Biology subjects

Dunham, M. J.

Publications and source records attributed to Dunham, M. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Beyond ERCs: exploring catastrophic forms of rDNA instability in aging yeast

Single-celled organisms grown in identical conditions have variable life spans. Identifying the factors that drive the inherent variability in life span is crucial for our understanding of aging at a fundamental level. Here, we revisit the role of chromosome XII instability as a source of life span variability in aging populations of the budding yeast, Saccharomyces cerevisiae. We followed populations of mother cells as they aged and quantified changes in karyotype, DNA content, and aberrant DNA structures, including the production of extrachromosomal rDNA circles (ERCs). We found that cells massively amplified their rDNA both as ERCs and as a structural form that could not be resolved on CHEF gels. We propose a model describing how these unresolved structures are generated. Our model, which we call CICR (Catastrophic IntraChromosomal Recombination), describes the consequences of recombination between repeats of different replication status. At the completion of replication, when all other replication forks have successfully terminated, CICR events leave behind a single, unopposed replication fork in a branched form of Chr XII that has profound consequences during mitosis and/or subsequent cycles. This form of instability within the ribosomal DNA can lead to a myriad of toxic recombination products that may contribute to the life span variability in isogenic populations of aging yeast. AUTHOR SUMMARYAging is an uphill battle against entropy. The wear and tear on biological processes in cells require surveillance mechanisms and repair processes to stave off the inevitable. A cells genome is constantly under external and internal insults that can ultimately lead to death. Known events that impact a cells DNA are damage from free radicals (produced by the very oxygen needed for survival), shortening of the caps on chromosomes (called telomeres) every time a cell divides, and mutations that accumulate over time that affect essential cell processes. Yeast is an excellent tool to examine specific causes of aging because of its asymmetric mode of cell division: a mother cell can divide a limited, but variable number of times before senescing. We have isolated cohorts of mother cells, examined their genomes over the course of their life span and find a new intrinsic insult that the genome must endure. We find, in addition to the accumulation of extrachromosomal copies of the genes that encode the RNA portion of ribosomes (rDNA), that cells accumulate a toxic product of genetic recombination within the rDNA. The chromosome that contains the rDNA becomes branched and/or fragmented which leads to cell death.

genetics↗

bioRxiv2026

Telomeric amplicons of SUL1 and Y' in yeast are generated by microhomology-mediated break induced replication occurring in cis

Gene amplification is a potent driver of evolution and is thought to contribute to genetic diseases, including cancer. The yeast Saccharomyces cerevisiae is a powerful organism for understanding amplification mechanisms. When yeast is grown long term in sulfate-limiting chemostats, amplification of the gene that encodes the primary sulfate transporter, SUL1, is a common outcome. Here we describe a form of SUL1 amplification in which multiple copies of the right terminal region of chromosome II are appended in tandem to a native telomere. We find this form of amplicon when we delete the origin of replication next to SUL1 or delete a variety of genes involved in DNA metabolism. It is the only form of amplification found in a yku70{Delta} mutant suggesting that unprotected telomeres are involved. We propose that these terminal addition events occur when the unprotected 3 G1-3T telomeric sequence invades a short ([~]7 bp) internal telomere sequence (ITS) to begin a form of microhomology-mediated break-induced replication (mmBIR) that has been documented in type-I survivors of telomerase mutants. In addition to amplification of the right end of chromosome II we also find that telomeres containing the sub-telomeric repeat Y experience similar tandem amplification events and show that their formation is reduced in a pol32{Delta} mutant, a gene required for mmBIR. Within individual amplicons the ITSs and Ys are nearly identical, suggesting that the multiple copies of the amplified region are generated in a single mmBIR event that we describe as pseudo-rolling circle mmBIR. A similar amplification event at the P-telomere of human chromosome 18 has four copies of a [~]54 kb region separated by ITSs of nearly identical size. This finding suggests that these additional copies of the terminal fragment of human chromosome 18 arose by the same pseudo-rolling circle mechanism, perhaps during a period of telomeric stress. AUTHOR SUMMARYThe human genome is peppered with duplicates (or higher numbers) of segments that are located at sites both nearby and distant from the original, ancestral segments. These Copy Number Variants, or CNVs, appear to be highly variable among different individuals and are being examined with great interest as potential loci associated with genetic disease. Experimentally determining how these CNVs arise and become distributed across the genome is nearly impossible using humans. We are using budding yeast as the model organism to explore mechanisms of gene amplification. In this work we show that by destabilizing the ends of yeast chromosomes (telomeres) or by interfering with genes involved in the replication, repair, or recombination of DNA results in a specific form of segmental copy number increase that is initiated at telomeres. We propose that a telomere invades an internal chromosome site and sets up a pseudo-circular template for conservative DNA replication. The outcome is a chromosome with multiple, identical copies of a chromosome end arranged in tandem. We believe that it is also a major mechanism used by cells to repair telomeres that have become eroded during aging.

genetics↗

bioRxiv2025

Using Experimental Evolution to Correct Mother-Daughter Separation Defects in Brewing Yeast

The budding yeast, Saccharomyces cerevisiae, is the workhorse of the brewing industry. Brewers have domesticated a vast array of different strains with traits that complement the beers they wish to brew. Yet, some domesticated strains also harbor traits that are undesirable. One example of an undesirable trait in brewing strains is the mother-daughter separation defect (MDSD). MDSDs are a reproductive flaw present in a widely used brewing strain, London Ale III. MDSDs cause cells to form large clusters, possibly leading to the known requirement for more headspace during London Ale III fermentations that result in a lower fermentative yield. Because MDSDs can be caused by mutations to a number of genes, targeted genetic approaches to reduce MDSDs are experimentally challenging, especially for a tetraploid strain like London Ale III. To improve MDSDs in this strain, we employed experimental evolution and passaged populations from three biological replicates for over 200 generations to generate three independent evolved strains that form fewer clusters than the ancestral strain, as seen by microscopy. To confirm these results, we used flow cytometry to measure the average size of the clusters in clones of our evolved replicates and found them to be smaller on average than the ancestor. We also qualitatively assessed the aggregation phenotype using a settling assay and found that our evolved replicates settle slower than the ancestor. Finally, we characterized the mutations in our evolved replicates using whole genome sequencing and identified increased copy numbers of chromosome 1 and chromosome 14 in all three evolved clones. The best-performing strain generated by this project is now available commercially. This project demonstrates how experimental evolution can be used to select against less desirable traits in industrial yeast strains when targeted genetic approaches present considerable challenges. Future research could implement a similar approach to improve other traits in widely used brewing and baking strains.

genetics↗

bioRxiv2025

The discovery and patterns of the underlying long-standing mild-effect mutator alleles in S. cerevisiae populations

Most mutations are neutral or deleterious, and mutator alleles that substantially increase the mutation rate of an organism are believed to be short-lived in natural populations. Theory suggests that mutator alleles of modest effect have the potential to segregate nearly neutrally, but this prediction has been difficult to test experimentally. Here, we report strong genomic signatures consistent with the transmission and long-term maintenance of at least one natural mutator allele in Saccharomyces cerevisiae isolates. Specifically, such signatures are consistent with segregation of mutator alleles that disproportionately increase A>C mutations in the African Beer strains of S. cerevisiae. Remarkably, the mutation spectra of African Beer strains deviate from that of other S. cerevisiae natural isolates more than some Saccharomyces species differ from each other. Furthermore, computational analysis suggests mutator allele introgression from the African Beer population into a subset of French Dairy yeast, motivating us to experimentally measure the de novo mutation rates and spectra of these strains. We observed a consistent but weak enrichment of A>C and A>G de novo mutations in the African Beer population, with the A>C enrichment also evident in the French Dairy subset. Unexpectedly, other de novo mutation types varied more substantially among strains, including one outlier African Beer strain (AFL) with an excess of C>A mutations. Overall, our measurements reveal that de novo mutation spectra show greater variability among populations compared to the mutation spectra of rare polymorphisms, which in turn show greater variability than the spectra of all polymorphisms. Together, while additional mutator alleles may segregate in natural populations, mild enrichment of A>C and A>G types in African Beer strains reflects mutator alleles with weak effects that can persist and accumulate mutations through evolution.

evolutionary biology↗

bioRxiv2025

Functional evidence for G6PD variant classification from mutational scanning

G6PD deficiency is one of the most common enzyme deficiencies worldwide, and increases the likelihood of adverse reactions to certain drugs and foods. Identifying people at risk is challenging, since most are asymptomatic until they encounter a trigger. This is further complicated since over 60% of 1,559 known genetic variants in G6PD are variants of uncertain significance and thus cannot guide drug prescribing and dosing. To resolve which variants are clinically meaningful and avoid harm from adverse drug reactions, we conducted two high-throughput functional assays: one for G6PD activity, and one for abundance. We measured the function of 9,527 missense, nonsense, and synonymous G6PD variants. The patterns of variant effect on activity and abundance confirmed the importance of structural NADP+ for G6PD activity and abundance, and G6PD dimerization for G6PD activity. Based on the ability of our functional assay scores to accurately classify G6PD variants of known clinical effect, we generated evidence that 4,870 missense variants contribute to G6PD deficiency and 2,245 are unlikely to contribute to G6PD deficiency. Our data can be used to deepen our understanding of G6PD as a protein, and to close the gap in classification for variants of uncertain significance to improve implementation of genetic medicine for G6PD deficiency.

genetics↗

bioRxiv2025

The yEvo Mutation Browser: Enhancing student understanding of experimental evolution and genomics through interactive data visualization

Experimental evolution is a powerful method for studying the relationship between genotype and phenotype by observing how populations genetically adapt to controlled selective pressures. In educational settings, this approach also offers a dynamic way for students to engage with molecular genetics. One such educational effort, known as "yEvo" (yeast evolution), introduces experimental evolution into high school classrooms, allowing students to evolve the bakers yeast Saccharomyces cerevisiae under various stressors and investigate the resulting genetic changes. While the hands-on experiments have been successful in fostering student interest and understanding of evolution, the downstream data analysis (interpreting whole-genome sequencing results of evolved yeast compared to the ancestor) remains a challenge. Students often struggle to grasp the significance of their mutated genes and lack the broader context to determine which mutations are most phenotypically relevant. To address these issues, we developed the yEvo Mutation Browser, an intuitive web tool designed to assist students and researchers alike in visualizing and contextualizing genome sequencing data. Developed using R Shiny, this tool features an interactive chromosome map displaying mutated genes, graphs categorizing mutation types, a gene viewer illustrating specific mutation sites within genes, and a protein view that maps specific mutations onto protein structures. The app also features an option for non-yEvo-affiliated users to upload their own experimental evolution or genetic screen datasets and compare them with all yEvo data collected since 2018. The yEvo Mutation Browser streamlines data interpretation, helping students understand how organisms employ diverse genetic strategies to adapt to environmental stress. In the future, this framework could be adapted for use with other model organisms, offering a valuable resource for both genetics research and education. SignificanceBringing novel research into the classroom can transform how students learn science. The yEvo project engages high school students in experimental evolution, enabling them to witness natural selection in action and connect genetic mutations to evolutionary outcomes. By providing an accessible web-based tool, the "yEvo Mutation Browser," this work lowers barriers to interpreting genome sequencing data, allowing students, teachers, and researchers alike to explore how yeast populations adapt to diverse stress conditions. This resource not only strengthens STEM education by making cutting-edge genetics more approachable, but also builds a shared dataset that enriches the broader yeast genetics community.

scientific communication and education↗

bioRxiv2025

A cross-species rescue by mating method to interrogate gene essentiality across the Saccharomyces genus.

The field of budding yeast research has long been empowered by the vast array of genetic tools and community resources available. Rescue by mating is one useful tool that entails mating of meiotic spores directly post-germination. This minimal cell division mating process facilitates mating based screens studying the effects of otherwise haploid lethal gene deletions. In this study, we describe the successful application of rescue by mating across two different Saccharomyces species: S. cerevisiae and S. uvarum. This novel inter-species tool enables studies on the evolution of essential genes within the broader Saccharomyces genus.

genetics↗

bioRxiv2025

Meiotic null MSH2 and SGS1 alleles in S. cerevisiae x S. uvarum hybrids result in near-haploid offspring with mixed parental chromosomal inheritance

In the natural world, genetic isolation between closely related species is often maintained by direct F1 hybrid sterility or hybrid breakdown in subsequent generations. This phenomenon is also observed in microorganisms within species of the Saccharomyces clade, including the model organism Saccharomyces cerevisiae. Members of Saccharomyces intermate and historical hybridization events have left genomic footprints, but these hybrids are themselves sterile and produce very few viable offspring. Previous work suggests a main cause of F1 hybrid sterility is the action of mismatch repair machinery. The sequence divergence between two parent Saccharomyces species--from [~]10% to [~]20%--is recognized by the cell as an aberrant attempt to recombine nonhomologous chromosomes during meiosis I, leading to massive nondisjunction and aneuploidy. Deletion of mismatch repair machinery improves fertility in hybrids of S. cerevisiae and its sister species S. paradoxus but leads to increased mutation accumulation during normal mitotic growth. To overcome this drawback, conditional alleles that specifically repress expression of mismatch repair machinery during meiosis have been applied in S. cerevisiae x S. paradoxus hybrids with success. Here, we describe the application of the meiotic null system to a different hybrid: S. cerevisiae x S. uvarum. The system does not increase hybrid fertility likely due to increased sequence divergence and chromosomal translocations, but mass spore recovery and flow cytometry enable the recovery of near-haploid offspring which inherit an assortment of parental chromosomes. These populations of mixed hybrid genomes can be leveraged in phenotypic studies to further understand the effects of hybrid genomes on organismal fitness.

genetics↗

bioRxiv2024

The Prevalence of Killer Yeasts in the Gardens of Fungus-Growing Ants and the Discovery of Novel Killer Toxin named Ksino.

Killer toxins are proteinaceous antifungal molecules produced by yeasts, with activity against a wide range of human and plant pathogenic fungi. Fungus gardens of attine ants in Brazil were surveyed to determine the presence of killer toxin-producing yeasts and to define their antifungal activities and ecological importance. Our results indicate that 10 out of 59 yeasts species isolated from fungal gardens are killer yeasts. Killer yeasts were less likely to inhibit the growth of yeasts isolated from the same environment but more effective at inhibiting yeast isolated from other environments, supporting a role for killer yeasts in shaping community composition. All killer yeasts harbored genome-encoded killer toxins lacked cytoplasmic toxin-encoding elements (i.e., double-stranded RNA satellites and linear double-stranded DNAs). Of all the killer yeasts associated with attine ants, Candida sinolaborantium (strain LESF 1467) showed a broad spectrum of antifungal activities against 39 out of 69 57% of yeast strains tested for toxin susceptibility. The complete genome sequence of C. sinolaborantium LESF 1467 identified a new killer toxin, Ksino, with similarities in primary sequence and tertiary structure to the Saccharomyces cerevisiae killer toxin named Klus. Surveys of publicly available genome databases identified homologs of Ksino in the genomes of yeast strains of Saccharomycetes and Pichiomycetes, as well as other species of Ascomycota and Basidiomycota filamentous fungi. This demonstrates that killer yeasts can be widespread in attine ant fungus gardens, possibly influencing fungal community composition and the importance of these complex microbial communities for discovering novel antifungal molecules. ImportanceAttine ants perform essential ecosystem services through the harvesting of substrates for fungiculture. The cultured fungi are a food source for attine ants. Characterizing antifungal toxin-producing yeasts (killer yeasts) is vital to understanding how they might protect gardens from invasion by unwanted fungal species. This study describes a new toxin named Ksino from the yeast Candida sinolaborantium, a member of a new group of toxins found across many different species of fungi. This work supports the role of killer yeasts in the ecology of fungicultures and competition between fungi. The observed high prevalence of killer yeasts in fungal gardens also enables the discovery of novel antifungal molecules with the potential to be applied against disease-causing fungi.

microbiology↗

bioRxiv2024

URA6 mutations provide an alternative mechanism for 5-FOA resistance in Saccharomyces cerevisiae

The URA3 gene is frequently used in the budding yeast community as the mutation target for 5-fluoroorotic acid (5-FOA) resistance. We identified a class of ura6 mutants that can grow in the presence of 5-FOA. Unlike ura3 mutants, ura6 mutants remain prototrophic and are able to grow in the absence of uracil. In addition to 5-FOA resistance, we found that ura6 mutants are also resistant to 5-fluorocytosine (5-FC) and 5-fluorouracil (5-FU). In total, we identified 50 unique missense mutations across 32 unique amino acid positions of Ura6 which confer resistance to 5-FOA. We found that 28 out of the 32 affected positions are located in regions conserved between Saccharomyces cerevisiae and three clinically relevant pathogenic fungi. Metabolic analysis revealed a build-up of uridine monophosphate (UMP) and 5-fluorouridine monophosphate (5-FUMP) in ura6 mutants, indicating a reduction in Ura6 activity. Despite the accumulation of UMP in ura6 mutants, uridine diphosphate and triphosphate (UDP, UTP) levels were similar across mutants and wild type. These findings suggest that missense mutations to URA6, can result in reduction of uridylate kinase activity and lead to cross resistance to fluorinated prodrugs that incorporate into both the de novo pyrimidine synthesis and the pyrimidine salvage pathway.

genetics↗

bioRxiv2024

Experimental evolution of S. cerevisiae for caffeine tolerance alters multidrug resistance and TOR signaling pathways

Caffeine is a natural compound that inhibits the major cellular signaling regulator TOR, leading to widespread effects including growth inhibition. S. cerevisiae yeast can adapt to tolerate high concentrations of caffeine in coffee and cacao fermentations and in experimental systems. While many factors affecting caffeine tolerance and TOR signaling have been identified, further characterization of their interactions and regulation remain to be studied. We used experimental evolution of S. cerevisiae to study the genetic contributions to caffeine tolerance in yeast, through a collaboration between high school students evolving yeast populations coupled with further research exploration in university labs. We identified multiple evolved yeast populations with mutations in PDR1 and PDR5, which contribute to multidrug resistance, and showed that gain-of-function mutations in multidrug resistance family transcription factors PDR1, PDR3, and YRR1 differentially contribute to caffeine tolerance. We also identified loss-of-function mutations in TOR effectors SIT4, SKY1, and TIP41, and show that these mutations contribute to caffeine tolerance. These findings support the importance of both the multidrug resistance family and TOR signaling in caffeine tolerance, and can inform future exploration of networks affected by caffeine and other TOR inhibitors in model systems and industrial applications.

genetics↗

bioRxiv2023

A unifying model that explains the origins of human inverted copy number variants

With the release of the telomere-to-telomere human genome sequence and the availability of both long-read sequencing and optical genome mapping techniques, the identification of copy number variants and other structural variants is providing new insights into human genetic disease. Different mechanisms have been proposed to account for the novel junctions in these complex architectures, including aberrant forms of DNA replication, non-allelic homologous recombination and various pathways that repair DNA breaks. Here we have focused on a set of structural variants that include an inverted segment and propose that they share a common initiating event: an inverted triplication with long, unstable palindromic junctions. The secondary rearrangement of these palindromes gives rise to the various forms of inverted structural variants. We postulate that this same mechanism (ODIRA: Origin Dependent Inverted Repeat Amplification) that creates the inverted copy number variants in inherited syndromes also generates the palindromes found in cancers.

genetics↗

bioRxiv2023

Systematic Profiling of Ale Yeast Protein Dynamics across Fermentation and Repitching

Studying the genetic and molecular characteristics of brewing yeast strains is crucial for understanding their domestication history and adaptations accumulated over time in fermentation environments, and for guiding optimizations to the brewing process itself. Saccharomyces cerevisiae (brewing yeast) is amongst the most profiled organisms on the planet, yet the temporal molecular changes that underlie industrial fermentation and beer brewing remain understudied. Here, we characterized the genomic makeup of a Saccharomyces cerevisiae ale yeast widely used in the production of Hefeweizen beers, and applied shotgun mass spectrometry to systematically measure the proteomic changes throughout two fermentation cycles which were separated by 14 rounds of serial repitching. The resulting brewing yeast proteomics resource includes 64,740 protein abundance measurements. We found that this strain possesses typical genetic characteristics of Saccharomyces cerevisiae ale strains and displayed progressive shifts in molecular processes during fermentation based on protein abundance changes. We observed protein abundance differences between early fermentation batches compared to those separated by 14 rounds of serial repitching. The observed abundance differences occurred mainly in proteins involved in the metabolism of ergosterol and isobutyraldehyde. Our systematic profiling serves as a starting point for deeper characterization of how the yeast proteome changes during commercial fermentations and additionally serves as a resource to guide fermentation protocols, strain handling, and engineering practices in commercial brewing and fermentation environments. Finally, we created a web interface (https://brewing-yeast-proteomics.ccbb.utexas.edu/) to serve as a valuable resource for yeast geneticists, brewers, and biochemists to provide insights into the global trends underlying commercial beer production.

systems biology↗

bioRxiv2023

Template switching between the leading and lagging strands at replication forks generates inverted copy number variants through hairpin-capped extrachromosomal DNA

Inherited and de novo copy number variants (CNVs) are increasingly found to be correlated with human developmental and cancerous phenotypes. Several models for template switching during replication have been proposed to explain the generation of these gross chromosomal rearrangements. We proposed a model of template switching (ODIRA--origin dependent inverted repeat amplification) in which simultaneous ligation of the leading and lagging strands at diverging replication forks could generate segmental inverted triplications through an extrachromosomal inverted circular intermediate. Here, we created a genetic assay using split-ura3 cassettes to trap the proposed inverted intermediate. However, instead of recovering circular inverted intermediates, we found inverted linear chromosomal fragments ending in native telomeres--suggesting that a template switch had occurred at the centromere-proximal fork of a replication bubble. As telomeric inverted hairpin fragments can also be created through double strand breaks we tested whether replication errors or repair of double stranded DNA breaks were the most likely initiating event. The results from CRISPR/Cas9 cleavage experiments and growth in the replication inhibitor hydroxyurea indicate that it is a replication error, not a double stranded break that creates the inverted junctions. Since inverted amplicons of the SUL1 gene occur during long-term growth in sulfate-limited chemostats, we sequenced evolved populations to look for evidence of linear intermediates formed by an error in replication. All of the data are compatible with a two-step version of the ODIRA model in which sequential template switching at short inverted repeats between the leading and lagging strands at replication forks generates inverted interstitial triplications. AUTHOR SUMMARYChromosomal rearrangements are a potent source of genetic variation in humans and other organisms. One specific type of rearrangement involves the increase in copies of segments of the genome. The variation in gene dosage that these rearrangements can cause has been associated with a wide range of neurological and other human disorders. A specific puzzling form of copy number increase consists of three tandem copies with the central copy in inverted orientation. How this rearrangement occurs is of great interest, yet the mechanisms responsible are only inferred by examining the sequence of final inverted products. Yeast provides a unique model system to explore the underlying molecular defects that give rise to inverted triplications. While the favored hypothesis suggests that double stranded DNA repair is the causative agent, we find that a particular form of template switching between strands at the replication fork, not a double stranded DNA break, is the initiating event. Using the awesome power of yeast genetics, we provide evidence in two different assays for this unique replication error that we call ODIRA (for Origin Dependent Inverted Repeat Amplification) and propose that it can also explain this form of copy number variant seen in human evolution and disease.

genomics↗

bioRxiv2023

Condition-dependent fitness effects of large synthetic chromosome amplifications

Whole-chromosome aneuploidy and large segmental amplifications can have devastating effects in multicellular organisms, from developmental disorders and miscarriage to cancer. Aneuploidy in single-celled organisms such as yeast also results in proliferative defects and reduced viability. Yet, paradoxically, CNVs are routinely observed in laboratory evolution experiments with microbes grown in stressful conditions. The defects associated with aneuploidy are often attributed to the imbalance of many differentially expressed genes on the affected chromosomes, with many genes each contributing incremental effects. An alternate hypothesis is that a small number of individual genes are large effect drivers of these fitness changes when present in an altered copy number. To test these two views, we have employed a collection of strains bearing large chromosomal amplifications that we previously assayed in nutrient-limited chemostat competitions. In this study, we focus on conditions known to be poorly tolerated by aneuploid yeast--high temperature, treatment with the Hsp90 inhibitor radicicol, and growth in extended stationary phase. To identify potential genes with a large impact on fitness, we fit a piecewise constant model to fitness data across chromosome arms, filtering breakpoints in this model by magnitude to focus on regions with a large impact on fitness in each condition. While fitness generally decreased as the length of the amplification increased, we were able to identify 91 candidate regions that disproportionately impacted fitness when amplified. Consistent with our previous work with this strain collection, nearly all candidate regions were condition specific, with only five regions impacting fitness in multiple conditions.

genetics↗

bioRxiv2023

Multidimensional proteomics identifies molecular trajectories of cellular aging and rejuvenation

The declining capacity of cells to maintain a functional proteome is a major driver of cellular dysfunction and decreased fitness in aging. Here we assess the impact of aging on multiple proteome dimensions, which are reflective of function, across the replicative lifespan of Saccharomyces cerevisiae. We quantified protein abundance, protein turnover, protein thermal stability, and protein phosphorylation in mother yeast cells and their derived progeny at different ages. We find progressive and cumulative proteomic alterations that are reflective of dysregulation of complex assemblies, mitochondrial remodeling, post-translational activation of the AMPK/Snf1 energy sensor in mother cells, and an overall shift from biosynthetic to energy-metabolic processes. Our multidimensional proteomic study systematically corroborates previous findings of asymmetric segregation and daughter cell rejuvenation, and extends these concepts to protein complexes, protein phosphorylation, and activation of signaling pathways. Lastly, profiling age-dependent proteome changes in a caloric restriction model of yeast provided mechanistic insights into longevity, revealing minimal remodeling of energy-metabolic pathways, improved mitochondrial maintenance, ameliorated protein biogenesis, and decreased stress responses. Taken together, our study provides thousands of age-dependent molecular events that can be used to gain a holistic understanding of mechanisms of aging.

systems biology↗

bioRxiv2023

Caffeine-tolerant mutations selected through an at-home yeast experimental evolution teaching lab

yEvo is a curriculum for high school students centered around evolution experiments in S. cerevisiae. To adapt the curriculum for remote instruction, we created a new protocol to evolve non-GMO yeast in the presence of caffeine. Evolved strains had increased caffeine tolerance and distinct colony morphologies. Many possessed copy number variations, transposon insertions, and mutations affecting genes with known relationships to caffeine and TOR signaling - which is inhibited by caffeine - and in other genes not previously connected with caffeine. This demonstrates that our accessible, at-home protocol is sufficient to permit novel insights into caffeine tolerance.

genetics↗

bioRxiv2022

Functional interpretation, cataloging, and analysis of 1,341 known and new glucose-6-phosphate dehydrogenase variants

Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency affects over 500 million individuals who can experience anemia in response to oxidative stressors such as certain foods and drugs. Recently, the World Health Organization (WHO) called for revisiting G6PD variant classification as a priority to implement genetic medicine in low- and middle-income countries. Towards this goal, we sought to collect reports of G6PD variants and provide interpretations. We identified 1,341 G6PD variants in population and clinical databases. Using the ACMG standards and guidelines for the interpretation of sequence variants, we provided interpretations for 268 variants, including 186 variants that were not reported or of uncertain significance in ClinVar, bringing the total number of variants with non-conflicting interpretations to 400. For 414 variants with functional or clinical data, we analyzed associations between activity, stability, and current classification systems, including the new 2022 WHO Classification. We corroborated known challenges with classification systems, including phenotypic variation, emphasizing the importance of comparing variant effects across patients and studies. Biobank data made available by All of Us illustrate the benefit of large-scale sequencing and phenotyping by adding additional support connecting variants to G6PD-deficient anemia. By leveraging available data and interpretation guidelines, we created a repository for information on G6PD variants and nearly doubled the number of variants with clinical interpretations. These tools enable better interpretation of G6PD variants for the implementation of genetic medicine.

genetics↗