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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Haploidy and aneuploidy in switchgrass mediated by misexpression of CENH3

Cross bred species such as switchgrass may benefit from advantageous breeding strategies requiring inbred lines. Doubled haploid production methods offer several ways that these lines can be produced that often involve uniparental genome elimination as the rate limiting step. We have used a centromere-mediated genome elimination strategy in which modified CENH3 is expressed to induce the process. Transgenic tetraploid switchgrass lines coexpressed Cas9, a poly-cistronic tRNA-gRNA tandem array containing eight guide RNAs that target two CENH3 genes, and different chimeric versions of CENH3 with alterations to the N-terminal tail region. Genotyping of CENH3 genes in transgenics identified edits including frameshift mutations and deletions in one or both copies of the two CENH3 genes. Flow cytometry of T 1 seedlings identified two T 0 lines that produced five haploid individuals representing an induction rate of 0.5% and 1.4%. Eight different T0 lines produced aneuploids at rates ranging from 2.1 to 14.6%. A sample of aneuploid lines were sequenced at low coverage and aligned to the reference genome, revealing missing chromosomes and chromosome arms.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2011

The Distribution of Chromosomal Aberrations in Human Cells Predicted by a Generalized Time-Dependent Model of Radiation-Induced Formation of Aberrations

New experimental data show how chromosomal aberrations for low- and high-LET radiation are dependent on DSB repair deficiencies in wild-type, AT and NBS cells. We simulated the development of chromosomal aberrations in these cells lines in a stochastic track-structure-dependent model, in which different cells have different kinetics of DSB repair. We updated a previously formulated model of chromosomal aberrations, which was based on a stochastic Monte Carlo approach, to consider the time-dependence of DSB rejoining. The previous version of the model had an assumption that all DSBs would rejoin, and therefore we called it a time-independent model. The chromosomal-aberrations model takes into account the DNA and track structure for low- and high-LET radiations, and provides an explanation and prediction of the statistics of rare and more complex aberrations. We compared the program-simulated kinetics of DSB rejoining to the experimentally-derived bimodal exponential curves of the DSB kinetics. We scored the formation of translocations, dicentrics, acentric and centric rings, deletions, and inversions. The fraction of DSBs participating in aberrations was studied in relation to the rejoining time. Comparisons of simulated dose dependence for simple aberrations to the experimental dose-dependence for HF19, AT and NBS cells will be made.

Ponomarev, Artem L.↗
DOE OSTI2023

The histone demethylase KdmB is part of a trimeric protein complex and mediates virulence and mycotoxin production in Penicillium expansum

Epigenetic modification of chromosome structure has increasingly been associated with alterations in secondary metabolism and sporulation defects in filamentous fungal pathogens. Recently, the epigenetic reader protein SntB was shown to govern virulence, spore production and mycotoxin synthesis in the fruit pathogen Penicillium expansum. Through immunoprecipitation-coupled mass spectrometry, we found that SntB is a member of a protein complex with KdmB, a histone demethylase and the essential protein RpdA, a histone deacetylase. Deletion of kdmB phenocopied some but not all characteristics of the ΔsntB mutant. KdmB deletion strains exhibited reduced lesion development on Golden Delicious apples and this was accompanied by decreased production of patulin and citrinin in host tissue. In addition, ΔkdmB mutants were sensitive to several cell wall stressors which possibly contributed to the decreased virulence observed on apples. In conclusion, slight differences in spore production and germination rates of ΔkdmB mutants in vitro did not impact overall diameter growth in culture.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Genomic and phenotypic characterization of multidrug-resistant Salmonella enterica serovar Reading isolates involved in a turkey-associated foodborne outbreak

Salmonella is a global bacterial foodborne pathogen associated with a variety of contaminated food products. Poultry products are a common source of Salmonella -associated foodborne illness, and an estimated 7% of human illnesses in the United States are attributed to turkey products. From November 2017 to March 2019, the Centers for Disease Control and Prevention reported a turkey-associated outbreak of multidrug-resistant (MDR; resistant to ≥3 antimicrobial classes) Salmonella enterica serovar Reading ( S . Reading) linked to 358 human infections in 42 US states and Canada. Since S . Reading was seldom linked to human illness prior to this outbreak, the current study compared genomic sequences of S . Reading isolates prior to the outbreak (pre-outbreak) to isolates identified during the outbreak period, focusing on genes that were different between the two groups but common within a group. Following whole-genome sequence analysis of five pre-outbreak and five outbreak-associated turkey/turkey product isolates of S . Reading, 37 genes located within two distinct chromosomal regions were identified only in the pre-outbreak isolates: (1) an ~5 kb region containing four protein-coding genes including uidA which encodes beta-glucuronidase, pgdA encoding peptidoglycan deacetylase, and two hypothetical proteins and (2) an ~28 kb region comprised of 32 phage-like genes and the xerC gene, which encodes tyrosine recombinase (frequently associated with phage genes). The five outbreak isolates also had a deletional event within the cirA gene, introducing a translational frame shift and premature stop codon. The cirA gene encodes a protein with dual receptor functions: a siderophore receptor for transport of dihydroxybenzoylserine as well as a colicin Ia/b receptor. Significant differences for the identified genetic variations were also detected in 75 S . Reading human isolates. Of the 41 S . Reading isolates collected before or in 2017, 81 and 90% of the isolates contained the uidA and pgdA genes, respectively, but only 24% of the isolates collected after 2017 harbored the uidA and pgdA genes. The truncation event within the cirA gene was also significantly higher in isolates collected after 2017 (74%) compared to before or in 2017 (5%). Phenotypic analysis of the S . Reading isolates for colicin and cefiderocol sensitivities (CirA) and β-methyl-D-glucuronic acid utilization (UidA and accessory proteins) supported the genomic data. Overall, a similar genome reduction pattern was generally observed in both the turkey and human isolates of S . Reading during the outbreak period, and the genetic differences were present in genes that could potentially promote pathogen dissemination due to variation in Salmonella colonization, fitness, and/or virulence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

De Novo Assembly and Annotation of 11 Diverse Shrub Willow (Salix) Genomes Reveals Novel Gene Organization in Sex-Linked Regions

Poplar and willow species in the Salicaceae are dioecious, yet have been shown to use different sex determination systems located on different chromosomes. Willows in the subgenus Vetrix are interesting for comparative studies of sex determination systems, yet genomic resources for these species are still quite limited. Only a few annotated reference genome assemblies are available, despite many species in use in breeding programs. Here we present de novo assemblies and annotations of 11 shrub willow genomes from six species. Copy number variation of candidate sex determination genes within each genome was characterized and revealed remarkable differences in putative master regulator gene duplication and deletion. We also analyzed copy number and expression of candidate genes involved in floral secondary metabolism, and identified substantial variation across genotypes, which can be used for parental selection in breeding programs. Lastly, we report on a genotype that produces only female descendants and identified gene presence/absence variation in the mitochondrial genome that may be responsible for this unusual inheritance.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1999

Three-Dimensional, Transgenic Cell Models to Quantify Space Genotoxic Effects

The space environment contains radiation and chemical agents known to be mutagenic and carcinogenic to humans. Additionally, microgravity is a complicating factor that may modify or synergize induced genotoxic effects. Most in vitro models fail to use human cells (making risk extrapolation to humans more difficult), overlook the dynamic effect of tissue intercellular interactions on genotoxic damage, and lack the sensitivity required to measure low-dose effects. Currently a need exists for a model test system that simulates cellular interactions present in tissue, and can be used to quantify genotoxic damage induced by low levels of radiation and chemicals, and extrapolate assessed risk to humans. A state-of-the-art, three-dimensional, multicellular tissue equivalent cell culture model will be presented. It consists of mammalian cells genetically engineered to contain multiple copies of defined target genes for genotoxic assessment,. NASA-designed bioreactors were used to coculture mammalian cells into spheroids, The cells used were human mammary epithelial cells (H184135) and Stratagene's (Austin, Texas) Big Blue(TM) Rat 2 lambda fibroblasts. The fibroblasts were genetically engineered to contain -a high-density target gene for mutagenesis (60 copies of lacl/LacZ per cell). Tissue equivalent spheroids were routinely produced by inoculation of 2 to 7 X 10(exp 5) fibroblasts with Cytodex 3 beads (150 micrometers in diameter). at a 20:1 cell:bead ratio, into 50-ml HARV bioreactors (Synthecon, Inc.). Fibroblasts were cultured for 5 days, an equivalent number of epithelial cells added, and the fibroblast/epithelial cell coculture continued for 21 days. Three-dimensional spheroids with diameters ranging from 400 to 600 micrometers were obtained. Histological and immunohistochemical Characterization revealed i) both cell types present in the spheroids, with fibroblasts located primarily in the center, surrounded by epithelial cells; ii) synthesis of extracellular matrix; and iii,, mitotic cells located throughout the spheroids. Spheroidal integrity and cell viability were retained for the 30-day test period after removal of spheroids from the bioreactor. Potential utility of this three-dimensional, transgenic model for genotoxicity was initially assessed by exposure of spheroids to 0-2 Gy neon at dose rates of 0.3 to 1.5 Gy/min (National Institute of Radiological Sciences, Chiba, Japan). Quantification of mutation at the lacl gene revealed a linear dose response for mutation induction. Limited sequencing analysis of mutant clones revealed higher frequencies of deletions and multiple base sequence changes with increasing dose. These results suggest that our three-dimensional, transgenic model is applicable to a wide variety of studies involving the quantification, identification, and characterization of genotoxicity incurred in space and on Earth. This model uniquely allows investigation of the interaction of relevant factors, namely cell-to-cell interactions and the mechanistic interaction of microgravity with radiation insults and DNA repair. Using this three-dimensional model will allow us to obtain dual genotoxic information (i.e., mutation rate plus chromosome aberration data) from the same system so that one endpoint can be used to reference the other, thereby increasing the fidelity of the data set. Moreover, the tissue-equivalent nature of the three-dimensional model provides high confidence for relevance of risk assessment, i.e., the establishment of quality factors directly applicable to the microgravity environment.

Gonda, S. R.↗
NASA NTRS1998

Mutagenicity of arsenic in mammalian cells: role of reactive oxygen species

Arsenite, the trivalent form of arsenic present in the environment, is a known human carcinogen that lacked mutagenic activity in bacterial and standard mammalian cell mutation assays. We show herein that when evaluated in an assay (AL cell assay), in which both intragenic and multilocus mutations are detectable, that arsenite is in fact a strong dose-dependent mutagen and that it induces mostly large deletion mutations. Cotreatment of cells with the oxygen radical scavenger dimethyl sulfoxide significantly reduces the mutagenicity of arsenite. Thus, the carcinogenicity of arsenite can be explained at least in part by it being a mutagen that depends on reactive oxygen species for its activity.

Non-NASA Center↗
DOE OSTI2025

Targeted seed EMS mutagenesis reveals a basic helix–loop–helix transcription factor underlying male sterility in sorghum

Abstract Forward genetic screens of mutant populations are fundamental for functional genomics studies. However, isolating independent mutant alleles to molecularly identify causal genes is challenging in species recalcitrant to genetic manipulation. Here, we demonstrate that classic seed ethyl methanesulfonate (EMS) mutagenesis coupled with genome sequencing can overcome this limitation in sorghum. We used this method to generate new mutant alleles of sorghum MALE STERILE 8 (MS8) and identified the causal locus for the ms8 phenotype as Sobic.004G270900, which encodes the sorghum ortholog of maize bhlh122, a basic helix–loop–helix (bHLH) transcription factor required for male fertility in maize. Bulked segregant analysis mapped ms8-1 to a region on chromosome 4 containing Sobic.004G270900. Seeds from heterozygous MS8/ms8-1 plants were mutagenized and screened for chimeric inflorescences containing sectors with white, sterile anthers resembling the ms8-1 homozygous phenotype. DNA sequencing of sterile and fertile sectors from a single chimeric inflorescence revealed two mutations in Sobic.004G270900 within the sterile sector, but not the fertile sector. Isolation of this loss-of-function allele (ms8-2) established Sobic.004G270900 as the causative locus for male sterility in the ms8 mutant. We generated additional alleles of MS8 in a different genetic background using CRISPR/Cas9-based gene editing, where deletions in Sobic.004G270900 also resulted in male sterility. Our work identified a gene underlying male sterility in sorghum and provides a novel and straightforward genetic tool for researchers who lack access to advanced transformation facilities to validate gene candidates. Unlike gene editing, no prior knowledge of candidate genes is required for targeted seed EMS mutagenesis to aid identification of causal loci.

Genetics & Heredity↗
DOE OSTI2022

The Chlamydomonas Genome Project, version 6: reference assemblies for mating type plus and minus strains reveal extensive structural mutation in the laboratory

Five versions of the Chlamydomonas reinhardtii reference genome have been produced over the last two decades. Here we present version 6, bringing significant advances in assembly quality and structural annotations. PacBio-based chromosome-level assemblies for two laboratory strains, CC-503 and CC-4532, provide resources for the plus and minus mating type alleles. We corrected major misassemblies in previous versions and validated our assemblies via linkage analyses. Contiguity increased over ten-fold and >80% of filled gaps are within genes. We used Iso-Seq and deep RNA-seq datasets to improve structural annotations, and updated gene symbols and textual annotation of functionally characterized genes via extensive manual curation. We discovered that the cell wall-less classical reference strain CC-503 exhibits genomic instability potentially caused by deletion of the helicase RECQ3, with major structural mutations identified that affect >100 genes. We therefore present the CC-4532 assembly as the primary reference, although this strain also carries unique structural mutations and is experiencing rapid proliferation of a Gypsy retrotransposon. We expect all laboratory strains to harbor gene-disrupting mutations, which should be considered when interpreting and comparing experimental results. Collectively, the resources presented here herald a new era of Chlamydomonas genomics and will provide the foundation for continued research in this important reference organism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Strategies to identify and edit improvements in synthetic genome segments episomally

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments at low copy number. However, all stages of whole-genome engineering have the potential to impose mutations on the synthetic genome that can reduce or eliminate the fitness of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to recoding an Escherichia coli strain to replace seven codons with synonymous alternatives genome wide. Ten 44 402–47 179 bp de novo synthesized DNA segments contained in a BAC from the recoded strain were unable to complement deletion of the corresponding 33–61 wild-type genes using a single antibiotic resistance marker. Next-generation sequencing (NGS) was used to identify 1–7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the recoded segment contained in the BAC when both the recoded and wild-type copies of the mutated genes had to exist by necessity during the repair process. Finally, two web-based tools were used to predict the impact of a subset of non-recoding missense mutations on strain fitness using protein structure and function calls.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1996

A low, adaptive dose of gamma-rays reduced the number and altered the spectrum of S1- mutants in human-hamster hybrid AL cells

We examined the effects of a low, adaptive dose of 137Cs-gamma-irradiation (0.04 Gy) on the number and kinds of mutants induced in AL human-hamster hybrid cells by a later challenge dose of 4 Gy. The yield of S1- mutants was significantly less (by 53%) after exposure to both the adaptive and challenge doses compared to the challenge dose alone. The yield of hprt- mutants was similarly decreased. Incubation with cycloheximide (CX) or 3-aminobenzamide largely negated the decrease in mutant yield. The adaptive dose did not perturb the cell cycle, was not cytotoxic, and did not of itself increase the mutant yield above background. The adaptive dose did, however, alter the spectrum of S1- mutants from populations exposed only to the adaptive dose, as well as affecting the spectrum of S1- mutants generated by the challenge dose. The major change in both cases was a significant increase in the proportion of complex mutations compared to small mutations and simple deletions.

Non-NASA Center↗
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 OSTI2022

Transformation and gene editing in the bioenergy grass Miscanthus

Miscanthus, a C4 member of Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies M. x giganteus and its parental species M. sacchariflorus and M. sinensis. Use of biotechnology-based procedures to genetically improve Miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites. We developed gene editing procedures for Miscanthus using CRISPR/Cas9 that enabled the mutation of a specific (targeted) endogenous gene to knock out its function. Classified as paleo-allopolyploids (duplicated ancient Sorghum-like DNA plus chromosome fusion event), design of guide RNAs (gRNAs) for Miscanthus needed to target both homeologs and their alleles to account for functional redundancy. Prior research in Zea mays demonstrated that editing the lemon white1 (lw1) gene, involved in chlorophyll and carotenoid biosynthesis, via CRISPR/Cas9 yielded pale green/yellow, striped or white leaf phenotypes making lw1 a promising target for visual confirmation of editing in other species. Using sequence information from both Miscanthus and sorghum, orthologs of maize lw1 were identified; a multi-step screening approach was used to select three gRNAs that could target homeologs of lw1. Embryogenic calli of M. sacchariflorus, M. sinensis and M. x giganteus were transformed via particle bombardment (biolistics) or Agrobacterium tumefaciens introducing the Cas9 gene and three gRNAs to edit lw1. Leaves on edited Miscanthus plants displayed the same phenotypes noted in maize. Sanger sequencing confirmed editing; deletions in lw1 ranged from 1 to 26 bp in length, and one deletion (433 bp) encompassed two target sites. Confocal microscopy verified lack of autofluorescence (chlorophyll) in edited leaves/sectors. We developed procedures for gene editing via CRISPR/Cas9 in Miscanthus and, to the best of our knowledge, are the first to do so. This included five genotypes representing three Miscanthus species. Designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles); results also confirmed lw1 made a good editing target in species other than Z. mays. The ability to target specific loci to enable endogenous gene editing presents a new avenue for genetic improvement of this important biomass crop.

09 BIOMASS FUELS↗
NASA NTRS1994

Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts

The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 micrometer2 and 0.09 to 5.56 x 10(-3) micrometer2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(-5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

NASA Center JSC↗
NASA NTRS1994

Charged-particle mutagenesis 2. Mutagenic effects of high energy charged particles in normal human fibroblasts

The biological effects of high Linear Energy Transfer (LET) charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 sq micrometer and 0.09 to 5.56 x 10(exp -3) sq micrometer respectively. The maximum values were obtained by Fe-56 with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(exp -5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

Chen, D. J.↗