More Like this

1. Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome

   https://doi.org/10.1038/s41467-024-55318-3  

   Goold, Hugh D; Kroukamp, Heinrich; Erpf, Paige E; Zhao, Yu; Kelso, Philip; Calame, Julie; Timmins, John_J B; Wightman, Elizabeth_L I; Peng, Kai; Carpenter, Alexander C; et al (December 2025, Nature Communications)

   Abstract The Sc2.0 global consortium to design and construct a synthetic genome based on theSaccharomyces cerevisiaegenome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp syntheticSaccharomyces cerevisiaechromosomesynXVIof the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5’ UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects andsynXVI, anin-silicoredesign of thesynXVIchromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. Thein-silicoredesign ofsynXVIincludes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms. 

   more » « less
2. Telomere Roles in Fungal Genome Evolution and Adaptation

   https://doi.org/10.3389/fgene.2021.676751  

   Rahnama, Mostafa; Wang, Baohua; Dostart, Jane; Novikova, Olga; Yackzan, Daniel; Yackzan, Andrew; Bruss, Haley; Baker, Maray; Jacob, Haven; Zhang, Xiaofei; et al (August 2021, Frontiers in Genetics)

   Telomeres form the ends of linear chromosomes and usually comprise protein complexes that bind to simple repeated sequence motifs that are added to the 3′ ends of DNA by the telomerase reverse transcriptase (TERT). One of the primary functions attributed to telomeres is to solve the “end-replication problem” which, if left unaddressed, would cause gradual, inexorable attrition of sequences from the chromosome ends and, eventually, loss of viability. Telomere-binding proteins also protect the chromosome from 5′ to 3′ exonuclease action, and disguise the chromosome ends from the double-strand break repair machinery whose illegitimate action potentially generates catastrophic chromosome aberrations. Telomeres are of special interest in the blast fungus, Pyricularia , because the adjacent regions are enriched in genes controlling interactions with host plants, and the chromosome ends show enhanced polymorphism and genetic instability. Previously, we showed that telomere instability in some P. oryzae strains is caused by novel retrotransposons (MoTeRs) that insert in telomere repeats, generating interstitial telomere sequences that drive frequent, break-induced rearrangements. Here, we sought to gain further insight on telomeric involvement in shaping Pyricularia genome architecture by characterizing sequence polymorphisms at chromosome ends, and surrounding internalized MoTeR loci (relics) and interstitial telomere repeats. This provided evidence that telomere dynamics have played historical, and likely ongoing, roles in shaping the Pyricularia genome. We further demonstrate that even telomeres lacking MoTeR insertions are poorly preserved, such that the telomere-adjacent sequences exhibit frequent presence/absence polymorphism, as well as exchanges with the genome interior. Using TERT knockout experiments, we characterized chromosomal responses to failed telomere maintenance which suggested that much of the MoTeR relic-/interstitial telomere-associated polymorphism could be driven by compromised telomere function. Finally, we describe three possible examples of a phenomenon known as “Adaptive Telomere Failure,” where spontaneous losses of telomere maintenance drive rapid accumulation of sequence polymorphism with possible adaptive advantages. Together, our data suggest that telomere maintenance is frequently compromised in Pyricularia but the chromosome alterations resulting from telomere failure are not as catastrophic as prior research would predict, and may, in fact, be potent drivers of adaptive polymorphism. 

   more » « less
3. Mining the Utricularia gibba genome for insulator-like elements for genetic engineering

   https://doi.org/10.3389/fpls.2023.1279231  

   Laspisa, Daniel; llla-Berenguer, Eudald; Bang, Sohyun; Schmitz, Robert J.; Parrott, Wayne; Wallace, Jason (November 2023, Frontiers in Plant Science)

   IntroductionGene expression is often controlled via cis-regulatory elements (CREs) that modulate the production of transcripts. For multi-gene genetic engineering and synthetic biology, precise control of transcription is crucial, both to insulate the transgenes from unwanted native regulation and to prevent readthrough or cross-regulation of transgenes within a multi-gene cassette. To prevent this activity, insulator-like elements, more properly referred to as transcriptional blockers, could be inserted to separate the transgenes so that they are independently regulated. However, only a few validated insulator-like elements are available for plants, and they tend to be larger than ideal. MethodsTo identify additional potential insulator-like sequences, we conducted a genome-wide analysis ofUtricularia gibba(humped bladderwort), one of the smallest known plant genomes, with genes that are naturally close together. The 10 best insulator-like candidates were evaluated in vivo for insulator-like activity. ResultsWe identified a total of 4,656 intergenic regions with expression profiles suggesting insulator-like activity. Comparisons of these regions across 45 other plant species (representing Monocots, Asterids, and Rosids) show low levels of syntenic conservation of these regions. Genome-wide analysis of unmethylated regions (UMRs) indicates ~87% of the targeted regions are unmethylated; however, interpretation of this is complicated becauseU. gibbahas remarkably low levels of methylation across the genome, so that large UMRs frequently extend over multiple genes and intergenic spaces. We also could not identify any conserved motifs among our selected intergenic regions or shared with existing insulator-like elements for plants. Despite this lack of conservation, however, testing of 10 selected intergenic regions for insulator-like activity found two elements on par with a previously published element (EXOB) while being significantly smaller. DiscussionGiven the small number of insulator-like elements currently available for plants, our results make a significant addition to available tools. The high hit rate (2 out of 10) also implies that more useful sequences are likely present in our selected intergenic regions; additional validation work will be required to identify which will be most useful for plant genetic engineering. 

   more » « less
4. Architectural groups of a subtelomeric gene family evolve along distinct paths in Candida albicans

   https://doi.org/10.1093/g3journal/jkac283  

   Dunn, Matthew J; Shazib, Shahed U; Simonton, Emily; Slot, Jason C; Anderson, Matthew Z (October 2022, G3 Genes|Genomes|Genetics)

   Rokas, A (Ed.)

   Abstract Subtelomeres are dynamic genomic regions shaped by elevated rates of recombination, mutation, and gene birth/death. These processes contribute to formation of lineage-specific gene family expansions that commonly occupy subtelomeres across eukaryotes. Investigating the evolution of subtelomeric gene families is complicated by the presence of repetitive DNA and high sequence similarity among gene family members that prevents accurate assembly from whole genome sequences. Here, we investigated the evolution of the telomere-associated (TLO) gene family in Candida albicans using 189 complete coding sequences retrieved from 23 genetically diverse strains across the species. Tlo genes conformed to the 3 major architectural groups (α/β/γ) previously defined in the genome reference strain but significantly differed in the degree of within-group diversity. One group, Tloβ, was always found at the same chromosome arm with strong sequence similarity among all strains. In contrast, diverse Tloα sequences have proliferated among chromosome arms. Tloγ genes formed 7 primary clades that included each of the previously identified Tloγ genes from the genome reference strain with 3 Tloγ genes always found on the same chromosome arm among strains. Architectural groups displayed regions of high conservation that resolved newly identified functional motifs, providing insight into potential regulatory mechanisms that distinguish groups. Thus, by resolving intraspecies subtelomeric gene variation, it is possible to identify previously unknown gene family complexity that may underpin adaptive functional variation. 

   more » « less
5. The Genome Architecture of the Copepod Eurytemora carolleeae —  the Highly Invasive Atlantic Clade of the Eurytemora affinis Species Complex

   https://doi.org/10.1093/gpbjnl/qzae066  

   Du, Zhenyong; Gelembiuk, Gregory; Moss, Wynne; Tritt, Andrew; Lee, Carol Eunmi (October 2024, Genomics, Proteomics & Bioinformatics)

   Jiang, Yu (Ed.)

   Abstract Copepods are among the most abundant organisms on the planet and play critical functions in aquatic ecosystems. Among copepods, populations of the Eurytemora affinis species complex are numerically dominant in many coastal habitats and serve as food sources for major fisheries. Intriguingly, certain populations possess the unusual capacity to invade novel salinities on rapid time scales. Despite their ecological importance, high-quality genomic resources have been absent for calanoid copepods, limiting our ability to comprehensively dissect the genome architecture underlying the highly invasive and adaptive capacity of certain populations. Here, we present the first chromosome-level genome of a calanoid copepod, from the Atlantic clade (Eurytemora carolleeae) of the E. affinis species complex. This genome was assembled using high-coverage PacBio long-read and Hi-C sequences of an inbred line, generated through 30 generations of full-sib mating. This genome, consisting of 529.3 Mb (contig N50 = 4.2 Mb, scaffold N50 = 140.6 Mb), was anchored onto four chromosomes. Genome annotation predicted 20,262 protein-coding genes, of which ion transport-related gene families were substantially expanded based on comparative analyses of 12 additional arthropod genomes. Also, we found genome-wide signatures of historical gene body methylation of the ion transport-related genes and the significant clustering of these genes on each chromosome. This genome represents one of the most contiguous copepod genomes to date and is among the highest quality marine invertebrate genomes. As such, this genome provides an invaluable resource to help yield fundamental insights into the ability of this copepod to adapt to rapidly changing environments. 

   more » « less