rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants

Abstract

The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae.

Keywords: CNV; Saccharomyces cerevisiae; lithium; rDNA; transformation.

Figure 1

Detection of rDNA copy number variation between wild-type clones and YKO deletion strains. (A) The 150 repeats of the wild-type S. cerevisiae rDNA locus comprise ∼1.4 Mb of Chromosome XII. Relative locations of the rDNA-flanking BamHI sites and single copy RNH203 probe sequence used for this study are as indicated. (B) Top: ethidium bromide-stained CHEF gels of 20 independent wild-type clones and 16 YKO strains (wild-type control indicated by arrow) shows positions of all 16 S. cerevisiae chromosomes, with chromosome XII generally being the largest. Bottom: Southern blots of the same gels hybridized with an RNH203 probe, a single copy gene on chromosome XII. (C) rDNA copy number distribution of 40 wild-type clones and the 434 YKO collection strains examined by CHEF gel analysis (Wilcoxon rank-sum: P < 0.0001). (D) rDNA copy number distribution of long-lived YKO strains and control YKO strains (Wilcoxon rank-sum: P = 0.196). CHEF, contour-clamped homogenous electric field; Chr, chromosome; EtBr, ethidium bromide; rDNA, ribosomal DNA; Tel, telomere; wt, wild-type; YKO, Yeast Knock-Out.

Figure 2

Assessing the causality of YKO mutations on rDNA copy number phenotypes: de novo gene deletion, serial passaging, and meiotic segregation. Southern blots of CHEF gels probed with RNH203 (chromosome XII). Wild-type and original YKO strain samples were included for comparison. (A) Left panel: chromosome XII sizes of chosen candidate strains taken directly from the YKO collection. Right panels: Southern blots showing chromosome XII sizes of multiple isolates from each de novo gene deletion: erv25Δ, bil1Δ, tpm1Δ, rpp1bΔ, and rpp2bΔ. (B) De novo gene deletion strains of hsp82Δ and rim1Δ were examined by CHEF gel electrophoresis over the course of 80–100 generations of growth. (C) Left panel: CHEF gel examining chromosome XII in the rim1Δ strain from the MATα YKO collection, wild-type MATa strain BY4741, and the resulting rim1Δ/RIM1 heterozygous diploid strain. Right panels: chromosome XII sizes of rim1Δ and RIM1 haploid strains generated from diploid sporulation examined after 40 and 150 generations. rDNA copy number randomly segregated in both rim1Δ and RIM1 spores and the inherited rDNA copy number was maintained after 150 generations in all but one rim1Δ strain, which reduced rDNA copy number. CHEF, contour-clamped homogenous electric field; rDNA, ribosomal DNA; wt, wild-type; YKO, Yeast Knock-Out.

Figure 3

The Magic Marker haploid deletion collection exhibits rDNA copy number heterogeneity. (A) Strains from the Magic Marker collection were inoculated directly from the library wells into 96-well plates, grown overnight, and collected for CHEF gel and Southern blot analysis. Multiple chromosome XII bands were present in many Magic Marker collection strains such as pta1Δ, spo7Δ, fun12Δ, and pam18Δ. (B) Single colonies were isolated from Magic Marker strains for CHEF gel analysis. Each colony from strains pta1Δ or spo7Δ possessed a single chromosome XII band, whereas fun12Δ or pam18Δ colonies each maintained more than one chromosome XII band. (C) Flow cytometry analysis of strains from the Magic Marker collection cultures indicate that the pta1Δ and spo7Δ strains were haploid while fun12Δ and pam18Δ were diploid. CHEF, contour-clamped homogenous electric field; rDNA, ribosomal DNA; wt, wild-type.

Figure 4

rDNA copy number variants are generated during transformation after lithium acetate exposure. (A) CHEF gel electrophoresis of 19 single colonies isolated from an untreated asynchronous culture, after transformation with the plasmid pRS426, or after mock transformation without selectable DNA. A common untreated control sample was run on each gel (arrows). (B) The plots show chromosomes XII and IV variation in wild-type untreated control isolates, YKO collection strains, de novo gene deletion transformants, plasmid pRS426 transformants, and mock transformants (*** P < 0.0001 in comparison to the untreated wild-type controls). The distribution of chromosome IV sizes indicated that there was little variation in this chromosome between isolates (plotted on the same scale). (C) The plots show chromosomes XII and IV variation in isolates from different stages of a mock transformation protocol. Each transformation group was compared to untreated controls [Wilcoxon Rank-Sum: P < 0.0001 (***), < 0.01 (**), < 0.05 (*)]. CHEF, contour-clamped homogenous electric field; Chr., chromosome; LiAc, lithium acetate; PEG, polyethylene glycol; rDNA, ribosomal DNA; ssDNA, single-stranded DNA; TE, Tris-EDTA; wt, wild-type; YKO, Yeast Knock-Out.