scholarly journals Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

1998 ◽  
Vol 9 (5) ◽  
pp. 1007-1023 ◽  
Author(s):  
Peter Lorenz ◽  
Brenda F. Baker ◽  
C. Frank Bennett ◽  
David L. Spector
Keyword(s):  
Antisense Oligonucleotides ◽  
De Novo ◽  
Regulation Of Gene Expression ◽  
Nuclear Body ◽  
Nuclear Bodies ◽  
Phosphorothioate Oligodeoxynucleotides ◽  
Nuclear Structures ◽  
In Cells

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.

Glycan Labeling and Analysis in Cells and In Vivo

2021 ◽  
Vol 14 (1) ◽  
pp. 363-387
Author(s):  
Bo Cheng ◽  
Qi Tang ◽  
Che Zhang ◽  
Xing Chen
Keyword(s):  
Chemical Modification ◽  
Biological Function ◽  
Biological Processes ◽  
Affinity Tags ◽  
Functional Roles ◽  
Antibody Conjugates ◽  
Living Organisms ◽  
In Cells

As one of the major types of biomacromolecules in the cell, glycans play essential functional roles in various biological processes. Compared with proteins and nucleic acids, the analysis of glycans in situ has been more challenging. Herein we review recent advances in the development of methods and strategies for labeling, imaging, and profiling of glycans in cells and in vivo. Cellular glycans can be labeled by affinity-based probes, including lectin and antibody conjugates, direct chemical modification, metabolic glycan labeling, and chemoenzymatic labeling. These methods have been applied to label glycans with fluorophores, which enables the visualization and tracking of glycans in cells, tissues, and living organisms. Alternatively, labeling glycans with affinity tags has enabled the enrichment of glycoproteins for glycoproteomic profiling. Built on the glycan labeling methods, strategies enabling cell-selective and tissue-specific glycan labeling and protein-specific glycan imaging have been developed. With these methods and strategies, researchers are now better poised than ever to dissect the biological function of glycans in physiological or pathological contexts.

scholarly journals Improving 3-methylphenol (m-cresol) production in yeast via in vivo glycosylation or methylation

2020 ◽  
Vol 20 (8) ◽  
Author(s):  
Julia Hitschler ◽  
Eckhard Boles
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Wild Type ◽  
Biotechnological Production ◽  
Yeast Saccharomyces Cerevisiae ◽  
In Situ Extraction ◽  
Methylsalicylic Acid Synthase ◽  
In The Wild

ABSTRACT Heterologous expression of 6-methylsalicylic acid synthase (MSAS) together with 6-MSA decarboxylase enables de novo production of the platform chemical and antiseptic additive 3-methylphenol (3-MP) in the yeast Saccharomyces cerevisiae. However, toxicity of 3-MP prevents higher production levels. In this study, we evaluated in vivo detoxification strategies to overcome limitations of 3-MP production. An orcinol-O-methyltransferase from Chinese rose hybrids (OOMT2) was expressed in the 3-MP producing yeast strain to convert 3-MP to 3-methylanisole (3-MA). Together with in situ extraction by dodecane of the highly volatile 3-MA this resulted in up to 211 mg/L 3-MA (1.7 mM) accumulation. Expression of a UDP-glycosyltransferase (UGT72B27) from Vitis vinifera led to the synthesis of up to 533 mg/L 3-MP as glucoside (4.9 mM). Conversion of 3-MP to 3-MA and 3-MP glucoside was not complete. Finally, deletion of phosphoglucose isomerase PGI1 together with methylation or glycosylation and feeding a fructose/glucose mixture to redirect carbon fluxes resulted in strongly increased product titers, with up to 897 mg/L 3-MA/3-MP (9 mM) and 873 mg/L 3-MP/3-MP as glucoside (8.1 mM) compared to less than 313 mg/L (2.9 mM) product titers in the wild type controls. The results show that methylation or glycosylation are promising tools to overcome limitations in further enhancing the biotechnological production of 3-MP.

scholarly journals Clastosome: A Subtype of Nuclear Body Enriched in 19S and 20S Proteasomes, Ubiquitin, and Protein Substrates of Proteasome

2002 ◽  
Vol 13 (8) ◽  
pp. 2771-2782 ◽  
Author(s):  
Miguel Lafarga ◽  
Maria Teresa Berciano ◽  
Emma Pena ◽  
Isabel Mayo ◽  
Jose G. Castaño ◽  
...  
Keyword(s):  
Proteasome Inhibitors ◽  
Cell Types ◽  
Nuclear Body ◽  
Nuclear Bodies ◽  
26S Proteasome ◽  
High Concentration ◽  
Protein Substrates ◽  
Adenovirus E1a ◽  
Ubiquitin Proteasome ◽  
Nuclear Structures

Nuclear bodies represent a heterogeneous class of nuclear structures. Herein, we describe that a subset of nuclear bodies is highly enriched in components of the ubiquitin–proteasome pathway of proteolysis. We coined the term clastosome (from the Greekklastos, broken and soma, body) to refer to this type of nuclear body. Clastosomes contain a high concentration of 1) ubiquitin conjugates, 2) the proteolytically active 20S core and the 19S regulatory complexes of the 26S proteasome, and 3) protein substrates of the proteasome. Although detected in a variety of cell types, clastosomes are scarce under normal conditions; however, they become more abundant when proteasomal activity is stimulated. In contrast, clastosomes disappear when cells are treated with proteasome inhibitors. Protein substrates of the proteasome that are found concentrated in clastosomes include the short-lived transcription factors c-Fos and c-Jun, adenovirus E1A proteins, and the PML protein. We propose that clastosomes are sites where proteolysis of a variety of protein substrates is taking place.

scholarly journals Correction: A ratiometric fluorescent probe for peroxynitrite prepared by de novo synthesis and its application in assessing the mitochondrial oxidative stress status in cells and in vivo

2018 ◽  
Vol 54 (93) ◽  
pp. 13159-13159 ◽  
Author(s):  
Dong-Ye Zhou ◽  
Yongfei Li ◽  
Wen-Li Jiang ◽  
Yang Tian ◽  
Junjie Fei ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Fluorescent Probe ◽  
De Novo ◽  
De Novo Synthesis ◽  
Mitochondrial Oxidative Stress ◽  
Oxidative Stress Status ◽  
Ratiometric Fluorescent Probe ◽  
In Cells

Correction for ‘A ratiometric fluorescent probe for peroxynitrite prepared by de novo synthesis and its application in assessing the mitochondrial oxidative stress status in cells and in vivo’ by Dong-Ye Zhou et al., Chem. Commun., 2018, 54, 11590–11593.

scholarly journals Guiding biomolecular interactions in cells using de novo protein-protein interfaces

2018 ◽  
Author(s):  
Abigail J Smith ◽  
Franziska Thomas ◽  
Deborah Shoemark ◽  
Derek N Woolfson ◽  
Nigel J Savery
Keyword(s):  
Rational Design ◽  
De Novo ◽  
Gene Activation ◽  
Low Complexity ◽  
Coiled Coils ◽  
Biomolecular Interactions ◽  
Biomolecular Systems ◽  
Binding Domains ◽  
In Cells

An improved ability to direct and control biomolecular interactions in living cells would impact on synthetic biology. A key issue is the need to introduce interacting components that act orthogonally to endogenous proteomes and interactomes. Here we show that low-complexity, de novo designed protein-protein-interaction (PPI) domains can substitute for natural PPIs and guide engineered protein-DNA interactions in Escherichia coli. Specifically, we use de novo homo- and hetero-dimeric coiled coils to reconstitute a cytoplasmic split adenylate cyclase; to recruit RNA polymerase to a promoter and activate gene expression; and to oligomerize both natural and designed DNA-binding domains to repress transcription. Moreover, the stabilities of the heterodimeric coiled coils can be modulated by rational design and, thus, adjust the levels of gene activation and repression in vivo. These experiments demonstrate the possibilities for using designed proteins and interactions to control biomolecular systems such as enzyme cascades and circuits in cells.

5-lipoxygenase expression in dendritic cells generated from CD34+ hematopoietic progenitors and in lymphoid organs

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3857-3865
Author(s):  
Rainer Spanbroek ◽  
Markus Hildner ◽  
Dieter Steinhilber ◽  
Norbert Fusenig ◽  
Kozo Yoneda ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Lymphocyte ◽  
Transforming Growth Factor ◽  
Leukotriene B4 ◽  
Lymphoid Tissues ◽  
Ionophore A23187 ◽  
In Cells

The 5-lipoxygenase (5-LO) pathway in human CD34+ hematopoietic progenitor cells, which were induced to differentiate into dendritic cells (DCs) by cytokines in vitro and in DCs of lymphoid tissues in situ, was examined. Extracts prepared from HPCs contained low levels of 5-LO or 5-LO–activating protein. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plus tumor necrosis factor–α (TNF-α) promoted DC differentiation and induced a strong rise in 5-LO and FLAP expression. Fluorescence-activated cell sorter (FACS) analyses identified a major DC population coexpressing human leukocyte antigen (HLA)-DR/CD80 and monocytic or Langerhans cell markers. Transforming growth factor–β1 (TGF-β–1), added to support DC maturation, strongly promoted the appearance of CD1a+/Lag+ Langerhans-type cells as well as mature CD83+ DCs. TGF-β–1 further increased 5-LO and FLAP expression, recruited additional cells into the 5-LO+DC population, and promoted production of 5-hydroxyeicosatetraenoic acid and leukotriene B4 in response to calcium (Ca++) ionophore A23187. These in vitro findings were corroborated by 5-LO expression in distinct DC phenotypes in vivo. Scattered 5-LO and FLAP in situ hybridization signals were recorded in cells of paracortical T-lymphocyte–rich areas and germinal centers (GCs) of lymph nodes (LNs) and tonsil and in cells of mucosae overlying the Waldeyer tonsillar ring. 5-LO protein localized to both CD1a+ immature DCs and to CD83+ mature interdigitating DCs of T-lymphocyte–rich areas of LNs and tonsil. As DCs have the unique ability to initiate naive lymphocyte activation, our data support the hypothesis that leukotrienes act at proximal steps of adaptive immune responses.

scholarly journals A convolutional neural network for the prediction and forward design of ribozyme-based gene-control elements

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Calvin M Schmidt ◽  
Christina D Smolke
Keyword(s):  
Machine Learning ◽  
Rational Design ◽  
De Novo ◽  
Regulation Of Gene Expression ◽  
Regulatory Activity ◽  
Rna Sequences ◽  
Genetic Switch ◽  
Gene Regulatory ◽  
Functional Rnas

Ribozyme switches are a class of RNA-encoded genetic switch that support conditional regulation of gene expression across diverse organisms. An improved elucidation of the relationships between sequence, structure, and activity can improve our capacity for de novo rational design of ribozyme switches. Here, we generated data on the activity of hundreds of thousands of ribozyme sequences. Using automated structural analysis and machine learning, we leveraged these large datasets to develop predictive models that estimate the in vivo gene-regulatory activity of a ribozyme sequence. These models supported the de novo design of ribozyme libraries with low mean basal gene-regulatory activities and new ribozyme switches that exhibit changes in gene-regulatory activity in the presence of a target ligand, producing functional switches for four out of five aptamers. Our work examines how biases in the model and the dataset that affect prediction accuracy can arise and demonstrates that machine learning can be applied to RNA sequences to predict gene-regulatory activity, providing the basis for design tools for functional RNAs.

scholarly journals Phase separation and DAXX redistribution contribute to LANA nuclear body and KSHV genome dynamics during latency and reactivation

2021 ◽  
Vol 17 (1) ◽  
pp. e1009231
Author(s):  
Olga Vladimirova ◽  
Alessandra De Leo ◽  
Zhong Deng ◽  
Andreas Wiedmer ◽  
James Hayden ◽  
...  
Keyword(s):  
Phase Separation ◽  
Nuclear Body ◽  
Lytic Replication ◽  
Nuclear Bodies ◽  
Nuclear Antigen ◽  
Morphological Transformation ◽  
Chromosome Conformation ◽  
Lytic Reactivation ◽  
Morphological Transitions ◽  
Nuclear Structures

Liquid-liquid phase separation (LLPS) can drive formation of diverse and essential macromolecular structures, including those specified by viruses. Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) genomes associate with the viral encoded Latency-Associated Nuclear Antigen (LANA) to form stable nuclear bodies (NBs) during latent infection. Here, we show that LANA-NB formation and KSHV genome conformation involves LLPS. Using LLPS disrupting solvents, we show that LANA-NBs are partially disrupted, while DAXX and PML foci are highly resistant. LLPS disruption altered the LANA-dependent KSHV chromosome conformation but did not stimulate lytic reactivation. We found that LANA-NBs undergo major morphological transformation during KSHV lytic reactivation to form LANA-associated replication compartments encompassing KSHV DNA. DAXX colocalizes with the LANA-NBs during latency but is evicted from the LANA-associated lytic replication compartments. These findings indicate the LANA-NBs are dynamic super-molecular nuclear structures that partly depend on LLPS and undergo morphological transitions corresponding the different modes of viral replication.

scholarly journals Exploring Mammalian Genome within Phase-Separated Nuclear Bodies: Implications for the Regulation of Gene Expression

2019 ◽  
Author(s):  
Annick Lesne ◽  
Marie-Odile Baudement ◽  
Cosette Rebouissou ◽  
Thierry Forné
Keyword(s):  
Gene Expression ◽  
Self Assembly ◽  
Genome Organization ◽  
Regulation Of Gene Expression ◽  
Physical Nature ◽  
Mammalian Genome ◽  
Nuclear Bodies ◽  
Control Of Gene Expression ◽  
Nuclear Structures ◽  
Genomic Regions

The importance of genome organization at the supranucleosomal scale in the control of gene expression is increasingly recognized today. In mammals, Topologically Associating Domains (TADs) and the active / inactive chromosomal compartments are two of the main nuclear structures that contribute to this organization level. However, recent works reviewed here indicate that, at specific loci, chromatin interactions with nuclear bodies could also be crucial to regulate genome functions, in particular transcription. They moreover suggest that these nuclear bodies are membrane-less organelles dynamically self-assembled and disassembled through mechanisms of phase separation. We have recently developed a novel genome-wide experimental method, High-salt Recovered Sequences sequencing (HRS-seq), designed to identify chromatin regions associated with large ribonucleoprotein (RNP) complexes and nuclear bodies. We argue that the physical nature of such RNP complexes and nuclear bodies appears to be central in their ability to promote efficient interactions between distant genomic regions. The development of novel experimental approaches, including our HRS-seq method, is opening new avenues to understand how self-assembly of phase separated nuclear bodies possibly contributes to mammalian genome organization and gene expression.

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