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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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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Inference of Gene Regulatory Network Uncovers the Linkage between Circadian Clock and Crassulacean Acid Metabolism in Kalanchoë fedtschenkoi

The circadian clock drives time-specific gene expression, enabling biological processes to be temporally controlled. Plants that conduct crassulacean acid metabolism (CAM) photosynthesis represent an interesting case of circadian regulation of gene expression as stomatal movement is temporally inverted relative to stomatal movement in C3 plants. The mechanisms behind how the circadian clock enabled physiological differences at the molecular level is not well understood. Recently, the rescheduling of gene expression was reported as a mechanism to explain how CAM evolved from C3. Therefore, we investigated whether core circadian clock genes in CAM plants were re-phased during evolution, or whether networks of phase-specific genes were simply re-wired to different core clock genes. We identified candidate core clock genes based on gene expression features and then applied the Local Edge Machine (LEM) algorithm to infer regulatory relationships between this new set of core candidates and known core clock genes in Kalanchoë fedtschenkoi. We further inferred stomata-related gene targets for known and candidate core clock genes and constructed a gene regulatory network for core clock and stomata-related genes. Our results provide new insight into the mechanism of circadian control of CAM-related genes in K. fedtschenkoi, facilitating the engineering of CAM machinery into non-CAM plants for sustainable crop production in water-limited environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii

Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4 . The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas , land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The Ancient Salicoid Genome Duplication Event: A Platform for Reconstruction of De Novo Gene Evolution in Populus trichocarpa

Orphan genes are characteristic genomic features that have no detectable homology to genes in any other species and represent an important attribute of genome evolution as sources of novel genetic functions. Here, we identified 445 genes specific to Populus trichocarpa. Of these, we performed deeper reconstruction of 13 orphan genes to provide evidence of de novo gene evolution. Populus and its sister genera Salix are particularly well suited for the study of orphan gene evolution because of the Salicoid whole-genome duplication event which resulted in highly syntenic sister chromosomal segments across the Salicaceae. We leveraged this genomic feature to reconstruct de novo gene evolution from intergenera, interspecies, and intragenomic perspectives by comparing the syntenic regions within the P. trichocarpa reference, then P. deltoides, and finally Salix purpurea. Furthermore, we demonstrated that 86.5% of the putative orphan genes had evidence of transcription. Additionally, we also utilized the Populus genome-wide association mapping panel, a collection of 1,084 undomesticated P. trichocarpa genotypes to further determine putative regulatory networks of orphan genes using expression quantitative trait loci (eQTL) mapping. Functional enrichment of these eQTL subnetworks identified common biological themes associated with orphan genes such as response to stress and defense response. We also identify a putative cis-element for a de novo gene and leverage conserved synteny to describe evolution of a putative transcription factor binding site. Overall, 45% of orphan genes were captured in trans-eQTL networks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Identification of important invasion and proliferation related genes in adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is an end‐stage hormonal syndrome. Although profound attempts have been made to illuminate the pathogenesis, the molecular mechanisms of ACC remain to be clarified. To identify the important genes in the progression of ACC, microarray datasets GSE19775 was downloaded from the gene expression omnibus database. The differentially-expressed genes (DEGs) were identified, and pathway and GO enrichment analyses were performed. The protein–protein interaction (PPI) network was constructed and the module analysis was performed using the protein interaction network analysis and Cytoscape. Also constructed target genes–miRNA regulatory network and target genes–TF regulatory network. Correlation of the hub genes were analyzed in The Cancer Genome Atlas. The prognostic values of hub genes were further validated by online tool UALCAN. Mutation analysis was done by online tool CBio Cancer Genomics Portal. A total of 884 DEGs were identified, with 441 in up regulation and 443 in down regulation. Pathways in catecholamine biosynthesis, aldosterone synthesis and secretion, pyrimidine deoxyribonucleosides salvage and systemic lupus erythematosus were the most significantly enriched for DEGs (up and down regulated). Blood vessel morphogenesis and cell cycle phase transition were the most significantly enriched term in biological processes, while extracellular matrix and chromosome, centromeric region were in cellular component and heparin binding and protein dimerization activity were in molecular function. Among the PPI networks and its module, target genes–miRNA regulatory network and target genes–TF regulatory network, hub genes were YWHAZ, FN1, GRK5, VCAM1, GATA6, TXNIP, HSPA1A, and F11R. Hub genes such as YWHAZ, STAT1, ICAM1, SH3BP5, CD83, FN1, TK1, HIST1H1C, CABLES1, and MCM3 were associated with poor overall survival, while hub genes such as STAT1, ICAM1, CD83, FN1, TK1, HIST1H1C, and MCM3 were highly expressed in stage 4. In conclusion, DEGs and hub genes diagnosed in this study may deepen our understanding of molecular mechanisms underlying the progression of ACC, and provide important targets for diagnosis and treatment of ACC.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2020

Co-locality to co-functionality: Eukaryotic gene neighborhoods as a resource for function

Diverging from the classic paradigm of random gene order in eukaryotes, gene proximity can be leveraged to systematically identify functionally related gene neighborhoods in eukaryotes, utilizing techniques pioneered in bacteria. Current methods of identifying gene neighborhoods typically rely on sequence similarity to characterized gene products. However, this approach is not robust for non model organisms like algae, which are evolutionarily distant from well-characterized model organisms. Here, we utilize a comparative genomic approach to identify evolutionarily conserved Proximal Orthologous Gene (POG) pairs conserved across at least two taxonomic classes of green algae. A total of 317 gene neighborhoods were identified. In some cases, gene proximity appears to have been conserved since before the streptophyte-chlorophyte split, 1,000 million years ago. Using functional inferences derived from reconstructed evolutionary relationships, we identified several novel functional clusters. A putative mycosporine-like amino acid (MAA), "sunscreen", neighborhood contains genes similar to either vertebrate or cyanobacterial pathways, suggesting a novel mosaic biosynthetic pathway in green algae. One of two putative arsenic-detoxification neighborhoods includes an organoarsenical transporter (ArsJ), a glyceraldehyde 3-phosphate dehydrogenase-like gene, homologs of which are involved in arsenic detoxification in bacteria, and a novel algal-specific phosphoglycerate kinase-like gene (PGK). Mutants of the ArsJ-like transporter and PGK-like genes in Chlamydomonas reinhardtii were found to be sensitive to arsenate, providing experimental support for the role of these identified neighbors in resistance to arsenate. Potential evolutionary origins of neighborhoods are discussed, and updated annotations for formerly poorly annotated genes are presented, highlighting the potential of this strategy for functional annotation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Machine learning analysis of RB-TnSeq fitness data predicts functional gene modules in Pseudomonas putida KT2440

ABSTRACT There is growing interest in engineering Pseudomonas putida KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of P. putida relies on the understanding of the functional relationships between genes. In this work, independent component analysis (ICA) was applied to a compendium of existing fitness data from randomly barcoded transposon insertion sequencing (RB-TnSeq) of P. putida KT2440 grown in 179 unique experimental conditions. ICA identified 84 independent groups of genes, which we call fModules (“functional modules”), where gene members displayed shared functional influence in a specific cellular process. This machine learning-based approach both successfully recapitulated previously characterized functional relationships and established hitherto unknown associations between genes. Selected gene members from fModules for hydroxycinnamate metabolism and stress resistance, acetyl coenzyme A assimilation, and nitrogen metabolism were validated with engineered mutants of P. putida . Additionally, functional gene clusters from ICA of RB-TnSeq data sets were compared with regulatory gene clusters from prior ICA of RNAseq data sets to draw connections between gene regulation and function. Because ICA profiles the functional role of several distinct gene networks simultaneously, it can reduce the time required to annotate gene function relative to manual curation of RB-TnSeq data sets. IMPORTANCE This study demonstrates a rapid, automated approach for elucidating functional modules within complex genetic networks. While Pseudomonas putida randomly barcoded transposon insertion sequencing data were used as a proof of concept, this approach is applicable to any organism with existing functional genomics data sets and may serve as a useful tool for many valuable applications, such as guiding metabolic engineering efforts in other microbes or understanding functional relationships between virulence-associated genes in pathogenic microbes. Furthermore, this work demonstrates that comparison of data obtained from independent component analysis of transcriptomics and gene fitness datasets can elucidate regulatory-functional relationships between genes, which may have utility in a variety of applications, such as metabolic modeling, strain engineering, or identification of antimicrobial drug targets.

09 BIOMASS FUELS↗
DOE OSTI2022

Functional Divergence in a Multi-gene Family Is a Key Evolutionary Innovation for Anaerobic Growth in Saccharomyces cerevisiae

The amplification and diversification of genes into large multi-gene families often mark key evolutionary innovations, but this process often creates genetic redundancy that hinders functional investigations. When the model budding yeast Saccharomyces cerevisiae transitions to anaerobic growth conditions, the cell massively induces the expression of seven serine/threonine-rich anaerobically-induced cell wall mannoproteins (anCWMPs): TIP1, TIR1, TIR2, TIR3, TIR4, DAN1, and DAN4. Here, we show that these genes likely derive evolutionarily from a single ancestral anCWMP locus, which was duplicated and translocated to new genomic contexts several times both prior to and following the budding yeast whole genome duplication (WGD) event. Based on synteny and their phylogeny, we separate the anCWMPs into four gene subfamilies. To resolve prior inconclusive genetic investigations of these genes, we constructed a set of combinatorial deletion mutants to determine their contributions toward anaerobic growth in S. cerevisiae. We found that two genes, TIR1 and TIR3, were together necessary and sufficient for the anCWMP contribution to anaerobic growth. Overexpressing either gene alone was insufficient for anaerobic growth, implying that they encode non-overlapping functional roles in the cell during anaerobic growth. We infer from the phylogeny of the anCWMP genes that these two important genes derive from an ancient duplication that predates the WGD event, whereas the TIR1 subfamily experienced gene family amplification after the WGD event. Taken together, the genetic and molecular evidence suggests that one key anCWMP gene duplication event, several auxiliary gene duplication events, and functional divergence underpin the evolution of anaerobic growth in budding yeasts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Gene Regulation Shifts Shed Light on Fungal Adaption in Plant Biomass Decomposers

ABSTRACT Fungi dominate the recycling of carbon sequestered in woody biomass. This process of organic turnover was first evolved among “white rot” fungi that degrade lignin to access carbohydrates and later evolved multiple times toward more efficient strategies to selectively target carbohydrates—“brown rot.” The brown rot adaption was often explained by mechanisms to deploy reactive oxygen species (ROS) to oxidatively attack wood structures. However, its genetic basis remains unclear, especially in the context of gene contractions of conventional carbohydrate-active enzymes (CAZYs) relative to white rot ancestors. Here, we hypothesized that these apparent gains in brown rot efficiency despite gene losses were due, in part, to upregulation of the retained genes. We applied comparative transcriptomics to multiple species of both rot types grown across a wood wafer to create a gradient of progressive decay and to enable tracking temporal gene expression. Dozens of “decay-stage-dependent” ortho-genes were isolated, narrowing a pool of candidate genes with time-dependent regulation unique to brown rot fungi. A broad comparison of the expression timing of CAZY families indicated a temporal regulatory shift of lignocellulose-oxidizing genes toward early stages in brown rot compared to white rot, enabling the segregation of oxidative treatment ahead of hydrolysis. These key brown rot ROS-generating genes with iron ion binding functions were isolated. Moreover, transcription energy was shifted to be invested on the retained GHs in brown rot fungi to strengthen carbohydrate conversion. Collectively, these results support the hypothesis that gene regulation shifts played a pivotal role in brown rot adaptation. IMPORTANCE Fungi dominate the turnover of wood, Earth’s largest pool of aboveground terrestrial carbon. Fungi first evolved this capacity by degrading lignin to access and hydrolyze embedded carbohydrates (white rot). Multiple lineages, however, adapted faster reactive oxygen species (ROS) pretreatments to loosen lignocellulose and selectively extract sugars (brown rot). This brown rot “shortcut” often coincided with losses (>60%) of conventional lignocellulolytic genes, implying that ROS adaptations supplanted conventional pathways. We used comparative transcriptomics to further pursue brown rot adaptations, which illuminated the clear temporal expression shift of ROS genes, as well as the shift toward synthesizing more GHs in brown rot relative to white rot. These imply that gene regulatory shifts, not simply ROS innovations, were key to brown rot fungal evolution. These results not only reveal an important biological shift among these unique fungi, but they may also illuminate a trait that restricts brown rot fungi to certain ecological niches.

plant biomas↗
DOE OSTI2019

Expression profiles of cell-wall related genes vary broadly between two common maize inbreds during stem development

The cellular machinery for cell wall synthesis and metabolism is encoded by members of large multi-gene families. Maize is both a genetic model for grass species and a potential source of lignocellulosic biomass from crop residues. Genetic improvement of maize for its utility as a bioenergy feedstock depends on identification of the specific gene family members expressed during secondary wall development in stems. High-throughput sequencing of transcripts expressed in developing rind tissues of stem internodes provided a comprehensive inventory of cell wall-related genes in maize ( Zea mays , cultivar B73). Of 1239 of these genes, 854 were expressed among the internodes at =95 reads per 20 M, and 693 of them at =500 reads per 20 M. Grasses have cell wall compositions distinct from non-commelinid species; only one-quarter of maize cell wall-related genes expressed in stems were putatively orthologous with those of the eudicot Arabidopsis. Using a slope-metric algorithm, five distinct patterns for sub-sets of co-expressed genes were defined across a time course of stem development. For the subset of genes associated with secondary wall formation, fifteen sequence motifs were found in promoter regions. The same members of gene families were often expressed in two maize inbreds, B73 and Mo17, but levels of gene expression between them varied, with 30% of all genes exhibiting at least a 5-fold difference at any stage. Although presence-absence and copy-number variation might account for much of these differences, fold-changes of expression of a CADa and a FLA11 gene were attributed to polymorphisms in promoter response elements. Large genetic variation in maize as a species precludes the extrapolation of cell wall-related gene expression networks even from one common inbred line to another. Elucidation of genotype-specific expression patterns and their regulatory controls will be needed for association panels of inbreds and landraces to fully exploit genetic variation in maize and other bioenergy grass species.

09 BIOMASS FUELS↗
DOE OSTI2021

Leptin modulates gene expression in the heart, cardiomyocytes and the adipose tissue thus mitigating LPS-induced damage

Highlights: • LPS enhanced expression of the leptin gene in mouse tissues and cardiomyocytes. • Leptin downregulated pro-inflammatory genes in vivo and in cardiomyocytes after LPS treatment. • Leptin upregulated expression of antioxidant genes in vivo and in cardiomyocytes after LPS treatment. • Leptin reduced ROS levels in cardiomyocytes after LPS treatment. • Leptin acts to counteract LPS-induced damage in the heart and cardiomyocytes by modulating gene expression. • Anti-inflammatory effects of leptin in transgenic aMUPA mice overexpressing leptin surpassed that of wild type mice. Leptin is an adipokine of pleiotropic effects linked to energy metabolism, satiety, the immune response, and cardioprotection. We have recently shown that leptin causally conferred resistance to myocardial infarction-induced damage in transgenic αMUPA mice overexpressing leptin compared to their wild type (WT) ancestral mice FVB/N. Prompted by these findings, we have investigated here if leptin can counteract the inflammatory response triggered after LPS administration in tissues in vivo and in cardiomyocytes in culture. The results have shown that LPS upregulated in vivo and in vitro all genes examined here, both pro-inflammatory and antioxidant, as well as the leptin gene. Pretreating mice with leptin neutralizing antibodies further upregulated the expression of TNFα and IL-1β in the adipose tissue of both mouse types, and in the αMUPA heart. The antibodies also increased the levels of serum markers for cell toxicity in both mouse types. These results indicate that under LPS, leptin actually reduced the levels of these inflammatory-related parameters. In addition, pretreatment with leptin antibodies reduced the levels of HIF-1α and VEGF mRNAs in the heart, indicating that under LPS leptin increased the levels of these mRNAs. In cardiomyocytes, pretreatment with exogenous leptin prior to LPS reduced the expression of both pro-inflammatory genes, enhanced the expression of the antioxidant genes HO-1, SOD2 and HIF-1α, and lowered ROS staining. In addition, results obtained with leptin antibodies and the SMLA leptin antagonist indicated that endogenous and exogenous leptin can inhibit leptin gene expression. Together, these findings have indicated that under LPS, leptin concomitantly downregulated pro-inflammatory genes, upregulated antioxidant genes, and lowered ROS levels. These results suggest that leptin can counteract inflammation in the heart and adipose tissue by modulating gene expression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Single‐parent expression drives dynamic gene expression complementation in maize hybrids

SUMMARY Single‐parent expression (SPE) is defined as gene expression in only one of the two parents. SPE can arise from differential expression between parental alleles, termed non‐presence/absence (non‐PAV) SPE, or from the physical absence of a gene in one parent, termed PAV SPE. We used transcriptome data of diverse Zea mays (maize) inbreds and hybrids, including 401 samples from five different tissues, to test for differences between these types of SPE genes. Although commonly observed, SPE is highly genotype and tissue specific. A positive correlation was observed between the genetic distance of the two inbred parents and the number of SPE genes identified. Regulatory analysis showed that PAV SPE and non‐PAV SPE genes are mainly regulated by cis effects, with a small fraction under trans regulation. Polymorphic transposable element insertions in promoter sequences contributed to the high level of cis regulation for PAV SPE and non‐PAV SPE genes. PAV SPE genes were more frequently expressed in hybrids than non‐PAV SPE genes. The expression of parentally silent alleles in hybrids of non‐PAV SPE genes was relatively rare but occurred in most hybrids. Non‐PAV SPE genes with expression of the silent allele in hybrids are more likely to exhibit above high parent expression level than hybrids that do not express the silent allele, leading to non‐additive expression. This study provides a comprehensive understanding of the nature of non‐PAV SPE and PAV SPE genes and their roles in gene expression complementation in maize hybrids.

Li, Zhi↗
DOE OSTI2020

Desulfatiglans anilini Initiates Degradation of Aniline With the Production of Phenylphosphoamidate and 4-Aminobenzoate as Intermediates Through Synthases and Carboxylases From Different Gene Clusters

The anaerobic degradation of aniline was studied in the sulfate-reducing bacterium Desulfatiglans anilini. Our aim was to identify the genes and their proteins that are required for the initial activation of aniline as well as to characterize intermediates of this reaction. Aniline-induced genes were revealed by comparison of the proteomes of D. anilini grown with different substrates (aniline, 4- aminobenzoate, phenol, and benzoate). Most genes encoding proteins that were highly abundant in aniline- or 4-aminobenzoate-grown D. anilini cells but not in phenol- or benzoate-grown cells were located in the putative gene clusters ani (aniline degradation), hcr (4-hydroxybenzoyl-CoA reductase) and phe (phenol degradation). Of these putative gene clusters, only the phe gene cluster has been studied previously. Based on the differential proteome analysis, four candidate genes coding for kinase subunits and carboxylase subunits were suspected to be responsible for the initial conversion of aniline to 4-aminobenzoate. These genes were cloned and overproduced in E. coli. The recombinant proteins were obtained in inclusion bodies but could be refolded successfully. Two subunits of phenylphosphoamidate synthase and two carboxylase subunits converted aniline to 4-aminobenzoate with phenylphosphoamidate as intermediate under consumption of ATP. Only when both carboxylase subunits, one from gene cluster ani and the other from gene cluster phe, were combined, phenylphosphoamidate was converted to 4-aminobenzoate in vitro, with Mn 2+ , K + , and FMN as co-factors. Thus, aniline is degraded by the anaerobic bacterium D. anilini only by recruiting genes for the enzymatic machinery from different gene clusters. We conclude, that D. anilini carboxylates aniline to 4-aminobenzoate via phenylphosphoamidate as an energy rich intermediate analogous to the degradation of phenol to 4-hydroxybenzoate via phenylphosphate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Identification of new marker genes from plant single‐cell RNA‐seq data using interpretable machine learning methods

Summary An essential step in the analysis of single‐cell RNA sequencing data is to classify cells into specific cell types using marker genes. In this study, we have developed a machine learning pipeline called single‐cell predictive marker (SPmarker) to identify novel cell‐type marker genes in the Arabidopsis root. Unlike traditional approaches, our method uses interpretable machine learning models to select marker genes. We have demonstrated that our method can: assign cell types based on cells that were labelled using published methods; project cell types identified by trajectory analysis from one data set to other data sets; and assign cell types based on internal GFP markers. Using SPmarker, we have identified hundreds of new marker genes that were not identified before. As compared to known marker genes, the new marker genes have more orthologous genes identifiable in the corresponding rice single‐cell clusters. The new root hair marker genes also include 172 genes with orthologs expressed in root hair cells in five non‐ Arabidopsis species, which expands the number of marker genes for this cell type by 35–154%. Our results represent a new approach to identifying cell‐type marker genes from scRNA‐seq data and pave the way for cross‐species mapping of scRNA‐seq data in plants.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Using online tools at the Bovine Genome Database to manually annotate genes in the new reference genome

With the availability of a new highly contiguous Bos taurus reference genome assembly (ARS-UCD1.2), it is the opportune time to upgrade the bovine gene set by seeking input from researchers. Furthermore, advances in graphical genome annotation tools now make it possible for researchers to leverage sequence data generated with the latest technologies to collaboratively curate genes. For many years the Bovine Genome Database (BGD) has provided tools such as the APOLLO genome annotation editor to support manual bovine gene curation. The goal of this paper is to explain the reasoning behind the decisions made in the manual gene curation process while providing examples using the existing BGD tools. We will describe the sources of gene annotation evidence provided at the BGD, including RNAseq and Iso-Seq data. We will also explain how to interpret various data visualizations when curating gene models, and will demonstrate the value of manual gene annotation. The process described here can be applied to manual gene curation for other species with similar tools. With a better understanding of manual gene annotation, researchers will be encouraged to edit gene models and contribute to the enhancement of livestock gene sets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A High-efficacy CRISPRi System for Gene Function Discovery in Zymomonas mobilis

Zymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized relative to those of model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is a programmable gene knockdown system that can precisely control the timing and extent of gene repression, thus enabling targeting of essential genes. Here, we establish a stable, high-efficacy CRISPRi system in Z. mobilis that is capable of perturbing all genes—including essential genes. We show that Z. mobilis CRISPRi causes either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes or partial knockdowns using spacers with mismatches. We demonstrate the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, are key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance. Our Z. mobilis CRISPRi system will enable comprehensive gene function discovery, opening a path to rational design of biofuel production strains with improved yields.

lignocellulosic hydrolysate↗
DOE OSTI2019

Protein expression and gene editing in monocots using foxtail mosaic virus vectors

Abstract Plant viruses can be engineered to carry sequences that direct silencing of target host genes, expression of heterologous proteins, or editing of host genes. A set of foxtail mosaic virus (FoMV) vectors was developed that can be used for transient gene expression and single guide RNA delivery for Cas9‐mediated gene editing in maize, Setaria viridis , and Nicotiana benthamiana . This was accomplished by duplicating the FoMV capsid protein subgenomic promoter, abolishing the unnecessary open reading frame 5A, and inserting a cloning site containing unique restriction endonuclease cleavage sites immediately after the duplicated promoter. The modified FoMV vectors transiently expressed green fluorescent protein (GFP) and bialaphos resistance (BAR) protein in leaves of systemically infected maize seedlings. GFP was detected in epidermal and mesophyll cells by epifluorescence microscopy, and expression was confirmed by Western blot analyses. Plants infected with FoMV carrying the bar gene were temporarily protected from a glufosinate herbicide, and expression was confirmed using a rapid antibody‐based BAR strip test. Expression of these proteins was stabilized by nucleotide substitutions in the sequence of the duplicated promoter region. Single guide RNAs expressed from the duplicated promoter mediated edits in the N. benthamiana Phytoene desaturase gene, the S. viridis Carbonic anhydrase 2 gene, and the maize HKT1 gene encoding a potassium transporter. The efficiency of editing was enhanced in the presence of synergistic viruses and a viral silencing suppressor. This work expands the utility of FoMV for virus‐induced gene silencing (VIGS), virus‐mediated overexpression (VOX), and virus‐enabled gene editing (VEdGE) in monocots.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The quantitative genetics of gene expression in Mimulus guttatus

Gene expression can be influenced by genetic variants that are closely linked to the expressed gene (cis eQTLs) and variants in other parts of the genome (trans eQTLs). We created a multiparental mapping population by sampling genotypes from a single natural population of Mimulus guttatus and scored gene expression in the leaves of 1,588 plants. We find that nearly every measured gene exhibits cis regulatory variation (91% have FDR < 0.05). cis eQTLs are usually allelic series with three or more functionally distinct alleles. The cis locus explains about two thirds of the standing genetic variance (on average) but varies among genes and tends to be greatest when there is high indel variation in the upstream regulatory region and high nucleotide diversity in the coding sequence. Despite mapping over 10,000 trans eQTL / affected gene pairs, most of the genetic variance generated by trans acting loci remains unexplained. This implies a large reservoir of trans acting genes with subtle or diffuse effects. Mapped trans eQTLs show lower allelic diversity but much higher genetic dominance than cis eQTLs. Several analyses also indicate that trans eQTLs make a substantial contribution to the genetic correlations in expression among different genes. They may thus be essential determinants of “gene expression modules,” which has important implications for the evolution of gene expression and how it is studied by geneticists.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A compendium of human gene functions derived from evolutionary modelling

A comprehensive, computable representation of the functional repertoire of all macromolecules encoded within the human genome is a foundational resource for biology and biomedical research. The Gene Ontology Consortium has been working towards this goal by generating a structured body of information about gene functions, which now includes experimental findings reported in more than 175,000 publications for human genes and genes in experimentally tractable model organisms 1,2 . Here, we describe the results of a large, international effort to integrate all of these findings to create a representation of human gene functions that is as complete and accurate as possible. Specifically, we apply an expert-curated, explicit evolutionary modelling approach to all human protein-coding genes. This approach integrates available experimental information across families of related genes into models that reconstruct the gain and loss of functional characteristics over evolutionary time. The models and the resulting set of 68,667 integrated gene functions cover approximately 82% of human protein-coding genes. The functional repertoire reveals a marked preponderance of molecular regulatory functions, and the models provide insights into the evolutionary origins of human gene functions. We show that our set of descriptions of functions can improve the widely used genomic technique of Gene Ontology enrichment analysis. The experimental evidence for each functional characteristic is recorded, thereby enabling the scientific community to help review and improve the resource, which we have made publicly available.

59 BASIC BIOLOGICAL SCIENCES↗