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At least 451 records · Page 25

Exploring genetic diversity, population structure, and subgenome differences in the allopolyploid Camelina sativa : implications for future breeding and research studies

Abstract Camelina (Camelina sativa), an allohexaploid species, is an emerging aviation biofuel crop that has been the focus of resurgent interest in recent decades. To guide future breeding and crop improvement efforts, the community requires a deeper comprehension of subgenome dominance, often noted in allopolyploid species, “alongside an understanding of the genetic diversity” and population structure of material present within breeding programs. We conducted population genetic analyses of a C. sativa diversity panel, leveraging a new genome, to estimate nucleotide diversity and population structure, and analyzed for patterns of subgenome expression dominance among different organs. Our analyses confirm that C. sativa has relatively low genetic diversity and show that the SG3 subgenome has substantially lower genetic diversity compared to the other two subgenomes. Despite the low genetic diversity, our analyses identified 13 distinct subpopulations including two distinct wild populations and others putatively representing founders in existing breeding populations. When analyzing for subgenome composition of long non-coding RNAs, which are known to play important roles in (a)biotic stress tolerance, we found that the SG3 subgenome contained significantly more lincRNAs compared to other subgenomes. Similarly, transcriptome analyses revealed that expression dominance of SG3 is not as strong as previously reported and may not be universal across all organ types. From a global analysis, SG3 “was only significant higher expressed” in flower, flower bud, and fruit organs, which is an important discovery given that the crop yield is associated with these organs. Collectively, these results will be valuable for guiding future breeding efforts in camelina.

Agriculture↗

Thermophilic site-specific recombination system for rapid insertion of heterologous DNA into the Clostridium thermocellum chromosome

Clostridium thermocellum is an anaerobic thermophile capable of producing ethanol and other commodity chemicals from lignocellulosic biomass. The insertion of heterologous DNA into the C. thermocellum chromosome is currently achieved via a time-consuming homologous recombination process, where a single stable insertion can take 2–4 weeks or more to construct. In this work, we developed a thermostable version of the Serine recombinase Assisted Genome Engineering (tSAGE) approach for gene insertion in C. thermocellum utilizing a site-specific recombinase from Geobacillus sp. Y412MC61, enabling quick and easy insertion of DNA into the chromosome for accelerated genetic tool screening and heterologous gene expression. Using tSAGE, chromosomal insertion of plasmid DNA occurred at a maximum transformation efficiency of 5 × 10 3 CFU/µg, which is comparable to the transformation efficiency of a replicating control plasmid in C. thermocellum. Using tSAGE, we chromosomally integrated and characterized 17 reporter genes, 15 homologous and 31 heterologous constitutive promoters of varying strengths, 4 inducible promoters, and 5 riboswitches in C. thermocellum. We also determined that a 6–7 nucleotide gap between the ribosome binding site (RBS) and the start codon is optimal for high expression by employing a library of superfolder green fluorescent protein expression constructs driven by our strongest tested promoter (P clo1313_1194 ) with different distances between the RBS and start codon. The tools developed here will aid in accelerating C. thermocellum strain engineering for producing sustainable fuels and chemicals directly from plant biomass.

Biofuels↗

Unveiling shared genetic regulators of plant architectural and biomass yield traits in the Sorghum Association Panel

Abstract Sorghum is emerging as an ideal genetic model for designing high-biomass bioenergy crops. Biomass yield, a complex trait influenced by various plant architectural characteristics, is typically regulated by numerous genes. This study aimed to dissect the genetic regulators underlying 14 plant architectural traits and 10 biomass yield traits in the Sorghum Association Panel across two growing seasons. We identified 321 associated loci through genome-wide association studies (GWAS), involving 234 264 single nucleotide polymorphisms (SNPs). These loci include genes with known associations to biomass traits, such as maturity, dwarfing (Dw), and leafbladeless1, as well as several uncharacterized loci not previously linked to these traits. We also identified 22 pleiotropic loci associated with variation in multiple phenotypes. Three of these loci, located on chromosomes 3 (S03_15463061), 6 (S06_42790178; Dw2), and 9 (S09_57005346; Dw1), exerted significant and consistent effects on multiple traits across both growing seasons. Additionally, we identified three genomic hotspots on chromosomes 6, 7, and 9, each containing multiple SNPs associated with variation in plant architecture and biomass yield traits. Chromosome-wise correlation analyses revealed multiple blocks of positively associated SNPs located near or within the same genomic regions. Finally, genome-wide correlation-based network analysis showed that loci associated with flowering, plant height, leaf traits, plant density, and tiller number per plant were highly interconnected with other genetic loci influencing plant architectural and biomass yield traits. The pyramiding of favorable alleles related to these traits holds promise for enhancing the future development of bioenergy sorghum crops.

Singh, Anuradha (ORCID:0000000197149095)↗

Genomic description of human clinical Aspergillus fumigatus isolates, California, 2020

Abstract Aspergillus fumigatus, an environmental mold, causes life-threatening infections. Studies on the phylogenetic structure of human clinical A. fumigatus isolates are limited. Here, we performed whole genome sequencing of 24 A. fumigatus isolates collected from 18 patients in U.S. healthcare facilities in California. Single-nucleotide polymorphism (SNP) differences between patient isolates ranged from 187 to 70 829 SNPs. For five patients with multiple isolates, we calculated the within-host diversities. Three patients had a within-host diversity that ranged from 4 to 10 SNPs and two patients ranged from 2 to 16 977 SNPs. Findings revealed highly diverse A. fumigatus strains among patients and two patterns of diversity for isolates that come from the same patient, low and extremely high diversity.

Misas, Elizabeth (ORCID:0000000162437716)↗

Occurrence and molecular characterization of Enterocytozoon bieneusi in wild and domestic animal species in Portugal

Abstract The phylum Microsporidia encompasses a diverse group of obligate, intracellular, and spore-forming organisms able to infect a wide range of animal hosts. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and animals. Little is known about the presence and epidemiology of E. bieneusi in wildlife. We investigated E. bieneusi occurrence and genetic diversity in wild and domestic mammals, through molecular-detection methods, from different regions across Portugal. A total of 756 samples were collected from 288, 242, and 226 wild carnivores, wild ungulates, and domestic animals, respectively. Overall, eight specimens were E. bieneusi-positive (1.1%, 8/756) obtained from five wild (Iberian lynx, Iberian wolf, red fox, stone marten, and wild boar) and one domestic (sheep) host. Nucleotide sequence analysis identified four genotypes of E. bieneusi, Type IV, Wildboar3, BEB6, and PtEbIX. Three of those genotypes belong to Groups 1 (Type IV and Wildboar3) and 2 (BEB6), which are known to contain genotypes capable of infecting a variety of hosts, including humans, highlighting their public health importance. PtEbIX belongs to the dog-specific Group 11. This study represents the first, largest, and most comprehensive molecular-based epidemiology survey carried out in Portugal in wild and domestic animals to date and the first worldwide identification of E. bieneusi in wolf species. Our study showed that wild carnivores and ungulates may act as reservoirs of zoonotic genotypes of E. bieneusi, establishing their role in maintaining the sylvatic cycle of this parasite while representing a potential source of infection for humans and domestic animals.

Figueiredo, Ana M. (ORCID:0000000226236340)↗

Brassica rapa Domestication: Untangling Wild and Feral Forms and Convergence of Crop Morphotypes

The study of domestication contributes to our knowledge of evolution and crop genetic resources. Human selection has shaped wild Brassica rapa into diverse turnip, leafy, and oilseed crops. Despite its worldwide economic importance and potential as a model for understanding diversification under domestication, insights into the number of domestication events and initial crop(s) domesticated in B. rapa have been limited due to a lack of clarity about the wild or feral status of conspecific noncrop relatives. To address this gap and reconstruct the domestication history of B. rapa, we analyzed 68,468 genotyping-by-sequencing-derived single nucleotide polymorphisms for 416 samples in the largest diversity panel of domesticated and weedy B. rapa to date. To further understand the center of origin, we modeled the potential range of wild B. rapa during the mid-Holocene. Our analyses of genetic diversity across B. rapa morphotypes suggest that noncrop samples from the Caucasus, Siberia, and Italy may be truly wild, whereas those occurring in the Americas and much of Europe are feral. Clustering, tree-based analyses, and parameterized demographic inference further indicate that turnips were likely the first crop type domesticated, from which leafy types in East Asia and Europe were selected from distinct lineages. These findings clarify the domestication history and nature of wild crop genetic resources for B. rapa, which provides the first step toward investigating cases of possible parallel selection, the domestication and feralization syndrome, and novel germplasm for Brassica crop improvement.

59 BASIC BIOLOGICAL SCIENCES↗

Structural insights into the substrate specificity of the endonuclease activity of the influenza virus cap-snatching mechanism

Abstract The endonuclease activity within the influenza virus cap-snatching process is a proven therapeutic target. The anti-influenza drug baloxavir is highly effective, but is associated with resistance mutations that threaten its clinical efficacy. The endonuclease resides within the N-terminal domain of the PA subunit (PAN) of the influenza RNA dependent RNA polymerase, and we report here complexes of PAN with RNA and DNA oligonucleotides to understand its specificity and the structural basis of baloxavir resistance mutations. The RNA and DNA oligonucleotides bind within the substrate binding groove of PAN in a similar fashion, explaining the ability of the enzyme to cleave both substrates. The individual nucleotides occupy adjacent conserved pockets that flank the two-metal active site. However, the 2′ OH of the RNA ribose moieties engage in additional interactions that appear to optimize the binding and cleavage efficiency for the natural substrate. The major baloxavir resistance mutation at position 38 is at the core of the substrate binding site, but structural studies and modeling suggest that it maintains the necessary virus fitness via compensating interactions with RNA. These studies will facilitate the development of new influenza therapeutics that spatially match the substrate and are less likely to elicit resistance mutations.

59 BASIC BIOLOGICAL SCIENCES↗

DXO/Rai1 enzymes remove 5′-end FAD and dephospho-CoA caps on RNAs

Abstract In eukaryotes, the DXO/Rai1 enzymes can eliminate most of the incomplete and non-canonical NAD caps through their decapping, deNADding and pyrophosphohydrolase activities. Here, we report that these enzymes can also remove FAD and dephospho-CoA (dpCoA) non-canonical caps from RNA, and we have named these activities deFADding and deCoAping. The crystal structures of mammalian DXO with 3′-FADP or CoA and fission yeast Rai1 with 3′-FADP provide elegant insight to these activities. FAD and CoA are accommodated in the DXO/Rai1 active site by adopting folded conformations. The flavin of FAD and the pantetheine group of CoA contact the same region at the bottom of the active site tunnel, which undergoes conformational changes to accommodate the different cap moieties. We have developed FAD-capQ to detect and quantify FAD-capped RNAs and determined that FAD caps are present on short RNAs (with less than ∼200 nucleotides) in human cells and that these RNAs are stabilized in the absence of DXO.

59 BASIC BIOLOGICAL SCIENCES↗

Base pairing, structural and functional insights into N 4 -methylcytidine (m 4 C) and N 4 ,N 4 -dimethylcytidine (m 4 2 C) modified RNA

The N 4 -methylation of cytidine (m 4 C and m 4 2 C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m 4 C and m 4 2 C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m 4 C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m 4 2 C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m 4 2 C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N 4 -position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.

59 BASIC BIOLOGICAL SCIENCES↗

A minimal motif for sequence recognition by mitochondrial transcription factor A (TFAM)

Abstract Mitochondrial transcription factor A (TFAM) plays a critical role in mitochondrial transcription initiation and mitochondrial DNA (mtDNA) packaging. Both functions require DNA binding, but in one case TFAM must recognize a specific promoter sequence, while packaging requires coating of mtDNA by association with non sequence-specific regions. The mechanisms by which TFAM achieves both sequence-specific and non sequence-specific recognition have not yet been determined. Existing crystal structures of TFAM bound to DNA allowed us to identify two guanine-specific interactions that are established between TFAM and the bound DNA. These interactions are observed when TFAM is bound to both specific promoter sequences and non-sequence specific DNA. These interactions are established with two guanine bases separated by 10 random nucleotides (GN10G). Our biochemical results demonstrate that the GN10G consensus is essential for transcriptional initiation and contributes to facilitating TFAM binding to DNA substrates. Furthermore, we report a crystal structure of TFAM in complex with a non sequence-specific sequence containing a GN10G consensus. The structure reveals a unique arrangement in which TFAM bridges two DNA substrates while maintaining the GN10G interactions. We propose that the GN10G consensus is key to facilitate the interaction of TFAM with DNA.

59 BASIC BIOLOGICAL SCIENCES↗

Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination

Abstract In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion. The offspring displayed complex inheritance patterns with one of the parents consistently contributing the major part of the chromosome backbone and multiple unselected fragments originating from the second parent. Our results demonstrate that this bias was in part due to the action of restriction–modification systems, whereas genome features like GC content and local nucleotide identity did not affect distribution of recombination events around the chromosome. Furthermore, we found that recombination occurred uniformly across the genome without concentration into hotspots. Notably, our results show that species-level genetic distance did not affect genome-wide recombination. This study provides a new insight into the dynamics of recombination in bacteria and a platform for studying recombination patterns in diverse bacterial species.

59 BASIC BIOLOGICAL SCIENCES↗

Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome

The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

59 BASIC BIOLOGICAL SCIENCES↗

Uncovering translation roadblocks during the development of a synthetic tRNA

Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNA UTu1 , a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNA UTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.

59 BASIC BIOLOGICAL SCIENCES↗

The IMG/M data management and analysis system v.7: content updates and new features

The Integrated Microbial Genomes & Microbiomes system at the Department of Energy (DOE) Joint Genome Institute (JGI) continues to provide support for users to perform comparative analysis of isolate and single cell genomes, metagenomes, and metatranscriptomes. In addition to datasets produced by the JGI, IMG v.7 also includes datasets imported from public sources such as NCBI Genbank, SRA, and the DOE National Microbiome Data Collaborative (NMDC), or submitted by external users. In the past couple years, we have continued our effort to help the user community by improving the annotation pipeline, upgrading the contents with new reference database versions, and adding new analysis functionalities such as advanced scaffold search, Average Nucleotide Identity (ANI) for high-quality metagenome bins, new cassette search, improved gene neighborhood display, and improvements to metatranscriptome data display and analysis. Here, we also extended the collaboration and integration efforts with other DOE-funded projects such as NMDC and DOE Biology Knowledgebase (KBase).

59 BASIC BIOLOGICAL SCIENCES↗

Quantum biological insights into CRISPR-Cas9 sgRNA efficiency from explainable-AI driven feature engineering

Abstract CRISPR-Cas9 tools have transformed genetic manipulation capabilities in the laboratory. Empirical rules-of-thumb have been developed for only a narrow range of model organisms, and mechanistic underpinnings for sgRNA efficiency remain poorly understood. This work establishes a novel feature set and new public resource, produced with quantum chemical tensors, for interpreting and predicting sgRNA efficiency. Feature engineering for sgRNA efficiency is performed using an explainable-artificial intelligence model: iterative Random Forest (iRF). By encoding quantitative attributes of position-specific sequences for Escherichia coli sgRNAs, we identify important traits for sgRNA design in bacterial species. Additionally, we show that expanding positional encoding to quantum descriptors of base-pair, dimer, trimer, and tetramer sequences captures intricate interactions in local and neighboring nucleotides of the target DNA. These features highlight variation in CRISPR-Cas9 sgRNA dynamics between E. coli and H. sapiens genomes. These novel encodings of sgRNAs enhance our understanding of the elaborate quantum biological processes involved in CRISPR-Cas9 machinery.

59 BASIC BIOLOGICAL SCIENCES↗

Interaction of N-methylmesoporphyrin IX with a hybrid left-/right-handed G-quadruplex motif from the promoter of the SLC2A1 gene

Abstract Left-handed G-quadruplexes (LHG4s) belong to a class of recently discovered noncanonical DNA structures under the larger umbrella of G-quadruplex DNAs (G4s). The biological relevance of these structures and their ability to be targeted with classical G4 ligands is underexplored. Here, we explore whether the putative LHG4 DNA sequence from the SLC2A1 oncogene promoter maintains its left-handed characteristics upon addition of nucleotides in the 5′- and 3′-direction from its genomic context. We also investigate whether this sequence interacts with a well-established G4 binder, N-methylmesoporphyrin IX (NMM). We employed biophysical and X-ray structural studies to address these questions. Our results indicate that the sequence d[G(TGG)3TGA(TGG)4] (termed here as SLC) adopts a two-subunit, four-tetrad hybrid left-/right-handed G4 (LH/RHG4) topology. Addition of 5′-G or 5′-GG abolishes the left-handed fold in one subunit, while the addition of 3′-C or 3′-CA maintains the original fold. X-ray crystal structure analyses show that SLC maintains the same hybrid LH/RHG4 fold in the solid state and that NMM stacks onto the right-handed subunit of SLC. NMM binds to SLC with a 1:1 stoichiometry and a moderate-to-tight binding constant of 15 μM−1. This work deepens our understanding of LHG4 structures and their binding with traditional G4 ligands.

Seth, Paul↗

Self-assembly and condensation of intermolecular poly(UG) RNA quadruplexes

Abstract Poly(UG) or ‘pUG’ dinucleotide repeats are highly abundant sequences in eukaryotic RNAs. In Caenorhabditis elegans, pUGs are added to RNA 3′ ends to direct gene silencing within Mutator foci, a germ granule condensate. Here, we show that pUG RNAs efficiently self-assemble into gel condensates through quadruplex (G4) interactions. Short pUG sequences form right-handed intermolecular G4s (pUG G4s), while longer pUGs form left-handed intramolecular G4s (pUG folds). We determined a 1.05 Å crystal structure of an intermolecular pUG G4, which reveals an eight stranded G4 dimer involving 48 nucleotides, 7 different G and U quartet conformations, 7 coordinated potassium ions, 8 sodium ions and a buried water molecule. A comparison of the intermolecular pUG G4 and intramolecular pUG fold structures provides insights into the molecular basis for G4 handedness and illustrates how a simple dinucleotide repeat sequence can form complex structures with diverse topologies.

Biochemistry & Molecular Biology↗

Processing of DNA single-strand breaks with oxidatively damaged ends by LIG1

Abstract DNA ligase 1 (LIG1) seals broken strand breaks by joining two adjacent ends during DNA replication and repair transactions. We previously reported atomic-level insight into the strategies that LIG1 uses to discriminate mismatches or ribonucleotides. However, how LIG1 processes strand breaks with oxidatively damaged ends in the absence and presence of a “wrong” sugar remains unknown. Here, we determined the crystal structures of LIG1/nick DNA complexes with 3′-8-oxodG and 3′-8-oxorG templating A or C during the pre- and post-catalytic steps of the ligation reaction. Our structures demonstrated differences in the distances at the +1 and +2 nucleotides relative to the 3′-end of the nick and a shift in the template base position to accommodate the oxidative lesion depending on the dual coding potential of 8-oxoG, which forms Hoogsteen or Watson–Crick base pairing in -syn or -anti conformation. Furthermore, these structural adjustments lead to mutagenic ligation or non-mutagenic end joining of the nick substrates. Overall, our findings provide mechanistic insight into how LIG1 processes nicks harboring oxidative damage and ribonucleotides to ensure fidelity at the final ligation step of DNA repair and replication to maintain genome integrity.

Balu, Kanal E [Department of Biochemistry and Mole↗