The chromosomal context for <i>SUL1</i> amplification.

<p>(A) The inverted repeat sequences in <i>CTP1</i> and <i>PCA1</i> that define the breakpoints of a specific <i>SUL1</i> amplification event <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002016#pgen.1002016-Araya1" target="_blank">[7]</a>. (B) The structure of the wild type <i>SUL1</i> locus that includes the nearby origin of replication, <i>ARS228</i>. (C) The inferred structure of the head-to-head/tail-to-tail 5× <i>SUL1</i> amplification product recovered after selective growth of a haploid yeast strain in medium limiting for sulfur.</p

The Fitness Consequences of Aneuploidy Are Driven by Condition-Dependent Gene Effects

<div><p>Aneuploidy is a hallmark of tumor cells, and yet the precise relationship between aneuploidy and a cell’s proliferative ability, or cellular fitness, has remained elusive. In this study, we have combined a detailed analysis of aneuploid clones isolated from laboratory-evolved populations of <i>Saccharomyces cerevisiae</i> with a systematic, genome-wide screen for the fitness effects of telomeric amplifications to address the relationship between aneuploidy and cellular fitness. We found that aneuploid clones rise to high population frequencies in nutrient-limited evolution experiments and show increased fitness relative to wild type. Direct competition experiments confirmed that three out of four aneuploid events isolated from evolved populations were themselves sufficient to improve fitness. To expand the scope beyond this small number of exemplars, we created a genome-wide collection of >1,800 diploid yeast strains, each containing a different telomeric amplicon (Tamp), ranging in size from 0.4 to 1,000 kb. Using pooled competition experiments in nutrient-limited chemostats followed by high-throughput sequencing of strain-identifying barcodes, we determined the fitness effects of these >1,800 Tamps under three different conditions. Our data revealed that the fitness landscape explored by telomeric amplifications is much broader than that explored by single-gene amplifications. As also observed in the evolved clones, we found the fitness effects of most Tamps to be condition specific, with a minority showing common effects in all three conditions. By integrating our data with previous work that examined the fitness effects of single-gene amplifications genome-wide, we found that a small number of genes within each Tamp are centrally responsible for each Tamp’s fitness effects. Our genome-wide Tamp screen confirmed that telomeric amplifications identified in laboratory-evolved populations generally increased fitness. Our results show that Tamps are mutations that produce large, typically condition-dependent changes in fitness that are important drivers of increased fitness in asexually evolving populations.</p></div

High-Throughput Identification of Adaptive Mutations in Experimentally Evolved Yeast Populations

<div><p>High-throughput sequencing has enabled genetic screens that can rapidly identify mutations that occur during experimental evolution. The presence of a mutation in an evolved lineage does not, however, constitute proof that the mutation is adaptive, given the well-known and widespread phenomenon of genetic hitchhiking, in which a non-adaptive or even detrimental mutation can co-occur in a genome with a beneficial mutation and the combined genotype is carried to high frequency by selection. We approximated the spectrum of possible beneficial mutations in <i>Saccharomyces cerevisiae</i> using sets of single-gene deletions and amplifications of almost all the genes in the <i>S</i>. <i>cerevisiae</i> genome. We determined the fitness effects of each mutation in three different nutrient-limited conditions using pooled competitions followed by barcode sequencing. Although most of the mutations were neutral or deleterious, ~500 of them increased fitness. We then compared those results to the mutations that actually occurred during experimental evolution in the same three nutrient-limited conditions. On average, ~35% of the mutations that occurred during experimental evolution were predicted by the systematic screen to be beneficial. We found that the distribution of fitness effects depended on the selective conditions. In the phosphate-limited and glucose-limited conditions, a large number of beneficial mutations of nearly equivalent, small effects drove the fitness increases. In the sulfate-limited condition, one type of mutation, the amplification of the high-affinity sulfate transporter, dominated. In the absence of that mutation, evolution in the sulfate-limited condition involved mutations in other genes that were not observed previously—but were predicted by the systematic screen. Thus, gross functional screens have the potential to predict and identify adaptive mutations that occur during experimental evolution.</p></div

Driver mutations.

<p>(<b>A</b>) Boxplot representing the ratio of driver to total mutations detected in evolved clones and populations. The significance of the difference between clones and populations was estimated using a Wilcoxon-ranked test.</p

Alternative beneficial mutations are selected in the absence of the main driver.

<p>(<b>A</b>) The copy number of <i>SUL1</i> was assessed using qPCR of samples taken from two independent experiments in which <i>SUL1</i> was not amplified (green and pink) and compared with previously published data from wild-type strains (in grey) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006339#pgen.1006339.ref040" target="_blank">40</a>]. (<b>B</b>) The fitness coefficient as compared to the ancestral strain of population samples at generations 5, 50, and 200 and the fitness of two clones isolated at generation 200. (<b>C</b>) A small deletion (~5kb) encompassing four genes on chromosome IV was detected in a population from one experiment (between brackets); polyT sequences are present at the breakpoints. The colors of the boxes represent the orientation of the genes (yellow: gene on the Watson strand, grey: genes on the Crick strand). (<b>D</b>) Fitness coefficients of the deletion strains <i>ipt1</i>Δ and <i>snf11</i>Δ and those of both deletion strains complemented with <i>IPT1</i> or <i>SNF11</i> on a low-copy plasmid grown in sulfate limitation.</p

Point mutations in evolved clones identified by WGS.

<p>Point mutations in evolved clones identified by WGS.</p

The genetic basis for aneuploidy’s effect on cellular fitness.

<p>A) The fitness landscape explored by Tamps is much broader than that explored by single-gene amplification. CEN = fitness density distribution of a genome-wide collection of yeast strains with each gene cloned into a low-copy-number CEN plasmid (raw data from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.ref034" target="_blank">34</a>], <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s010" target="_blank">S2 Table</a>), 2 μ = fitness density distribution of a genome-wide of yeast strains with each gene cloned into a high-copy-number 2 μ plasmid (raw data from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.ref034" target="_blank">34</a>], <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s010" target="_blank">S2 Table</a>), Tamp = fitness density distribution of Tamp as determined by the Tamp screen described in this study (raw data from this study <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s014" target="_blank">S6 Table</a>). B) Tamps are more pleiotropic than single-gene amplifications. Pleiotropy is defined here as the between-condition variance in fitness [See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s029" target="_blank">S1 Text</a>]. CEN = density distribution of variance in fitness of a genome-wide collection of yeast strains with each gene cloned into a low-copy-number CEN plasmid (raw data from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.ref034" target="_blank">34</a>], <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s010" target="_blank">S2 Table</a>), 2 μ = density distribution of variance in fitness of a genome-wide collection of yeast strains with each gene cloned into a high-copy-number 2 μ plasmid (raw data from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.ref034" target="_blank">34</a>], <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s010" target="_blank">S2 Table</a>), Tamp = density distribution of variance in fitness of Tamps as determined by the Tamp screen described in this study (raw data from this study <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s014" target="_blank">S6 Table</a>). C) The average fitness effects of all single-gene amplifications overlapping a fitness breakpoint is greater for Downsteps than for Upsteps (unpaired, two-tailed <i>t</i> test, <i>p</i> = 0.008). Raw data for individual single-gene amplifications is from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.ref034" target="_blank">34</a>], <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s010" target="_blank">S2 Table</a>. Raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s025" target="_blank">S17 Table</a>. D) Upsteps in glucose- and phosphate-limiting conditions are enriched for genes mutated in glucose- and phosphate-limited evolution experiments. Raw data can be found in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.ref034" target="_blank">34</a>], <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s012" target="_blank">S4 Table</a>; from this study, Upstep and Downstep genes can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s025" target="_blank">S17 Table</a>. E) Our Tamp screen predicted amplification of the left arm of chromosome XIV to increase fitness under glucose-limiting conditions. Our Tamp screen identified six Downsteps along this region, all but one of which have a candidate driver gene associated with them. The yellow and blue boxes represent the average relative fitness, positive or negative, respectively, due to amplification of the region enclosed. Raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s014" target="_blank">S6 Table</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s017" target="_blank">S9 Table</a>, and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s019" target="_blank">S11 Table</a>.</p

Experimental design for genome-wide screen for the fitness effects of Tamps.

<p>A) A genome-wide pool of telomeric amplicon strains (Tamps) was constructed. Each Tamp initiates at the <i>KanMX</i> cassette and extends to the proximal telomere, creating a strain that has two chromosomal copies (2N) at most genomic locations, one copy (1N) in the region replaced by the <i>KanMX</i> cassette in the deletion collection, and three copies (3N) at locations telomeric of the deleted gene. The Tamp BC and a portion of KanMX are also present at 2 copies. Each strain contains two barcodes: one identifying the Tamp and a second identifying the unique biological replicate. A third barcode was incorporated during the generation of the barcode sequencing libraries which allowed for multiplexing of experimental samples. Large black arrows represent telomeres; large black circles represent centromeres. The primers represent the barseq primers used to create libraries for sequencing. B) Genome-wide pooled competition experimental design.</p

Distribution of the fitness effects of single-gene amplifications and deletions.

<p>Fitness distributions of the five yeast collections in glucose-limited, sulfate-limited, and phosphate-limited continuous-growth conditions. The fitness of each strain is shown as a small line. The fitness distribution of the control collection is shown in grey. The thick black line represents the mean. Dashed grey lines indicate the cutoff of ±10% measured using the control collection.</p

Aneuploidy variably affects fitness of evolved clones.

<p>The individual fitness effects of point mutations and aneuploid events were determined for all mutations identified in the evolved clones S8c2 (A), P5c3 (B), and P6c1 (C). The supernumerary chromosome(s) in each clone are labeled by their identifying translocation or, in the case of the chromosome XIII disomy, as “Chr XIII.” Aneuploid and euploid clones are color-coded according to the legend. As described in the text, “All” for P5c3 and P6c1 and “None” for P5c3 represent backcrossed segregants that contain all or none of the mutations present in the original evolved clone. “No <i>SUL1</i> amp” for S8c2 is a backcrossed segregant that contains all the mutations identified in S8c2 except for the <i>SUL1</i> amplicon. Raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002155#pbio.1002155.s010" target="_blank">S2 Table</a>.</p