Tractable Genetic System Research Articles

Modulation of fungal phosphate homeostasis by the plant hormone strigolactone

Inter-kingdom communication through small molecules is essential to the coexistence of organisms in an ecosystem. In soil communities, the plant root is a nexus of interactions for a remarkable number of fungi and is a source of small-molecule plant hormones that shape fungal compositions. Although hormone signaling pathways are established in plants, how fungi perceive and respond to molecules is unclear because many plant-associated fungi are recalcitrant to experimentation. Here, we develop an approach using the model fungus, Saccharomyces cerevisiae, to elucidate mechanisms of fungal response to plant hormones. Two plant hormones, strigolactone and methyl jasmonate, produce unique transcript profiles in yeast, affecting phosphate and sugar metabolism, respectively. Genetic analysis in combination with structural studies suggests that SLs require the high-affinity transporter Pho84 to modulate phosphate homeostasis. The ability to study small-molecule plant hormones in a tractable genetic system should have utility in understanding fungal-plant interactions.

Limiting factors in the operation of photosystems I and II in cyanobacteria.

Cyanobacteria are important targets for biotechnological applications due to their ability to grow in a wide variety of environments, rapid growth rates, and tractable genetic systems. They and their bioproducts can be used as bioplastics, biofertilizers, and in carbon capture and produce important secondary metabolites that can be used as pharmaceuticals. However, the photosynthetic process in cyanobacteria can be limited by a wide variety of environmental factors such as light intensity and wavelength, exposure to UV light, nutrient limitation, temperature, and salinity. Carefully considering these limitations, modifying the environment, and/or selecting cyanobacterial species will allow cyanobacteria to be used in biotechnological applications.

Site-Specific Profiling of N-Glycans in Drosophila melanogaster.

Drosophila melanogaster is a well-studied and highly tractable genetic model system for deciphering the molecular mechanisms underlying various biological processes. Although being one of the most critical post-translational modifications of proteins, the understanding of glycosylation in Drosophila is still lagging behind compared with that of other model organisms. In this study, we systematically investigated the site-specific N-glycan profile of Drosophila melanogaster using intact glycopeptide analysis technique. This approach identified the glycans, proteins, and their glycosites in Drosophila, as well as information on site-specific glycosylation, which allowed us to know which glycans are attached to which glycosylation sites. The results showed that the majority of N-glycans in Drosophila were high-mannose type (69.3%), consistent with reports in other insects. Meanwhile, fucosylated N-glycans were also highly abundant (22.7%), and the majority of them were mono-fucosylated. In addition, 24 different sialylated glycans attached with 16 glycoproteins were identified, and these proteins were mainly associated with developmental processes. Gene ontology analysis showed that N-glycosylated proteins in Drosophila were involved in multiple biological processes, such as axon guidance, N-linked glycosylation, cell migration, cell spreading, and tissue development. Interestingly, we found that seven glycosyltransferases and four glycosidases were N-glycosylated, which suggested that N-glycans may play a regulatory role in the synthesis and degradation of N-glycans and glycoproteins. To our knowledge, this work represents the first comprehensive analysis of site-specific N-glycosylation in Drosophila, thereby providing new perspectives for the understanding of biological functions of glycosylation in insects.

Employing Cloning-Independent Mutagenesis of Parvimonas micra for the Study of Cell Wall Biogenesis.

The cell wall plays an important structural role for bacteria and is intimately tied to a variety of critical processes ranging from growth and differentiation to pathogenesis. Our understanding of cell wall biogenesis is primarily derived from a relatively small number of heavily studied model organisms. Consequently, these processes can only be inferred for the vast majority of prokaryotes, especially among groups of uncharacterized and/or genetically intractable organisms. Recently, we developed the first tractable genetic system for Parvimonas micra, which is a ubiquitous Gram-positive pathobiont of the human microbiome involved in numerous types of inflammatory infections as well as a variety of malignant tumors. P. micra is also the first, and currently only, member of the entire Tissierellia class of the Bacillota phylum in which targeted genetic manipulation has been demonstrated. Thus, it is now possible to study cell wall biogenesis mechanisms within a member of the Tissierellia, which may also reveal novel aspects of P. micra pathobiology. Herein, we describe a procedure for cloning-independent genetic manipulation of P. micra, including allelic replacement mutagenesis and genetic complementation. The described techniques are also similarly applicable for the study of other aspects of P. micra pathobiology and physiology.

Techniques for Quantifying Bacterially Induced Carbonate Mineralization in Escherichia coli

The lack of reliable techniques to accurately quantify bacterial carbonate production and a tractable genetic system limits our ability to thoroughly understand the metabolic basis for bacterial calcification. In this work, we demonstrate bacterially induced CaCO3 precipitation in the model organism, Escherichia coli K-12, and develop new quantitative methods for carbonate mineral production using inductively coupled plasma-optical emission spectroscopy (ICP-OES) and an image analysis approach repurposed for agar cultures. These assays allowed us to assess the impact of various calcium sources and environmental factors on crystalline CaCO3 formation in E. coli. Our data demonstrate that Ca-acetate and Ca-succinate yield the greatest CaCO3 output in E. coli, while a preliminary study demonstrated microclimatic influences on crystal growth and size. This work helps lay the foundation for using E. coli as a model bacterial species to probe the genetic elements that promote calcification in Bacteria, with the potential to identify previously undescribed pathways for precipitation.

Human-specific gene CT47 blocks PRMT5 degradation to lead to meiosis arrest

Exploring the functions of human-specific genes (HSGs) is challenging due to the lack of a tractable genetic model system. Testosterone is essential for maintaining human spermatogenesis and fertility, but the underlying mechanism is unclear. Here, we identified Cancer/Testis Antigen gene family 47 (CT47) as an essential regulator of human-specific spermatogenesis by stabilizing arginine methyltransferase 5 (PRMT5). A humanized mouse model revealed that CT47 functions to arrest spermatogenesis by interacting with and regulating CT47/PRMT5 accumulation in the nucleus during the leptotene/zygotene-to-pachytene transition of meiosis. We demonstrate that testosterone induces nuclear depletion of CT47/PRMT5 and rescues leptotene-arrested spermatocyte progression in humanized testes. Loss of CT47 in human embryonic stem cells (hESCs) by CRISPR/Cas9 led to an increase in haploid cells but blocked the testosterone-induced increase in haploid cells when hESCs were differentiated into haploid spermatogenic cells. Moreover, CT47 levels were decreased in nonobstructive azoospermia. Together, these results established CT47 as a crucial regulator of human spermatogenesis by preventing meiosis initiation before the testosterone surge.

Genetic analysis of Hsp90 function in Cryptococcus neoformans highlights key roles in stress tolerance and virulence.

The opportunistic human fungal pathogen Cryptococcus neoformans has tremendous impact on global health, causing 181,000 deaths annually. Current treatment options are limited, and the frequent development of drug resistance exacerbates the challenge of managing invasive cryptococcal infections. In diverse fungal pathogens, the essential molecular chaperone Hsp90 governs fungal survival, drug resistance, and virulence. Therefore, targeting this chaperone has emerged as a promising approach to combat fungal infections. However, the role of Hsp90 in supporting C. neoformans pathogenesis remains largely elusive due to a lack of genetic characterization. To help dissect the functions of Hsp90 in C. neoformans, we generated a conditional expression strain in which HSP90 is under control of the copper-repressible promoter CTR4-2. Addition of copper to culture medium depleted Hsp90 transcript and protein levels in this strain, resulting in compromised fungal growth at host temperature; increased sensitivity to stressors, including the azole class of antifungals; altered C. neoformans morphology; and impaired melanin production. Finally, leveraging the fact that copper concentrations vary widely in different mouse tissues, we demonstrated attenuated virulence for the CTR4-2p-HSP90 mutant specifically in an inhalation model of Cryptococcus infection. During invasion and establishment of infection in this mouse model, the pathogen is exposed to the relatively high copper concentrations found in the lung as compared to blood. Overall, this work generates a tractable genetic system to study the role of Hsp90 in supporting the pathogenicity of C. neoformans and provides proof-of-principle that targeting Hsp90 holds great promise as a strategy to control cryptococcal infection.

Genetics of white color and iridophoroma in "Lemon Frost" leopard geckos.

The squamates (lizards and snakes) are close relatives of birds and mammals, with more than 10,000 described species that display extensive variation in a number of important biological traits, including coloration, venom production, and regeneration. Due to a lack of genomic tools, few genetic studies in squamates have been carried out. The leopard gecko, Eublepharis macularius, is a popular companion animal, and displays a variety of coloration patterns. We took advantage of a large breeding colony and used linkage analysis, synteny, and homozygosity mapping to investigate a spontaneous semi-dominant mutation, "Lemon Frost", that produces white coloration and causes skin tumors (iridophoroma). We localized the mutation to a single locus which contains a strong candidate gene, SPINT1, a tumor suppressor implicated in human skin cutaneous melanoma (SKCM) and over-proliferation of epithelial cells in mice and zebrafish. Our work establishes the leopard gecko as a tractable genetic system and suggests that a tumor suppressor in melanocytes in humans can also suppress tumor development in iridophores in lizards.

Exploiting the Fruitfly, Drosophila melanogaster, to Identify the Molecular Basis of Cryptochrome-Dependent Magnetosensitivity

The flavoprotein CRYPTOCHROME (CRY) is now generally believed to be a magnetosensor, providing geomagnetic information via a quantum effect on a light-initiated radical pair reaction. Whilst there is considerable physical and behavioural data to support this view, the precise molecular basis of animal magnetosensitivity remains frustratingly unknown. A key reason for this is the difficulty in combining molecular and behavioural biological experiments with the sciences of magnetics and spin chemistry. In this review, we highlight work that has utilised the fruit fly, Drosophila melanogaster, which provides a highly tractable genetic model system that offers many advantages for the study of magnetosensitivity. Using this “living test-tube”, significant progress has been made in elucidating the molecular basis of CRY-dependent magnetosensitivity.

Effects of 3D culturing conditions on the transcriptomic profile of stem-cell-derived neurons.

Understanding neurological diseases requires tractable genetic systems. Engineered 3D neural tissues are an attractive choice, but how the cellular transcriptomic profiles in these tissues are affected by the encapsulating materials and are related to the human-brain transcriptome is not well understood. Here, we report the characterization of the effects of culturing conditions on the transcriptomic profiles of induced neuronal cells, as well as a method for the rapid generation of 3D co-cultures of neuronal and astrocytic cells from the same pool of human embryonic stem cells. By comparing the gene-expression profiles of neuronal cells in culture conditions relevant to the developing human brain, we found that modifying the degree of crosslinking of composite hydrogels can tune expression patterns so they correlate with those of specific brain regions and developmental stages. Moreover, by using single-cell sequencing, we show that our engineered tissues recapitulate transcriptional patterns of cell types in the human brain. The analysis of culturing conditions will inform the development of 3D neural tissues for use as tractable models of brain diseases.

Assays to Study Repair of Inducible DNA Double-Strand Breaks at Telomeres.

The ends of linear chromosomes are constituted of repetitive DNA sequences called telomeres. Telomeres, nearby regions called subtelomeres, and their associated factors prevent chromosome erosion over cycles of DNA replication and prevent chromosome ends from being recognized as DNA double-strand breaks (DSBs). This raises the question of how cells repair DSBs that actually occur near chromosome ends. One approach is to edit the genome and engineer cells harboring inducible DSB sites within the subtelomeric region of different chromosome ends. This provides a reductionist and tractable genetic model system in which mechanisms mediating repair can be dissected via genetics, molecular biology, and microscopy tools.

Understanding Synaptogenesis and Functional Connectome in C. elegans by Imaging Technology.

Formation of functional synapses is a fundamental process for establishing neural circuits and ultimately for expressing complex behavior. Extensive research has interrogated how such functional synapses are formed and how synapse formation contributes to the generation of neural circuitry and behavior. The nervous system of Caenorhabditis elegans, due to its relatively simple structure, the transparent body, and tractable genetic system, has been adapted as an excellent model to investigate synapses and the functional connectome. Advances in imaging technology together with the improvement of genetically encoded molecular tools enabled us to visualize synapses and neural circuits of the animal model, which provide insights into our understanding of molecules and their signaling pathways that mediate synapse formation and neuronal network modulation. Here, we review synaptogenesis in active zones and the mapping of local connectome in C. elegans nervous system whose understandings have been extended by the advances in imaging technology along with the genetic molecular tools.

Editorial: Non-coding RNA Regulation: Lessons from Model Organisms and Impact on Human Health

This research topic highlights the great interest and cautious optimism surrounding our constantly expanding understanding of the roles played by non-coding RNA (ncRNA) molecules in fundamental biology as well as in human health, aging, and disease. In this topic, five review articles and one hypothesis/theory paper cover in breadth and depth the established, emerging, and controversial aspects of ncRNA biology. Claycomb discusses how we owe, at least in large part, today's broad interest in this area of biology to the discovery and characterization of RNA interference in the tiny soil worm Caenorhabditis elegans, a simple and elegant model organism that continues to be at the forefront of ncRNA research (Youngman and Claycomb). She also discusses how this worm provides a tractable genetic system in which researchers continue to study aging, epigenetic inheritance, and the role of ncRNA in such processes. Continuing with contributions from genetic model organisms to the field, Mekhail then discusses how yeast, fly, mouse, and human systems have helped uncover roles of ncRNA molecules in neural function and neurological disorders across the lifespan (Szafranski et al.). In particular, this article discusses the role of different classes of ncRNA including miRNA and lncRNA in healthy neural function and aging before highlighting how deleterious processes can also be triggered by ncRNA and/or its regulators in neurodegenerative diseases especially as we age. Suh then summarizes the rich connections between a great number of disease-linked miRNAs and the insulin growth factor 1 (IGF-1) signaling pathway, which regulates diverse processes from development to aging (Jung and Suh). Discussed connections between miRNAs and diseases including cancers, diabetes, and aging are astounding. This certainly highlights the therapeutic potential of miRNAs and miRNA-targeting nucleotides. Fish then discusses how networks of miRNAs and lncRNAs modulate NFkB signaling, a major driver of blood vessel inflammation (Cheng et al.). Such inflammation can start at a younger age but lead to heart attack and stroke later in life highlighting the therapeutic potential of ncRNA modulation to treat vascular inflammation and its associated complications across the lifespan. Mhlanga discusses how ncRNAs can be manipulated by pathogens such as the human immunodeficiency virus (HIV) (Barichievy et al.). This article reveals how ncRNA molecules are at the center of a power struggle between virus and host fighting for the regulatory potential of ncRNAs. Finally, Palazzo helps us take a step back and carefully assess the possibility that most ncRNA is junk transcriptional noise (Palazzo and Lee). Contrary to the first impression that this article is anti-ncRNA, this hypothesis and theory article should help the field better distinguish functional from junk ncRNA. In conclusion, I thank the authors and my co-guest editor for their contributions to this research topic. I also extend my most sincere thanks to the tireless researchers in the ncRNA field for their dedication to decipher what continues to be one of the most fascinating areas of biology. In addition to revealing secrets of human health, disease, and aging, this field is helping us better understand the evolution of processes that are so vital to our existence as a human species. By uncovering these processes, we learn more about who we are in an effort to extend our health span.

GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport.

The GGGGCC (G4C2) repeat expansion in a noncoding region of C9orf72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis and frontotemporal dementia. The basis for pathogenesis is unknown. To elucidate the consequences of G4C2 repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 G4C2-repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein to detect repeat-associated non-AUG (RAN) translation. We show that these transgenic animals display dosage-dependent, repeat-length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins, as observed in patients with C9orf72-related disease. This model was used in a large-scale, unbiased genetic screen, ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC), as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results, we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats in vitro and in vivo. Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged induced pluripotent stem-cell-derived neurons from patients with C9orf72-related disease. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration.

Chimeric Tumor and Organ Transplantation Models.

Mouse models of cancer development and progression provide a means to study tumor response in appropriate physiological contexts. However, mouse cancer progression and therapy models have traditionally suffered from many of the same problems as human clinical cancer research, including genetic heterogeneity and tumor-stage variability at the time of treatment. Additionally, most mouse models are not tractable genetic systems, making it difficult to recapitulate the diverse set of alterations that regularly occur during tumor development. The recent development of chimeric and tumor transplantation techniques address many of the limitations of conventional mouse genetics. These strategies allow for the somatic introduction of complex genetic alterations into a subset of cells in reconstituted tumors or organ systems. Moreover, these different approaches can be combined in such a way that tumors with multiple genotypes are rapidly produced. These matched pairs can be systemically introduced into recipient mice for the rapid ex vivo modification of preestablished malignancies allows the generation of "matched pairs" of tumors differing in a single defined lesion (i.e., aliquots of the same primary malignancy with and without a gene of interest). Thus, treatment studies can be performed (1) in the context of an otherwise normal organ system, (2) on tumors that are in their appropriate anatomical context, and (3) on tumors that are essentially identical besides the presence of defined experimentally introduced alterations. Here, we will introduce procedures for modifying both normal and transformed cells and their adaptation to study in vivo tumor biology.

Genome-wide DNA binding pattern of the homeodomain transcription factor Sine oculis (So) in the developing eye of Drosophila melanogaster.

The eye of the fruit fly Drosophila melanogaster provides a highly tractable genetic model system for the study of animal development, and many genes that regulate Drosophila eye formation have homologs implicated in human development and disease. Among these is the homeobox gene sine oculis (so), which encodes a homeodomain transcription factor (TF) that is both necessary for eye development and sufficient to reprogram a subset of cells outside the normal eye field toward an eye fate. We have performed a genome-wide analysis of So binding to DNA prepared from developing Drosophila eye tissue in order to identify candidate direct targets of So-mediated transcriptional regulation, as described in our recent article [20]. The data are available from NCBI Gene Expression Omnibus (GEO) with the accession number GSE52943. Here we describe the methods, data analysis, and quality control of our So ChIP-seq dataset.

Model of Yeast Actin Cable Distribution and Dynamics

The growth of fission yeast relies on the polymerization of actin filaments at cell tips. These filaments are nucleated by formin proteins that localize at tip cortical sites. These actin filaments bundle to form actin cables that span the cell and guide the movement of vesicles toward the cell tips. Since actin cables are structures whose dynamics can be monitored by fluorescence microscopy, and since yeast is a tractable genetic system, comparison of the results of theoretical models of actin cables to experiment could enable quantitative tests of the mechanisms of actin polymerization in cells. We used computer simulations to study the spatial and dynamical properties of actin cables. We simulated actin individual filaments as semiflexible polymers in 3D, composed of beads connected with springs. Formin polymerization was simulated as filament growth out of cortical sites located at cell tips. Actin filament severing by cofilin was simulated as filament turnover. We added attractive interactions between beads to simulate filament bundling by actin cross-linkers such as fimbrin. Comparison of the results of the model to prior experiments suggests that filament severing, nucleation and crosslinking are sufficient to describe the many features of actin cables. We found bundled and unbundled phases as cross-linking strength was varied and propose experiments to test the model predictions.

Establishment of a Tractable Genetic Transformation System in Veillonella spp

We have constructed the first Escherichia coli-Veillonella shuttle vector based on an endogenous plasmid (pVJL1) isolated from a clinical Veillonella strain. A highly transformable Veillonella strain was also identified. Both the shuttle vector and the transformable strain should be valuable tools for future Veillonella genetic studies.

Chlamydia persistence – a tool to dissect chlamydia–host interactions

Under stress, chlamydiae can enter a non-infectious but viable state termed persistence. In the absence of a tractable genetic system, persistence induction provides an important experimental tool with which to study these fascinating organisms. This review will discuss examples of: i) persistence studies that have illuminated critical chlamydiae/host interactions; and ii) novel persistence models that will do so in the future.

The Obligate Intracellular Lifestyle

The Obligate Intracellular Lifestyle