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GFP Fluorescence (gfp + fluorescence)
Selected AbstractsLarge-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA libraryGENES TO CELLS, Issue 3 2000Da-Qiao Ding Background Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green fluorescent protein (GFP)-fusion genomic DNA library of S. pombe. Results We constructed the S. pombe GFP-fusion genomic DNA library by fusing, in all three reading frames, random fragments of genomic DNA to the 5, end of the GFP gene in such a way that expression of potential GFP-fusion proteins would be under the control of the own promoters contained in the genomic DNA fragments. Fission yeast cells were transformed with this plasmid library, and microscopic screening of 49 845 transformants yielded 6954 transformants which exhibited GFP fluorescence, of which 728 transformants showed fluorescence localized to distinct intracellular structures such as the nucleus, the nuclear membrane, and cytoskeletal structures. Plasmids were isolated from 516 of these transformants, and a determination of their DNA sequences identified 250 independent genes. The intracellular localizations of the 250 GFP-fusion constructs was categorized as an image database; using this database, DNA sequences can be searched for based on the localizations of their products. Conclusions A number of new intracellular structural components were found in this library. The library of GFP-fusion constructs also provides useful fluorescent markers for various intracellular structures and cellular activities, which can be readily used for microscopic observation in living cells. [source] E1-Ngn2/Cre is a new line for regional activation of Cre recombinase in the developing CNSGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 4 2004Joachim Berger Abstract We generated a transgenic mouse line named E1-Ngn2/Cre that expresses Cre recombinase and GFP under the control of the E1 enhancer element of the gene Ngn2 (Scardigli et al.: Neuron 31:203,217, 2001). Cre-recombinase activity and GFP fluorescence are consistent with the reported expression pattern controlled by the E1-Ngn2 enhancer. Recombination was detected in the progenitor domains p1 and p2 in the ventricular zone of the neural tube and in distinct domains of the pretectum, the dorsal and ventral thalamus, the tegmentum of the mesencephalon, and the hindbrain. In the developing cortex, Cre-recombinase activity is confined to a subpopulation of progenitors predominantly in the region of the ventral and lateral pallium. The E1-Ngn2/Cre mouse line thus provides an excellent novel tool for a region-specific conditional mutagenesis in the developing CNS. genesis 40:195,199, 2004. © 2004 Wiley-Liss, Inc. [source] ,-Aminobutyric acid-mediated regulation of the activity-dependent olfactory bulb dopaminergic phenotypeJOURNAL OF NEUROSCIENCE RESEARCH, Issue 10 2009Yosuke Akiba Abstract ,-Aminobutyric acid (GABA) regulates the proliferation and migration of olfactory bulb (OB) interneuron progenitors derived from the subventricular zone (SVZ), but the role of GABA in the differentiation of these progenitors has been largely unexplored. This study examines the role of GABA in the differentiation of OB dopaminergic interneurons using neonatal forebrain organotypic slice cultures prepared from transgenic mice expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (Th) gene promoter (ThGFP). KCl-mediated depolarization of the slices induced ThGFP expression. The addition of GABA to the depolarized slices further increased GFP fluorescence by inducing ThGFP expression in an additional set of periglomerular cells. These findings show that GABA promoted differentiation of SVZ-derived OB dopaminergic interneurons and suggest that GABA indirectly regulated Th expression and OB dopaminergic neuron differentiation through an acceleration of the maturation rate for the dopaminergic progenitors. Additional studies revealed that the effect of GABA on ThGFP expression required activation of L- and P/Q-type Ca2+ channels as well as GABAA and GABAB receptors. These voltage-gated Ca2+ channels and GABA receptors have previously been shown to be required for the coexpressed GABAergic phenotype in the OB interneurons. Together, these findings suggest that Th expression and the differentiation of OB dopaminergic interneurons are coupled to the coexpressed GABAergic phenotype and demonstrate a novel role for GABA in neurogenesis. © 2009 Wiley-Liss, Inc. [source] Imaging OmpR localization in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 6 2006Eric Batchelor Summary We have used a fusion of GFP to the response regulator OmpR to image the spatial distribution of OmpR in live cells of Escherichia coli. We observed foci of increased OmpR,GFP fluorescence that appear to be due to interactions with the histidine kinase EnvZ. We also observed colocalization of OmpR,GFP with clusters of plasmids carrying OmpR binding sites, which enabled us to develop a simple method for imaging the binding of OmpR to DNA in live cells. We used the peak fluorescence intensity within cells to quantify the extent of OmpR,GFP localization either due to interactions with EnvZ or due to binding DNA. With these assays we compared the effects of osmolarity and procaine, both of which are believed to modulate EnvZ activity. Our results suggest that, at least under our growth conditions, procaine activates EnvZ,OmpR signalling whereas osmolarity has, at best, a weak effect on the EnvZ,OmpR system. [source] Probing Pineal-specific Gene Expression with Transgenic Zebrafish,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Daisuke Kojima The pineal gland of zebrafish (Danio rerio) contains light-sensitive photoreceptor cells and plays an important role in the neuroendocrine system. The zebrafish exorhodopsin gene encodes a pineal-specific photoreceptive protein, whose promoter region harbors a cis -acting element, pineal expression-promoting element (PIPE), directing pineal-specific gene expression. For in vivo genetic studies on PIPE-binding proteins and their regulatory mechanisms, we generated a transgenic zebrafish line, Tg(P20 -rh/P:gfp), that expresses green fluorescent protein (GFP) under the control of the zebrafish rhodopsin promoter fused with 20 PIPE repeats. In Tg(P20 -rh/P:gfp) fish, PIPE-dependent gene expression is visualized by GFP fluorescence in the pineal gland along with PIPE-independent GFP signals in the retinal rod photoreceptors. The transgenic fish exhibit detectable and reproducible GFP fluorescence in the larval pineal gland by 5 days postfertilization. Antisense morpholino-mediated knock-down of a pineal transcription factor gene, otx5, suppresses pineal GFP expression in the transgenic line. In a pilot screen of N -ethyl- N -nitrosourea-treated fish of the GFP transgenic line, we isolated potential dominant mutations that cause attenuation of pineal GFP fluorescence with a marginal effect on the retinal GFP signal. The results suggest that the Tg(P20 -rh/P:gfp) line will be useful for detecting deficits in PIPE-dependent gene expression in the pineal gland. [source] Green fluorescence induced by EF-hand assembly in a split GFP systemPROTEIN SCIENCE, Issue 6 2009Stina Lindman Abstract The affinity between the 1,157 and 158,238 fragments of green fluorescent protein (GFP) is too low for spontaneous in vivo reassembly of the protein upon co-expression of the two fragments. This prevents chromophore maturation and the cells lack GFP fluorescence. We have utilized the very high affinity between the two EF-hands of calbindin D9k to facilitate GFP assembly from its fragments and to introduce a calcium dependent molecular switch. In GFPN-EF1, residues 1,157 of GFP are fused to residues 1,43 of calbindin, and in EF2-GFPC, residues 44,75 of calbindin are fused to residues 158,238 of GFP. When co-expressed, GFPN-EF1 and EF2-GFPC associate spontaneously and rapidly resulting in a folded reconstituted protein with bright GFP fluorescence. The high affinity of GFPN-EF1 for EF2-GFPC leads to brighter fluorescence of the cells compared to cells with a control constructs carrying leucine zippers (Wilson et al., Nature Methods 2004;3:255). The complex of GFPN-EF1 and EF2-GFPC was purified from cells using metal-ion chelate chromatography and the temperature dependence of GFP fluorescence was found to be calcium dependent. The GFPN-EF1 and EF2-GFPC fragments were separated by ion exchange chromatography. The assembly of the fragments was found to be reversible and the complex was regained upon mixing, as evidenced by surface plasmon resonance (SPR) data. The affinity between GFPN-EF1 and EF2-GFPC as well as rates of association and dissociation were found to be Ca2+ -dependent. [source] The use of green fluorescent protein as a marker for monitoring a probiotic Bacillus S11 in the black tiger shrimp Penaeus monodonAQUACULTURE NUTRITION, Issue 3 2009S. RENGPIPAT Abstract To monitor the probiotic Bacillus S11 (BS11) in vivo, wild-type cells were transformed with the green fluorescent protein (GFP)-expressing plasmid, pAD44-12, carrying the gfpmut3a gene under the constitutive Bacillus cereus UW85 promoter, and its non-GFP control plasmid pAD. Transformants with pAD44-12 (BS11-GFP), not pAD (BS11-pAD), expressed detectable but not too high levels of green fluorescence. Chloramphenicol resistance (BS11-GFP, BS11-pAD) and GFP fluorescence (BS11-GFP) as markers suggested that plasmid retention was 78,79% for both BS11-GFP and BS11-pAD cells after approximately 50 generations of growth without antibiotic selection. When mixed into shrimp feed at a final concentration ,105 CFU g,1, the inclusion of viable transformed bacteria in fed shrimps was observed. After feeding shrimp three times daily in 400-L cement tanks for 9 weeks, no significant differences in the average shrimp weight, the number of BS11 in either the culture water or in the shrimp's gut were seen between shrimp fed BS11-GFP and BS11-pAD or BS11, suggesting that expression of the gfpmut3a gene has no detectable effect on BS11 properties and shrimp growth. Histological examination of sections of shrimp's intestines following feeding with BS11-GFP demonstrated that BS11-GFP in shrimp feed survived and adhered onto the shrimp intestines' surface. BS11-GFP thus has good potential as a non-invasive marker tag for short-term experiments. [source] | ||||||||||