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NASA NTRSDate not supplied

Preparation for and Performance of a Pseudomonas aeruginosa Biofilm Experiment On Board the International Space Station

Biofilms are a problem on Earth given their ability to degrade the materials upon which they grow and due to their relevance to infections. Remarkably, 65% and 80% of infections and chronic diseases on Earth are associated with biofilms, respectively. In space, these problems' impact is higher because the crew's lives and mission success depend on nominal operation of mechanical systems. Furthermore, the isolated confined environment nature of spaceflight may increase the rates of disease transmission. In the case of the International Space Station (ISS), biofilms are an identified problem on the Environmental Control and Life Support System (ECLSS), namely on the water processor assembly (WPA). In late 2019, the Space Biofilms experiment launched towards ISS to (i) characterize the mass, thickness, morphology, and gene expression of biofilms formed in space with respect to matched Earth controls, (ii) interrogate the expression of antimicrobial resistance genes, and (iii) test novel materials as potential biofilm control strategies for future ECLSS components. For this, 288 bacterial samples were prepared prior to the launch of the Northrop Grumman CRS-12 mission from NASA's Wallops Flight Facility. The samples were integrated into the spaceflight hardware, BioServe's Fluid Processing Apparatus (FPA) packed in sets of eight in Group Activation Packs (GAP). Half of these samples were activated and terminated on orbit by NASA astronauts Jessica Meir and Christina Koch, while the remaining half were processed equivalently on Earth. The spaceflight bacterial samples of Space Biofilms returned on board SpaceX' CRS-19 Dragon spacecraft, in early 2020. We here describe the test campaign implemented to verify the experiment design and confirm it would enable us to achieve the project's scientific goals. This campaign ended with the Experiment Verification Test (EVT), from which we here present example morphology and transcriptomic results. We describe in detail the sample preparation prior to flight, including cleaning and sterilization of the coupons of six materials (SS316, passivated-SS316, lubricant impregnated surface, catheter-grade silicone with and without a nanotopography, and cellulose membrane), loading and integration of growth media, bacterial inoculum, fixative and preservative to enable experiment termination on orbit. Additionally, we describe the performance of the experiment on board the ISS, including crew activities, use of assets, temperature profile, and experiment timeline; all leading to a successful spaceflight experiment.

Pamela Flores↗
DOE OSTI2025

Mining Thermophile Photosynthesis Genes: A Synthetic Operon Expressing Chloroflexota Species Reaction Center Genes in Rhodobacter sphaeroides

Photosynthesis is the foundation of the vast majority of life systems, and is therefore the most important bioenergetic process on earth. The greatest diversity of photosynthetic systems is found in microorganisms. However, our understanding of the biophysical and biochemical processes that transduce light into chemical energy is derived from a relatively small subset of proteins from microbes that are amenable to cultivation, in contrast to the huge number of predicted proteins that catalyze the initial photochemical reactions deposited in databases, such as from metagenomics. We describe the use of a Rhodobacter sphaeroides laboratory strain for the expression of heterologous photosynthesis genes to demonstrate the feasibility of mining this resource, focusing on hot spring Chloroflexota gene sequences. Using a synthetic operon of genes, we produced a photochemically active complex of reaction center proteins in our biological system. We also present bioinformatic analyses of anoxygenic type II reaction center sequences from metagenomic samples collected from hot (42–90 °C) springs available through the JGI IMG database, to generate a resource of diverse sequences that are potentially adapted to photosynthesis at such temperatures. These data provide a view into the natural diversity of anoxygenic photosynthesis, through a lens focused on high-temperature environments. The approach we took to express such genes can be applied for potential biotechnology purposes as well as for studies of fundamental catalytic properties of these heretofore inaccessible protein complexes.

Chloroflexota↗
DOE OSTI2024

Central transcriptional regulator controls photosynthetic growth and carbon storage in response to high light

Carbon capture and biochemical storage are some of the primary drivers of photosynthetic yield and productivity. To elucidate the mechanisms governing carbon allocation, we designed a photosynthetic light response test system for genetic and metabolic carbon assimilation tracking, using microalgae as simplified plant models. The systems biology mapping of high light-responsive photophysiology and carbon utilization dynamics between two variants of the same Picochlorum celeri species, TG1 and TG2 elucidated metabolic bottlenecks and transport rates of intermediates using instationary 13 C-fluxomics. Simultaneous global gene expression dynamics showed 73% of the annotated genes responding within one hour, elucidating a singular, diel-responsive transcription factor, closely related to the CCA1/LHY clock genes in plants, with significantly altered expression in TG2. Transgenic P. celeri TG1 cells expressing the TG2 CCA1/LHY gene, showed 15% increase in growth rates and 25% increase in storage carbohydrate content, supporting a coordinating regulatory function for a single transcription factor.

09 BIOMASS FUELS↗
DOE OSTI2023

Comparative transcriptome analysis of rainbow trout gonadal cells (RTG-2) infected with U and J genogroup infectious hematopoietic necrosis virus

Infectious hematopoietic necrosis virus (IHNV) is the causative pathogen of infectious hematopoietic necrosis, outbreaks of which are responsible for significant losses in rainbow trout aquaculture. Strains of IHNV isolated worldwide have been classified into five major genogroups, J, E, L, M, and U. To date, comparative transcriptomic analysis has only been conducted individually for the J and M genogroups. In this study, we compared the transcriptome profiles in U genogroup and J genogroup IHNV-infected RTG-2 cells with mock-infected RTG-2 cells. The RNA-seq results revealed 17,064 new genes, of which 7,390 genes were functionally annotated. Differentially expressed gene (DEG) analysis between U and J IHNV-infected cells revealed 2,238 DEGs, including 1,011 downregulated genes and 1,227 upregulated genes. Among the 2,238 DEGs, 345 new genes were discovered. The DEGs related to immune responses, cellular signal transduction, and viral diseases were further analyzed. RT-qPCR validation confirmed that the changes in expression of the immune response-related genes trpm2 , sting , itgb7 , ripk2 , and irf1 , cellular signal transduction-related genes irl , cacnb2 , bmp2l , gadd45α , and plk2 , and viral disease-related genes mlf1, mtor, armc5 , pik3r1 , and c-myc were consistent with the results of transcriptome analysis. Taken together, our findings provide a comprehensive transcriptional analysis of the differential virulence of the U and J genogroups of IHNV, and shed new light on the pathogenic mechanisms of IHNV strains.

Zhao, Jing-Zhuang↗
DOE OSTI2022

OperonSEQer: A set of machine-learning algorithms with threshold voting for detection of operon pairs using short-read RNA-sequencing data

Operon prediction in prokaryotes is critical not only for understanding the regulation of endogenous gene expression, but also for exogenous targeting of genes using newly developed tools such as CRISPR-based gene modulation. A number of methods have used transcriptomics data to predict operons, based on the premise that contiguous genes in an operon will be expressed at similar levels. While promising results have been observed using these methods, most of them do not address uncertainty caused by technical variability between experiments, which is especially relevant when the amount of data available is small. In addition, many existing methods do not provide the flexibility to determine the stringency with which genes should be evaluated for being in an operon pair. We present OperonSEQer, a set of machine learning algorithms that uses the statistic and p-value from a non-parametric analysis of variance test (Kruskal-Wallis) to determine the likelihood that two adjacent genes are expressed from the same RNA molecule. We implement a voting system to allow users to choose the stringency of operon calls depending on whether your priority is high recall or high specificity. In addition, we provide the code so that users can retrain the algorithm and re-establish hyperparameters based on any data they choose, allowing for this method to be expanded as additional data is generated. We show that our approach detects operon pairs that are missed by current methods by comparing our predictions to publicly available long-read sequencing data. OperonSEQer therefore improves on existing methods in terms of accuracy, flexibility, and adaptability.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Productivity and bioproduct formation in phototropic knock/out mutants in micro algae

Phototropin is a blue light receptor, which mediates a variety of blue-light elicited physiological processes in plants and algae. In higher plants these processes include phototropism, chloroplast movement and stomatal opening. In the green alga Chlamydomonas reinhardtii, phototropin plays a vital role in progression of the sexual life cycle and in the control of the eye spot size and light sensitivity. Phototropin is also involved in blue-light mediated changes in the synthesis of chlorophylls, carotenoids, chlorophyll binding proteins. We compared the transcriptome of phototropin knock out (PHOT KO) mutant and wild-type parent to analyze differences in gene expression in high light grown cultures (500 μmol photons m⁻²s⁻¹). Our results indicate the up-regulation of genes involved in photosynthetic electron transport chain, carbon fixation pathway, starch, lipid, and cell cycle control genes. With respect to photosynthetic electron transport genes, genes encoding proteins of the cytochrome b6f and ATP synthase complex were up regulated potentially facilitating proton-coupled electron transfer. In addition genes involved in limiting steps in the Calvin cycle Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO), Sidoheptulose 1,7 biophosphatase (SBPase), Glyceraldehyde-3-phosphate dehydrogenase (3PGDH) and that mediate cell-cycle control (CDK) were also up regulated along with starch synthase and fatty acid biosynthesis genes involved in starch and lipid synthesis. In addition, transmission electron micrographs show increased accumulation of starch granules in PHOT mutant compared to wild type, which is consistent with the higher expression of starch synthase genes. Collectively, the altered patterns of gene expression in the PHOT mutants were associated with a two-fold increase in growth and biomass accumulation compared to wild type when grown in environmental photobioreactors (Phenometrics) that simulate a pond environment. In conclusion, our studies suggest that phototropin may be a master gene regulator that suppresses rapid cell growth and promotes gametogenesis and sexual recombination in wild type strains.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2006

Immune Response in Microgravity: Genetic Basis and Countermeasure Development Implications

Impairment of the immunity in astronauts and cosmonauts even in shortterm flights is a recognized risk. Longterm orbital space missions and anticipated interplanetary flights increase the concern for more pronounced effects on the immune system with potential clinical consequences. Studies in true and modeled microgravity (MG) have demonstrated that MG directly affects numerous lymphocyte functions. The purpose of this study was to screen for genes involved in lymphocytes response to modeled microgravity (MMG) that could explain the functional and structural changes observed earlier. The microgravity-induced changes in gene expression were analyzed by microarray DNA chip technology. CD3and IL2activated Tcells were cultured in 1g (static) and modeled microgravity (NASA Rotating Wall Vessel bioreactor) conditions for 24 hours. Total RNA was extracted using the RNeasy isolation kit (Qiagen, Valencia, CA). Microarray experiments were performed utilizing Affymetrix Gene Chips (U133A), allowing testing for 18,400 human genes. To decrease the biological variation and aid in detecting microgravity-associated changes, experiments were performed in triplicate using cells obtained from three different donors. Exposure to modeled microgravity resulted in alteration of 89 genes, 10 of which were upregulated and 79 down-regulated. Altered genes were categorized by their function, structural role and by association with metabolic and regulatory pathways. A large proportion was found to be involved in fundamental cellular processes: signal transduction, DNA repair, apoptosis, and multiple metabolic pathways. There was a group of genes directly related to immune and inflammatory responses (IL7R, granulysin, proteasome activator subunit 2, peroxiredoxin 4, HLADRA, lymphocyte antigen 75, IL18R and DOCK2 genes). Among these genes only one (IL7R) was upregulated, the rest were downregulated. The upregulation of the IL7 receptor gene was confirmed by RT PCR. Three genes with altered expression were identified in the apoptosis related group (Granzyme B, APO2 ligand and Beta3endonexin). All of them were downregulated. Gene expression changes in MG might appear pivotal in identifying potential molecular targets for countermeasure development. (Supported by NRA OLMSA02 and NSCORT NAG54072 grants).

Risin, Diana↗
DOE OSTI2026

YAP1 Dysfunction Promotes Molecular Properties Linked to Breast Cancer Susceptibility

YAP1 is a cotranscription factor that promotes malignant and stem cell properties in cancer. We previously found that YAP1 dysregulation is associated with aging in human mammary epithelia. With increased age, YAP1 expression changes in luminal epithelial cells, the prospective breast cancer cell of origin. Because age is a significant risk factor for breast cancer, we tested whether YAP1 dysregulation acted early in cancer progression by conferring cellular states associated with increased cancer susceptibility. In this study, we find that with increased age and genetic risk for developing cancer, human breast tissues showed significantly increased YAP1 expression, and cultured primary human mammary epithelial cells (HMEC) showed significantly increased expression of both YAP1 and its transcriptional targets. Increased YAP1 expression in cultured HMEC induced gene expression changes associated with increased cancer susceptibility, such as genes associated with stem cell states, increased telomerase activity, breast cancer progression, and increased age and genetic breast cancer risk. Furthermore, overexpression of YAP1 in post-stasis HMEC—finite lifespan cells that have bypassed a retinoblastoma-mediated senescence barrier—promoted properties related to increased growth potential. We found that YAP1 dysregulation in finite epithelial cells allows for access to gene programs and functions that are typically thought to be restricted to stem cells. We hypothesize that YAP1 acts early in breast cancer progression, long before the development of a tumor, to impose cancer-susceptible molecular states.

YAP1↗
DOE OSTI2023

Generating Co-expression Networks for Three Cyanobacteria: Synechococcus sp. PCC 7942, Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803

Cyanobacteria are photosynthetic organisms capable of high growth rate and represent a promising bioplatform for harnessing the sun’s energy to make biofuel. Additionally, the process of photosynthesis absorbs CO2 from the environment. Understanding the metabolic processes involved in photosynthesis could lead to solutions to the recent rise of CO2 concentration in Earth’s atmosphere and the associated climate change. More research on the transcriptional regulation of these cells is needed to learn how to harness the untapped potential of cyanobacteria for these applications. Transcriptional analysis via RNA-seq provides an understanding of how gene expression changes at the mRNA level under diverse growing conditions. I systematically collected and analyzed RNA-Seq data obtained under a variety of conditions and available on the NCBI database for three cyanobacteria model organisms: Synechococcus elongatus sp. PCC 7942, Synechococcus sp. PCC 7002, and Synechocystis sp. PCC 6803. For each organism, the data was mapped to a reference genome to characterize the RNA expression profile. Samples were checked for quality based on the number of reads and the correlation of the expression profile between labeled replicates. All samples were transformed into transcripts per million reads, followed by a log transformation to account for the wide range of sample sizes. Gene co-expression networks were generated and analyzed for each species using Cytoscape. These networks provide a base level of gene expression for each species. The network topology and high-betweeness nodes of these networks need to be analyzed further to provide insight on potential ways to harness cyanobacteria genetics. Additionally, these datasets can be used together to form a core genome network analysis- one that includes only the genes that are homologous between the three species. This project has prepared the way for a more in-depth study on photosynthetic microbes on a genetic level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Optimal dimensionality selection for independent component analysis of transcriptomic data

Independent component analysis is an unsupervised machine learning algorithm that separates a set of mixed signals into a set of statistically independent source signals. Applied to high-quality gene expression datasets, independent component analysis effectively reveals both the source signals of the transcriptome as co-regulated gene sets, and the activity levels of the underlying regulators across diverse experimental conditions. Two major variables that affect the final gene sets are the diversity of the expression profiles contained in the underlying data, and the user-defined number of independent components, or dimensionality, to compute. Availability of high-quality transcriptomic datasets has grown exponentially as high-throughput technologies have advanced; however, optimal dimensionality selection remains an open question. We computed independent components across a range of dimensionalities for four gene expression datasets with varying dimensions (both in terms of number of genes and number of samples). We computed the correlation between independent components across different dimensionalities to understand how the overall structure evolves as the number of user-defined components increases. We then measured how well the resulting gene clusters reflected known regulatory mechanisms, and developed a set of metrics to assess the accuracy of the decomposition at a given dimension. We found that over-decomposition results in many independent components dominated by a single gene, whereas under-decomposition results in independent components that poorly capture the known regulatory structure. From these results, we developed a new method, called OptICA, for finding the optimal dimensionality that controls for both over- and under-decomposition. Specifically, OptICA selects the highest dimension that produces a low number of components that are dominated by a single gene. We show that OptICA outperforms two previously proposed methods for selecting the number of independent components across four transcriptomic databases of varying sizes. OptICA avoids both over-decomposition and under-decomposition of transcriptomic datasets resulting in the best representation of the organism’s underlying transcriptional regulatory network.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Two promoters integrate multiple enhancer inputs to drive wild-type knirps expression in the Drosophila melanogaster embryo

Abstract Proper development depends on precise spatiotemporal gene expression patterns. Most developmental genes are regulated by multiple enhancers and often by multiple core promoters that generate similar transcripts. We hypothesize that multiple promoters may be required either because enhancers prefer a specific promoter or because multiple promoters serve as a redundancy mechanism. To test these hypotheses, we studied the expression of the knirps locus in the early Drosophila melanogaster embryo, which is mediated by multiple enhancers and core promoters. We found that one of these promoters resembles a typical “sharp” developmental promoter, while the other resembles a “broad” promoter usually associated with housekeeping genes. Using synthetic reporter constructs, we found that some, but not all, enhancers in the locus show a preference for one promoter, indicating that promoters provide both redundancy and specificity. By analyzing the reporter dynamics, we identified specific burst properties during the transcription process, namely burst size and frequency, that are most strongly tuned by the combination of promoter and enhancer. Using locus-sized reporters, we discovered that enhancers with no promoter preference in a synthetic setting have a preference in the locus context. Our results suggest that the presence of multiple promoters in a locus is due both to enhancer preference and a need for redundancy and that “broad” promoters with dispersed transcription start sites are common among developmental genes. They also imply that it can be difficult to extrapolate expression measurements from synthetic reporters to the locus context, where other variables shape a gene’s overall expression pattern.

Li, Lily (ORCID:0000000301986521)↗
NASA NTRS1997

Development of Plant Gene Vectors for Tissue-Specific Expression Using GFP as a Reporter Gene

Reporter genes are widely employed in plant molecular biology research to analyze gene expression and to identify promoters. Gus (UidA) is currently the most popular reporter gene but its detection requires a destructive assay. The use of jellyfish green fluorescent protein (GFP) gene from Aequorea Victoria holds promise for noninvasive detection of in vivo gene expression. To study how various plant promoters are expressed in sweet potato (Ipomoea batatas), we are transcriptionally fusing the intron-modified (mGFP) or synthetic (modified for codon-usage) GFP coding regions to these promoters: double cauliflower mosaic virus 35S (CaMV 35S) with AMV translational enhancer, ubiquitin7-intron-ubiquitin coding region (ubi7-intron-UQ) and sporaminA. A few of these vectors have been constructed and introduced into E. coli DH5a and Agrobacterium tumefaciens EHA105. Transient expression studies are underway using protoplast-electroporation and particle bombardment of leaf tissues.

Jackson, Jacquelyn↗
DOE OSTI2023

Expression of macromolecular organic nitrogen degrading enzymes identifies potential mediators of soil organic N availability to an annual grass

Abstract Nitrogen (N) is frequently limiting to plant growth, in part because most soil N is present as polymeric organic compounds that are not readily taken up by plants. Microbial depolymerization of these large macromolecular N-substrates gradually releases available inorganic N. While many studies have researched and modeled controls on soil organic matter formation and bulk N mineralization, the ecological—spatial, temporal and phylogenetic—patterns underlying organic N degradation remain unclear. We analyzed 48 time-resolved metatranscriptomes and quantified N-depolymerization gene expression to resolve differential expression by soil habitat and time in specific taxonomic groups and gene-based guilds. We observed much higher expression of extracellular serine-type proteases than other extracellular N-degrading enzymes, with protease expression of predatory bacteria declining with time and other taxonomic patterns driven by the presence (Gammaproteobacteria) or absence (Thermoproteota) of live roots and root detritus (Deltaproteobacteria and Fungi). The primary chitinase chit1 gene was more highly expressed by eukaryotes near root detritus, suggesting predation of fungi. In some lineages, increased gene expression over time suggests increased competitiveness with rhizosphere age (Chloroflexi). Phylotypes from some genera had protease expression patterns that could benefit plant N nutrition, for example, we identified a Janthinobacterium phylotype and two Burkholderiales that depolymerize organic N near young roots and a Rhizobacter with elevated protease levels near mature roots. These taxon-resolved gene expression results provide an ecological read-out of microbial interactions and controls on N dynamics in specific soil microhabitats and could be used to target potential plant N bioaugmentation strategies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1994

Prostanoid-induced expression of matrix metalloproteinase-1 messenger ribonucleic acid in rat osteosarcoma cells

Individual prostanoids have distinct potencies in activating intracellular signaling pathways and regulating gene expression in osteoblastic cells. The E-series prostaglandins (PGs) are known to stimulate matrix metalloproteinase-1 (MMP-1) synthesis and secretion in certain rodent and human osteoblastic cells, yet the intracellular events involved remain unclear. To further characterize this response and its signal transduction pathway(s), we examined prostanoid-induced expression of the MMP-1 gene in the rat osteoblastic osteosarcoma cell line UMR 106-01. Northern blot analysis demonstrated that prostaglandin E2 (PGE2) and PGE1 were very potent stimulators (40-fold) of MMP-1 transcript abundance, PGF2 alpha and prostacyclin were weak stimulators (4-fold), and thromboxane-B2 had no effect. The marked increase in MMP-1 transcript abundance after PGE2 treatment was first detected at 2 h, became maximal at 4 h, and persisted beyond 24 h. This response was dose dependent and elicited maximal and half-maximal effects with concentrations of 10(-6) and 0.6 x 10(-7) M, respectively. Cycloheximide, a protein synthesis inhibitor, completely blocked this effect of PGE2, suggesting that the expression of other genes is required. Nuclear run-on experiments demonstrated that PGE2 rapidly activates MMP-1 gene transcription, with a maximal increase at 2-4 h. The second messenger analog, 8-bromo-cAMP, mimicked the effects of PGE2 by stimulating a dose-dependent increase in MMP-1 messenger RNA (mRNA) levels, with a maximal effect quantitatively similar to that observed with PGE2. Thus, in UMR 106-01 cells, different prostanoids have distinct potencies in stimulating MMP-1 mRNA abundance. Our data suggest that PGE2 stimulation of MMP-1 synthesis is due to activation of MMP-1 gene transcription and a subsequent marked increase in MMP-1 mRNA abundance. This effect is dependent on de novo protein synthesis and is mimicked by protein kinase-A activation.

NASA Discipline Musculoskeletal↗
NASA NTRS2012

Effects of Dietary Iron and Gamma Radiation on the Rat Retina

A health risk of concern for NASA relates to radiation exposure and its synergistic effects with other space environmental factors, includi ng nutritional status of the crew. Astronauts consume almost three times the recommended daily allowance of iron due to the use of fortifie d foods aboard the International Space Station, with iron intake occa sionally exceeding six times the recommended values. Recently, NASA has become concerned with visual changes associated with spaceflight, a nd research is being conducted to elucidate the etiology of eye structure alterations in the spaceflight environment. Terrestrially, iron o verload is also associated with certain optic neuropathies. In additi on, due to its role in Fenton reactions, iron can potentiate oxidative stress, which is a recognized cause of cataract formation. As part o f a study investigating the combined effects of radiation exposure an d iron overload on multiple physiological systems, we focused on defining the effects of both treatments on eye biology. In this study, 12- week-old Sprague-Dawley rats were assigned to one of four experimental groups: normal iron/no radiation (Control/Sham), high iron/no radiat ion (Fe/Sham), normal iron/gamma radiation (3 Gy cumulative dose, fra ctionated at 0.375 Gy/d every other day for 16 d) (Control/Rad), and high iron/gamma radiation (Fe/Rad). Oxidative stress-induced DNA damag e, measured as concentration of the marker 8-hydroxy-2'-deoxyguanosine (8OHdG) in eye retinal tissue by enzyme-immunoanalysis did not show significant changes among treatments. However, there was an overall i ncrease in 8OHdG immunostaining density in retina sections due to radiation exposure (P = 0.05). Increased dietary iron and radiation expos ure had an interactive effect (P = 0.02) on 8OHdG immunostaining of t he retinal ganglion cell layer with iron diet increasing the signal in the group not exposed to radiation (P = 0.05). qPCR gene expression profiling of relevant target genes indicated upregulation of ferritin light chain (P = 0.09) as a result of dietary iron but no change in e xpression of the gene for ferritin heavy chain. Immunolocalization of light chain and heavy chain of the iron storage protein ferritin showed the expected distribution in the choroid, photoreceptor layer, inn er nuclear layer and in the inner plexiform layer that corresponded t o the synaptic terminals of bipolar cells. Evidence of stress and damage in the retina was also suggested by a decrease in expression of th e survival marker Bcl2 (P = 0.01) and the protective proteins clusterin (P = 0.04) and heat shock factor 1 (Hsf1, P < 0.001), as a result o f increased dietary iron. The effect of increased iron on expression of the antioxidant enzyme heme oxygenase 1 (Hmox1) had a significant interaction with the effect of radiation (P < 0.001). In summary, the results of this study indicate that both gamma radiation exposure and a moderate increase in dietary iron can contribute to deleterious cha nges in retinal health and physiology.

Morgan, Jennifer↗
DOE OSTI2021

Rapid Cis–Trans Coevolution Driven by a Novel Gene Retroposed from a Eukaryotic Conserved CCR4–NOT Component in Drosophila

Young, or newly evolved, genes arise ubiquitously across the tree of life, and they can rapidly acquire novel functions that influence a diverse array of biological processes. Previous work identified a young regulatory duplicate gene in Drosophila, Zeus that unexpectedly diverged rapidly from its parent, Caf40, an extremely conserved component in the CCR4–NOT machinery in post-transcriptional and post-translational regulation of eukaryotic cells, and took on roles in the male reproductive system. This neofunctionalization was accompanied by differential binding of the Zeus protein to loci throughout the Drosophila melanogaster genome. However, the way in which new DNA-binding proteins acquire and coevolve with their targets in the genome is not understood. Here, by comparing Zeus ChIP-Seq data from D. melanogaster and D. simulans to the ancestral Caf40 binding events from D. yakuba, a species that diverged before the duplication event, we found a dynamic pattern in which Zeus binding rapidly coevolved with a previously unknown DNA motif, which we term Caf40 and Zeus-Associated Motif (CAZAM), under the influence of positive selection. Interestingly, while both copies of Zeus acquired targets at male-biased and testis-specific genes, D. melanogaster and D. simulans proteins have specialized binding on different chromosomes, a pattern echoed in the evolution of the associated motif. Using CRISPR-Cas9-mediated gene knockout of Zeus and RNA-Seq, we found that Zeus regulated the expression of 661 differentially expressed genes (DEGs). Our results suggest that the evolution of young regulatory genes can be coupled to substantial rewiring of the transcriptional networks into which they integrate, even over short evolutionary timescales. Our results thus uncover dynamic genome-wide evolutionary processes associated with new genes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structural insights into acetylated histone ligand recognition by the $\mathrm{BDP1}$ bromodomain of Plasmodium falciparum

Plasmodium falciparum requires a two-host system, moving between Anopheles mosquito and humans, to complete its life cycle. To overcome such dynamic growth conditions its histones undergo various post-translational modifications to regulate gene expression. The P. falciparum Bromodomain Protein 1 (PfBDP1) has been shown to interact with acetylated lysine modifications on histone H3 to regulate the expression of invasion-related genes. Here, we investigated the ability of the PfBDP1 bromodomain to interact with acetyllsyine modifications on additional core and variant histones. A crystal structure of the PfBDP1 bromodomain (PfBDP1-BRD) reveals it contains the conserved bromodomain fold, but our comparative analysis between the PfBDP1-BRD and human bromodomain families indicates it has a unique binding mechanism. Solution NMR spectroscopy and ITC binding assays carried out with acetylated histone ligands demonstrate that it preferentially recognizes tetra-acetylated histone H4, and we detected weaker interactions with multi-acetylated H2A.Z in addition to the previously reported interactions with acetylated histone H3. Our findings indicate PfBDP1 may play additional roles in the P. falciparum life cycle, and the distinctive features of its bromodomain binding pocket could be leveraged for the development of new therapeutic agents to help overcome the continuously evolving resistance of P. falciparum against currently available drugs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Analyses of transcriptomes and the first complete genome of Leucocalocybe mongolica provide new insights into phylogenetic relationships and conservation

In this study, we report a de novo assembly of the first high-quality genome for a wild mushroom species Leucocalocybe mongolica (LM). We performed high-throughput transcriptome sequencing to analyze the genetic basis for the life history of LM. Our results show that the genome size of LM is 46.0 Mb, including 26 contigs with a contig N50 size of 3.6 Mb. In total, we predicted 11,599 protein-coding genes, of which 65.7% (7630) could be aligned with high confidence to annotated homologous genes in other species. We performed phylogenetic analyses using genes form 3269 single-copy gene families and showed support for distinguishing LM from the genus Tricholoma (L.) P.Kumm., in which it is sometimes circumscribed. We believe that one reason for limited wild occurrences of LM may be the loss of key metabolic genes, especially carbohydrate-active enzymes (CAZymes), based on comparisons with other closely related species. The results of our transcriptome analyses between vegetative (mycelia) and reproductive (fruiting bodies) organs indicated that changes in gene expression among some key CAZyme genes may help to determine the switch from asexual to sexual reproduction. Taken together, our genomic and transcriptome data for LM comprise a valuable resource for both understanding the evolutionary and life history of this species.

54 ENVIRONMENTAL SCIENCES↗