Overdrive is essential for targeted sperm elimination by Segregation Distorter.

Intra-genomic conflict driven by selfish chromosomes is a powerful force that shapes the evolution of genomes and species. In the male germline, many selfish chromosomes bias transmission in their own favor by eliminating spermatids bearing the competing homologous chromosomes. However, the mechanisms of targeted gamete elimination remain mysterious. Here, we show that Overdrive (Ovd), a gene required for both segregation distortion and male sterility in Drosophila pseudoobscura hybrids, is broadly conserved in Dipteran insects but dispensable for viability and fertility. In D. melanogaster, Ovd is required for targeted Responder spermatid elimination after the histone-to-protamine transition in the classical Segregation Distorter system. We propose that Ovd functions as a general spermatid quality checkpoint that is hijacked by independent selfish chromosomes to eliminate competing gametes.

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

Inherited and germ-line 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 a replication fork, followed by integration via homologous recombination, generates inverted interstitial triplications.

yEvo: experimental evolution in high school classrooms selects for novel mutations that impact clotrimazole resistance in Saccharomyces cerevisiae.

Antifungal resistance in pathogenic fungi is a growing global health concern. Nonpathogenic laboratory strains of Saccharomyces cerevisiae are an important model for studying mechanisms of antifungal resistance that are relevant to understanding the same processes in pathogenic fungi. We have developed a series of laboratory modules in which high school students used experimental evolution to study antifungal resistance by isolating azole-resistant S. cerevisiae mutants and examining the genetic basis of resistance. We have sequenced 99 clones from these experiments and found that all possessed mutations previously shown to impact azole resistance, validating our approach. We additionally found recurrent mutations in an mRNA degradation pathway and an uncharacterized mitochondrial protein (Csf1) that have possible mechanistic connections to azole resistance. The scale of replication in this initiative allowed us to identify candidate epistatic interactions, as evidenced by pairs of mutations that occur in the same clone more frequently than expected by chance (positive epistasis) or less frequently (negative epistasis). We validated one of these pairs, a negative epistatic interaction between gain-of-function mutations in the multidrug resistance transcription factors Pdr1 and Pdr3. This high school-university collaboration can serve as a model for involving members of the broader public in the scientific process to make meaningful discoveries in biomedical research.

Directed evolution as an approach to increase fructose utilization in synthetic grape juice by wine yeast AWRI 796.

In winemaking, slow or stuck alcoholic fermentation can impact processing efficiency and wine quality. Residual fructose in the later stages of fermentation can leave the wine 'out of specification' unless removed, which requires reinoculation or use of a more fructophilic yeast. As such, robust, fermentation efficient strains are still highly desirable to reduce this risk. We report on a combined EMS mutagenesis and Directed Evolution (DE) approach as a 'proof of concept' to improve fructose utilization and decrease fermentation duration. One evolved isolate, Tee 9, was evaluated against the parent, AWRI 796 in defined medium (CDGJM) and Semillon juice. Interestingly, Tee 9 exhibited improved fermentation in CDGJM at several nitrogen contents, but not in juice. Genomic comparison between AWRI 796 and Tee 9 identified 371 mutations, but no chromosomal copy number variation. A total of 95 noncoding and 276 coding mutations were identified in 297 genes (180 of which encode proteins with one or more substitutions). Whilst introduction of two of these, Gid7 (E726K) or Fba1 (G135S), into AWRI 796 did not lead to the fermentation improvement seen in Tee 9, similar allelic swaps with the other mutations are needed to understand Tee 9's adaption to CDGJM. Furthermore, the 378 isolates, potentially mutagenized but with the same genetic background, are likely a useful resource for future phenotyping and genome-wide association studies.

Effect of the Ala234Asp replacement in mitochondrial branched-chain amino acid aminotransferase on the production of BCAAs and fusel alcohols in yeast.

In the yeast Saccharomyces cerevisiae, the mitochondrial branched-chain amino acid (BCAA) aminotransferase Bat1 plays an important role in the synthesis of BCAAs (valine, leucine, and isoleucine). Our upcoming study (Large et al. bioRχiv. 10.1101/2020.06.26.166157, Large et al. 2020) will show that the heterozygous tetraploid beer yeast strain, Wyeast 1056, which natively has a variant causing one amino acid substitution of Ala234Asp in Bat1 on one of the four chromosomes, produced higher levels of BCAA-derived fusel alcohols in the brewer's wort medium than a derived strain lacking this mutation. Here, we investigated the physiological role of the A234D variant Bat1 in S. cerevisiae. Both bat1∆ and bat1A234D cells exhibited the same phenotypes relative to the wild-type Bat1 strain-namely, a repressive growth rate in the logarithmic phase; decreases in intracellular valine and leucine content in the logarithmic and stationary growth phases, respectively; an increase in fusel alcohol content in culture medium; and a decrease in the carbon dioxide productivity. These results indicate that amino acid change from Ala to Asp at position 234 led to a functional impairment of Bat1, although homology modeling suggests that Asp234 in the variant Bat1 did not inhibit enzymatic activity directly. KEY POINTS: • Yeast cells expressing Bat1A234D exhibited a slower growth phenotype. • The Val and Leu levels were decreased in yeast cells expressing Bat1A234D. • The A234D substitution causes a loss-of-function in Bat1. • The A234D substitution in Bat1 increased fusel alcohol production in yeast cells.

Temperature preference can bias parental genome retention during hybrid evolution.

Interspecific hybridization can introduce genetic variation that aids in adaptation to new or changing environments. Here, we investigate how hybrid adaptation to temperature and nutrient limitation may alter parental genome representation over time. We evolved Saccharomyces cerevisiae x Saccharomyces uvarum hybrids in nutrient-limited continuous culture at 15°C for 200 generations. In comparison to previous evolution experiments at 30°C, we identified a number of responses only observed in the colder temperature regime, including the loss of the S. cerevisiae allele in favor of the cryotolerant S. uvarum allele for several portions of the hybrid genome. In particular, we discovered a genotype by environment interaction in the form of a loss of heterozygosity event on chromosome XIII; which species' haplotype is lost or maintained is dependent on the parental species' temperature preference and the temperature at which the hybrid was evolved. We show that a large contribution to this directionality is due to a temperature dependent fitness benefit at a single locus, the high affinity phosphate transporter gene PHO84. This work helps shape our understanding of what forces impact genome evolution after hybridization, and how environmental conditions may promote or disfavor the persistence of hybrids over time.

Phenotypic and Genotypic Consequences of CRISPR/Cas9 Editing of the Replication Origins in the rDNA of Saccharomyces cerevisiae.

The complex structure and repetitive nature of eukaryotic ribosomal DNA (rDNA) is a challenge for genome assembly, thus the consequences of sequence variation in rDNA remain unexplored. However, renewed interest in the role that rDNA variation may play in diverse cellular functions, aside from ribosome production, highlights the need for a method that would permit genetic manipulation of the rDNA. Here, we describe a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based strategy to edit the rDNA locus in the budding yeast Saccharomyces cerevisiae, developed independently but similar to one developed by others. Using this approach, we modified the endogenous rDNA origin of replication in each repeat by deleting or replacing its consensus sequence. We characterized the transformants that have successfully modified their rDNA locus and propose a mechanism for how CRISPR/Cas9-mediated editing of the rDNA occurs. In addition, we carried out extended growth and life span experiments to investigate the long-term consequences that altering the rDNA origin of replication have on cellular health. We find that long-term growth of the edited clones results in faster-growing suppressors that have acquired segmental aneusomy of the rDNA-containing region of chromosome XII or aneuploidy of chromosomes XII, II, or IV. Furthermore, we find that all edited isolates suffer a reduced life span, irrespective of their levels of extrachromosomal rDNA circles. Our work demonstrates that it is possible to quickly, efficiently, and homogeneously edit the rDNA origin via CRISPR/Cas9.