G3: Genes, Genomes, Genetics

Journal: G3: Genes, Genomes, Genetics

Abbreviation

G3

Publisher

Oxford University Press

ISSN

2160-1836

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Publications 1 - 9 of 9

Insect High Five™ cell line development using site-specific flipase recombination technology
Dias, Mafalda M.; Vidigal, João; Sequeira, Daniela P.; et al. (2021)
G3: Genes, Genomes, Genetics
Insect Trichoplusia ni High Five™ (Hi5) cells have been widely explored for production of heterologous proteins, traditionally mostly using the lytic baculovirus expression vector system (BEVS), and more recently using virus-free transient gene expression systems. Stable expression in such host cells would circumvent the drawbacks associated with both systems when it comes to scale-up and implementation of more efficient high-cell density process modes for the manufacturing of biologics. In this study, we combined Flipase (Flp) recombinase-mediated cassette exchange (RMCE) with fluorescence-activated cell sorting (FACS) for generating a stable master clonal Hi5 cell line with the flexibility to express single or multiple proteins of interest from a tagged genomic locus. The 3-step protocol herein implemented consisted of (i) introducing the RMCE docking cassette into the cell genome by random integration followed by selection in Hygromycin B and FACS (Hi5-tagging population), (ii) eliminating cells tagged in loci with low recombination efficiency by transfecting the tagging population with an eGFP-containing target cassette followed by selection in G418 and FACS (Hi5-RMCE population), and (iii) isolation of pure eGFP-expressing cells by FACS and expansion to suspension cultures (Hi5-RMCE master clone). Exchangeability of the locus in the master clone was demonstrated in small-scale suspension cultures by replacing the target cassette by one containing a single protein (i.e., iCherry, as an intracellular protein model) or two proteins (i.e., influenza HA and M1 for virus-like particles production, as an extracellular protein model). Overall, the stable insect Hi5 cell platform herein assembled has the potential to assist and accelerate biologics development.
Genome sequences of Rhizopogon roseolus, Mariannaea elegans, Myrothecium verrucaria, and Sphaerostilbella broomeana and the identification of biosynthetic gene clusters for fungal peptide natural products
Vogt, Eva; Field, Christopher; Sonderegger, Lukas; et al. (2022)
G3: Genes, Genomes, Genetics
In recent years, a variety of fungal cyclic peptides with interesting bioactivities have been discovered. For many of these peptides, the biosynthetic pathways are unknown and their elucidation often holds surprises. The cyclic and backbone N-methylated omphalotins from Omphalotus olearius were recently shown to constitute a novel class (borosins) of ribosomally synthesized and posttranslationally modified peptides, members of which are produced by many fungi, including species of the genus Rhizopogon. Other recently discovered fungal peptide macrocycles include the mariannamides from Mariannaea elegans and the backbone N-methylated verrucamides and broomeanamides from Myrothecium verrucaria and Sphaerostilbella broomeana, respectively. Here, we present draft genome sequences of four fungal species Rhizopogon roseolus, Mariannaea elegans, Myrothecium verrucaria, and Sphaerostilbella broomeana. We screened these genomes for precursor proteins or gene clusters involved in the mariannamide, verrucamide, and broomeanamide biosynthesis including a general screen for borosin-producing precursor proteins. While our genomic screen for potential ribosomally synthesized and posttranslationally modified peptide precursor proteins of mariannamides, verrucamides, broomeanamides, and borosins remained unsuccessful, antiSMASH predicted nonribosomal peptide synthase gene clusters that may be responsible for the biosynthesis of mariannamides, verrucamides, and broomeanamides. In M. verrucaria, our antiSMASH search led to a putative NRPS gene cluster with a predicted peptide product of 20 amino acids, including multiple nonproteinogenic isovalines. This cluster likely encodes a member of the peptaibols, an antimicrobial class of peptides previously isolated primarily from the Genus Trichoderma. The nonribosomal peptide synthase gene clusters discovered in our screenings are promising candidates for future research.
Systematic profiling of ale yeast protein dynamics across fermentation and repitching
Garge, Riddhiman K.; Geck, Renee C.; Armstrong, Joseph O.; et al. (2024)
G3: Genes, Genomes, Genetics
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 among 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 2 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.
iProteinDB: An Integrative Database of Drosophila Post-translational Modifications
Hu, Yanhui; Sopko, Richelle; Chung, Verena; et al. (2019)
G3: Genes, Genomes, Genetics
Post-translational modification (PTM) serves as a regulatory mechanism for protein function, influencing their stability, interactions, activity and localization, and is critical in many signaling pathways. The best characterized PTM is phosphorylation, whereby a phosphate is added to an acceptor residue, most commonly serine, threonine and tyrosine in metazoans. As proteins are often phosphorylated at multiple sites, identifying those sites that are important for function is a challenging problem. Considering that any given phosphorylation site might be non-functional, prioritizing evolutionarily conserved phosphosites provides a general strategy to identify the putative functional sites. To facilitate the identification of conserved phosphosites, we generated a large-scale phosphoproteomics dataset from Drosophila embryos collected from six closely-related species. We built iProteinDB (https://www.flyrnai.org/tools/iproteindb/), a resource integrating these data with other high-throughput PTM datasets, including vertebrates, and manually curated information for Drosophila. At iProteinDB, scientists can view the PTM landscape for any Drosophila protein and identify predicted functional phosphosites based on a comparative analysis of data from closely-related Drosophila species. Further, iProteinDB enables comparison of PTM data from Drosophila to that of orthologous proteins from other model organisms, including human, mouse, rat, Xenopus tropicalis, Danio rerio, and Caenorhabditis elegans.
Dynamics of transposable elements in recently diverged fungal pathogens: lineage-specific transposable element content and efficiency of genome defenses
Lorrain, Cécile; Feurtey, Alice; Möller, Mareike; et al. (2021)
G3: Genes, Genomes, Genetics
Transposable elements (TEs) impact genome plasticity, architecture, and evolution in fungal plant pathogens. The wide range of TE content observed in fungal genomes reflects diverse efficacy of host-genome defense mechanisms that can counter-balance TE expansion and spread. Closely related species can harbor drastically different TE repertoires. The evolution of fungal effectors, which are crucial determinants of pathogenicity, has been linked to the activity of TEs in pathogen genomes. Here, we describe how TEs have shaped genome evolution of the fungal wheat pathogen Zymoseptoria tritici and four closely related species. We compared de novo TE annotations and repeat-induced point mutation signatures in 26 genomes from the Zymoseptoria species-complex. Then, we assessed the relative insertion ages of TEs using a comparative genomics approach. Finally, we explored the impact of TE insertions on genome architecture and plasticity. The 26 genomes of Zymoseptoria species reflect different TE dynamics with a majority of recent insertions. TEs associate with accessory genome compartments, with chromosomal rearrangements, with gene presence/absence variation, and with effectors in all Zymoseptoria species. We find that the extent of RIP-like signatures varies among Z. tritici genomes compared to genomes of the sister species. The detection of a reduction of RIP-like signatures and TE recent insertions in Z. tritici reflects ongoing but still moderate TE mobility.
Quantitative trait locus mapping of osmotic stress response in the fungal wheat pathogen Zymoseptoria tritici
Stapley, Jessica; McDonald, Bruce (2023)
G3: Genes, Genomes, Genetics
Osmotic stress is a ubiquitous and potent stress for all living organisms, but few studies have investigated the genetic basis of salt tolerance in filamentous fungi. The main aim of this study was to identify regions of the genome associated with tolerance to potassium chloride (KCl) in the wheat pathogen Zymoseptoria tritici. A secondary aim was to identify candidate genes affecting salt tolerance within the most promising chromosomal regions. We achieved these aims with a quantitative trait locus (QTL) mapping study using offspring from 2 crosses grown in vitro in the presence or absence of osmotic stress imposed by 0.75 M KCl. We identified significant QTL for most of the traits in both crosses. Several QTLs overlapped with QTL identified in earlier studies for other traits, and some QTL explained trait variation in both the control and salt stress environments. A significant QTL on chromosome 3 explained variation in colony radius at 8-day postinoculation (dpi) in the KCl environment as well as colony radius KCl tolerance at 8 dpi. The QTL peak had a high logarithm of the odds ratio (LOD) and encompassed an interval containing only 36 genes. Six of these genes present promising candidates for functional analyses. A gene ontology (GO) enrichment analysis of QTL unique to the KCl environment found evidence for the enrichment of functions involved in osmotic stress responses.
The genetic basis of apple shape and size unraveled by digital phenotyping
Keller, Beat; Jung, Michaela; Bühlmann-Schütz, Simone; et al. (2024)
G3: Genes, Genomes, Genetics
Great diversity of shape, size, and skin color is observed among the fruits of different apple genotypes. These traits are critical for consumers and therefore interesting targets for breeding new apple varieties. However, they are difficult to phenotype and their genetic basis, especially for fruit shape and ground color, is largely unknown. We used the FruitPhenoBox to digitally phenotype 525 genotypes of the apple reference population (apple REFPOP) genotyped for 303,148 single nucleotide polymorphism (SNP) markers. From the apple images, 573 highly heritable features describing fruit shape and size as well as 17 highly heritable features for fruit skin color were extracted to explore genotype-phenotype relationships. Out of these features, seven principal components (PCs) and 16 features with the Pearson's correlation r < 0.75 (selected features) were chosen to carry out genome-wide association studies (GWAS) for fruit shape and size. Four PCs and eight selected features were used in GWAS for fruit skin color. In total, 69 SNPs scattered over all 17 apple chromosomes were significantly associated with round, conical, cylindrical, or symmetric fruit shapes and fruit size. Novel associations with major effect on round or conical fruit shapes and fruit size were identified on chromosomes 1 and 2. Additionally, 16 SNPs associated with PCs and selected features related to red overcolor as well as green and yellow ground color were found on eight chromosomes. The identified associations can be used to advance marker-assisted selection in apple fruit breeding to systematically select for desired fruit appearance.
Genome assemblies of the simultaneously hermaphroditic flatworms Macrostomum cliftonense and Macrostomum hystrix
Wiberg, R. Axel W.; Brand, Jeremias N.; Viktorin, Gudrun; et al. (2023)
G3: Genes, Genomes, Genetics
The free-living, simultaneously hermaphroditic flatworms of the genus Macrostomum are increasingly used as model systems in various contexts. In particular, Macrostomum lignano, the only species of this group with a published genome assembly, has emerged as a model for the study of regeneration, reproduction, and stem-cell function. However, challenges have emerged due to M. lignano being a hidden polyploid, having recently undergone whole-genome duplication and chromosome fusion events. This complex genome architecture presents a significant roadblock to the application of many modern genetic tools. Hence, additional genomic resources for this genus are needed. Here, we present such resources for Macrostomum cliftonense and Macrostomum hystrix, which represent the contrasting mating behaviors of reciprocal copulation and hypodermic insemination found in the genus. We use a combination of PacBio long-read sequencing and Illumina shot-gun sequencing, along with several RNA-Seq data sets, to assemble and annotate highly contiguous genomes for both species. The assemblies span ∼227 and ∼220 Mb and are represented by 399 and 42 contigs for M. cliftonense and M. hystrix, respectively. Furthermore, high BUSCO completeness (∼84–85%), low BUSCO duplication rates (8.3–6.2%), and low k-mer multiplicity indicate that these assemblies do not suffer from the same assembly ambiguities of the M. lignano genome assembly, which can be attributed to the complex karyology of this species. We also show that these resources, in combination with the prior resources from M. lignano, offer an excellent foundation for comparative genomic research in this group of organisms.
Mapping genomic regions associated with temperature stress in the wheat pathogen Zymoseptoria tritici
Stapley, Jessica; Zhong, Ziming; McDonald, Bruce (2025)
G3: Genes, Genomes, Genetics
Climate change can alter interactions between plants and their pathogens, which could adversely affect crop production. To better understand the molecular mechanisms underlying the responses of pathogenic fungi to temperature stress, we conducted a quantitative trait loci (QTL) mapping study in the wheat pathogen Zymoseptoria tritici to identify genomic regions associated with colony growth and melanization at 3 temperatures (10, 18, and 27 °C). We then identified likely candidate genes for thermal adaptation within these intervals by combining information regarding gene function, gene ontology (GO) annotation enrichment, transcriptional profile, and results from previous genome-wide association studies investigating responses to climate, temperature, and thermal adaptation. The QTL mapping, conducted for 2 separate crosses involving 4 Swiss parents, found significant QTL uniquely associated with traits measured in high and low temperatures. These intervals contained many genes known to regulate responses to temperature stress, including heat-shock proteins and proteins involved in the mitogen-activated protein kinase (MAPK) pathways, and were enriched for genes with a zinc ion binding GO annotation. We highlight the most promising candidate genes for thermal adaptation, including an ammonium transporter gene, a stress response factor (Whi1) and 2 MAPK pathway genes-SSk2 and Opy2. Future validation work on these candidate genes could provide novel insight into the molecular mechanisms underlying temperature adaptation in this important wheat pathogen.

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Journal: G3: Genes, Genomes, Genetics

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