Changes in the distribution of fitness effects and adaptive mutational spectra following a single first step towards adaptation.

Historical contingency and diminishing returns epistasis have been typically studied for relatively divergent genotypes and/or over long evolutionary timescales. Here, we use Saccharomyces cerevisiae to study the extent of diminishing returns and the changes in the adaptive mutational spectra following a single first adaptive mutational step. We further evolve three clones that arose under identical conditions from a common ancestor. We follow their evolutionary dynamics by lineage tracking and determine adaptive outcomes using fitness assays and whole genome sequencing. We find that diminishing returns manifests as smaller fitness gains during the 2nd step of adaptation compared to the 1st step, mainly due to a compressed distribution of fitness effects. We also find that the beneficial mutational spectra for the 2nd adaptive step are contingent on the 1st step, as we see both shared and diverging adaptive strategies. Finally, we find that adaptive loss-of-function mutations, such as nonsense and frameshift mutations, are less common in the second step of adaptation than in the first step

Adaptive roles of SSY1 and SIR3 during cycles of growth and starvation in Saccharomyces cerevisiae populations enriched for quiescent or nonquiescent cells

Over its evolutionary history, Saccharomyces cerevisiae has evolved to be well-adapted to fluctuating nutrient availability. In the presence of sufficient nutrients, yeast cells continue to proliferate, but upon starvation haploid yeast cells enter stationary phase and differentiate into nonquiescent (NQ) and quiescent (Q) cells. Q cells survive stress better than NQ cells and show greater viability when nutrient-rich conditions are restored. To investigate the genes that may be involved in the differentiation of Q and NQ cells, we serially propagated yeast populations that were enriched for either only Q or only NQ cell types over many repeated growth–starvation cycles. After 30 cycles (equivalent to 300 generations), each enriched population produced a higher proportion of the enriched cell type compared to the starting population, suggestive of adaptive change. We also observed differences in each population’s fitness suggesting possible tradeoffs: clones from NQ lines were better adapted to logarithmic growth, while clones from Q lines were better adapted to starvation. Whole-genome sequencing of clones from Q- and NQ-enriched lines revealed mutations in genes involved in the stress response and survival in limiting nutrients (ECM21, RSP5, MSN1, SIR4, and IRA2) in both Q and NQ lines, but also differences between the two lines: NQ line clones had recurrent independent mutations affecting the Ssy1p-Ptr3p-Ssy5p (SPS) amino acid sensing pathway, while Q line clones had recurrent, independent mutations in SIR3 and FAS1. Our results suggest that both sets of enriched-cell type lines responded to common, as well as distinct, selective pressures

Novel Interactions between Actin and the Proteasome Revealed by Complex Haploinsufficiency

Saccharomyces cerevisiae has been a powerful model for uncovering the landscape of binary gene interactions through whole-genome screening. Complex heterozygous interactions are potentially important to human genetic disease as loss-of-function alleles are common in human genomes. We have been using complex haploinsufficiency (CHI) screening with the actin gene to identify genes related to actin function and as a model to determine the prevalence of CHI interactions in eukaryotic genomes. Previous CHI screening between actin and null alleles for non-essential genes uncovered ∼240 deleterious CHI interactions. In this report, we have extended CHI screening to null alleles for essential genes by mating a query strain to sporulations of heterozygous knock-out strains. Using an act1Δ query, knock-outs of 60 essential genes were found to be CHI with actin. Enriched in this collection were functional categories found in the previous screen against non-essential genes, including genes involved in cytoskeleton function and chaperone complexes that fold actin and tubulin. Novel to this screen was the identification of genes for components of the TFIID transcription complex and for the proteasome. We investigated a potential role for the proteasome in regulating the actin cytoskeleton and found that the proteasome physically associates with actin filaments in vitro and that some conditional mutations in proteasome genes have gross defects in actin organization. Whole-genome screening with actin as a query has confirmed that CHI interactions are important phenotypic drivers. Furthermore, CHI screening is another genetic tool to uncover novel functional connections. Here we report a previously unappreciated role for the proteasome in affecting actin organization and function

Paths to adaptation under fluctuating nitrogen starvation: The spectrum of adaptive mutations in Saccharomyces cerevisiae is shaped by retrotransposons and microhomology-mediated recombination.

There are many mechanisms that give rise to genomic change: while point mutations are often emphasized in genomic analyses, evolution acts upon many other types of genetic changes that can result in less subtle perturbations. Changes in chromosome structure, DNA copy number, and novel transposon insertions all create large genomic changes, which can have correspondingly large impacts on phenotypes and fitness. In this study we investigate the spectrum of adaptive mutations that arise in a population under consistently fluctuating nitrogen conditions. We specifically contrast these adaptive alleles and the mutational mechanisms that create them, with mechanisms of adaptation under batch glucose limitation and constant selection in low, non-fluctuating nitrogen conditions to address if and how selection dynamics influence the molecular mechanisms of evolutionary adaptation. We observe that retrotransposon activity accounts for a substantial number of adaptive events, along with microhomology-mediated mechanisms of insertion, deletion, and gene conversion. In addition to loss of function alleles, which are often exploited in genetic screens, we identify putative gain of function alleles and alleles acting through as-of-yet unclear mechanisms. Taken together, our findings emphasize that how selection (fluctuating vs. non-fluctuating) is applied also shapes adaptation, just as the selective pressure (nitrogen vs. glucose) does itself. Fluctuating environments can activate different mutational mechanisms, shaping adaptive events accordingly. Experimental evolution, which allows a wider array of adaptive events to be assessed, is thus a complementary approach to both classical genetic screens and natural variation studies to characterize the genotype-to-phenotype-to-fitness map

Actin staining of complex hemizygotes.

<p><i>ACT1/act1Δ::Nat^R</i> hemizygotes carrying additional hemizygous mutations for (A) <i>yal066wΔ::kan^R</i> (control gene deletion), (B) <i>pfy1</i>Δ<i>::kan^R</i>, (C) <i>rpn5</i>Δ<i>::kan^R</i>, and (D) <i>tub1</i>Δ<i>::kan^R</i> were fixed and stained with rhodamine phalloidin after growth at 30° in YPD medium.</p

The structure of the proteasome and the locations of subunits whose null alleles are CHI with <i>act1Δ</i>.

<p>(A) Cartoon of the structure of the 26S, doubly capped proteasome showing the general locations of subunits whose null alleles are CHI (bold and underlined) or are not CHI with <i>act1Δ</i>; based on cryo-EM reconstructions of the 26S proteasome <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002288#pgen.1002288-Walz1" target="_blank">[47]</a>. (B) and (C) Surface rendering of the 20S core proteasome X-ray structure (1RYP.pdb; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002288#pgen.1002288-Groll1" target="_blank">[25]</a>); panels are rotated 180° with respect to each other. Proteins whose null alleles are CHI with <i>act1Δ</i> have been rendered in color: red for Scl1p, gold for Pre10p, orange for Pre5p, teal for Pup1p, light blue for Pre1p, and dark blue for Pre2p. Rendering was performed with Chimera (<a href="http://www.cgl.ucsf.edu/chimera" target="_blank">http://www.cgl.ucsf.edu/chimera</a>).</p

Growth and actin organization defects in conditional mutants of proteasome components.

<p>(A) Growth of strains carrying temperature sensitive alleles of proteasome genes were monitored and compared to the growth of a wild-type strain (BY4741) in a TECAN microplate reader at 37°C for 24 hr. (B and C) Strains carrying Ts⁻ alleles in proteasome component genes were grown to mid-log, shifted to 37°C for 2 hr, fixed, stained with rhodamine phalloidin and visualized by fluorescence microscopy. (B) Proteasome mutant strains with actin organization defects. (C) Proteasome mutant strains that do not have actin organization defects.</p

Strains used in this study.

<p>Strains used in this study.</p

Cells treated with the proteasome inhibitor MG132 have normal actin cytoskeleton organization.

<p>(A) To facilitate drug permeability or accumulation, <i>erg6Δ</i> and <i>pdr5Δ</i> strains were grown to mid-log, treated with 100 µM MG132 for 2 hr, fixed, stained with rhodamine phalloidin and visualized by fluorescence microscopy. (B) Wild-type (BY4742), <i>erg6Δ</i>, and <i>pdr5Δ</i> cells were treated with 50 µM MG132 and protein samples were isolated 0, 30, 75, and 120 min after MG132 addition to the medium. Protein concentrations were determined by Bradford assays and ∼45 µg of protein were separated by SDS-PAGE and analyzed by Western blotting with an anti-ubiquitin antibody.</p