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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
NASA NTRS1997

Mechanical continuity and reversible chromosome disassembly within intact genomes removed from living cells

Chromatin is thought to be structurally discontinuous because it is packaged into morphologically distinct chromosomes that appear physically isolated from one another in metaphase preparations used for cytogenetic studies. However, analysis of chromosome positioning and movement suggest that different chromosomes often behave as if they were physically connected in interphase as well as mitosis. To address this paradox directly, we used a microsurgical technique to physically remove nucleoplasm or chromosomes from living cells under isotonic conditions. Using this approach, we found that pulling a single nucleolus or chromosome out from interphase or mitotic cells resulted in sequential removal of the remaining nucleoli and chromosomes, interconnected by a continuous elastic thread. Enzymatic treatments of interphase nucleoplasm and chromosome chains held under tension revealed that mechanical continuity within the chromatin was mediated by elements sensitive to DNase or micrococcal nuclease, but not RNases, formamide at high temperature, or proteases. In contrast, mechanical coupling between mitotic chromosomes and the surrounding cytoplasm appeared to be mediated by gelsolin-sensitive microfilaments. Furthermore, when ion concentrations were raised and lowered, both the chromosomes and the interconnecting strands underwent multiple rounds of decondensation and recondensation. As a result of these dynamic structural alterations, the mitotic chains also became sensitive to disruption by restriction enzymes. Ion-induced chromosome decondensation could be blocked by treatment with DNA binding dyes, agents that reduce protein disulfide linkages within nuclear matrix, or an antibody directed against histones. Fully decondensed chromatin strands also could be induced to recondense into chromosomes with pre-existing size, shape, number, and position by adding anti-histone antibodies. Conversely, removal of histones by proteolysis or heparin treatment produced chromosome decondensation which could be reversed by addition of histone H1, but not histones H2b or H3. These data suggest that DNA, its associated protein scaffolds, and surrounding cytoskeletal networks function as a structurally-unified system. Mechanical coupling within the nucleoplasm may coordinate dynamic alterations in chromatin structure, guide chromosome movement, and ensure fidelity of mitosis.

NASA Discipline Cell Biology↗
DOE OSTI2026

The small protein SbtC is a functional component of the CO 2 concentrating mechanism in Synechocystis sp. PCC 6803

Oxygenic phototrophs fix CO 2 via the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), which shows relatively low CO 2 affinity and specificity. To circumvent low and fluctuating CO 2 concentrations in aquatic systems, cyanobacteria and algae have evolved sophisticated inorganic carbon (Ci) concentrating mechanisms (CCMs). Bicarbonate transporters such as SbtA play a crucial role in the cyanobacterial CCM and hence display multiple layers of tight regulation. Control of sbtA gene expression and corresponding transporter activity involves the PII-like protein SbtB, whose gene is frequently co-transcribed with sbtA. A previously non-annotated gene located upstream of the sbtAB operon in the model Synechocystis sp. PCC 6803 encodes the small protein SbtC, composed of 80 amino acids. Presence of SbtC was confirmed by immunoblotting of the sbtC-coding sequence fused to a Flag-tag. Similar to sbtAB , transcription of the sbtC locus is induced by low CO 2 availability; however, it is controlled independently. Mutation of the sbtC locus in a wild-type background produced only a mild phenotype, even under low CO 2 , but impaired diurnal growth resembled that of the mutant ΔsbtB . Biochemical analysis indicated a trimeric SbtABC complex in the membrane. Bicarbonate leakage from cells was strongly elevated when either sbtB or sbtC was deleted from recombinant Synechocystis strains harboring only SbtA as single Ci uptake system. Here, our results provide evidence that SbtC contributes to the formation of the SbtAB complex, thereby regulating bicarbonate exchange at the cytoplasmic membrane. Well-conserved SbtC-like proteins encoded in the neighborhood of sbtAB exist in many cyanobacterial genomes, pointing toward an important role in the cyanobacterial CCM.

Walke, Peter [Univ. of Rostock (Germany)] (ORCID:0↗
DOE OSTI2026

Artificial intelligence tools for enzyme engineering and metabolic engineering

Enzyme engineering and metabolic engineering drive innovation in energy biotechnology. In recent years, artificial intelligence (AI) has supported successful applications in designing effective enzymes and productive microbial cell factories. This review summarizes recent advances in enzyme redesign using protein language models, de novo enzyme design with generative models, and AI tools for engineering metabolism and related cellular phenotypes. Across these areas, AI models are shifting from single modality inputs to integrated representations of protein function, metabolic pathways, and cell states. We emphasize that unifying the diverse data representations across scales will be necessary for advancements in energy biotechnology.

Volk, Michael [Univ. of Illinois at Urbana-Champai↗
NASA NTRS1999

Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

NASA Discipline Plant Biology↗
DOE OSTI2023

BUZZ : an essential gene for postinitiation root hair growth and a mediator of root architecture in Brachypodium distachyon

Here, we discover a player in root development. Recovered from a forward-genetic screen in Brachypodium distachyon, the buzz mutant initiates root hairs but they fail to elongate. In addition, buzz roots grow twice as fast as wild-type roots. Also, lateral roots show increased sensitivity to nitrate, whereas primary roots are less sensitive to nitrate. Using whole-genome resequencing, we identified the causal single nucleotide polymorphism as occurring in a conserved but previously uncharacterized cyclin-dependent kinase (CDK)-like gene. The buzz mutant phenotypes are rescued by the wild-type B. distachyon BUZZ coding sequence and by an apparent homolog in Arabidopsis thaliana. Moreover, T-DNA mutants in A. thaliana BUZZ have shorter root hairs. BUZZ mRNA localizes to epidermal cells and develops root hairs and, in the latter, partially colocalizes with the NRT1.1A nitrate transporter. Based on qPCR and RNA-Seq, buzz overexpresses ROOT HAIRLESS LIKE SIX-1 and -2 and misregulates genes related to hormone signaling, RNA processing, cytoskeletal, and cell wall organization, and to the assimilation of nitrate. Overall, these data demonstrate that BUZZ is required for tip growth after root hair initiation and root architectural responses to nitrate.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2003

Polyomavirus JCV excretion and genotype analysis in HIV-infected patients receiving highly active antiretroviral therapy

OBJECTIVE: To assess the frequency of shedding of polyomavirus JC virus (JCV) genotypes in urine of HIV-infected patients receiving highly active antiretroviral therapy (HAART). METHODS: Single samples of urine and blood were collected prospectively from 70 adult HIV-infected patients and 68 uninfected volunteers. Inclusion criteria for HIV-infected patients included an HIV RNA viral load < 1000 copies, CD4 cell count of 200-700 x 106 cells/l, and stable HAART regimen. PCR assays and sequence analysis were carried out using JCV-specific primers against different regions of the virus genome. RESULTS: JCV excretion in urine was more common in HIV-positive patients but not significantly different from that of the HIV-negative group [22/70 (31%) versus 13/68 (19%); P = 0.09]. HIV-positive patients lost the age-related pattern of JCV shedding (P = 0.13) displayed by uninfected subjects (P = 0.01). Among HIV-infected patients significant differences in JCV shedding were related to CD4 cell counts (P = 0.03). Sequence analysis of the JCV regulatory region from both HIV-infected patients and uninfected volunteers revealed all to be JCV archetypal strains. JCV genotypes 1 (36%) and 4 (36%) were the most common among HIV-infected patients, whereas type 2 (77%) was the most frequently detected among HIV-uninfected volunteers. CONCLUSION: These results suggest that JCV shedding is enhanced by modest depressions in immune function during HIV infection. JCV shedding occurred in younger HIV-positive persons than in the healthy controls. As the common types of JCV excreted varied among ethnic groups, JCV genotypes associated with progressive multifocal leukoencephalopathy may reflect demographics of those infected patient populations.

Non-NASA Center↗
DOE OSTI2022

Improved genome editing by an engineered CRISPR-Cas12a

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis - and trans -DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A cross-study analysis of drug response prediction in cancer cell lines

Abstract To enable personalized cancer treatment, machine learning models have been developed to predict drug response as a function of tumor and drug features. However, most algorithm development efforts have relied on cross-validation within a single study to assess model accuracy. While an essential first step, cross-validation within a biological data set typically provides an overly optimistic estimate of the prediction performance on independent test sets. To provide a more rigorous assessment of model generalizability between different studies, we use machine learning to analyze five publicly available cell line-based data sets: National Cancer Institute 60, ancer Therapeutics Response Portal (CTRP), Genomics of Drug Sensitivity in Cancer, Cancer Cell Line Encyclopedia and Genentech Cell Line Screening Initiative (gCSI). Based on observed experimental variability across studies, we explore estimates of prediction upper bounds. We report performance results of a variety of machine learning models, with a multitasking deep neural network achieving the best cross-study generalizability. By multiple measures, models trained on CTRP yield the most accurate predictions on the remaining testing data, and gCSI is the most predictable among the cell line data sets included in this study. With these experiments and further simulations on partial data, two lessons emerge: (1) differences in viability assays can limit model generalizability across studies and (2) drug diversity, more than tumor diversity, is crucial for raising model generalizability in preclinical screening.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Variation of Structure and Cellular Functions of Type IA Topoisomerases across the Tree of Life

Topoisomerases regulate the topological state of cellular genomes to prevent impediments to vital cellular processes, including replication and transcription from suboptimal supercoiling of double-stranded DNA, and to untangle topological barriers generated as replication or recombination intermediates. The subfamily of type IA topoisomerases are the only topoisomerases that can alter the interlinking of both DNA and RNA. In this article, we provide a review of the mechanisms by which four highly conserved N-terminal protein domains fold into a toroidal structure, enabling cleavage and religation of a single strand of DNA or RNA. We also explore how these conserved domains can be combined with numerous non-conserved protein sequences located in the C-terminal domains to form a diverse range of type IA topoisomerases in Archaea, Bacteria, and Eukarya. There is at least one type IA topoisomerase present in nearly every free-living organism. The variation in C-terminal domain sequences and interacting partners such as helicases enable type IA topoisomerases to conduct important cellular functions that require the passage of nucleic acids through the break of a single-strand DNA or RNA that is held by the conserved N-terminal toroidal domains. In addition, this review will exam a range of human genetic disorders that have been linked to the malfunction of type IA topoisomerase.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Novel Mutant Alleles Reveal a Role of the Extra-Large G Protein in Rice Grain Filling, Panicle Architecture, Plant Growth, and Disease Resistance

Plant growth and grain filling are the key agronomical traits for grain weight and yield of rice. The continuous improvement in rice yield is required for a future sustainable global economy and food security. The heterotrimeric G protein complex containing a canonical α subunit (RGA1) couples extracellular signals perceived by receptors to modulate cell function including plant development and grain weight. We hypothesized that, besides RGA1, three atypical, extra-large GTP-binding protein (XLG) subunits also regulate panicle architecture, plant growth, development, grain weight, and disease resistance. Here, we identified a role of XLGs in agronomic traits and stress tolerance by genetically ablating all three rice XLGs individually and in combination using the CRISPR/Cas9 genome editing in rice. For this study, eight (three single, two double, and three triple) null mutants were selected. Three XLG proteins combinatorically regulate seed filling, because loss confers a decrease in grain weight from 14% with loss of one XLG and loss of three to 32% decrease in grain weight. Null mutations in XLG2 and XLG4 increase grain size. The mutants showed significantly reduced panicle length and number per plant including lesser number of grains per panicle compared to the controls. Loss-of-function of all individual XLGs contributed to 9% more aerial biomass compared to wild type (WT). The double mutant showed improved salinity tolerance. Moreover, loss of the XLG gene family confers hypersensitivity to pathogens. Our findings suggest that the non-canonical XLGs play important roles in regulating rice plant growth, grain filling, panicle phenotype, stress tolerance, and disease resistance. Genetic manipulation of XLG s has the potential to improve agronomic properties in rice.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Analysis of zebrafish periderm enhancers facilitates identification of a regulatory variant near human KRT8/18

Genome-wide association studies for non-syndromic orofacial clefting (OFC) have identified single nucleotide polymorphisms (SNPs) at loci where the presumed risk-relevant gene is expressed in oral periderm. The functional subsets of such SNPs are difficult to predict because the sequence underpinnings of periderm enhancers are unknown. We applied ATAC-seq to models of human palate periderm, including zebrafish periderm, mouse embryonic palate epithelia, and a human oral epithelium cell line, and to complementary mesenchymal cell types. We identified sets of enhancers specific to the epithelial cells and trained gapped-kmer support-vector-machine classifiers on these sets. We used the classifiers to predict the effects of 14 OFC-associated SNPs at 12q13 near KRT18. All the classifiers picked the same SNP as having the strongest effect, but the significance was highest with the classifier trained on zebrafish periderm. Reporter and deletion analyses support this SNP as lying within a periderm enhancer regulating KRT18/KRT8 expression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Ultra-Structural Imaging Provides 3D Organization of 46 Chromosomes of a Human Lymphocyte Prophase Nucleus

Three dimensional (3D) ultra-structural imaging is an important tool for unraveling the organizational structure of individual chromosomes at various stages of the cell cycle. Performing hitherto uninvestigated ultra-structural analysis of the human genome at prophase, we used serial block-face scanning electron microscopy (SBFSEM) to understand chromosomal architectural organization within 3D nuclear space. Acquired images allowed us to segment, reconstruct, and extract quantitative 3D structural information about the prophase nucleus and the preserved, intact individual chromosomes within it. Our data demonstrate that each chromosome can be identified with its homolog and classified into respective cytogenetic groups. Thereby, we present the first 3D karyotype built from the compact axial structure seen on the core of all prophase chromosomes. The chromosomes display parallel-aligned sister chromatids with familiar chromosome morphologies with no crossovers. Furthermore, the spatial positions of all 46 chromosomes revealed a pattern showing a gene density-based correlation and a neighborhood map of individual chromosomes based on their relative spatial positioning. A comprehensive picture of 3D chromosomal organization at the nanometer level in a single human lymphocyte cell is presented.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2020

Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break

Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase μ (Polμ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polμ engaging a DSB substrate. Synapsis is mediated solely by Polμ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol μ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polμ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2018

Coalescence of DNA Double Strand Breaks Induced by Galactic Cosmic Radiation is Modulated by Genetics in 15 Inbred Strains of Mice

In this manuscript we address the challenges associated with the ability to predict radiation sensitivity associated with exposure to either cosmic radiation or X-rays in a population study, by monitoring DNA damage sensing protein 53BP1 forming small nuclear radiation-induced foci (RIF) as a surrogate biomarker of DNA double strand breaks (DSB). 76 primary skin fibroblasts were isolated from 10 collaborative cross strains and five reference inbred mice (C57Bl/6, BALB/CByJ, B6C3, C3H and CBA/CaJ) and exposed to three different charged nuclei of increasing LET (350 MeV/n Si, 350 MeV/n Ar and 600 MeV/n Fe) and X-ray. Our data brings strong evidence against the classic "contact-first" model where DSBs are assumed to be immobile and repaired at the lesion site. In contrast, our model suggests nearby DSBs move into single repair unit characterized by large RIF before the repair machinery kicks in. Such model has the advantage of being much more efficient molecularly but is poorly suited to deal with cosmic radiation, where energy is concentrated along the particle trajectory, inducing a large density of DSBs along each particle track. In accordance with this model, RIF quantification after X-ray exposition showed a saturated dose response for early time points post-irradiation for all strains. Similarly, the high-LET response showed that RIF number matched the number of track per cell, not the number of expected DSB per cell (1). At the temporal level, we noted that the percentage of unrepaired high-LET tracks over a 48 hour time-course increased with LET, confirming that the DNA repair process becomes more difficult as more DSB coalesce into single RIF. There was also good agreement between persistent RIF levels measured in-vitro in the primary skin cultures and survival levels of T-cells and B-cells collected in blood samples from 10 CC strains 24 hours after 0.1 Gy whole-body dose of X-ray. This suggests that persistent RIF 24 hour post-IR is a good surrogate in-vitro biomarker for in-vivo radiation toxicity. Finally, at the genomic level, large differences in repair rates between strains for high-LET allowed us to identify suggestive genetic loci associated with radiation sensitivity. Interestingly, the two highest LETs provided the most strain variation with a common locus on Chromosome 10 highly enriched for DNA repair associated genes we discussed in detail.

Radiation-Induced Foci↗
DOE OSTI2021

Remarkably coherent population structure for a dominant Antarctic Chlorobium species

Background: In Antarctica, summer sunlight enables phototrophic microorganisms to drive primary production, thereby “feeding” ecosystems to enable their persistence through the long, dark winter months. In Ace Lake, a stratified marine-derived system in the Vestfold Hills of East Antarctica, a Chlorobium species of green sulphur bacteria (GSB) is the dominant phototroph, although its seasonal abundance changes more than 100-fold. Here, we analysed 413 Gb of Antarctic metagenome data including 59 Chlorobium metagenome-assembled genomes (MAGs) from Ace Lake and nearby stratified marine basins to determine how genome variation and population structure across a 7-year period impacted ecosystem function. Results: A single species, Candidatus Chlorobium antarcticum (most similar to Chlorobium phaeovibrioides DSM265) prevails in all three aquatic systems and harbours very little genomic variation (≥ 99% average nucleotide identity). A notable feature of variation that did exist related to the genomic capacity to biosynthesize cobalamin. The abundance of phylotypes with this capacity changed seasonally ~ 2-fold, consistent with the population balancing the value of a bolstered photosynthetic capacity in summer against an energetic cost in winter. The very high GSB concentration (> 10 8 cells ml –1 in Ace Lake) and seasonal cycle of cell lysis likely make Ca. Chlorobium antarcticum a major provider of cobalamin to the food web. Analysis of Ca. Chlorobium antarcticum viruses revealed the species to be infected by generalist (rather than specialist) viruses with a broad host range (e.g., infecting Gammaproteobacteria) that were present in diverse Antarctic lakes. The marked seasonal decrease in Ca. Chlorobium antarcticum abundance may restrict specialist viruses from establishing effective lifecycles, whereas generalist viruses may augment their proliferation using other hosts. Conclusion: The factors shaping Antarctic microbial communities are gradually being defined. In addition to the cold, the annual variation in sunlight hours dictates which phototrophic species can grow and the extent to which they contribute to ecosystem processes. The Chlorobium population studied was inferred to provide cobalamin, in addition to carbon, nitrogen, hydrogen, and sulphur cycling, as critical ecosystem services. The specific Antarctic environmental factors and major ecosystem benefits afforded by this GSB likely explain why such a coherent population structure has developed in this Chlorobium species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Elioraea tepida , sp. nov., a Moderately Thermophilic Aerobic Anoxygenic Phototrophic Bacterium Isolated from the Mat Community of an Alkaline Siliceous Hot Spring in Yellowstone National Park, WY, USA

Strain MS-P2 T was isolated from microbial mats associated with Mushroom Spring, an alkaline siliceous hot spring in Yellowstone National Park, WY, USA. The isolate grows chemoheterotrophically by oxygen-dependent respiration, and light stimulates photoheterotrophic growth under strictly oxic conditions. Strain MS-P2 T synthesizes bacteriochlorophyll a and the carotenoid spirilloxanthin. However, photoautotrophic growth did not occur under oxic or anoxic conditions, suggesting that this strain should be classified as an aerobic anoxygenic phototrophic bacterium. Strain MS-P2 T cells are motile, curved rods about 0.5 to 1.0 μm wide and 1.0 to 1.5 μm long. The optimum growth temperature is 45–50 °C, and the optimum pH for growth is circum-neutral (pH 7.0–7.5). Sequence analysis of the 16S rRNA gene revealed that strain MS-P2 T is closely related to Elioraea species, members of the class Alphaproteobacteria, with a sequence identity of 96.58 to 98%. The genome of strain MS-P2 T is a single circular DNA molecule of 3,367,643 bp with a mol% guanine-plus-cytosine content of 70.6%. Based on phylogenetic, physiological, biochemical, and genomic characteristics, we propose this bacteriochlorophyll a-containing isolate is a new species belonging to the genus Elioraea, with the suggested name Elioraeatepida. The type-strain is strain MS-P2 T (= JCM33060 T = ATCC TSD-174 T ).

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2007

High-LET Patterns of DSBs in DNA Loops, the HPRT Gene and Phosphorylation Foci

We present new results obtained with our model based on the track structure and chromatin geometry that predicts the DSB spatial and genomic distributions in a cell nucleus with the full genome represented. The model generates stochastic patterns of DSBs in the physical space of the nucleus filled with the realistic configuration of human chromosomes. The model was re-used to find the distribution of DSBs in a physical volume corresponding to a visible phosphorylation focus believed to be associated with a DSB. The data shows whether there must more than one DSB per foci due to finite size of the visible focus, even if a single DSB is radiochemically responsible for the phosphorylation of DNA in its vicinity. The same model can predict patterns of closely located DSBs in a given gene, or in a DNA loop, one of the large-scale chromatin structures. We demonstrated for the example of the HPRT gene, how different sorts of radiation lead to proximity effect in DSB locations, which is important for modeling gene deletions. The spectrum of intron deletions and total gene deletions was simulated for the HPRT gene. The same proximity effect of DSBs in a loop can hinder DSB restitutions, as parts of the loop between DSBs is deleted with a higher likelihood. The distributions of DSBs and deletions of DNA in a loop are presented.

Ponomarev, Artem L.↗
DOE OSTI2020

Photosynthetic Co-production of Succinate and Ethylene in a Fast-Growing Cyanobacterium, Synechococcus elongatus PCC 11801

Cyanobacteria are emerging as hosts for photoautotrophic production of chemicals. Recent studies have attempted to stretch the limits of photosynthetic production, typically focusing on one product at a time, possibly to minimise the additional burden of product separation. Here, we explore the simultaneous production of two products that can be easily separated: ethylene, a gaseous product, and succinate, an organic acid that accumulates in the culture medium. This was achieved by expressing a single copy of the ethylene forming enzyme (efe) under the control of P cpcB , the inducer-free super-strong promoter of phycocyanin β subunit. We chose the recently reported, fast-growing and robust cyanobacterium, Synechococcus elongatus PCC 11801, as the host strain. A stable recombinant strain was constructed using CRISPR-Cpf1 in a first report of markerless genome editing of this cyanobacterium. Under photoautotrophic conditions, the recombinant strain shows specific productivities of 338.26 and 1044.18 μmole/g dry cell weight/h for ethylene and succinate, respectively. These results compare favourably with the reported productivities for individual products in cyanobacteria that are highly engineered. Metabolome profiling and 13 C labelling studies indicate carbon flux redistribution and suggest avenues for further improvement. Our results show that S. elongatus PCC 11801 is a promising candidate for metabolic engineering.

13C isotopic labelling↗