Intercellular trafficking of aKNOTTED1green fluorescent protein fusion in the leaf and shoot meristem ofArabidopsis

ABSTRACT Modified single-stranded DNA oligonucleotides can direct nucleotide exchange in Saccharomyces cerevisiae. Point and frameshift mutations are corrected in a reaction catalyzed by cellular enzymes involved in various DNA repair processes. The present model centers on the annealing of the vector to one strand of the helix, followed by the correction of the designated base. The choice of which strand to target is a reaction parameter that can be controlled, so here we investigate the properties of strand bias in targeted gene repair. An in vivo system has been established in which a plasmid containing an actively transcribed, but mutated, hygromycin-enhanced green fluorescent protein fusion gene is targeted for repair and upon conversion will confer hygromycin resistance on the cell. Overall transcriptional activity has a positive influence on the reaction, elevating the frequency. If the targeting vector is synthesized so that it directs nucleotide repair on the nontranscribed strand, the level of gene repair is higher than if the template strand is targeted. We provide data showing that the targeting vector can be displaced from the template strand by an active T7 phage RNA polymerase. The strand bias is not influenced by which strand serves as the leading or lagging strand during DNA synthesis. These results may provide an explanation for the enhancement of gene repair observed when the nontemplate strand is targeted.

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Microtubule-dependent distribution of mRNA in adult cardiocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01275.2007 ◽

2008 ◽

Vol 294 (3) ◽

pp. H1135-H1144 ◽

Cited By ~ 17

Author(s):

Dimitri Scholz ◽

Catalin F. Baicu ◽

William J. Tuxworth ◽

Lin Xu ◽

Harinath Kasiganesan ◽

...

Keyword(s):

Protein Synthesis ◽

Fluorescent Protein ◽

Average Distance ◽

Yellow Fluorescent Protein ◽

Pressure Overload ◽

Structural Protein ◽

Protein Fusion ◽

Microtubule Depolymerization ◽

Microtubule Network ◽

Hypertrophic Growth

Synthesis of myofibrillar proteins in the diffusion-restricted adult cardiocyte requires microtubule-based active transport of mRNAs as part of messenger ribonucleoprotein particles (mRNPs) to translation sites adjacent to nascent myofibrils. This is especially important for compensatory hypertrophy in response to hemodynamic overloading. The hypothesis tested here is that excessive microtubule decoration by microtubule-associated protein 4 (MAP4) after cardiac pressure overloading could disrupt mRNP transport and thus hypertrophic growth. MAP4-overexpressing and pressure-overload hypertrophied adult feline cardiocytes were infected with an adenovirus encoding zipcode-binding protein 1-enhanced yellow fluorescent protein fusion protein, which is incorporated into mRNPs, to allow imaging of these particles. Speed and distance of particle movement were measured via time-lapse microscopy. Microtubule depolymerization was used to study microtubule-based transport and distribution of mRNPs. Protein synthesis was assessed as radioautographic incorporation of [3H]phenylalanine. After microtubule depolymerization, mRNPs persist only perinuclearly and apparent mRNP production and protein synthesis decrease. Reestablishing microtubules restores mRNP production and transport as well as protein synthesis. MAP4 overdecoration of microtubules via adenovirus infection in vitro or following pressure overloading in vivo reduces the speed and average distance of mRNP movement. Thus cardiocyte microtubules are required for mRNP transport and structural protein synthesis, and MAP4 decoration of microtubules, whether directly imposed or accompanying pressure-overload hypertrophy, causes disruption of mRNP transport and protein synthesis. The dense, highly MAP4-decorated microtubule network seen in severe pressure-overload hypertrophy both may cause contractile dysfunction and, perhaps even more importantly, may prevent a fully compensatory growth response to hemodynamic overloading.

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Adaptation of the Endogenous Salmonella enterica Serovar Typhi clyA-Encoded Hemolysin for Antigen Export Enhances the Immunogenicity of Anthrax Protective Antigen Domain 4 Expressed by the Attenuated Live-Vector Vaccine Strain CVD 908-htrA

Infection and Immunity ◽

10.1128/iai.72.12.7096-7106.2004 ◽

2004 ◽

Vol 72 (12) ◽

pp. 7096-7106 ◽

Cited By ~ 45

Author(s):

James E. Galen ◽

Licheng Zhao ◽

Magaly Chinchilla ◽

Jin Yuan Wang ◽

Marcela F. Pasetti ◽

...

Keyword(s):

Salmonella Enterica ◽

Fluorescent Protein ◽

Protective Antigen ◽

Expression System ◽

Dna Encoding ◽

Protein Fusion ◽

Vector Vaccine ◽

Interleukin 5 ◽

Export System ◽

Domain 4

ABSTRACT Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs. We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins. Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of Salmonella enterica serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated Salmonella serovar Typhi live-vector vaccine strains. We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in Salmonella serovar Typhi CVD 908-htrA, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis. The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of Bacillus anthracis protective antigen (PA). A total of 11 of 15 mice immunized intranasally with Salmonella serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (P = 0.0002). In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes. This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines.

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Specific expression of an oxytocin-enhanced cyan fluorescent protein fusion transgene in the rat hypothalamus and posterior pituitary

Journal of Endocrinology ◽

10.1677/joe-09-0289 ◽

2009 ◽

Vol 204 (3) ◽

pp. 275-285 ◽

Cited By ~ 20

Author(s):

Akiko Katoh ◽

Hiroaki Fujihara ◽

Toyoaki Ohbuchi ◽

Tatsushi Onaka ◽

W Scott Young ◽

...

Keyword(s):

Fluorescent Protein ◽

Fusion Gene ◽

Plasma Osmolality ◽

Cyan Fluorescent Protein ◽

Posterior Pituitary ◽

Protein Fusion ◽

Transgenic Rats ◽

Salt Loading ◽

Wide Range ◽

Enhanced Cyan Fluorescent Protein

We have generated rats bearing an oxytocin (OXT)-enhanced cyan fluorescent protein (eCFP) fusion transgene designed from a murine construct previously shown to be faithfully expressed in transgenic mice. In situ hybridisation histochemistry revealed that the Oxt–eCfp fusion gene was expressed in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) in these rats. The fluorescence emanating from eCFP was observed only in the SON, the PVN, the internal layer of the median eminence and the posterior pituitary (PP). In in vitro preparations, freshly dissociated cells from the SON and axon terminals showed clear eCFP fluorescence. Immunohistochemistry for OXT and arginine vasopressin (AVP) revealed that the eCFP fluorescence co-localises with OXT immunofluorescence, but not with AVP immunofluorescence in the SON and the PVN. Although the expression levels of the Oxt–eCfp fusion gene in the SON and the PVN showed a wide range of variations in transgenic rats, eCFP fluorescence was markedly increased in the SON and the PVN, but decreased in the PP after chronic salt loading. The expression of the Oxt gene was significantly increased in the SON and the PVN after chronic salt loading in both non-transgenic and transgenic rats. Compared with wild-type animals, euhydrated and salt-loaded male and female transgenic rats showed no significant differences in plasma osmolality, sodium concentration and OXT and AVP levels, suggesting that the fusion gene expression did not disturb any physiological processes. These results suggest that our new transgenic rats are a valuable new tool to identify OXT-producing neurones and their terminals.

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Renilla luciferase and green fluorescent protein fusion genes (A Szalay et al, US)

Biofutur ◽

10.1016/s0294-3506(98)80200-7 ◽

1998 ◽

Vol 1998 (181) ◽

pp. 50

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Renilla Luciferase ◽

Fusion Genes ◽

Green Fluorescent Protein Fusion ◽

Protein Fusion ◽

Green Fluorescent

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Both Binding and Activation of p38 Mitogen-Activated Protein Kinase (MAPK) Play Essential Roles in Regulation of the Nucleocytoplasmic Distribution of MAPK-Activated Protein Kinase 5 by Cellular Stress

Molecular and Cellular Biology ◽

10.1128/mcb.22.20.6931-6945.2002 ◽

2002 ◽

Vol 22 (20) ◽

pp. 6931-6945 ◽

Cited By ~ 61

Author(s):

Ole Morten Seternes ◽

Bjarne Johansen ◽

Beate Hegge ◽

Mona Johannessen ◽

Stephen M. Keyse ◽

...

Keyword(s):

Protein Kinase ◽

P38 Mapk ◽

Nuclear Export ◽

Fluorescent Protein ◽

Mapk Pathway ◽

Nuclear Export Signal ◽

Mitogen Activated Protein Kinase ◽

Protein Fusion ◽

Translational Machinery ◽

Mitogen Activated Protein

ABSTRACT The p38 mitogen-activated protein kinase (MAPK) pathway is an important mediator of cellular responses to environmental stress. Targets of p38 include transcription factors, components of the translational machinery, and downstream serine/threonine kinases, including MAPK-activated protein kinase 5 (MK5). Here we have used enhanced green fluorescent protein fusion proteins to analyze the subcellular localization of MK5. Although this protein is predominantly nuclear in unstimulated cells, MK5 shuttles between the nucleus and the cytoplasm. Furthermore, we have shown that the C-terminal domain of MK5 contains both a functional nuclear localization signal (NLS) and a leucine-rich nuclear export signal (NES), indicating that the subcellular distribution of this kinase reflects the relative activities of these two signals. In support of this, we have shown that stress-induced activation of the p38 MAPK stimulates the chromosomal region maintenance 1 protein-dependent nuclear export of MK5. This is regulated by both binding of p38 MAPK to MK5, which masks the functional NLS, and stress-induced phosphorylation of MK5 by p38 MAPK, which either activates or unmasks the NES. These properties may define the ability of MK5 to differentially phosphorylate both nuclear and cytoplasmic targets or alternatively reflect a mechanism whereby signals initiated by activation of MK5 in the nucleus may be transmitted to the cytoplasm.

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Compartmentalized Cell Envelope Biosynthesis in Mycobacterium tuberculosis

10.1101/2022.01.07.475471 ◽

2022 ◽

Author(s):

Julia Puffal ◽

Ian L. Sparks ◽

James R. Brenner ◽

Xuni Li ◽

John D. Leszyk ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Proteomic Analysis ◽

Fluorescent Protein ◽

Cell Envelope ◽

Membrane Domain ◽

Protein Fusion ◽

Final Maturation ◽

Associated Proteins ◽

Gradient Fractionation ◽

Membrane Compartmentalization

The intracellular membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in Mycobacterium smegmatis. The IMD correlates both temporally and spatially with the polar cell envelope elongation in M. smegmatis. Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here we show that the IMD is a conserved membrane structure found in Mycobacterium tuberculosis. We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in M. tuberculosis. Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as peptidoglycan, arabinogalactan, and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the polar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, α1,3-fucosyltransferase and fucosyl 4-O-methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both M. tuberculosis and M. smegmatis, and M. tuberculosis utilizes this membrane location for the synthesis of its surface-exposed lipid virulence factors.

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