Large chromosomal amplicons and deletions or copy number variation (CNV) are found in many different cancers. However, the inheritance, stability, and fixation of these amplicons and deletions within a population are widely debated and poorly understood. In an effort to better understand how the stability of this large chromosomal rearrangement, I analyzed the stability of a large segmental amplification in the yeast S. cerevisiae. This amplification is commonly observed in clones evolved under sulfate-limited conditions and contains the sulfate transporter SUL1. This amplification is an important adaptive strategy used by the cells to improve their ability to extract the limited supply of sulfate available in the media. High copy number of this gene confers a competitive fitness advantage over other clones that have only one copy of the gene. We used a GFP marker integrated next to SUL1 to differentiate green clones, which contain a single copy of the SUL1 gene, from “super green” clones that have multiple copies of the gene. A super green evolved clone was grown in steady state growth vessels for ~30 generations in media that is non-selective for adaptation and our selective media as a control. I monitored the population using flow cytometry and quantitative PCR to find clones that have lost the amplification. These green clones were isolated using flow sorting and then had their genome sequenced to look at the scar that was left behind. This tells us how efficiently the cells can remove amplifications without causing detrimental effects to their fitness.

We are interested in identifying the spectrum of mutations that arise when performing the same evolution experiment many times. We have previously grown 32 replicates of haploid yeast cells in continuous cultures under constant sulfur limiting environments for 300 generations. Mutants with increased fitness will increase in prevalence and can be characterized using an array of comparative genomic hybridization (aCGH) or next generation sequencing (NGS). To date we have used aCGH on a subset of our evolutions, and in 16/16 cultures, found amplification of SUL1, a high affinity sulfate ion transporter gene used by the cells to aid in increasing import of sulfate. We are also interested in identifying alternative means for adaptation in this environment and have evolved a a strain in which we'd knocked out SUL1 (sul1Δ). With this background, we observed SUL2 amplification in 4/4 cases in the absence of SUL1. A second focus of our work is to track changes in fitness as specific mutations arise. To assay fitness, we periodically compete an evolving culture against a Green Fluorescent Protein (GFP) expressing ancestral strain of yeast. This work has shown that the dynamics of adaptation of wt and sul1Δ yeast differ, which suggests very different fitness landscapes during the evolution for these two genotypes. Collectively these data show that SUL1 amplification is a first target of evolutionary selection and that in its absence, amplification of SUL2 becomes the primary means for adaptation in constant sulfate limitation.