scholarly journals nucGEMs probe the biophysical properties of the nucleoplasm

2021 ◽  
Author(s):  
Tamas Szoradi ◽  
Tong Shu ◽  
Gururaj R Kidiyoor ◽  
Ying Xie ◽  
Nora L Herzog ◽  
...  
Keyword(s):  
Physical Properties ◽  
Rna Polymerase Ii ◽  
Mitotic Chromosome ◽  
Single Gene ◽  
Continuous Production ◽  
Reaction Rates ◽  
Fluorescent Nanoparticles ◽  
Mammalian Cell Lines ◽  
Evolutionary Comparison ◽  
Mitotic Chromosome Condensation

The cell interior is highly crowded and far from thermodynamic equilibrium. This environment can dramatically impact molecular motion and assembly, and therefore influence sub-cellular organization and biochemical reaction rates. These effects depend strongly on length-scale, with the least information available at the important mesoscale (10-100 nanometers), which corresponds to the size of crucial regulatory molecules such as RNA polymerase II. It has been challenging to study the mesoscale physical properties of the nucleoplasm because previous methods were labor-intensive and perturbative. Here, we report nuclear Genetically Encoded Multimeric nanoparticles (nucGEMs). Introduction of a single gene leads to continuous production and assembly of protein-based bright fluorescent nanoparticles of 40 nm diameter. We implemented nucGEMs in budding and fission yeasts and in mammalian cell lines. We found that the nucleus is more crowded than the cytosol at the mesoscale, that mitotic chromosome condensation ejects nucGEMs from the nucleus, and that nucGEMs are excluded from heterochromatin and the nucleolus. nucGEMs enable hundreds of nuclear rheology experiments per hour, and allow evolutionary comparison of the physical properties of the cytosol and nucleoplasm.

Linking transcription, RNA polymerase II elongation and alternative splicing

2020 ◽  
Vol 477 (16) ◽  
pp. 3091-3104 ◽  
Author(s):  
Luciana E. Giono ◽  
Alberto R. Kornblihtt
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Rna Polymerase Ii ◽  
Environmental Changes ◽  
Transcription Elongation ◽  
Single Gene ◽  
Regulation Of Gene Expression ◽  
Regulation Of Transcription ◽  
Stepping Stones ◽  
Eukaryotic Transcription

Gene expression is an intricately regulated process that is at the basis of cell differentiation, the maintenance of cell identity and the cellular responses to environmental changes. Alternative splicing, the process by which multiple functionally distinct transcripts are generated from a single gene, is one of the main mechanisms that contribute to expand the coding capacity of genomes and help explain the level of complexity achieved by higher organisms. Eukaryotic transcription is subject to multiple layers of regulation both intrinsic — such as promoter structure — and dynamic, allowing the cell to respond to internal and external signals. Similarly, alternative splicing choices are affected by all of these aspects, mainly through the regulation of transcription elongation, making it a regulatory knob on a par with the regulation of gene expression levels. This review aims to recapitulate some of the history and stepping-stones that led to the paradigms held today about transcription and splicing regulation, with major focus on transcription elongation and its effect on alternative splicing.

scholarly journals Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew N. Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Computational Models ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell

AbstractThe carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

scholarly journals Integration of biological data by kernels on graph nodes allows prediction of new genes involved in mitotic chromosome condensation

2014 ◽  
Vol 25 (16) ◽  
pp. 2522-2536 ◽  
Author(s):  
Jean-Karim Hériché ◽  
Jon G. Lees ◽  
Ian Morilla ◽  
Thomas Walter ◽  
Boryana Petrova ◽  
...  
Keyword(s):  
Experimental Validation ◽  
Mitotic Chromosome ◽  
Chromosome Condensation ◽  
Biological Data ◽  
Cell Functions ◽  
Functional Relationships ◽  
New Genes ◽  
Public Data ◽  
Mitotic Chromosome Condensation ◽  
Graph Nodes

The advent of genome-wide RNA interference (RNAi)–based screens puts us in the position to identify genes for all functions human cells carry out. However, for many functions, assay complexity and cost make genome-scale knockdown experiments impossible. Methods to predict genes required for cell functions are therefore needed to focus RNAi screens from the whole genome on the most likely candidates. Although different bioinformatics tools for gene function prediction exist, they lack experimental validation and are therefore rarely used by experimentalists. To address this, we developed an effective computational gene selection strategy that represents public data about genes as graphs and then analyzes these graphs using kernels on graph nodes to predict functional relationships. To demonstrate its performance, we predicted human genes required for a poorly understood cellular function—mitotic chromosome condensation—and experimentally validated the top 100 candidates with a focused RNAi screen by automated microscopy. Quantitative analysis of the images demonstrated that the candidates were indeed strongly enriched in condensation genes, including the discovery of several new factors. By combining bioinformatics prediction with experimental validation, our study shows that kernels on graph nodes are powerful tools to integrate public biological data and predict genes involved in cellular functions of interest.

Fuel Composition Effects on Forced Ignition of Liquid Fuel Sprays

2018 ◽  
Author(s):  
Sheng Wei ◽  
Brandon Sforzo ◽  
Jerry Seitzman
Keyword(s):  
Physical Properties ◽  
Liquid Fuel ◽  
Chemical Properties ◽  
Reaction Rates ◽  
High Energy ◽  
Fuel Spray ◽  
Successful Outcome ◽  
Fuel Composition ◽  
Ignition Probability ◽  
Fuel Sprays

In gas turbine combustors, ignition is achieved by using sparks from igniters to start a flame. The process of sparks interacting with fuel/air mixture and creating self-sustained flames is termed forced ignition. Physical and chemical properties of a liquid fuel can influence forced ignition. The physical effects manifest through processes such as droplet atomization, spray distribution, and vaporization rate. The chemical effects impact reaction rates and heat release. This study focuses on the effect of fuel composition on forced ignition of fuel sprays in a well-controlled flow with a commercial style igniter. A facility previously used to examine prevaporized, premixed liquid fuel-air mixtures is modified and employed to study forced ignition of liquid fuel sprays. In our experiments, a wall-mounted, high energy, recessed cavity discharge igniter operating at 15 Hz with average spark energy of 1.25 J is used to ignite liquid fuel spray produced by a pressure atomizer located in a uniform air coflow. The successful outcome of each ignition events is characterized by the (continued) presence of chemiluminescence 2 ms after spark discharge, as detected by a high-speed camera. The ignition probability is defined as the fraction of successful sparks at a fixed condition, with the number of events evaluated for each fuel typically in the range 600–1200. Ten fuels were tested, including standard distillate jet fuels (e.g., JP-8 and Jet-A), as well as many distillate and alternative fuel blends, technical grade n-dodecane, and surrogates composed of a small number of components. During the experiments, the air temperature is controlled at 27 C and the fuel temperature is controlled at 21 C. Experiments are conducted at a global equivalence ratio of 0.55. Results show that ignition probabilities correlate strongly to liquid fuel viscosity (presumably through droplet atomization) and vapor pressure (or recovery temperature), as smaller droplets of a more volatile fuel would lead to increased vaporization rates. This allows the kernel to transition to a self-sustained flame before entrainment reduces its temperature to a point where chemical rates are too slow. Chemical properties of the fuel showed little influence, except when the fuels had similar physical properties. This result demonstrates that physical properties of liquid fuels have dominating effects on forced ignition of liquid fuel spray in coflow air.

scholarly journals Centromeric Localization of Dispersed Pol III Genes in Fission Yeast

2010 ◽  
Vol 21 (2) ◽  
pp. 254-265 ◽  
Author(s):  
Osamu Iwasaki ◽  
Atsunari Tanaka ◽  
Hideki Tanizawa ◽  
Shiv I.S. Grewal ◽  
Ken-ichi Noma
Keyword(s):  
Genome Organization ◽  
Gene Locus ◽  
Mitotic Chromosome ◽  
Three Dimensional ◽  
Rrna Genes ◽  
Mitotic Chromosomes ◽  
Gene Encoding ◽  
5S Rrna Genes ◽  
Pol Iii ◽  
Mitotic Chromosome Condensation

The eukaryotic genome is a complex three-dimensional entity residing in the nucleus. We present evidence that Pol III–transcribed genes such as tRNA and 5S rRNA genes can localize to centromeres and contribute to a global genome organization. Furthermore, we find that ectopic insertion of Pol III genes into a non-Pol III gene locus results in the centromeric localization of the locus. We show that the centromeric localization of Pol III genes is mediated by condensin, which interacts with the Pol III transcription machinery, and that transcription levels of the Pol III genes are negatively correlated with the centromeric localization of Pol III genes. This centromeric localization of Pol III genes initially observed in interphase becomes prominent during mitosis, when chromosomes are condensed. Remarkably, defective mitotic chromosome condensation by a condensin mutation, cut3-477, which reduces the centromeric localization of Pol III genes, is suppressed by a mutation in the sfc3 gene encoding the Pol III transcription factor TFIIIC subunit, sfc3-1. The sfc3-1 mutation promotes the centromeric localization of Pol III genes. Our study suggests there are functional links between the process of the centromeric localization of dispersed Pol III genes, their transcription, and the assembly of condensed mitotic chromosomes.

Paraboeremia yungensis sp. nov., a new fungal species isolated from Las Yungas, South America, with promising tyrosinase production potential

Phytotaxa ◽  
2021 ◽  
Vol 528 (3) ◽  
pp. 191-201
Author(s):  
MARIA PATRICIA PERALTA ◽  
JOAQUÍN ALIAGA ◽  
OSVALDO DANIEL DELGADO ◽  
JULIA INÉS FARIÑA ◽  
BERNARDO ERNESTO LECHNER
Keyword(s):  
South America ◽  
Rna Polymerase Ii ◽  
Single Gene ◽  
Large Subunit ◽  
Fungal Species ◽  
Malt Extract ◽  
South American ◽  
South American Species ◽  
Morphological Studies ◽  
Identification Approach

In the context of a bioprospection programme for tyrosinase/L-DOPA- and melanin-producing fungal strains for biotechnological purposes, a hyperproducer isolate was obtained from Las Yungas rainforest, a relevant biodiverse ecoregion in North-Western Argentina. The selected strain was preliminarily identified as Paraboeremia sp. This is, to the best of our knowledge, the first native reported species of this genus in South America. Single-gene and multi-locus analyses of the internal transcribed spacer nuclear ribosomal RNA gene region (ITS), partial large subunit 28S nrDNA region (LSU), RNA polymerase II region (RPB2) and partial β-tubulin gene (TUB2) alignments were carried out to define the phylogenetic identity of this strain. As part of a polyphasic identification approach, these results were combined with morphological studies of active cultures growing on malt extract, oatmeal and potato dextrose agar plates. Incubation was performed under diverse conditions to stimulate sporulation for the subsequent micromorphological analysis. Microphotographs of pycnidia and conidia were taken with a scanning electron microscope. Maximum likelihood and Bayesian Inference analyses supported the location of the strain within the genus Paraboeremia, whilst morphological features allowed distinguishing it from previously described species within this genus. Based on the results herein reported, the new South-American species Paraboeremia yungensis is described and proposed.

Mammalian cell lines expressing functional RNA polymerase II tagged with the green fluorescent protein

2000 ◽  
Vol 113 (15) ◽  
pp. 2679-2683 ◽  
Author(s):  
K. Sugaya ◽  
M. Vigneron ◽  
P.R. Cook
Keyword(s):  
Green Fluorescent Protein ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Temperature Sensitive ◽  
Mammalian Cell Lines ◽  
Eukaryotic Genes ◽  
Green Fluorescent

RNA polymerase II is a multi-subunit enzyme responsible for transcription of most eukaryotic genes. It associates with other complexes to form enormous multifunctional ‘holoenzymes’ involved in splicing and polyadenylation. We wished to study these different complexes in living cells, so we generated cell lines expressing the largest, catalytic, subunit of the polymerase tagged with the green fluorescent protein. The tagged enzyme complements a deficiency in tsTM4 cells that have a temperature-sensitive mutation in the largest subunit. Some of the tagged subunit is incorporated into engaged transcription complexes like the wild-type protein; it both resists extraction with sarkosyl and is hyperphosphorylated at its C terminus. Remarkably, subunits bearing such a tag can be incorporated into the active enzyme, despite the size and complexity of the polymerizing complex. Therefore, these cells should prove useful in the analysis of the dynamics of transcription in living cells.

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