The Experts below are selected from a list of 261 Experts worldwide ranked by ideXlab platform
Eric Lecuyer - One of the best experts on this subject based on the ideXlab platform.
-
cefra seq systematic mapping of rna subCellular distribution properties through Cell Fractionation coupled to deep sequencing
Methods, 2017Co-Authors: Fabio Alexis Lefebvre, Neal A L Cody, Louis Philip Benoit Bouvrette, Julie Bergalet, Xiaofeng Wang, Eric LecuyerAbstract:The subCellular trafficking of RNA molecules is a conserved feature of eukaryotic Cells and plays key functions in diverse processes implicating polarised Cellular activities. Large-scale imaging and subCellular transcriptomic studies suggest that regulated RNA localization is a highly prevalent process that appears to be disrupted in several neuromuscular disorders. These features underline the importance and usefulness of implementing procedures to assess global transcriptome subCellular distribution properties. Here, we present a method combining biochemical Fractionation of Cells and high-throughput RNA sequencing (CeFra-seq) that enables rapid and efficient systematic mapping of RNA cytotopic distributions in Cells. The described procedure involves biochemical Fractionation to derive extracts of nuclear, cytosolic, endomembrane, cytoplasmic insoluble and extraCellular material from Cell culture lines. The RNA content of each fraction can then be profiled by deep-sequencing, revealing global subCellular signatures. We provide a detailed protocol for the CeFra-seq procedure along with relevant validation steps and data analysis guidelines to graphically represent RNA spatial distribution features. As a complement to imaging approaches, CeFra-seq represents a powerful and scalable tool to investigate global alterations in RNA trafficking.
Steven S. Broyles - One of the best experts on this subject based on the ideXlab platform.
-
Vaccinia virus transcription.
Journal of General Virology, 2003Co-Authors: Steven S. BroylesAbstract:Vaccinia virus replication takes place in the cytoplasm of the host Cell. The nearly 200 kbp genome owes part of its complexity to encoding most of the proteins involved in genome and mRNA synthesis. The multisubunit vaccinia virus RNA polymerase requires a separate set of virus-encoded proteins for the transcription of the early, intermediate and late classes of genes. Cell Fractionation studies have provided evidence for a role for host Cell proteins in the initiation and termination of vaccinia virus intermediate and late gene transcription. Vaccinia virus resembles nuclear DNA viruses in the integration of viral and host proteins for viral mRNA synthesis, yet is markedly less reliant on host proteins than its nuclear counterparts.
Gunter Blobel - One of the best experts on this subject based on the ideXlab platform.
-
Disruption of the nucleoporin gene NUP133 results in clustering of nuclear pore complexes.
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Lucy F. Pemberton, Michael P. Rout, Gunter BlobelAbstract:We have characterized a protein with an estimated molecular mass of 130 kDa that is contained in a highly enriched yeast nuclear pore complex (NPC) fraction. Partial amino acid sequence from this protein has led us to a previously identified open reading frame on chromosome XI of Saccharomyces cerevisiae encoding a protein of 133 kDa. Due to its coenrichment with NPCs during Cell Fractionation and the phenotype observed in the disrupted strain, we propose to term the gene encoding this protein NUP133. Cells carrying a disrupted copy of NUP133 were temperature sensitive for growth. In addition, abnormal clustering of NPCs was observed. This phenotype is similar to that previously observed in the disruption of another nucleoporin gene, NUP145. We speculate that the gene product of NUP133, Nup133p, may functionally overlap with the NUP145 gene product, Nup145p, and that these proteins may be involved in maintaining the position of the NPC within the nuclear envelope.
-
Colocalization of vertebrate lamin B and lamin B receptor (LBR) in nuclear envelopes and in LBR-induced membrane stacks of the yeast Saccharomyces cerevisiae
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Susan Smith, Gunter BlobelAbstract:Abstract We have expressed human lamin B and the chicken lamin B receptor (LBR), either separately or together, in yeast and have monitored the subCellular location of the expressed proteins by immunofluorescence microscopy, immunoelectron microscopy, and Cell Fractionation. At the light microscopic level, the heterologous lamin B localized to the yeast nuclear rim and at electron microscopic resolution was found subjacent to the yeast inner nuclear membrane. These data indicate that vertebrate lamin B was correctly targeted in yeast. Expression of the heterologous LBR, either alone or together with the heterologous lamin B, resulted in the formation of membrane stacks primarily adjacent to the nuclear envelope, but also projecting from the nuclear envelope into the cytoplasm or under the plasma membrane. Double immunoelectron microscopy showed colocalization of the heterologous lamin B and LBR in the yeast nuclear envelope and in the LBR-induced membrane stacks. Cell Fractionation showed the presence of the heterologous lamin B and LBR in a subnuclear fraction enriched in nuclear envelopes. The heterologous lamin B was extracted at 8 M urea, but not at 4 M urea, thus behaving as a peripheral membrane protein and indistinguishable from assembled lamins. The heterologous LBR was not extracted by 8 M urea, indicating that it was integrated into the membrane. The observed colocalization and coFractionation are consistent with previously reported in vitro binding data and suggest that heterologous lamin B and LBR interact with each other when coexpressed in yeast.
Andrew Camilli - One of the best experts on this subject based on the ideXlab platform.
-
pneumolysin localizes to the Cell wall of streptococcus pneumoniae
Journal of Bacteriology, 2009Co-Authors: Katherine E Price, Andrew CamilliAbstract:Streptococcus pneumoniae is the causative agent of multiple diseases, including otitis media, pneumonia, bacteremia, and meningitis. Pneumolysin (Ply), a member of the cholesterol-dependent cytolytic pore-forming toxins, is produced by virtually all clinical isolates of S. pneumoniae, and strains in which the Ply gene has been deleted are severely attenuated in mouse models of infection. In contrast to all other members of the cholesterol-dependent cytolysin family, Ply lacks a signal peptide for export. Instead, Ply has been hypothesized to be released upon autolysis or, alternatively, via a nonautolytic mechanism that remains ill defined. We determined by use of Cell Fractionation and Western blotting that, during in vitro growth, exported Ply is localized primarily to the Cell wall compartment in 18 different serotypes in the absence of detectable Cell lysis. Hemolytic assays revealed that this Cell wall-localized Ply is active. Additionally, Cell wall-localized Ply is accessible to extraCellular protease and is detergent releasable.
J. David Castle - One of the best experts on this subject based on the ideXlab platform.
-
Overview of Cell Fractionation.
Current Protocols in Protein Science, 2004Co-Authors: J. David CastleAbstract:This discussion unit describes the most common methods for Cell Fractionation which provides the essential ingredients for the increasing number of Cell-free assays now being used in test-tube reconstructions of complex Cellular events involving intercompartmental interactions. Gel filtration separates on the basis of size, centrifugation separates on the basis of size and density, and electrophoresis separates on the basis of surface charge density. Centrifugation is the most widely used procedure in Cell Fractionation and is the only approach commonly used to separate crude tissue homogenates (often having quite large volumes) into subfractions as starting material for more refined purification procedures. Therefore, this overview focuses primarily on Fractionation of organelles by centrifugation.
-
Current Protocols in Protein Science - Overview of Cell Fractionation
Current protocols in cell biology, 1998Co-Authors: J. David CastleAbstract:Cell Fractionation is a useful preparative and analytical method in Cell biology. It is essential for analysis of composition and function of Cellular compartments and it is used to prepare materials for in vitro reconstitution studies This overview discusses the basic principles of centrifugation, the instruments available, choice of media, evaluation of Fractionation, and procedure optimization.