Cell division without cell separation produces multicellular clusters in budding yeast. Two fundamental characteristics of these clusters are their size (the number of cells per cluster) and cellular composition: the fractions of cells with different phenotypes. However, we do not understand how different cellular features quantitatively influence these two phenotypes. Using cells as nodes and links between mother and daughter cells as edges, we model cluster growth and breakage by varying three parameters: the cell division rate, the rate at which intercellular connections break, and the kissing number (the maximum number of connections to one cell). We find that the kissing number sets the maximum possible cluster size. Below this limit, the ratio of the cell division rate to the connection breaking rate determines the cluster size. If links have a constant probability of breaking per unit time, the probability that a link survives decreases exponentially with its age. Modeling this behavior recapitulates experimental data. We then use this framework to examine synthetic,  differentiating clusters with two cell types, faster-growing germ cells and their somatic derivatives. The fraction of clusters that contain both cell types increases as either of two parameters increase: the kissing number and difference between the growth rate of germ and somatic cells. In a population of clusters, the variation in cellular composition is inversely correlated (r2=0.87) with the average fraction of somatic cells in clusters. Our results show how a small number of cellular features can control the phenotypes of multicellular clusters that were potentially the ancestors of more complex forms of multicellular development, organization, and reproduction.

The mating of fungi depends on pheromones that mediate communication between two mating types. Most species use short peptides as pheromones, which are either unmodified (e.g., a-factor in Saccharomyces cerevisiae) or C-terminally farnesylated (e.g., a-factor in S. cerevisiae). Peptide pheromones have been found by genetics or biochemistry in a small number of fungi, but their short sequences and modest conservation make it impossible to detect homologous sequences in most species. To overcome this problem, we used a four-step computational pipeline to identify candidate a-factor genes in sequenced genomes of the Saccharomycotina, the fungal clade that contains most of the yeasts: we require that candidate genes have a C-terminal prenylation motif, are fewer than 100 amino acids long, contain a proteolytic processing motif upstream of the potential mature pheromone sequence, and that closely related species contain highly conserved homologs of the potential mature pheromone sequence. Additional manual curation exploits the observation that many species carry more than one a-factor gene, encoding identical or nearly identical pheromones. From 332 Saccharomycotina genomes, we identified strong candidate pheromone genes in 241 genomes, covering 13 clades that are each separated from each other by at least 100 million years, the time required for evolution to remove detectable sequence homology among small pheromone genes. For one small clade, the Yarrowia, we demonstrated that our algorithm found the a-factor genes: deleting all four related genes in the a-mating type of Yarrowia lipolytica prevents mating.

The conserved regulon of Heat Shock Factor 1 in budding yeast contains chaperones

for general protein folding as well as Zinc Finger Protein Zpr1, whose essential role in

archaea and eukaryotes remains unknown. Here, we show that Zpr1 depletion causes

acute proteotoxicity driven by biosynthesis of misfolded eukaryotic translation

elongation factor 1A (eEF1A). Prolonged Zpr1 depletion leads to loss of eEF1A thereby

inducing the integrated stress response and inhibiting protein synthesis. Strikingly, we

show using two distinct biochemical reconstitution approaches that Zpr1 enables

eEF1A to achieve a conformational state resistant to protease digestion. Lastly, we use

a ColabFold model of the Zpr1-eEF1A complex to reveal a folding mechanism

mediated by Zpr1’s zinc finger and alpha helical hairpin structures. Our work uncovers

the long sought-after function of Zpr1 as a bespoke chaperone for one of the most

abundant proteins in the cell.

Comparisons of genomes of different species are used to identify lineage-specific genes, those genes that appear unique to one species or clade. Lineage-specific genes are often thought to represent genetic novelty that underlies unique adaptations. Identification of these genes depends not only on genome sequences, but also on inferred gene annotations. Comparative analyses typically use available genomes that have been annotated using different methods, increasing the risk that orthologous DNA sequences may be erroneously annotated as a gene in one species but not another, appearing lineage specific as a result. To evaluate the impact of such “annotation heterogeneity,” we identified four clades of species with sequenced genomes with more than one publicly available gene annotation, allowing us to compare the number of lineage-specific genes inferred when differing annotation methods are used to those resulting when annotation method is uniform across the clade. In these case studies, annotation heterogeneity increases the apparent number of lineage-specific genes by up to 15-fold, suggesting that annotation heterogeneity is a substantial source of potential artifact.

In haploid budding yeast, evolutionary adaptation to constitutive DNA replication stress alters three genome maintenance modules: DNA replication, the DNA damage checkpoint, and sister chromatid cohesion. We asked how these trajectories depend on genomic features by comparing the adaptation in three strains: haploids, diploids, and recombination deficient haploids. In all three, adaptation happens within 1000 generations at rates that are correlated with the initial fitness defect of the ancestors. Mutations in individual genes are selected at different frequencies in populations with different genomic features, but the benefits these mutations confer are similar in the three strains, and combinations of these mutations reproduce the fitness gains of evolved populations. Despite the differences in the selected mutations, adaptation targets the same three functional modules despite differences in genomic features, revealing a common evolutionary response to constitutive DNA replication stress.

Evolutionary adaptation to perturbations in DNA replication follows reproducible trajectories that lead to changes in three important aspects of genome maintenance: DNA replication, the DNA damage checkpoint, and sister chromatid cohesion. We asked how these trajectories depend on a population's genome architecture by testing whether ploidy or the ability to perform homologous recombination influence the evolutionary fate of the budding yeast, Saccharomyces cerevisiae, as it adapts to constitutive DNA replication stress, a condition that characterizes many cancer cells. In all three genome architectures, adaptation happens within 1000 generations at rates that are linearly correlated with the initial fitness defect of the ancestors. Which genes are mutated depends on the frequency at which mutations occur and the selective advantage they confer. The recombination-deficient strain amplifies adaptive chromosomal regions less often, whereas the selective advantage of loss-of-function mutations, such as those that inactivate the DNA damage checkpoint, is reduced in diploids because of the presence of a second, wild-type copy of the gene. Despite these differences, selection targets the same three functional modules in all three architectures, suggesting that genome architecture controls which genes are mutated but not which modules are modified.

Genes for which homologs can be detected only in a limited group of evolutionarily

related species, called “lineage-specific genes,” are pervasive: essentially every

lineage has them, and they often comprise a sizable fraction of the group’s total genes.

Lineage-specific genes are often interpreted as “novel” genes, representing genetic

novelty born anew within that lineage. Here, we develop a simple method to test an

alternative null hypothesis: that lineage-specific genes do have homologs outside of

the lineage that, even while evolving at a constant rate in a novelty-free manner, have

merely become undetectable by search algorithms used to infer homology. We show

that this null hypothesis is sufficient to explain the lack of detected homologs of a large

number of lineage-specific genes in fungi and insects. However, we also find that a

minority of lineage-specific genes in both clades are not well-explained by this noveltyfree

model. The method provides a simple way of identifying which lineage-specific

genes call for special explanations beyond homology detection failure, highlighting

them as interesting candidates for further study.

To grow and divide, cells must extract resources from dynamic and unpredictable environments. Many organisms use different metabolic strategies for distinct contexts. Budding yeast can produce ATP from carbon sources by mechanisms that prioritize either speed (fermentation) or yield (respiration). Withdrawing glucose from exponentially growing cells reveals variability in their ability to switch from fermentation to respiration. We observe two subpopulations of glucose-starved cells: recoverers, which rapidly adapt and resume growth, and arresters, which enter a shock state characterized by deformation of many cellular structures, including mitochondria. These states are heritable, and on high glucose, arresters grow and divide faster than recoverers. Recoverers have a fitness advantage during a carbon source shift but are less fit in a constant, high-glucose environment, and we observe natural variation in the frequency of the two states across wild yeast strains. These experiments suggest that bet hedging has evolved in budding yeast.

Sister chromatid cohesion essential for mitotic chromosome segregation is thought to involve the co-entrapment of sister DNAs within cohesin rings. Though cohesin can load onto chromosomes throughout the cell cycle, it normally only builds cohesion during S phase. A key question is whether cohesion is generated by conversion of cohesin complexes associate with un-replicated DNAs ahead of replication forks into cohesive structures behind them, or from nucleoplasmic cohesin that is loaded de novo onto nascent DNAs associated with forks, a process that would be dependent on cohesin’s Scc2 subunit. We show here that in S. cerevisiae, both mechanisms exist and that each requires a different set of non-essential replisome-associated proteins. Cohesion produced by cohesin conversion requires Tof1/Csm3, Ctf4 and Chl1 (TCCC) but not Scc2 while that created by Scc2-dependent de novo loading at replication forks requires the Ctf18-RFC complex. Though inactivation of either pathway individually merely reduces the efficiency of cohesion establishment, simultaneous inactivation resembles the effect of cohesin ablation and is lethal. The association of specific replisome proteins with different types of cohesion establishment opens the way to a mechanistic understanding of an aspect of DNA replication unique to eukaryotic cells.

Evolutionary novelty is difficult to define. It typically involves shifts in organismal or biochemical phenotypes that can be seen as qualitative as well as quantitative changes. In laboratory-based experimental evolution of novel phenotypes and the human domestication of crops, the majority of the mutations that lead to adaptation are loss of function mutations that impair or eliminate the function of genes rather than gain of function mutations that increase or qualitatively alter the function of proteins. I speculate that easier access to loss of function mutations has led them to play a major role in the adaptive radiations that occur when populations have access to many unoccupied ecological niches. I discuss four possible objections to this claim: that genes can only survive if they confer benefits to the organisms that bear them, antagonistic pleiotropy, the importance of pre-existing genetic variation in populations, and the danger that adaptation by breaking genes will, over long times, cause organisms to run out of genes.

Comparative genomics reveals an unexpected diversity in the molecular mechanisms underlying conserved cellular functions, such as DNA replication and cytokinesis. However, the genetic bases and evolutionary processes underlying this “molecular diversity” remain to be explained. Here, we review a tool to generate alternative mechanisms for conserved cellular functions and test hypotheses concerning the generation of molecular diversity: evolutionary repair experiments, in which laboratory microbial populations adapt in response to a genetic perturbation. We summarize the insights gained from evolutionary repair experiments, the spectrum and dynamics of compensatory mutations, and the alternative molecular mechanisms used to repair perturbed cellular functions. We relate these experiments to the modifications of conserved functions that have occurred outside the laboratory. We propose experimental strategies, especially those that establish a quantitative understanding of compensatory mutations and alternative molecular mechanisms, to improve evolutionary repair as a tool to explore the molecular diversity of life.