colony color). It is unlikely that the variation in transmission barriers observed are due to a prion other than [PSI+
] because the sequences of Sup35p are involved, and no yeast prion is known to arise at a frequency high enough to explain our results.
After crossing an intraspecies barrier, we find that the [PSI+ref] examined is unstable in its new host, emphasizing the effectiveness of these barriers. We also find that the rare [PSI+] prions found in wild strains are, in most cases, sensitive to the intraspecies barriers, suggesting that these barriers have evolved to protect yeast from the detrimental effects of this prion.
] in strain 779-6A, with the reference Sup35p sequence, showed a reproducible strong preference for the reference sequence, transferring only very inefficiently to the Δ19 or E9 Sup35 backgrounds. However, simple mitotic growth of this strain resulted in the mitotic segregation of at least four variants distinguished by their abilities to cross intraspecies barriers. These variants were stable and reproducible with limited expansion of the corresponding clones, but following many generations of growth, each of those tested gave rise again to the same four general classes of subclones. Prion mutation is well documented in mammals and in yeast under selective conditions 
, and Weissmann's group has suggested that prions resistant to a drug can arise during prion propagation in tissue culture cells in the absence of the drug 
. We observe changes in the predominant prion variant under non-selective conditions in vivo. Selection only happens during the test, when cytoplasm is passed by cytoduction from the subclones to be tested to the recipient expressing one of the three Sup35p polymorphs. A new prion variant, recently described by Sharma and Liebman 
, may represent a phenomenon similar to that described here. Certain induced [PSI+
] clones continually gave off subclones that were a mixture of strong and weak variants, what the authors called “unspecified [PSI+
Although multiple de novo prion generation events in forming amyloid in vitro result in multiple prion variants on transfection into yeast, even a [PIN+
] cell generates [PSI+
] clones too rarely to explain our results as de novo prion generation. Rather, mis-templating must be the mechanism of generation of variant diversity that we are observing. Our results imply that there must be a finite rate of amyloid mis-templating that is not due to a mismatch of two prion protein sequences. In spite of extensive purification by mitotic growth and subcloning, we were unable to obtain a prion variant that was completely stable in its transmission pattern to polymorphs. These results are consistent with the ‘prion cloud’ hypothesis 
, in which it is supposed that even a prion variant purified by end-point titration consists of a major variant as well as an array of minor variants. This production of new prion variants during non-selective growth is analogous to the generation of RNA virus mutants during viral replication (reviewed in ref. 
), in which a cloud of sequence variants accumulate because of the error-prone nature of RNA-dependent RNA polymerases.
The segregation of a mixed prion population could be considered analogous to the segregation of differently marked plasmids with the same replicon. The latter situation has been carefully examined by Novick and Hoppenstadt 
, who find that the fraction of cells remaining with a mixture of plasmids is H
, where H0
is the starting fraction of mixed cells, N is the copy number of the plasmid, and n is the number of generations 
. Random replication of plasmids and equal partition at mitosis is assumed. One result of this treatment is that after N generations, H≈0.36 H0
The copy number in the case of yeast prions might be taken as the ‘seed number’ determined by the methods developed by Cox et al. 
, found to be ~20–120 for the strains examined. The assumption of equipartition is probably not accurate here, since yeast daughter cells are smaller than mother cells 
. Moreover, the sticky nature of amyloids might suggest that progeny filaments might stick to parent filaments exaggerating this effect. We have propagated our [PSI+
] strains for a number of generations comparable to the presumed copy number, so segregation of different prions is not surprising.
However, we find that even when we have apparently purified a variant, further non-selective growth and subcloning leads to further appearance of the full range of variants among the progeny (). This indicates that we are not only observing segregation, but also the (repeated) generation of variants during growth. While varying with respect to transmission, they remain ‘strong’ variants, suggesting that the structural differences responsible for this transmission barrier differ from those involved in the strong vs. weak differences. King has shown that residues 1–61 are sufficient to propagate strong vs weak prion strains 
, but the sequence differences among the Sup35 polymorphs are outside this area, and transmission variants may thus largely differ in the region C-terminal to the 1–61 area, perhaps a region with more variable structure. Other studies have indicated effects of this region on propagation of some prion variants 
, and β-sheet structure of Sup35NM amyloid extends throughout N and even into M