[17]In vitro assays for characterization of RNA-protein complexes involved in pro-opiomelanocortin mRNA translation

1996 ◽  
pp. 321-332
Author(s):  
Stephanie D. Flagg ◽  
Corinne M. Spencer ◽  
James H. Eberwine
Keyword(s):  
Protein Complexes ◽  
Mrna Translation ◽  
In Vitro Assays ◽  
Rna Protein Complexes

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals Alu RNA-protein complexes formed in vitro react with a novel lupus autoantibody.

1985 ◽  
Vol 260 (21) ◽  
pp. 11781-11786
Author(s):  
R Kole ◽  
L D Fresco ◽  
J D Keene ◽  
P L Cohen ◽  
R A Eisenberg ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Alu Rna ◽  
Rna Protein Complexes

Abstract 3228: Development and characterization of in vitro assays to detect and quantitate tubulysin B hydrazide in biological samples

2017 ◽  
Author(s):  
Nikki L. Parker ◽  
Jonathan M. Shillingford ◽  
Melissa Nelson ◽  
Joseph A. Reddy ◽  
Christopher P. Leamon
Keyword(s):  
Biological Samples ◽  
In Vitro Assays

scholarly journals Method combining BAC film and positive staining for the characterization of DNA intermediates by dark-field electron microscopy

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Yann Benureau ◽  
Eliana Moreira Tavares ◽  
Ali-Akbar Muhammad ◽  
Sonia Baconnais ◽  
Eric Le Cam ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dark Field ◽  
In Vitro Assays ◽  
Positive Staining ◽  
Synthetic Dna ◽  
Dark Field Imaging ◽  
Alkyl Ammonium

Abstract DNA intermediate structures are formed in all major pathways of DNA metabolism. Transmission electron microscopy (TEM) is a tool of choice to study their choreography and has led to major advances in the understanding of these mechanisms, particularly those of homologous recombination (HR) and replication. In this article, we describe specific TEM procedures dedicated to the structural characterization of DNA intermediates formed during these processes. These particular DNA species contain single-stranded DNA regions and/or branched structures, which require controlling both the DNA molecules spreading and their staining for subsequent visualization using dark-field imaging mode. Combining BAC (benzyl dimethyl alkyl ammonium chloride) film hyperphase with positive staining and dark-field TEM allows characterizing synthetic DNA substrates, joint molecules formed during not only in vitro assays mimicking HR, but also in vivo DNA intermediates.

Determinants of platelet number and regulation of thrombopoiesis

Hematology ◽  
2009 ◽  
Vol 2009 (1) ◽  
pp. 147-152 ◽  
Author(s):  
Kenneth Kaushansky
Keyword(s):  
Stem Cell ◽  
Progenitor Cell ◽  
In Vitro Assays ◽  
Cellular Mechanisms ◽  
Hematopoietic Stem ◽  
Platelet Production ◽  
Progenitor Cell Proliferation ◽  
Basic Biology

Abstract Our understanding of thrombopoiesis has improved greatly in the last two decades with the availability of in vitro assays of megakaryocyte progenitor cell growth, with the cloning and characterization of stem cell factor (SCF) and thrombopoietin (Tpo), the latter the primary humoral regulator of this process, and with the generation of genetically altered murine models of thrombopoietic failure and excess. While SCF affects developmentally early aspects of megakaryocyte growth, Tpo affects nearly all aspects of platelet production, from hematopoietic stem cell (HSC) self-renewal and expansion, through stimulation of megakaryocyte progenitor cell proliferation, to supporting their maturation into platelet-producing cells. The molecular and cellular mechanisms through which the marrow microenvironment and humoral mediators affect platelet production provide new insights into the interplay between intrinsic and extrinsic influences on hematopoiesis, and highlight new opportunities to translate basic biology into clinical advances.

scholarly journals YPR139c/LOA1encodes a novel lysophosphatidic acid acyltransferase associated with lipid droplets and involved in TAG homeostasis

2012 ◽  
Vol 23 (2) ◽  
pp. 233-246 ◽  
Author(s):  
Sophie Ayciriex ◽  
Marina Le Guédard ◽  
Nadine Camougrand ◽  
Gisèle Velours ◽  
Mario Schoene ◽  
...  
Keyword(s):  
Lysophosphatidic Acid ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
In Vitro Assays ◽  
Metabolic Labeling ◽  
Lysophosphatidic Acid Acyltransferase ◽  
Escherichia Coli Cells ◽  
Type Strains

For many years, lipid droplets (LDs) were considered to be an inert store of lipids. However, recent data showed that LDs are dynamic organelles playing an important role in storage and mobilization of neutral lipids. In this paper, we report the characterization of LOA1 (alias VPS66, alias YPR139c), a yeast member of the glycerolipid acyltransferase family. LOA1 mutants show abnormalities in LD morphology. As previously reported, cells lacking LOA1 contain more LDs. Conversely, we showed that overexpression results in fewer LDs. We then compared the lipidome of loa1Δ mutant and wild-type strains. Steady-state metabolic labeling of loa1Δ revealed a significant reduction in triacylglycerol content, while phospholipid (PL) composition remained unchanged. Interestingly, lipidomic analysis indicates that both PLs and glycerolipids are qualitatively affected by the mutation, suggesting that Loa1p is a lysophosphatidic acid acyltransferase (LPA AT) with a preference for oleoyl-CoA. This hypothesis was tested by in vitro assays using both membranes of Escherichia coli cells expressing LOA1 and purified proteins as enzyme sources. Our results from purification of subcellular compartments and proteomic studies show that Loa1p is associated with LD and active in this compartment. Loa1p is therefore a novel LPA AT and plays a role in LD formation.

scholarly journals RBP-L, a transcription factor related to RBP-Jkappa.

1997 ◽  
Vol 17 (5) ◽  
pp. 2679-2687 ◽  
Author(s):  
S Minoguchi ◽  
Y Taniguchi ◽  
H Kato ◽  
T Okazaki ◽  
L J Strobl ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Notch Receptor ◽  
In Vitro Assays ◽  
Functional Interaction ◽  
Protein Protein Interaction ◽  
Isolation And Characterization ◽  
Signalling Molecule

RBP-Jkappa is a sequence-specific DNA binding protein which plays a central role in signalling downstream of the Notch receptor by physically interacting with its intracellular region. Although at least four Notch genes exist in mammals, it is unknown whether each Notch requires a specific downstream signalling molecule. Here we report isolation and characterization of a mouse RBP-Jkappa-related gene named RBP-L that is expressed almost exclusively in lung, in contrast to the ubiquitous expression of RBP-Jkappa. For simplicity, we propose to call RBP-Jkappa RBP-J. The RBP-L protein bound to a DNA sequence almost identical to that of RBP-J. Surprisingly, RBP-L did not interact with any of the known four mouse Notch proteins. Although we found that RBP-L and EBNA-2 cooperated in transcriptional activation, they did not show significantly strong protein-protein interaction that can be detected by several in vivo and in vitro assays. This is again in contrast to physical association of RBP-J with EBNA-2. Several models to explain functional interaction between RBP-L and EBNA-2 are discussed.

scholarly journals Watching cellular machinery in action, one molecule at a time

2016 ◽  
Vol 216 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Enrico Monachino ◽  
Lisanne M. Spenkelink ◽  
Antoine M. van Oijen
Keyword(s):  
Dynamic Behavior ◽  
Single Molecule ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Ensemble Averaging ◽  
Cellular Processes ◽  
Cellular Machinery

Single-molecule manipulation and imaging techniques have become important elements of the biologist’s toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

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