scholarly journals Labeling of peroxisomes with green fluorescent protein in living P. pastoris cells.

1996 ◽  
Vol 44 (6) ◽  
pp. 581-589 ◽  
Author(s):  
E Z Monosov ◽  
T J Wenzel ◽  
G H Lüers ◽  
J A Heyman ◽  
S Subramani
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Uv Light ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Ultrastructural Localization ◽  
Yeast Cells ◽  
Fluorescent Properties ◽  
Green Fluorescent

We exploited the light-activated fluorescent properties of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria for studies on the peroxisomal sorting of polypeptides. GFP and GFP-SKL (containing a C-terminal, tripeptide peroxisomal targeting signal, SKL) were expressed from a methanol-inducible, alcohol oxidase (AOX1) promoter in the methylotrophic yeast Pichia pastoris. GFP was cytosolic, whereas the GFP-SKL fusion protein was targeted to peroxisomes, as demonstrated by biochemical fractionation of organelles on Nycodenz gradients. Neither GFP nor GFP-SKL affected the viability of yeast cells but both were fluorescent on excitation with 395-nm UV light. The subcellular locations of GFP and GFP-SKL in living yeast cells were monitored by fluorescence microscopy and their fluorescence was coupled to photo-oxidation of diaminobenzidine (DAB), resulting in the deposition of electron-dense oxidized DAB at intracellular locations of GFP derivatives. This photooxidation procedure permitted facile ultrastructural localization of GFP in cells by electron microscopy, and provided further evidence that GFP produced in P. pastoris is cytosolic, whereas GFP-SKL is peroxisomal. The GFP-SKL fusion protein is therefore a versatile reporter for the peroxisomal compartment, with many applications for studies involving peroxisomal import and biogenesis.

scholarly journals Direct Visualization of the Translocation of the γ-Subspecies of Protein Kinase C in Living Cells Using Fusion Proteins with Green Fluorescent Protein

1997 ◽  
Vol 139 (6) ◽  
pp. 1465-1476 ◽  
Author(s):  
Norio Sakai ◽  
Keiko Sasaki ◽  
Natsu Ikegaki ◽  
Yasuhito Shirai ◽  
Yoshitaka Ono ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Green Fluorescent Protein ◽  
Nmda Receptor ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Kinase C ◽  
Gfp Fusion Protein ◽  
Green Fluorescent

We expressed the γ-subspecies of protein kinase C (γ-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal laser scanning fluorescent microscope. γ-PKC–GFP fusion protein had enzymological properties very similar to that of native γ-PKC. The fluorescence of γ-PKC– GFP was observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4α-phorbol 12, 13-didecanoate) induced a significant translocation of γ-PKC–GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induced a more rapid translocation of γ-PKC–GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca2+. TPA- induced translocation of γ-PKC–GFP was unidirected, while Ca2+ ionophore–induced translocation was reversible; that is, γ-PKC–GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of γ-PKC in translocation, we expressed mutant γ-PKC–GFP fusion protein in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca2+ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca2+ ionophore. To examine the translocation of γ-PKC–GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-d-aspartate (NMDA) receptor–transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K+ depolarization induced rapid translocation of γ-PKC–GFP. In NMDA receptor–transfected COS-7 cells, application of NMDA plus glycine also translocated γ-PKC–GFP. Furthermore, rapid translocation and sequential retranslocation of γ-PKC–GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affected the translocation of γ-PKC–GFP, indicating that translocation of γ-PKC was independent of actin and microtubule. γ-PKC–GFP fusion protein is a useful tool for investigating the molecular mechanism of γ-PKC translocation and the role of γ-PKC in the central nervous system.

Fluorescent properties of model chromophores of tyrosine-66 substituted mutants of Aequorea green fluorescent protein (GEP)

1998 ◽  
Vol 39 (29) ◽  
pp. 5239-5242 ◽  
Author(s):  
Satoshi Kojima ◽  
Hiroko Ohkawa ◽  
Takashi Hirano ◽  
Shojiro Maki ◽  
Haruki Niwa ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fluorescent Properties ◽  
Green Fluorescent

Application to immunoassays of the fusion protein between protein ZZ and enhanced green fluorescent protein

2006 ◽  
Vol 309 (1-2) ◽  
pp. 130-138 ◽  
Author(s):  
Qi-Lai Huang ◽  
Cheng Chen ◽  
Yun-Zi Chen ◽  
Chen-Guang Gong ◽  
Lin Cao ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Development and Application of a Green Fluorescent Protein Sentinel System for Identification of RNA Interference in Blastomyces dermatitidis Illuminates the Role of Septin in Morphogenesis and Sporulation

2007 ◽  
Vol 6 (8) ◽  
pp. 1299-1309 ◽  
Author(s):  
T. Krajaejun ◽  
G. M. Gauthier ◽  
C. A. Rappleye ◽  
T. D. Sullivan ◽  
B. S. Klein
Keyword(s):  
Green Fluorescent Protein ◽  
Rna Interference ◽  
Gene Silencing ◽  
Rapid Detection ◽  
Target Gene ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Blastomyces Dermatitidis ◽  
Dimorphic Fungus ◽  
Green Fluorescent

ABSTRACT A high-throughput strategy for testing gene function would accelerate progress in our understanding of disease pathogenesis for the dimorphic fungus Blastomyces dermatitidis, whose genome is being completed. We developed a green fluorescent protein (GFP) sentinel system of gene silencing to rapidly study genes of unknown function. Using Gateway technology to efficiently generate RNA interference plasmids, we cloned a target gene, “X,” next to GFP to create one hairpin to knock down the expression of both genes so that diminished GFP reports target gene expression. To test this approach in B. dermatitidis, we first used LACZ and the virulence gene BAD1 as targets. The level of GFP reliably reported interference of their expression, leading to rapid detection of gene-silenced transformants. We next investigated a previously unstudied gene encoding septin and explored its possible role in morphogenesis and sporulation. A CDC11 septin homolog in B. dermatitidis localized to the neck of budding yeast cells. CDC11-silenced transformants identified with the sentinel system grew slowly as flat or rough colonies on agar. Microscopically, they formed ballooned, distorted yeast cells that failed to bud, and they sporulated poorly as mold. Hence, this GFP sentinel system enables rapid detection of gene silencing and has revealed a pronounced role for septin in morphogenesis, budding, and sporulation of B. dermatitidis.

scholarly journals Fluorescently tagged canine adenovirus via modification with protein IX–enhanced green fluorescent protein

2005 ◽  
Vol 86 (12) ◽  
pp. 3201-3208 ◽  
Author(s):  
Long P. Le ◽  
Jing Li ◽  
Vladimir V. Ternovoi ◽  
Gene P. Siegal ◽  
David T. Curiel
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Neutralizing Antibodies ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Adenovirus Type ◽  
Canine Model ◽  
Heterologous Proteins ◽  
Green Fluorescent ◽  
Protein Ix

Canine adenovirus type 2 (CAV2) has become an attractive vector for gene therapy because of its non-pathogenicity and the lack of pre-existing neutralizing antibodies against this virus in the human population. Additionally, this vector has been proposed as a conditionally replicative adenovirus agent under the control of an osteocalcin promoter for evaluation in a syngeneic, immunocompetent canine model with spontaneous osteosarcoma. In this study, a CAV2 vector labelled with the fluorescent capsid fusion protein IX–enhanced green fluorescent protein (pIX–EGFP) was developed. Expression of the fluorescent fusion-protein label in infected cells with proper nuclear localization, and incorporation into virions, could be detected. The labelled virions could be visualized by fluorescence microscopy; this was applicable to the tracking of CAV2 infection, as well as localizing the distribution of the vector in tissues. Expression of pIX–EGFP could be exploited to detect the replication and spread of CAV2. These results indicate that pIX can serve as a platform for incorporation of heterologous proteins in the context of a canine adenovirus xenotype. It is believed that capsid-labelled CAV2 has utility for vector-development studies and for monitoring CAV2-based oncolytic adenovirus replication.

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