scholarly journals Genetic and transgenic reagents for Drosophila simulans, D. mauritiana, D. yakuba, D. santomea and D. virilis

2016 ◽  
Author(s):  
David L. Stern ◽  
Justin Crocker ◽  
Yun Ding ◽  
Nicolas Frankel ◽  
Gretchen Kappes ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Integration Site ◽  
Yellow Fluorescent Protein ◽  
Model Systems ◽  
Enhanced Yellow Fluorescent Protein ◽  
Functional Studies ◽  
Site Specific Integration ◽  
Genetic Strains ◽  
Fluorescent Molecules ◽  
Integration Efficiency

AbstractSpecies of the Drosophila melanogaster species subgroup, including the species D. simulans, D. mauritiana, D. yakuba, and D. santomea, have long served as model systems for studying evolution. Studies in these species have been limited, however, by a paucity of genetic and transgenic reagents. Here we describe a collection of transgenic and genetic strains generated to facilitate genetic studies within and between these species. We have generated many strains of each species containing mapped piggyBac transposons including an enhanced yellow fluorescent protein gene expressed in the eyes and a phiC31 attP site-specific integration site. We have tested a subset of these lines for integration efficiency and reporter gene expression levels. We have also generated a smaller collection of other lines expressing other genetically encoded fluorescent molecules in the eyes and a number of other transgenic reagents that will be useful for functional studies in these species. In addition, we have mapped the insertion locations of 58 transposable elements in D. virilis that will be useful for genetic mapping studies.

scholarly journals Pancreatic islet function in a transgenic mouse expressing fluorescent protein

2005 ◽  
Vol 186 (2) ◽  
pp. 333-341 ◽  
Author(s):  
Daniel Nyqvist ◽  
Göran Mattsson ◽  
Martin Köhler ◽  
Varda Lev-Ram ◽  
Arne Andersson ◽  
...  
Keyword(s):  
Pancreatic Islet ◽  
Glucose Homeostasis ◽  
Fluorescent Protein ◽  
Regional Blood Flow ◽  
Animal Health ◽  
Yellow Fluorescent Protein ◽  
Islet Function ◽  
Enhanced Yellow Fluorescent Protein ◽  
Isolated Pancreatic Islets ◽  
Arterial Blood

Pancreatic islet function and glucose homeostasis have been characterized in the transgenic YC-3.0 mouse, which expresses the yellow chameleon 3.0 (YC-3.0) protein under the control of the β-actin and the cytomegalovirus promoters. Fluorescence from the enhanced yellow fluorescent protein (EYFP), one part of the yellow chameleon protein, was used as a reporter of transgene expression. EYFP was expressed in different quantities throughout most cell types, including islet endocrine and stromal cells. No adverse effects of the transgene on animal health, growth or fertility were observed. Likewise, in vivo glucose homeostasis, mean arterial blood pressure and regional blood flow values were normal. Furthermore, the transgenic YC-3.0 mouse had a normal β-cell volume and mass as well as glucose-stimulated insulin release in vitro, compared with the C57BL/6 control mouse. Isolated islets from YC-3.0 animals continuously expressed the transgene and reversed hyperglycemia when transplanted under the renal capsule of alloxan-diabetic nude mice. We conclude that isolated pancreatic islets from YC-3.0 animals implanted into recipients without any EYFP expression, constitute a novel and versatile model for studies of islet engraftment.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

scholarly journals Acidocalcisomes and Polyphosphate Granules Are Different Subcellular Structures in Agrobacterium tumefaciens

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Celina Frank ◽  
Dieter Jendrossek
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Yellow Fluorescent Protein ◽  
Eukaryotic Cells ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Polyphosphate Granules

ABSTRACT Acidocalcisomes are membrane-enclosed, polyphosphate-containing acidic organelles in lower Eukaryota but have also been described for Agrobacterium tumefaciens (M. Seufferheld, M. Vieira, A. Ruiz, C. O. Rodrigues, S. Moreno, and R. Docampo, J Biol Chem 278:29971–29978, 2003, https://doi.org/10.1074/jbc.M304548200). This study aimed at the characterization of polyphosphate-containing acidocalcisomes in this alphaproteobacterium. Unexpectedly, fluorescence microscopic investigation of A. tumefaciens cells using fluorescent dyes and localization of constructed fusions of polyphosphate kinases (PPKs) and of vacuolar H+-translocating pyrophosphatase (HppA) with enhanced yellow fluorescent protein (eYFP) suggested that acidocalcisomes and polyphosphate are different subcellular structures. Acidocalcisomes and polyphosphate granules were frequently located close together, near the cell poles. However, they never shared the same position. Mutant strains of A. tumefaciens with deletions of both ppk genes (Δppk1 Δppk2) were unable to form polyphosphate but still showed cell pole-located eYFP-HppA foci and could be stained with MitoTracker. In conclusion, A. tumefaciens forms polyP granules that are free of a surrounding membrane and thus resemble polyP granules of Ralstonia eutropha and other bacteria. The composition, contents, and function of the subcellular structures that are stainable with MitoTracker and harbor eYFP-HppA remain unclear. IMPORTANCE The uptake of alphaproteobacterium-like cells by ancestors of eukaryotic cells and subsequent conversion of these alphaproteobacterium-like cells to mitochondria are thought to be key steps in the evolution of the first eukaryotic cells. The identification of acidocalcisomes in two alphaproteobacterial species some years ago and the presence of homologs of the vacuolar proton-translocating pyrophosphatase HppA, a marker protein of the acidocalcisome membrane in eukaryotes, in virtually all species within the alphaproteobacteria suggest that eukaryotic acidocalcisomes might also originate from related structures in ancestors of alphaproteobacterial species. Accordingly, alphaproteobacterial acidocalcisomes and eukaryotic acidocalcisomes should have similar features. Since hardly any information is available on bacterial acidocalcisomes, this study aimed at the characterization of organelle-like structures in alphaproteobacterial cells, with A. tumefaciens as an example.

scholarly journals Monitoring Biofilm Formation and Microbial Interactions that May Occur During a Salmonella Contamination Incident across the Network of a Water Bottling Plant

2019 ◽  
Vol 7 (8) ◽  
pp. 236
Author(s):  
Karampoula ◽  
Doulgeraki ◽  
Fotiadis ◽  
Tampakaki ◽  
Nychas
Keyword(s):  
Bacterial Communities ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Yellow Fluorescent Protein ◽  
Foodborne Pathogen ◽  
Microbial Interactions ◽  
Enhanced Yellow Fluorescent Protein ◽  
Serovar Typhimurium ◽  
Gradient Gel Electrophoresis

The present study aims to monitor the ability of Salmonella to colonize and compete as a member of the mixed species biofilm within key points at a water bottling plant, in case of a contamination incident with this major foodborne pathogen. To achieve this goal, bacterial communities throughout the production line were collected and their identities were investigated by microbial counts and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). These bacterial communities alone or along with constructed Salmonella enterica serovar Typhimurium (ST) fluorescence-based bioreporters were left to form a biofilm on stainless steel for 6 days at 20 °C. ST bioreporters were constructed by introducing plasmids expressing EYFP (enhanced yellow fluorescent protein) fusions of the genes csgB, csrA, sspH2, and fliD into ST 14028S. The bead vortexing-plate counting method was applied for the enumeration of the biofilm population, while the behavior of the bioreporters was evaluated by fluorescence microscopy. From a set of 16 samples that were collected from the plant, species of Citrobacter, Staphylococcus, Pseudomonas, Bacillus, and Exiguobacterium were identified. The presence of these indigenous bacteria neither inhibited nor enhanced the biofilm formation of ST in mixed bacterial communities (p > 0.05). Furthermore, the csrA-based bioreporter was shown to be induced in multispecies biofilms with Citrobacter. In conclusion, this study enhanced our knowledge of bacterial interactions occurring within a biofilm in a water bottling plant.

scholarly journals Enhancement of correct protein folding in vivo by a non-lytic baculovirus

2004 ◽  
Vol 382 (2) ◽  
pp. 695-702 ◽  
Author(s):  
Yu HO ◽  
Huei-Ru LO ◽  
Tzu-Ching LEE ◽  
Carol P. Y. WU ◽  
Yu-Chan CHAO
Keyword(s):  
Energy Transfer ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Random Mutagenesis ◽  
Resonance Energy ◽  
Vector System ◽  
Enhanced Yellow Fluorescent Protein

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP–LUC–ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

scholarly journals Use of Degradation Tags To Control Protein Levels in the Cyanobacterium Synechocystis sp. Strain PCC 6803

2013 ◽  
Vol 79 (8) ◽  
pp. 2833-2835 ◽  
Author(s):  
Brian P. Landry ◽  
Jana Stöckel ◽  
Himadri B. Pakrasi
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Terminal Protein ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Protein Levels ◽  
Steady State Fluorescence ◽  
Tunable Range ◽  
Control Protein ◽  
Synechocystis Sp

ABSTRACTWe generated a collection ofssrA-based C-terminal protein degradation tags with different degradation strengths. The steady-state fluorescence levels of different enhanced yellow fluorescent protein (eYFP) tag variants in aSynechocystissp. indicated a tunable range from 1% to 50% of untagged eYFP.

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