ABSTRACT
Aureobasidium pullulans is a ubiquitous polymorphic black yeast with industrial and agricultural applications. It has recently gained attention amongst cell biologists for its unconventional mode of proliferation in which multinucleate yeast cells make multiple buds within a single cell cycle. Here, we combine a chemical transformation method with genome-targeted homologous recombination to yield â¼60 transformants/µg of DNA in just 3 days. This protocol is simple, inexpensive, and requires no specialized equipment. We also describe vectors with codon-optimized green and red fluorescent proteins for A. pullulans and use these tools to explore novel cell biology. Quantitative imaging of a strain expressing cytosolic and nuclear markers showed that although the nuclear number varies considerably among cells of similar volume, total nuclear volume scales with cell volume over an impressive 70-fold size range. The protocols and tools described here expand the toolkit for A. pullulans biologists and will help researchers address the many other puzzles posed by this polyextremotolerant and morphologically plastic organism.
Subject(s)
Aureobasidium , Genetic Techniques , Transformation, Genetic , Aureobasidium/cytology , Aureobasidium/genetics , Aureobasidium/metabolism , Cell Nucleus/metabolism , Cell Nucleus/genetics , Genetic Vectors/metabolism , Green Fluorescent Proteins/metabolism , Green Fluorescent Proteins/genetics , Homologous Recombination , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Red Fluorescent ProteinABSTRACT
BACKGROUND: The three-dimensional (3D) genome architecture plays a critical role inregulating gene expression. However, the specific alterations in thisarchitecture within somatotroph tumors and their implications for gene expression remain largely unexplored. METHODS: We employed Hi-C and RNA-seq analyses to compare the 3D genomic structures of somatotroph tumors with normal pituitary tissue. This comprehensive approachenabled the characterization of A/B compartments, topologically associateddomains (TADs), and chromatin loops, integrating these with gene expression patterns. RESULTS: We observed a decrease in both the frequency of chromosomal interactions andthe size of TADs in tumor tissue compared to normal tissue. Conversely, the number of TADs and chromatin loops was found to be increased in tumors. Integrated analysis of Hi-C and RNA-seq data demonstrated that changes inhigher-order chromat in structure were associated with alterations in gene expression. Specifically, genes in A compartments showed higher density and increased expression relative to those in B compartments. Moreover, the weakand enhanced insulation boundaries were identified, and the associated genes were enriched in the Wnt/ß-Catenin signaling pathway. We identified the gainedand lost loops in tumor and integrated these differences with transcriptional changes to examine the functional relevance of the identified loops. Notably, we observed an enhanced insulation boundary and a greater number of loops in the TCF7L2 gene region within tumors, which was accompanied by an upregulation of TCF7L2 expression. Subsequently, TCF7L2 expression was confirmed through qRT-PCR, and upregulated TCF7L2 prompted cell proliferation and growth hormone (GH) secretion in vitro. CONCLUSION: Our results provide comprehensive 3D chromatin architecture maps of somatotroph tumors and offer a valuable resource for furthering the understanding of the underlying biology and mechanisms of gene expression regulation.
Subject(s)
Chromatin , Gene Expression Regulation, Neoplastic , Pituitary Neoplasms , Somatotrophs , Male , Female , Adult , Middle Aged , Aged , Somatotrophs/metabolism , Somatotrophs/pathology , Pituitary Neoplasms/genetics , Pituitary Neoplasms/pathology , Chromatin/metabolism , Sequence Analysis, RNA , Transcription Factor 7-Like 2 Protein/metabolism , Up-Regulation , Cell Proliferation , Growth Hormone/metabolism , Genetic TechniquesABSTRACT
Microbial products are essential for developing various therapeutic agents, including antibiotics, anticancer drugs, vaccines, and therapeutic enzymes. Genetic engineering techniques, functional genomics, and synthetic biology unlock previously uncharacterized natural products. This review highlights major advances in microbial biotechnology, focusing on gene-based technologies for medical applications.
Subject(s)
Biotechnology , Genetic Engineering , Biotechnology/methods , Genetic Techniques , Genomics , Synthetic BiologyABSTRACT
Brainbow is a genetic cell-labeling technique that allows random colorization of multiple cells and real-time visualization of cell fate within a tissue, providing valuable insights into understanding complex biological processes. However, fluorescent proteins (FPs) in Brainbow have distinct excitation spectra with peak difference greater than 35 nm, which requires sequential imaging under multiple excitations and thus leads to long acquisition times. In addition, they are not easily used together with other fluorophores due to severe spectral bleed-through. Here, we report the development of a single-wavelength excitable Brainbow, UFObow, incorporating three newly developed blue-excitable FPs. We have demonstrated that UFObow enables not only tracking the growth dynamics of tumor cells in vivo but also mapping spatial distribution of immune cells within a sub-cubic centimeter tissue, revealing cell heterogeneity. This provides a powerful means to explore complex biology in a simultaneous imaging manner at a single-cell resolution in organs or in vivo.
Subject(s)
Diagnostic Imaging , Genetic Techniques , Animals , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Coloring Agents , Mammals/geneticsABSTRACT
Modern medicine has produced large genetic datasets of high dimensions through advanced gene sequencing technology, and processing these data is of great significance for clinical decision-making. Gene selection (GS) is an important data preprocessing technique that aims to select a subset of feature information to improve performance and reduce data dimensionality. This study proposes an improved wrapper GS method based on forensic-based investigation (FBI). The method introduces the search mechanism of the slime mould algorithm in the FBI to improve the original FBI; the newly proposed algorithm is named SMA_FBI; then GS is performed by converting the continuous optimizer to a binary version of the optimizer through a transfer function. In order to verify the superiority of SMA_FBI, experiments are first executed on the 30-function test set of CEC2017 and compared with 10 original algorithms and 10 state-of-the-art algorithms. The experimental results show that SMA_FBI is better than other algorithms in terms of finding the optimal solution, convergence speed, and robustness. In addition, BSMA_FBI (binary version of SMA_FBI) is compared with 8 binary algorithms on 18 high-dimensional genetic data from the UCI repository. The results indicate that BSMA_FBI is able to obtain high classification accuracy with fewer features selected in GS applications. Therefore, SMA_FBI is considered an optimization tool with great potential for dealing with global optimization problems, and its binary version, BSMA_FBI, can be used for GS tasks.
Subject(s)
Algorithms , Physarum polycephalum , Clinical Decision-Making , Genetic Techniques , TechnologyABSTRACT
In fall 1972, Paul Berg's laboratory published articles in PNAS describing two methods for constructing recombinant DNAs in vitro. He received half of the 1980 Nobel Prize in Chemistry for this landmark accomplishment. Here, we describe how this discovery came about, revolutionizing both biological research and the pharmaceutical industry.
Subject(s)
DNA, Recombinant , Genetic Techniques , Nobel Prize , DNA, Recombinant/genetics , Drug IndustryABSTRACT
BACKGROUND: In recent years, researchers have made significant strides in understanding the heterogeneity of breast cancer and its various subtypes. However, the wealth of genomic and proteomic data available today necessitates efficient frameworks, instruments, and computational tools for meaningful analysis. Despite its success as a prognostic tool, the PAM50 gene signature's reliance on many genes presents challenges in terms of cost and complexity. Consequently, there is a need for more efficient methods to classify breast cancer subtypes using a reduced gene set accurately. RESULTS: This study explores the potential of achieving precise breast cancer subtype categorization using a reduced gene set derived from the PAM50 gene signature. By employing a "Few-Shot Genes Selection" method, we randomly select smaller subsets from PAM50 and evaluate their performance using metrics and a linear model, specifically the Support Vector Machine (SVM) classifier. In addition, we aim to assess whether a more compact gene set can maintain performance while simplifying the classification process. Our findings demonstrate that certain reduced gene subsets can perform comparable or superior to the full PAM50 gene signature. CONCLUSIONS: The identified gene subsets, with 36 genes, have the potential to contribute to the development of more cost-effective and streamlined diagnostic tools in breast cancer research and clinical settings.
Subject(s)
Breast Neoplasms , Humans , Female , Breast Neoplasms/genetics , Breast Neoplasms/diagnosis , Biomarkers, Tumor/genetics , Proteomics , Gene Expression Profiling/methods , Genetic TechniquesABSTRACT
Although fungi have been important model organisms for solving genetic, molecular, and ecological problems, recently, they are also becoming an important source of infectious disease. Despite their high medical burden, fungal pathogens are understudied, and relative to other pathogenic microbes, less is known about how their gene functions contribute to disease. This is due, in part, to a lack of powerful genetic tools to study these organisms. In turn, this has resulted in inappropriate treatments and diagnostics and poor disease management. There are a variety of reasons genetic studies were challenging in pathogenic fungi, but in recent years, most of them have been overcome or advances have been made to circumvent these barriers. In this minireview, we highlight how recent advances in genetic studies in fungal pathogens have resulted in the discovery of important biology and potential new antifungals and have created the tools to comprehensively study these important pathogens.
Subject(s)
Fungi , Mycoses , Fungi/genetics , Fungi/classification , Fungi/pathogenicity , Mycoses/microbiology , Genetic Techniques , Humans , Antifungal Agents/pharmacology , Antifungal Agents/therapeutic useABSTRACT
The microtubule motor dynein plays a key role in cellular organization. However, little is known about how dynein's biosynthesis, assembly, and functional diversity are orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a genome-wide gRNA library, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints revealed co-functional proteins involved in many cellular processes, including several candidate novel regulators of core dynein functions. Further analysis of one of these factors, the RNA-binding protein SUGP1, indicates that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our data represent a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organization captured by our high-content imaging.
Subject(s)
Dyneins , Microtubules , Humans , Dyneins/genetics , Microtubules/genetics , Peroxisomes/genetics , CRISPR-Cas Systems , Genetic TechniquesABSTRACT
This study investigates changes in the social valuation of the human genome over the more than 30 years since the establishment of the Human Genome Project. It offers a descriptive sociological analysis of the three waves of this valuation, mainly by considering three key UNESCO declarations and a relevant report. These waves represent a shifting balance between collectivism and individualism, starting with a broadly constructed valuation of the human genome as common human heritage and moving toward a valuation of dynamic applications within various social and medical contexts (e.g., personalized genomic medicine and genome editing). We seek to broaden the analytical perspective by examining how the declarations' ethical foci are framed within the context of rapidly evolving genetic technologies and their social applications. We conclude by discussing continuity and change in value balancing vis-à-vis changing genomic technologies.
Subject(s)
Genome, Human , Humans , Human Genome Project/ethics , Genomics/ethics , Genomics/methods , Genetic Techniques/ethics , Genetic Techniques/economics , Gene Editing/ethicsABSTRACT
Telomerase, the enzyme that maintains telomeres at natural chromosome ends, should be repressed at double-strand breaks (DSBs), where neotelomere formation can cause terminal truncations. We developed an assay to detect neotelomere formation at Cas9- or I-SceI-induced DSBs in human cells. Telomerase added telomeric repeats to DSBs, leading to interstitial telomeric repeat insertions or the formation of functional neotelomeres accompanied by terminal deletions. The threat that telomerase poses to genome integrity was minimized by ataxia telangiectasia and Rad3-related (ATR) kinase signaling, which inhibited telomerase at resected DSBs. In addition to acting at resected DSBs, telomerase used the extruded strand in the Cas9 enzyme-product complex as a primer for neotelomere formation. We propose that although neotelomere formation is detrimental in normal human cells, it may allow cancer cells to escape from breakage-fusion-bridge cycles.
Subject(s)
Ataxia Telangiectasia Mutated Proteins , DNA Breaks, Double-Stranded , Telomerase , Telomere , Humans , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Telomerase/genetics , Telomere/genetics , Telomere/metabolism , Genetic Techniques , CRISPR-Associated Protein 9 , HeLa CellsABSTRACT
Although the functional role of circular RNA (circRNA) interaction with microRNAs and proteins has been studied extensively, circRNA interactions with the protein-coding mRNAs in intact cells remain largely unknown. Here, by employing AMT-mediated proximity ligation of RNA-RNA duplexes followed by circRNA enrichment and deep sequencing, we report a novel Cross-Linking Poly(A) Pulldown RNase R Sequencing (CLiPPR-seq) technology which identified hundreds of mRNA-interacting circRNAs in three different cell types, including ßTC6, C2C12 and HeLa cells. Furthermore, CLiPP-seq without RNase R treatment was also performed to identify the mRNA expression in these cells. BLAST analysis of circRNAs in CLiPPR-seq sample with the mRNAs in CLiPP-seq samples determined their potential complementary sequences for circRNA-mRNA interaction. Pulldown of circRNAs and poly(A) RNAs confirmed the direct interaction of circRNAs with target mRNAs. Silencing of mRNA-interacting circRNAs led to the altered expression of target mRNAs in ßTC6 cells, suggesting the role of direct interaction of circRNAs with mRNAs in gene expression regulation. CLiPPR-seq thus represents a novel method for illuminating the myriad of uncharacterized circRNA-mRNA hybrids that may regulate gene expression.
Subject(s)
Genetic Techniques , MicroRNAs , RNA, Circular , Sequence Analysis, RNA , Humans , Gene Expression Profiling/methods , Gene Expression Regulation , HeLa Cells , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular/genetics , RNA, Circular/isolation & purification , RNA, Circular/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, RNA/methodsABSTRACT
A key attribute of some long noncoding RNAs (lncRNAs) is their ability to regulate expression of neighbouring genes in cis. However, such 'cis-lncRNAs' are presently defined using ad hoc criteria that, we show, are prone to false-positive predictions. The resulting lack of cis-lncRNA catalogues hinders our understanding of their extent, characteristics and mechanisms. Here, we introduce TransCistor, a framework for defining and identifying cis-lncRNAs based on enrichment of targets amongst proximal genes. TransCistor's simple and conservative statistical models are compatible with functionally defined target gene maps generated by existing and future technologies. Using transcriptome-wide perturbation experiments for 268 human and 134 mouse lncRNAs, we provide the first large-scale survey of cis-lncRNAs. Known cis-lncRNAs are correctly identified, including XIST, LINC00240 and UMLILO, and predictions are consistent across analysis methods, perturbation types and independent experiments. We detect cis-activity in a minority of lncRNAs, primarily involving activators over repressors. Cis-lncRNAs are detected by both RNA interference and antisense oligonucleotide perturbations. Mechanistically, cis-lncRNA transcripts are observed to physically associate with their target genes and are weakly enriched with enhancer elements. In summary, TransCistor establishes a quantitative foundation for cis-lncRNAs, opening a path to elucidating their molecular mechanisms and biological significance.
Subject(s)
Computational Biology , Genetic Techniques , RNA, Long Noncoding , Animals , Humans , Mice , RNA, Long Noncoding/genetics , RNA, Long Noncoding/isolation & purification , Transcription Factors/genetics , Transcriptome , Software/standards , Computational Biology/methodsABSTRACT
5-Methylcytosine (m5C), an abundant RNA modification, plays a crucial role in regulating RNA fate and gene expression. While recent progress has been made in understanding the biological roles of m5C, the inability to introduce m5C at specific sites within transcripts has hindered efforts to elucidate direct links between specific m5C and phenotypic outcomes. Here, we developed a CRISPR-Cas13d-based tool, named reengineered m5C modification system (termed 'RCMS'), for targeted m5C methylation and demethylation in specific transcripts. The RCMS editors consist of a nuclear-localized dCasRx conjugated to either a methyltransferase, NSUN2/NSUN6, or a demethylase, the catalytic domain of mouse Tet2 (ten-eleven translocation 2), enabling the manipulation of methylation events at precise m5C sites. We demonstrate that the RCMS editors can direct site-specific m5C incorporation and demethylation. Furthermore, we confirm their effectiveness in modulating m5C levels within transfer RNAs and their ability to induce changes in transcript abundance and cell proliferation through m5C-mediated mechanisms. These findings collectively establish RCMS editors as a focused epitranscriptome engineering tool, facilitating the identification of individual m5C alterations and their consequential effects.
Subject(s)
5-Methylcytosine , Genetic Techniques , Methylation , Methyltransferases , RNA Editing , Animals , Mice , 5-Methylcytosine/metabolism , Methyltransferases/genetics , Methyltransferases/metabolism , RNA, Transfer/metabolism , CRISPR-Cas Systems , HumansABSTRACT
Nonsense-mediated mRNA decay (NMD) is a network of pathways that degrades transcripts that undergo premature translation termination. In mammals, NMD can be divided into the exon junction complex (EJC)-enhanced and EJC-independent branches. Fluorescence- and luminescence-based reporters have long been effective tools to investigate NMD, yet existing reporters largely focus on the EJC-enhanced pathway. Here, we present a system of reporters for comparative studies of EJC-independent and EJC-enhanced NMD. This system also enables the study of NMD-associated outcomes such as premature termination codon (PTC) readthrough and truncated protein degradation. These reporters are compatible with fluorescence or luminescence-based readouts via transient transfection or stable integration. Using this reporter system, we show that EJC-enhanced NMD RNA levels are reduced by 2- or 9-fold and protein levels are reduced by 7- or 12-fold compared to EJC-independent NMD, depending on the reporter gene used. Additionally, the extent of readthrough induced by G418 and an NMD inhibitor (SMG1i), alone and in combination, varies across NMD substrates. When combined, G418 and SMG1i increase readthrough product levels in an additive manner for EJC-independent reporters, while EJC-enhanced reporters show a synergistic effect. We present these reporters as a valuable toolkit to deepen our understanding of NMD and its associated mechanisms.
Subject(s)
Exons , Genes, Reporter , Genetic Techniques , Nonsense Mediated mRNA Decay , Exons/genetics , Nonsense Mediated mRNA Decay/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Humans , HEK293 Cells , Genes, Reporter/geneticsABSTRACT
Natural killer (NK) cells are innate cytokine-producing and cytolytic effector lymphocytes. Their function is responsive to environmental factors, e.g., hypoxia, a frequent feature of inflamed tissues. Such responses require that the NK cells up-regulate HIF-1α (hypoxia inducible factor-1α), the major mediator of cellular responses to hypoxia that affects cell survival as well as immune responses. Thus, a major approach to the study of NK cell effector function under hypoxic conditions involves the ability to regulate HIF-1α levels in primary human NK cells. One difficulty with this approach, however, is that NK cells are difficult-to-transfect cells and common transfection methods, including electroporation or lipofection, suffer from variable transfection efficiency and cell viability. Moreover, the detection of HIF-1α is technically challenging because of the rapid degradation of the protein under normoxic conditions. Here, using the commercially available ExPERT ATx by MaxCyte, we report a workflow for the reliable delivery of small interfering RNA (siRNA) for targeting HIF-1α expression in primary human NK cells. We further provide a protocol for the detection of HIF-1α by immunoblot analysis demonstrating its efficient downregulation by siRNA. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of natural killer cells from human peripheral blood mononuclear cells Basic Protocol 2: Delivery of non-coding small interfering RNA and HIF-1α targeting siRNA into natural killer cells using ExPERT ATx Basic Protocol 3: Assessing the downregulation of HIF-1α protein using immunoblot analysis Support Protocol 1: Exemplary assessment of transfection efficiency using fluorescently labeled non-targeting siRNA Support Protocol 2: Exemplary assessment of NK cell viability 20 hr post-transfection Support Protocol 3: Exemplary assessment of HIF-1α knockdown using immunoblot analysis.
Subject(s)
Genetic Techniques , Killer Cells, Natural , RNA, Small Interfering , Humans , Cells, Cultured , Down-Regulation , Drug Delivery Systems , Hypoxia/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Killer Cells, Natural/metabolism , Leukocytes, Mononuclear/metabolism , RNA, Small Interfering/pharmacologyABSTRACT
Custom oligonucleotides (oligos) are widely used reagents in biomedical research. Some common applications of oligos include polymerase chain reaction (PCR), sequencing, hybridization, microarray, and library construction. The reliability of oligos in such applications depends on their purity and specificity. Here, we report that commercially available oligos are frequently contaminated with nonspecific sequences (i.e. other unrelated oligonucleotides). Most of the oligos that we designed to amplify clustered regularly interspersed palindromic repeats (CRISPR) guide sequences contained nonspecific CRISPR guides. These contaminants were detected in research-grade oligos procured from eight commercial oligo-suppliers located in three different geographic regions of the world. Deep sequencing of some of the oligos revealed a variety of contaminants. Given the wide range of applications of oligos, the impact of oligo cross-contamination varies greatly depending on the field and the experimental method. Incorporating appropriate control experiments in research design can help ensure that the quality of oligo reagents meets the intended purpose. This can also minimize risk depending on the purposes for which the oligos are used.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Drug Contamination , Indicators and Reagents , Oligonucleotides , Base Sequence , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Oligonucleotides/chemistry , Oligonucleotides/standards , Genetic Techniques , Indicators and Reagents/analysis , Indicators and Reagents/standards , Industry/standardsABSTRACT
Targeted spatial transcriptomics hold particular promise in analyzing complex tissues. Most such methods, however, measure only a limited panel of transcripts, which need to be selected in advance to inform on the cell types or processes being studied. A limitation of existing gene selection methods is their reliance on scRNA-seq data, ignoring platform effects between technologies. Here we describe gpsFISH, a computational method performing gene selection through optimizing detection of known cell types. By modeling and adjusting for platform effects, gpsFISH outperforms other methods. Furthermore, gpsFISH can incorporate cell type hierarchies and custom gene preferences to accommodate diverse design requirements.
Subject(s)
Gene Expression Profiling , Genetic Techniques , Single-Cell Analysis , Transcriptome , Sequence Analysis, RNAABSTRACT
Protein-specific Chromatin Conformation Capture (3C)-based technologies have become essential for identifying distal genomic interactions with critical roles in gene regulation. The standard techniques include Chromatin Interaction Analysis by Paired-End Tag (ChIA-PET), in situ Hi-C followed by chromatin immunoprecipitation (HiChIP) also known as PLAC-seq. To identify chromatin interactions from these data, a variety of computational methods have emerged. Although these state-of-art methods address many issues with loop calling, only few methods can fit different data types simultaneously, and the accuracy as well as the efficiency these approaches remains limited. Here we have generated a pipeline, MMCT-Loop, which ensures the accurate identification of strong loops as well as dynamic or weak loops through a mixed model. MMCT-Loop outperforms existing methods in accuracy, and the detected loops show higher activation functionality. To highlight the utility of MMCT-Loop, we applied it to conformational data derived from neural stem cell (NSCs) and uncovered several previously unidentified regulatory regions for key master regulators of stem cell identity. MMCT-Loop is an accurate and efficient loop caller for targeted conformation capture data, which supports raw data or pre-processed valid pairs as input, the output interactions are formatted and easily uploaded to a genome browser for visualization.