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At least 1,531 records · Page 85
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Robust, versatile DNA FISH probes for chromosome-specific repeats in Caenorhabditis elegans and Pristionchus pacificus

Repetitive DNA sequences are useful targets for chromosomal fluorescence in situ hybridization. We analyzed recent genome assemblies of Caenorhabditis elegans and Pristionchus pacificus to identify tandem repeats with a unique genomic localization. Based on these findings, we designed and validated sets of oligonucleotide probes for each species targeting at least 1 locus per chromosome. These probes yielded reliable fluorescent signals in different tissues and can easily be combined with the immunolocalization of cellular proteins. Synthesis and labeling of these probes are highly cost-effective and require no hands-on labor. The methods presented here can be easily applied in other model and nonmodel organisms with a sequenced genome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Metagenomics harvested genus-specific single-stranded DNA-annealing proteins improve and expand recombineering in Pseudomonas species

The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad set of valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we first employed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using a high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida and Pseudomonas aeruginosa, and expanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancing their potential for synthetic biology applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

DNA double-strand break movement in heterochromatin depends on the histone acetyltransferase dGcn5

Abstract Cells employ diverse strategies to repair double-strand breaks (DSBs), a dangerous form of DNA damage that threatens genome integrity. Eukaryotic nuclei consist of different chromatin environments, each displaying distinct molecular and biophysical properties that can significantly influence the DSB-repair process. DSBs arising in the compact and silenced heterochromatin domains have been found to move to the heterochromatin periphery in mouse and Drosophila to prevent aberrant recombination events. However, it is poorly understood how chromatin components, such as histone post-translational modifications, contribute to these DSB movements within heterochromatin. Using irradiation as well as locus-specific DSB induction in Drosophila tissues and cultured cells, we find enrichment of histone H3 lysine 9 acetylation (H3K9ac) at DSBs in heterochromatin but not euchromatin. We find this increase is mediated by the histone acetyltransferase dGcn5, which rapidly localizes to heterochromatic DSBs. Moreover, we demonstrate that in the absence of dGcn5, heterochromatic DSBs display impaired recruitment of the SUMO E3 ligase Nse2/Qjt and fail to relocate to the heterochromatin periphery to complete repair. In summary, our results reveal a previously unidentified role for dGcn5 and H3K9ac in heterochromatic DSB repair and underscore the importance of differential chromatin responses at heterochromatic and euchromatic DSBs to promote safe repair.

Biochemistry & Molecular Biology↗
DOE OSTI2014

The spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase β

To provide molecular-level insights into the spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase β (polβ), we report four crystal structures of polβ complexed with dG•dTTP and dA•dCTP mismatches in the presence of Mg 2+ or Mn 2+ . The Mg 2+ -bound ground-state structures show that the dA•dCTP-Mg 2+ complex adopts an ‘intermediate’ protein conformation while the dG•dTTP-Mg 2+ complex adopts an open protein conformation. The Mn 2+ -bound ‘pre-chemistry-state’ structures show that the dA•dCTP-Mn2+ complex is structurally very similar to the dA•dCTP-Mg 2+ complex, whereas the dG•dTTP-Mn 2+ complex undergoes a large-scale conformational change to adopt a Watson–Crick-like dG•dTTP base pair and a closed protein conformation. These structural differences, together with our molecular dynamics simulation studies, suggest that polβ increases replication fidelity via a two-stage mismatch discrimination mechanism, where one is in the ground state and the other in the closed conformation state. In the closed conformation state, polβ appears to allow only a Watson–Crick-like conformation for purine•pyrimidine base pairs, thereby discriminating the mismatched base pairs based on their ability to form the Watson–Crick-like conformation. Overall, the present studies provide new insights into the spontaneous replication error and the replication fidelity mechanisms of polβ.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

New Microviridae isolated from Sulfitobacter reveals two cosmopolitan subfamilies of single-stranded DNA phages infecting marine and terrestrial Alphaproteobacteria

The Microviridae family represents one of the major clades of single-stranded DNA (ssDNA) phages. Their cultivated members are lytic and infect Proteobacteria, Bacteroidetes, and Chlamydiae. Prophages have been predicted in the genomes from Bacteroidales, Hyphomicrobiales, and Enterobacteriaceae and cluster within the ‘Alpavirinae’, ‘Amoyvirinae’, and Gokushovirinae. We have isolated ‘Ascunsovirus oldenburgi’ ICBM5, a novel phage distantly related to known Microviridae. It infects Sulfitobacter dubius SH24-1b and uses both a lytic and a carrier-state life strategy. Using ICBM5 proteins as a query, we uncovered in publicly available resources sixty-five new Microviridae prophages and episomes in bacterial genomes and retrieved forty-seven environmental viral genomes (EVGs) from various viromes. Genome clustering based on protein content and phylogenetic analysis showed that ICBM5, together with Rhizobium phages, new prophages, episomes, and EVGs cluster within two new phylogenetic clades, here tentatively assigned the rank of subfamily and named ‘Tainavirinae’ and ‘Occultatumvirinae’. They both infect Rhodobacterales. Occultatumviruses also infect Hyphomicrobiales, including nitrogen-fixing endosymbionts from cosmopolitan legumes. A biogeographical assessment showed that tainaviruses and occultatumviruses are spread worldwide, in terrestrial and marine environments. The new phage isolated here sheds light onto new and diverse branches of the Microviridae tree, suggesting that much of the ssDNA phage diversity remains in the dark.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

DNA Sequence-Based Identification of Fusarium : A Work in Progress

Accurate species-level identification of an etiological agent is crucial for disease diagnosis and management because knowing the agent’s identity connects it with what is known about its host range, geographic distribution, and toxin production potential. This is particularly true in publishing peer-reviewed disease reports, where imprecise and/or incorrect identifications weaken the public knowledge base. This can be a daunting task for phytopathologists and other applied biologists that need to identify Fusarium in particular, because published and ongoing multilocus molecular systematic studies have highlighted several confounding issues. Paramount among these are: (i) this agriculturally and clinically important genus is currently estimated to comprise more than 400 phylogenetically distinct species (i.e., phylospecies), with more than 80% of these discovered within the past 25 years; (ii) approximately one-third of the phylospecies have not been formally described; (iii) morphology alone is inadequate to distinguish most of these species from one another; and (iv) the current rapid discovery of novel fusaria from pathogen surveys and accompanying impact on the taxonomic landscape is expected to continue well into the foreseeable future. To address the critical need for accurate pathogen identification, our research groups are focused on populating two web-accessible databases (FUSARIUM-ID v.3.0 and the nonredundant National Center for Biotechnology Information nucleotide collection that includes GenBank) with portions of three phylogenetically informative genes (i.e., TEF1, RPB1, and RPB2) that resolve at or near the species level in every Fusarium species. The objectives of this Special Report, and its companion in this issue ( Torres-Cruz et al. 2022 ), are to provide a progress report on our efforts to populate these databases and to outline a set of best practices for DNA sequence-based identification of fusaria.

Plant Sciences↗
DOE OSTI2014

The Rad50 hook domain regulates DNA damage signaling and tumorigenesis

The Mre11 complex (Mre11, Rad50, and Nbs1) is a central component of the DNA damage response (DDR), governing both double-strand break repair and DDR signaling. Rad50 contains a highly conserved Zn 2+ -dependent homodimerization interface, the Rad50 hook domain. Mutations that inactivate the hook domain produce a null phenotype. In this study, we analyzed mutants with reduced hook domain function in an effort to stratify hookdependent Mre11 complex functions. One of these alleles, Rad50 46 , conferred reduced Zn 2+ affinity and dimerization efficiency. Homozygous Rad50 46/46 mutations were lethal in mice. However, in the presence of wildtype Rad50, Rad50 46 exerted a dominant gain-of-function phenotype associated with chronic DDR signaling. At the organismal level, Rad50 +/46 exhibited hydrocephalus, liver tumorigenesis, and defects in primitive hematopoietic and gametogenic cells. These outcomes were dependent on ATM, as all phenotypes were mitigated in Rad50 +/46 Atm +/– mice. These data reveal that the murine Rad50 hook domain strongly influences Mre11 complex-dependent DDR signaling, tissue homeostasis, and tumorigenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Deciphering the crystal structure of a novel nanobody against the NEIL1 DNA glycosylase

Nanobodies (VHHs) are single-domain antibodies with three antigenic CDR regions and are used in diverse scientific applications. Here, an ∼14 kDa nanobody (A5) specific for the endonuclease VIII (Nei)-like 1 or NEIL1 DNA glycosylase involved in the first step of the base-excision repair pathway was crystallized and its structure was determined to 2.1 Å resolution. The crystals posed challenges due to potential twinning and anisotropic diffraction. Despite inconclusive twinning indicators, reprocessing in an orthorhombic setting and molecular replacement in space groupP2 1 2 1 2 enabled the successful modeling of 96% of residues in the asymmetric unit, with finalR work andR free values of 0.199 and 0.229, respectively.

Biochemistry & Molecular Biology↗
DOE OSTI2023

Particle size influences decay rates of environmental DNA in aquatic systems

Abstract Environmental DNA (eDNA) analysis is a powerful tool for remote detection of target organisms. However, obtaining quantitative and longitudinal information from eDNA data is challenging, requiring a deep understanding of eDNA ecology. Notably, if the various size components of eDNA decay at different rates, and we can separate them within a sample, their changing proportions could be used to obtain longitudinal dynamics information on targets. To test this possibility, we conducted an aquatic mesocosm experiment in which we separated fish‐derived eDNA components using sequential filtration to evaluate the decay rate and changing proportion of various eDNA particle sizes over time. We then fit four alternative mathematical decay models to the data, building towards a predictive framework to interpret eDNA data from various particle sizes. We found that medium‐sized particles (1–10 μm) decayed more slowly than other size classes (i.e., <1 and > 10 μm), and thus made up an increasing proportion of eDNA particles over time. We also observed distinct eDNA particle size distribution (PSD) between our Common carp and Rainbow trout samples, suggesting that target‐specific assays are required to determine starting eDNA PSDs. Additionally, we found evidence that different sizes of eDNA particles do not decay independently, with particle size conversion replenishing smaller particles over time. Nonetheless, a parsimonious mathematical model where particle sizes decay independently best explained the data. Given these results, we suggest a framework to discern target distance and abundance with eDNA data by applying sequential filtration, which theoretically has both metabarcoding and single‐target applications.

Brandão‐Dias, Pedro F. P.↗
DOE OSTI2025

eDNAjoint: An R package for interpreting paired or semi‐paired environmental DNA and traditional survey data in a Bayesian framework

Abstract Environmental DNA (eDNA) sampling is increasingly used in surveys of species distribution as a potentially sensitive and efficient monitoring method. Yet access to modelling tools designed specifically for interpreting this new data type lags behind its ubiquity. While occupancy modelling software has dominated the analytical landscape for eDNA data analysis of single species, this type of model may not always be the most appropriate. The rate of eDNA detection often corresponds to species density, rather than just occupancy, and researchers often have access to observations from non‐genetic sampling methods at the same sites. To provide users access to a modelling framework designed to maximize the use of all available data, we developed an R package, eDNAjoint . The package provides an easy‐to‐use interface for fitting a ‘joint’ model that integrates data from paired or semi‐paired eDNA and traditional surveys in a Bayesian framework. The model can be used to estimate parameters like the probability of a false positive eDNA detection and mean catch rate at a site, and the package allows access to multiple model variations and Bayesian prior customization. Additional functionality can be used for model selection, summarising posteriors and comparing the relative sensitivities of the two survey methods. We demonstrate the use of eDNAjoint by fitting a variation of the model with site‐level covariates that scale the sensitivity of eDNA sampling relative to traditional sampling. The example workflow uses binary eDNA and seine count data for the endangered tidewater goby ( Eucyclogobius newberryi ) from a study by Schmelzle and Kinziger (2016). This use case includes a prior sensitivity analysis and an evaluation of the relationship between detection rates and environmental variables. eDNAjoint has the potential to greatly increase the range of users who will be able to rigorously analyse eDNA and traditional survey data in a Bayesian framework, understand if and how eDNA can improve monitoring practices, and gain confidence in the interpretability of eDNA data.

Keller, Abigail G. [Department of Environment Scie↗
DOE OSTI2025

Environmental DNA as a tool for hydropower impact assessments: current status, special considerations, and future integration

Globally there is an urgent need to find sustainable solutions to balance energy production with the protection of vulnerable species and conservation of biodiversity. This is particularly critical for freshwater ecosystems, habitats, and species that may be impacted by hydropower development and operations needed to meet energy grid demands. Reliable and accurate environmental impact assessments (EIAs) that identify the biological, physical, or social impacts of hydropower are key to ensure biodiversity, ecosystem, and societal sustainability. The analysis of environmental DNA (eDNA) has the potential to transform hydropower EIAs, management and mitigation planning, and decision-making procedures. Further, the incorporation of eDNA surveys into EIAs during both hydropower planning and continued operations may streamline regulatory processes by improving our understanding of potentially impacted biota and habitats and evaluating environmental impacts mitigation. Here, we: (i) highlight current understanding and use of eDNA in freshwater environments; (ii) examine critical considerations for eDNA integration into hydropower EIAs and biological monitoring; (iii) identify knowledge gaps in eDNA analysis and applications unique to hydropower-regulated systems; and (iv) discuss future opportunities to bolster the incorporation of eDNA into hydropower research including regulatory acceptance and public engagement. While we acknowledge that there are several factors that may complicate the broad adoption of eDNA as a tool for assessing the impacts of hydropower, we anticipate that growing confidence in eDNA through hydropower-specific protocols, calibrations, and validations will overcome these inherent uncertainties.

aquatic biodiversity↗
DOE OSTI2020

Beyond regulations at DNA levels: A review of epigenetic therapeutics targeting cancer stem cells

Abstract In the past few years, the paramount role of cancer stem cells (CSCs), in terms of cancer initiation, proliferation, metastasis, invasion and chemoresistance, has been revealed by accumulating studies. However, this level of cellular plasticity cannot be entirely explained by genetic mutations. Research on epigenetic modifications as a complementary explanation for the properties of CSCs has been increasing over the past several years. Notably, therapeutic strategies are currently being developed in an effort to reverse aberrant epigenetic alterations using specific chemical inhibitors. In this review, we summarize the current understanding of CSCs and their role in cancer progression, and provide an overview of epigenetic alterations seen in CSCs. Importantly, we focus on primary cancer therapies that target the epigenetic modification of CSCs by the use of specific chemical inhibitors, such as histone deacetylase (HDAC) inhibitors, DNA methyltransferase (DNMT) inhibitors and microRNA‐based (miRNA‐based) therapeutics.

Zhang, Shunhao↗
DOE OSTI2022

Temperature modulation of CAMTA3 gene induction activity is mediated through the DNA binding domain

The calmodulin-binding transcription activator (CAMTA) proteins of Arabidopsis thaliana play a major role in cold acclimation, contributing to the rapid induction of the C-REPEAT BINDING FACTOR (CBF) genes and other genes that impart freezing tolerance in plants exposed to cold temperature (4°C). The goal of this study was to better understand how the gene induction activity of CAMTA3 is modulated by temperature. Our results indicate that a severely truncated version of CAMTA3, CAMTA3 334 , which includes the N-terminal CG-1 DNA binding domain and a newly identified transcriptional activation domain (TAD), was able to rapidly induce the expression of CBF2 and two newly identified target genes, EXPANSIN-LIKE A1 (EXPL1) and NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), in response to cold temperature. Additionally, CAMTA3 334 was able to restore freezing tolerance when expressed in a camta23 double mutant. The ability of CAMTA3 and CAMTA3 334 to induce target genes at cold temperature did not involve increased levels of these proteins or increased binding of these proteins to target gene promoters in cold-treated plants. Rather, domain-swapping experiments indicated that the CAMTA3 CG-1 domain conferred temperature dependence to the ability of the CAMTA3 TAD to induce gene expression. The CG-1 domain also enabled the TAD to induce the expression of target genes at a moderate temperature (22°C) in response to cycloheximide treatment, consistent with the TAD activity not being intrinsically temperature dependent. We propose a working model in which the temperature modulation of CAMTA3 gene induction activity occurs independently from the C-terminal calmodulin-binding domains that previously have been proposed to activate CAMTA3 transcriptional activity in response to cold temperature.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Haplotype‐resolved genome assembly of Populus tremula × P. alba reveals aspen‐specific megabase satellite DNA

SUMMARY Populus species play a foundational role in diverse ecosystems and are important renewable feedstocks for bioenergy and bioproducts. Hybrid aspen Populus tremula × P. alba INRA 717‐1B4 is a widely used transformation model in tree functional genomics and biotechnology research. As an outcrossing interspecific hybrid, its genome is riddled with sequence polymorphisms which present a challenge for sequence‐sensitive analyses. Here we report a telomere‐to‐telomere genome for this hybrid aspen with two chromosome‐scale, haplotype‐resolved assemblies. We performed a comprehensive analysis of the repetitive landscape and identified both tandem repeat array‐based and array‐less centromeres. Unexpectedly, the most abundant satellite repeats in both haplotypes lie outside of the centromeres, consist of a 147 bp monomer PtaM147, frequently span >1 megabases, and form heterochromatic knobs. PtaM147 repeats are detected exclusively in aspens (section Populus ) but PtaM147‐like sequences occur in LTR‐retrotransposons of closely related species, suggesting their origin from the retrotransposons. The genomic resource generated for this transformation model genotype has greatly improved the design and analysis of genome editing experiments that are highly sensitive to sequence polymorphisms. The work should motivate future hypothesis‐driven research to probe into the function of the abundant and aspen‐specific PtaM147 satellite DNA.

Zhou, Ran↗
DOE OSTI2016

SU-F-T-125: Radial Dose Distributions From Carbon Ions of Therapeutic Energies Calculated with Geant4-DNA

Purpose: Radial dose distribution D(r) is the dose as a function of lateral distance from the path of a heavy charged particle. Its main application is in modelling of biological effects of heavy ions, including applications to hadron therapy. It is the main physical parameter of a broad group of radiobiological models known as the amorphous track models. Our purpose was to calculate D(r) with Monte Carlo for carbon ions of therapeutic energies, find a simple formula for D(r) and fit it to the Monte Carlo data. Methods: All calculations were performed with Geant4-DNA code, for carbon ion energies from 10 to 400 MeV/u (ranges in water: ∼ 0.4 mm to 27 cm). The spatial resolution of dose distribution in the lateral direction was 1 nm. Electron tracking cut off energy was 11 eV (ionization threshold). The maximum lateral distance considered was 10 µm. Over this distance, D(r) decreases with distance by eight orders of magnitude. Results: All calculated radial dose distributions had a similar shape dominated by the well-known inverse square dependence on the distance. Deviations from the inverse square law were observed close to the beam path (r<10 nm) and at large distances (r >1 µm). At small and large distances D(r) decreased, respectively, slower and faster than the inverse square of distance. A formula for D(r) consistent with this behavior was found and fitted to the Monte Carlo data. The accuracy of the fit was better than 10% for all distances considered. Conclusion: We have generated a set of radial dose distributions for carbon ions that covers the entire range of therapeutic energies, for distances from the ion path of up to 10 µm. The latter distance is sufficient for most applications because dose beyond 10 µm is extremely low.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

NS1 DNA vaccination protects against Zika infection through T cell–mediated immunity in immunocompetent mice

The causal association of Zika virus (ZIKV) with microcephaly, congenital malformations in infants, and Guillain-Barré syndrome in adults highlights the need for effective vaccines. Thus far, efforts to develop ZIKV vaccines have focused on the viral envelope. ZIKV NS1 as a vaccine immunogen has not been fully explored, although it can circumvent the risk of antibody-dependent enhancement of ZIKV infection, associated with envelope antibodies. Here, we describe a novel DNA vaccine encoding a secreted ZIKV NS1, that confers rapid protection from systemic ZIKV infection in immunocompetent mice. We identify novel NS1 T cell epitopes in vivo and show that functional NS1-specific T cell responses are critical for protection against ZIKV infection. We demonstrate that vaccine-induced anti-NS1 antibodies fail to confer protection in the absence of a functional T cell response. This highlights the importance of using NS1 as a target for T cell–based ZIKV vaccines.

59 BASIC BIOLOGICAL SCIENCES↗