Most, but not all, yeast strains in the deletion library contain the [PIN(+)] prion

Abstract

The yeast deletion library is a collection of over 5100 single gene deletions that has been widely used by the yeast community. The presence of a non-Mendelian element, such as a prion, within this library could affect the outcome of many large-scale genomic studies. We previously showed that the deletion library parent strain contained the [PIN(+)] prion. [PIN(+)] is the misfolded infectious prion form of the Rnq1 protein that displays distinct fluorescent foci in the presence of RNQ1-GFP and exists in different physical conformations, called variants. Here, we show that over 97% of the library deletion strains are [PIN(+)]. Of the 141 remaining strains that have completely (58) or partially (83) lost [PIN(+)], 139 deletions were able to efficiently maintain three different [PIN(+)] variants despite extensive growth and storage at 4 degrees C. One strain, cue2Delta, displayed an alteration in the RNQ1-GFP fluorescent shape, but the Rnq1p prion aggregate shows no biochemical differences from the wild-type. Only strains containing a deletion of either HSP104 or RNQ1 are unable to maintain [PIN(+)], indicating that 5153 non-essential genes are not required for [PIN(+)] propagation.

Figure 1. Cytoduction using the kar1 plasmid donor

A. Each chromosome of the kar1 plasmid donor (top left) contains a wild type K. lactis URA3 gene and a neighboring GAL1 promoter that has been cloned adjacent to the centromere (arrow, Reid et al., 2008). The [PIN⁺] (small squares) lys2Δ kar1 plasmid donor that contains the LEU2 RNQ1:GFP plasmid forms heterokaryons (center) when mated to the LYS2 leu2Δ deletion library recipient strain (top right). While the cytoplasmic contents of donor and recipient cells mix, nuclear fusion is inhibited by the presence of the kar1Δ15 allele in the donor nucleus (see materials and methods). Upon plating on Gal-Lys-Leu, transcription from the GAL1 promoter through the centromeres destabilizes the chromosomes from the donor nucleus while −Lys selects against the donor cells and −Leu selects for the plasmid originally in the donor cell. Successive growth on media containing 5′ FOA (see part C) selects against any remaining chromosomes from the donor. The resulting cytoductants contain the Lys⁺ recipient nucleus with no donor cell chromosomes and the LEU2 RNQ1:GFP plasmid from the donor cell (bottom). B) A thin lawn of the kar1 plasmid donor was grown overnight, and the candidate strains were directly pinned onto the lawn and mated for six to eight hours. C). Mated cells were velveteen replicated onto Gal-Leu-Lys media and allowed to grow for five to seven days. While pilot studies have shown that the majority of cells within the spot are cytoductants, small amounts of donors and diploids were detected (data not shown). To eliminate remaining donors and diploids, cells were spotted again on Gal-Leu-Lys, and then spotted onto Gal-Leu-Lys+FOA followed by SD-Leu-Lys+FOA to remove any cytoductants carrying donor chromosomes from the population.

Figure 1. Cytoduction using the kar1 plasmid donor

A. Each chromosome of the kar1 plasmid donor (top left) contains a wild type K. lactis URA3 gene and a neighboring GAL1 promoter that has been cloned adjacent to the centromere (arrow, Reid et al., 2008). The [PIN⁺] (small squares) lys2Δ kar1 plasmid donor that contains the LEU2 RNQ1:GFP plasmid forms heterokaryons (center) when mated to the LYS2 leu2Δ deletion library recipient strain (top right). While the cytoplasmic contents of donor and recipient cells mix, nuclear fusion is inhibited by the presence of the kar1Δ15 allele in the donor nucleus (see materials and methods). Upon plating on Gal-Lys-Leu, transcription from the GAL1 promoter through the centromeres destabilizes the chromosomes from the donor nucleus while −Lys selects against the donor cells and −Leu selects for the plasmid originally in the donor cell. Successive growth on media containing 5′ FOA (see part C) selects against any remaining chromosomes from the donor. The resulting cytoductants contain the Lys⁺ recipient nucleus with no donor cell chromosomes and the LEU2 RNQ1:GFP plasmid from the donor cell (bottom). B) A thin lawn of the kar1 plasmid donor was grown overnight, and the candidate strains were directly pinned onto the lawn and mated for six to eight hours. C). Mated cells were velveteen replicated onto Gal-Leu-Lys media and allowed to grow for five to seven days. While pilot studies have shown that the majority of cells within the spot are cytoductants, small amounts of donors and diploids were detected (data not shown). To eliminate remaining donors and diploids, cells were spotted again on Gal-Leu-Lys, and then spotted onto Gal-Leu-Lys+FOA followed by SD-Leu-Lys+FOA to remove any cytoductants carrying donor chromosomes from the population.

Figure 2. All candidate strains, except for hsp104Δ and rnq1Δ, maintained three variants of [PIN⁺]

Medium [PIN⁺] (left), high [PIN⁺] (middle) or the library [PIN⁺] (right) was cytoduced into each candidate strain, along with the RNQ1:GFP inducible plasmid. Strains were induced for 16 hours with copper. Wild type strains (trp1Δ) show punctate fluorescence with all variants. rad6Δ and mso1Δ are representative phenotypes of 138 candidate strains that maintained [PIN⁺]. A library strain (YKL090w) containing a deletion of the cue2 gene shows spider-like fluorescence regardless of the [PIN⁺] variant.