Splicing signals and factors in plant intron removal

2002 ◽  
Vol 30 (2) ◽  
pp. 146-149 ◽  
Author(s):  
J. W. S. Brown ◽  
C. G. Simpson ◽  
G. Thow ◽  
G. P. Clark ◽  
S. N. Jennings ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Viral Vectors ◽  
Binding Proteins ◽  
Fluorescent Protein ◽  
Splicing Factors ◽  
Small Nuclear Rna ◽  
Intron Removal ◽  
Arabidopsis Proteins ◽  
Green Fluorescent

Constitutive splicing of the potato invertase miniexon 2 (9 nt long) requires a branchpoint sequence positioned around 50 nt upstream of the 5′ splice site of the adjacent intron and a U11 element found just downstream of the branchpoint in the upstream intron [Simpson, Hedley, Watters, Clark, McQuade, Machray and Brown (2000) RNA 6, 422–433]. The sensitivity of this in vivo plant splicing system has been used to demonstrate exon scanning in plants, and to characterize plant intronic elements, such as branchpoint and poly-pyrimidine tract sequences. Plant introns differ from their vertebrate and yeast couterparts in being UA- or U-rich (up to 85% UA). One of the key differences in splicing between plants and other eukaryotes lies in early intron recognition, which is thought to be mediated by UA-binding proteins. We are adopting three approaches to studying the RNA-protein interactions in plant splicing. First, overexpression of plant splicing factors and, in particular, UA-binding proteins, in conjunction with a range of mini-exon mutants. Secondly, the sequences of around 65% of vertebrate and yeast splicing factors have high-quality matches to Arabidopsis proteins, opening the door to identification and analysis of gene knockouts. Finally, to discover plant-specific proteins involved in splicing and in, for example, rRNA or small nuclear RNA processing, green fluorescent protein-cDNA fusion libraries in viral vectors are being screened.

scholarly journals In Vivo Quantitative Estimation of DNA-Dependent Interaction of Sox2 and Oct4 Using BirA-Catalyzed Site-Specific Biotinylation

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 142 ◽  
Author(s):  
Arman Kulyyassov ◽  
Vasily Ogryzko
Keyword(s):  
Transcription Factors ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Quantitative Estimation ◽  
Multiple Reaction Monitoring ◽  
Negative Control ◽  
Protein Protein Interactions ◽  
Close Proximity ◽  
Green Fluorescent

Protein–protein interactions of core pluripotency transcription factors play an important role during cell reprogramming. Cell identity is controlled by a trio of transcription factors: Sox2, Oct4, and Nanog. Thus, methods that help to quantify protein–protein interactions may be useful for understanding the mechanisms of pluripotency at the molecular level. Here, a detailed protocol for the detection and quantitative analysis of in vivo protein–protein proximity of Sox2 and Oct4 using the proximity-utilizing biotinylation (PUB) method is described. The method is based on the coexpression of two proteins of interest fused to a biotin acceptor peptide (BAP)in one case and a biotin ligase enzyme (BirA) in the other. The proximity between the two proteins leads to more efficient biotinylation of the BAP, which can be either detected by Western blotting or quantified using proteomics approaches, such as a multiple reaction monitoring (MRM) analysis. Coexpression of the fusion proteins BAP-X and BirA-Y revealed strong biotinylation of the target proteins when X and Y were, alternatively, the pluripotency transcription factors Sox2 and Oct4, compared with the negative control where X or Y was green fluorescent protein (GFP), which strongly suggests that Sox2 and Oct4 come in close proximity to each other and interact.

scholarly journals Docking and Assembly of the Type II Secretion Complex of Vibrio cholerae

2009 ◽  
Vol 191 (9) ◽  
pp. 3149-3161 ◽  
Author(s):  
Suzanne R. Lybarger ◽  
Tanya L. Johnson ◽  
Miranda D. Gray ◽  
Aleksandra E. Sikora ◽  
Maria Sandkvist
Keyword(s):  
Vibrio Cholerae ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Outer Membrane Proteins ◽  
Subcellular Location ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Protein Fusion ◽  
Green Fluorescent

ABSTRACT Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC- and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.

scholarly journals Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions

2010 ◽  
Vol 189 (4) ◽  
pp. 739-754 ◽  
Author(s):  
Nina C. Hubner ◽  
Alexander W. Bird ◽  
Jürgen Cox ◽  
Bianca Splettstoesser ◽  
Peter Bandilla ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Artificial Chromosome ◽  
Small Scale ◽  
Mitotic Spindles ◽  
Cellular Processes ◽  
Green Fluorescent ◽  
Highly Sensitive Detection ◽  
Interaction Proteomics

Protein interactions are involved in all cellular processes. Their efficient and reliable characterization is therefore essential for understanding biological mechanisms. In this study, we show that combining bacterial artificial chromosome (BAC) TransgeneOmics with quantitative interaction proteomics, which we call quantitative BAC–green fluorescent protein interactomics (QUBIC), allows specific and highly sensitive detection of interactions using rapid, generic, and quantitative procedures with minimal material. We applied this approach to identify known and novel components of well-studied complexes such as the anaphase-promoting complex. Furthermore, we demonstrate second generation interaction proteomics by incorporating directed mutational transgene modification and drug perturbation into QUBIC. These methods identified domain/isoform-specific interactors of pericentrin- and phosphorylation-specific interactors of TACC3, which are necessary for its recruitment to mitotic spindles. The scalability, simplicity, cost effectiveness, and sensitivity of this method provide a basis for its general use in small-scale experiments and in mapping the human protein interactome.

scholarly journals An internal ribosome entry site located upstream of the crucifer-infecting tobamovirus coat protein (CP) gene can be used for CP synthesis in vivo

2006 ◽  
Vol 87 (9) ◽  
pp. 2693-2697 ◽  
Author(s):  
Yu. L. Dorokhov ◽  
P. A. Ivanov ◽  
T. V. Komarova ◽  
M. V. Skulachev ◽  
J. G. Atabekov
Keyword(s):  
Coat Protein ◽  
Internal Ribosome Entry Site ◽  
Viral Vectors ◽  
Fluorescent Protein ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Cp Gene ◽  
Green Fluorescent

It was previously shown that, unlike the type member of the genus Tobamovirus (TMV U1), a crucifer-infecting tobamovirus (crTMV) contains a 148 nt internal ribosome entry site (IRES)CP,148 CR upstream of the coat protein (CP) gene. Here, viral vectors with substitutions in the stem–loop (SL) region of CP subgenomic promoters (TMV U1-CP–GFP/SL-mut and crTMV-CP–GFP/SL-mut) were constructed and the levels of CP synthesis in agroinoculation experiments were compared. No CP–GFP (green fluorescent protein) synthesis was detected in Nicotiana benthamiana leaves inoculated with TMV U1-CP–GFP/SL-mut, whereas a small amount of CP–GFP synthesis was obtained in crTMV-CP–GFP/SL-mut-injected leaves. Northern blots proved that both promoters were inactive. It could be hypothesized that IRES-mediated early production of the CP by crTMV is needed for realization of its crucifer-infecting capacity.

scholarly journals Evidence for Interaction of Schizophyllum commune Y Mating-Type Proteins in Vivo

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1461-1467
Author(s):  
C Ian Robertson ◽  
Alexander McMahon Kende ◽  
Kurt Toenjes ◽  
Charles P Novotny ◽  
Robert C Ullrich
Keyword(s):  
Mating Type ◽  
Protein Interactions ◽  
Sexual Development ◽  
Fluorescent Protein ◽  
Schizophyllum Commune ◽  
Nuclear Localization Sequence ◽  
Type Locus ◽  
Localization Sequence ◽  
Green Fluorescent

Abstract The Aα mating-type locus of Schizophyllum commune regulates sexual development and contains the code for two proteins, Y and Z, which are thought to form a complex and function as a transcription factor. Import of these proteins into the nucleus may be an essential step in Aα-regulated sexual development. The Y proteins contain a bipartite basic sequence, which is an excellent candidate for a nuclear localization sequence (NLS), while Z proteins contain no such sequence. Here we describe experiments in which deletions were made in the putative NLS sequence of Y4. We show that this putative NLS is essential to the function of the Y protein and capable of mislocalizing green fluorescent protein (GFP) to the nucleus in Saccharomyces cerevisiae. Further, we describe genetic experiments that demonstrate the first Y-Y protein interactions in vivo. These results are consistent with our previously postulated hypothesis that the Y-Z complex is likely to be of a higher order than dimer.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

scholarly journals Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Danielle Weber-Adrian ◽  
Rikke Hahn Kofoed ◽  
Joseph Silburt ◽  
Zeinab Noroozian ◽  
Kairavi Shah ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Intravenous Injection ◽  
Viral Vectors ◽  
Focused Ultrasound ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Peripheral Organs ◽  
Neuronal Expression ◽  
Green Fluorescent ◽  
The Brain

AbstractNon-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood–brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

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