Monitoring promoter activity in a single bacterial cell by using green and red fluorescent proteins

We investigated the possibility of monitoring promoter activity with flow cytometry by using green fluorescent protein (GFPmut2) and red fluorescent protein (drFP583) in a single bacterial cell. The drFP583 was used as an intrinsic marker of the bacterial cells, because it was expressed constantly in Escherichia coli MC1061 strain. The GFPmut2 expressed under the control of the Hg(2+) ion inducible mer promoter/operator, was used to study promoter activity. Over 75% of the cells were positive for red and green fluorescence in flow cytometric analysis. The average green fluorescence of the whole population increased from 6.7 to 1700 when the mercury concentration was increased from 0 to 1 x 10(-4) M, while the red fluorescence was unaffected by the mercury concentration. These results show that gfpmut2 and drFP583 could be expressed under different promoters in one bacterial cell and measured independently with a flow cytometer.     

A high-throughput approach to promoter study using green fluorescent protein

Green fluorescent protein (GFP) is a reporter that has had a significant impact due to its many advantages over other reporter genes: it is autofluorescent, it enables in situ detection, it is relatively small, and it is also nontoxic. By cloning a gene promoter upstream of the gfp gene and exposing the living cells transformed with the fusion to the specific inducer or repressor, gene expression can be real-time monitored by continuous quantitative measurement of the green fluorescence emitted by GFP. In this work, a promoter study using promoter-gfp fusions was conducted in 96-well plates. Because they were placed in an automated incubating and shaking microplate reader, the wells functioned as microscale bioreactors, allowing for parallel experiments and data analysis. In the study described here, an overexpression promoter (pBAD promoter) and two comparatively weak promoters (sodA and acnA in Escherichia coli SoxRS regulon) were studied in both endpoint and kinetics formats. Our results with the pBAD promoter revealed insight on its regulation, which is tightly controlled by levels of arabinose and glucose. Results on weak oxidative stress promoters (for sodA and acnA genes) were striking in that significant induction was observed when they were under a superoxide stress in plates. They both displayed dose-dependent induction to paraquat-generated superoxide anion, with sodA leading acnA in strength and time. These results, spanning highly inducible promoters for protein overexpression and weakly inducible promoters of metabolic interest, demonstrate that the approach is relatively easily executed and can be used for quantitative and temporal promoter studies in a high throughput format.     

The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein

Mycobacterium marinum , like Mycobacterium tuberculosis , is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200–1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2–20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis , Mycobacterium bovis BCG and Mycobacterium tuberculosis . These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.     

A simple assay and histochemical localization of transglutaminase activity using a derivative of green fluorescent protein as substrate.

Histidine-tagged green fluorescent protein (His(6)-Xpress-GFP), a widely used fluorescent probe, was found to be a good substrate for transglutaminase, an enzyme that catalyzes covalent crosslinking of proteins. GFP alone did not serve as a substrate but its derivative His(6)-Xpress-GFP was readily crosslinked through the Gln and Lys residues present in the short N-terminal extension (His(6)-Xpress). His(6)-Xpress-GFP was sensitive enough to detect the transglutaminase activity in guinea pig liver homogenates. The fluorescent substrate could also be used for activity staining of transglutaminase on histological tissue sections, and such applications revealed a surprisingly wide distribution of transglutaminase in the body, especially in the extracellular matrices of various tissues, suggesting an important role for transglutaminase in maintaining the integrity of the extracellular matrix and connective tissues by crosslinking its constituent proteins.(J Histochem Cytochem 49:247-258, 2001)     

Monitoring of conformational change in maltose binding protein using split green fluorescent protein

In this study, we describe a novel method for the detection of conformational changes in proteins, which is predicated on the reconstitution of split green fluorescent protein (GFP). We employed fluorescence complementation assays for the monitoring of the conformationally altered proteins. In particular, we used maltose binding protein (MBP) as a model protein, as MBP undergoes a characteristic hinge-twist movement upon substrate binding. The common feature of this approach is that GFP, as a reporter protein, splits into two non-fluorescent fragments, which are genetically fused to the N- and C-termini of MBP. Upon binding to maltose, the chromophores move closer together, resulting in the generation of fluorescence. This split GFP method also involves the reconstitution of GFP, which is determined via observations of the degree to which fluorescence intensity is restored. As a result, reconstituted GFP has been observed to generate fluorescence upon maltose binding in vitro, thereby allowing for the direct detection of changes in fluorescence intensity in response to maltose, in a concentration- and time-dependent fashion. Our findings showed that the fluorescence complementation assay can be used to monitor the conformational alterations of a target protein, and this ability may prove useful in a number of scientific and medical applications.     

Preimplantation screening for transgenesis using an embryonic specific promoter and green fluorescent protein

We report enrichment in the efficiency of generating mice transgenic for expression of a human protein in their milk using GFP-mediated preimplantation screening. The transgene array consisted of a functional gene (human alpha-1 antitrypsin under the control of the ovine BLG promoter) linked 5' to a reporter gene (GFP under the control of the murine Oct-4 promoter). GFP expression was detected in blastocysts by fluorescence microscopy and green and nongreen embryos were transferred to recipients in separate groups. In the first experiment, of seven pups that resulted from the transfer of blastocysts expressing GFP, five (71%) were transgenic. The experiment was repeated and of 12 pups that resulted from transfer of GFP-expressing blastocysts, 11 were transgenic (92%). The presence of the reporter cassette used for preimplantation screening did not affect the expression level of alpha-1-antitrypsin in the milk of the transgenic mice. In addition, in a related experiment wherein the GFP reporter gene was co-injected with a second mammary-specific transgene, pINC, no effect on transgene expression was observed. For mice transgenic for the mammary-specific gene alone, expression levels for four different lines were 192, 197, 382, and 415 microg/mL. For mice transgenic for both the mammary-specific transgene and the Oct4-GFP reporter cassette, expression levels for seven different lines were 282, 321, 468, 497, 499, 516, and 806 microg/mL.     

Analysis of apolipoprotein E nuclear localization using green fluorescent protein and biotinylation approaches

Previous results indicate that apoE (apolipoprotein E) may be associated with the nucleus in specific cell types, particularly under stress conditions such as serum starvation. In addition, nuclear apoE localization in ovarian cancer was recently shown to be correlated with patient survival. In order to better understand the factors associated with apoE nuclear localization, we examined intracellular apoE trafficking using live-cell imaging of CHO (Chinese-hamster ovary) cells that constitutively expressed apoE-GFP (green fluorescent protein). In addition, we used biotinylated apoE (in a lipid-free state and as a lipidated discoidal complex) to track the uptake and potential nuclear targeting of exogenous apoE. Our results indicate that a small proportion of apoE-GFP is detected in the nucleus of living apoE-GFP-expressing CHO cells and that the level of apoE-GFP in the nucleus is increased with serum starvation. Exposure of control CHO cells to exogenous apoE-GFP did not result in nuclear apoE-GFP localization in the recipient cells. Similarly, biotinylated apoE did not reach the nucleus of control CHO cells or SK-N-SH neurons. In contrast, when biotinylated apoE was delivered to recipient cells as a lipidated apoE disc, apoE was detected in the nucleus, suggesting that the lipoprotein complex alters the intracellular degradation or trafficking of apoE. Biotinylated apoE discs containing each of the three common human apoE isoforms (E2, E3 and E4) were also tested for nuclear trafficking. All three apoE isoforms were equally detected in the nucleus. These studies provide new evidence that apoE may be targeted to the nucleus and shed light on factors that regulate this process.     

Rapid protein-folding assay using green fluorescent protein.

Formation of the chromophore of green fluorescent protein (GFP) depends on the correct folding of the protein. We constructed a "folding reporter" vector, in which a test protein is expressed as an N-terminal fusion with GFP. Using a test panel of 20 proteins, we demonstrated that the fluorescence of Escherichia coli cells expressing such GFP fusions is related to the productive folding of the upstream protein domains expressed alone. We used this fluorescent indicator of protein folding to evolve proteins that are normally prone to aggregation during expression in E. coli into closely related proteins that fold robustly and are fully soluble and functional. This approach to improving protein folding does not require functional assays for the protein of interest and provides a simple route to improving protein folding and expression by directed evolution.     

Nuclear localization of enhanced green fluorescent protein homomultimers

The green fluorescent protein (GFP) and its variants are used in many studies to determine the subcellular localization of other proteins by analyzing fusion proteins. The main problem for nuclear localization studies is the fact that, to some extent, GFP translocates to the nucleus on its own. Because the nuclear import could be due to unspecific diffusion of the relatively small GFP through the nuclear pores, we analyzed the localization of multimers of a GFP variant, the enhanced GFP (EGFP). By detecting the fluorescence of the expressed proteins in gels after nonreducing SDS-PAGE, we demonstrate the integrity of the expressed proteins. Nevertheless, even EGFP homotetramers and homohexamers are found in the nuclei of the five analyzed mammalian cell lines. The use of fusion constructs of small proteins with multimeric EGFP alone, therefore, is not adequate to prove nuclear import processes. Fusion to tetrameric EGFP in combination with a careful quantification of the fluorescence intensities in the nucleus and cytoplasm might be sufficient in many cases to identify a significant difference between the fusion protein and tetrameric EGFP alone to deduce a nuclear localization signal.     

Monitoring the expression of green fluorescent protein in carrot

Green fluorescent protein (GFP) was successfully used as a visual reporter at various stages of carrot (Daucus carota L.) transformation. GFP-fluorescence was non-invasively observed in protoplasts, callus and plants after the delivery of mgfp5-er gene using two transformation methods: direct DNA transfer into polyethylene glycol (PEG) -treated protoplasts and inoculation of root discs with Agrobacterium rhizogenes. Transient GFP-expression was detected in the treated protoplasts and monitored during the first week of the cell culture until the stable level of expression was observed. It was useful for the comparison of protoplast susceptibility to DNA uptake and the transgene expression as the fluorescence declined with various rates depending on the used carrot genotype and PEG-concentration. GFP-monitoring in callus enabled the selection of stably expressing lines. It also allowed verification of the homogeneous tissue composition with regard to the expression of the transgene. In plants, GFP-performance depended on the assayed tissue and organ despite of the constitutive 35S promoter. The expression was visually detected in both vegetative and generative parts, but particularly strong fluorescence was observed in leaf marginal meristems, petioles, stems, and styles. Those tissues can be convenient for examination of the transgenic plants during their growth. The results encourage that GFP is a valuable reporter and can be routinely used for optimization of transformation protocol, selection of transformants and monitoring transgenic carrot.     

Charge transfer in green fluorescent protein.

Charge transfer reactions that contribute to the photoreactions of the wild type green fluorescent protein (GFP) do not occur in the isolated p-hydroxybenzylidene-imidazolidinone chromophore, demonstrating the role of the protein environment. The high quantum efficiency of the fluorescence photocycle that includes excited state proton transfer and the suppression of non-radiative pathways by the protein environment have been correlated with structural dynamics in the chromophore environment. A low quantum efficiency competing phototransformation reaction of GFP is accompanied by both proton and electron transfer, and closely mimics the charge redistribution that is occurring in the fluorescence photocycle. The protein response to this destabilising event has been demonstrated by cryo-trapping of early products in the reaction pathway and is found to be strong even at 100 K, including displacements of chromophore, protein, solvent and a photogenerated CO2 molecule derived from the decarboxylated Glu 222 side chain. We discuss the ramifications of the observation of strong conformational perturbations below the protein dynamical transition at approximately 200 K, in view of low temperature work on other light sensitive proteins such as myoglobin and bacteriorhodopsin. The proton and electron transfer in the phototransformation pathway mimics the proton and charge transfer which occurs during the fluorescence cycle, which leads to common structural responses in both photoreactions as shown by ultrafast spectroscopy. We review and discuss literature on light-induced and thermal charge transfer events, focusing on recent findings addressing conformational dynamics and implications for thermodynamic properties.     

Reliable use of green fluorescent protein in fluorescent pseudomonads.

When fluorescent pseudomonads are cultured on standard solid media under iron limiting conditions, they produce fluorescent, pigmented iron collating agents (siderophores). Siderophores can be readily identified by strong fluorescence seen under UV/blue light. The application of the eukaryotic green fluorescent protein (GFP) as a bacterial marker in microbial ecology is increasingly being used, particularly as it is a powerful method for non-destructive monitoring in situ. As gfp expressing bacteria have to be detected under UV/blue light, the fluorescence of siderophore-producing Pseudomonas spp. masks normal levels of GFP fluorescence when colonies are viewed on standard bacterial agar. Here, we describe a simple but effective way of identifying gfp-expressing Pseudomonas fluorescens using media supplemented with 0.45 mM FeSO(4).7H(2)O. This is of relevance for the screening of insertion libraries and in the application of GFP transposons as promoter probes.     

Recent advances using green and red fluorescent protein variants

Fluorescent proteins have proven to be excellent tools for live-cell imaging. In addition to green fluorescent protein (GFP) and its variants, recent progress has led to the development of monomeric red fluorescent proteins (mRFPs) that show improved properties with respect to maturation, brightness, and the monomeric state. This review considers green and red spectral variants, their paired use for live-cell imaging in vivo, in vitro, and in fluorescence resonance energy transfer (FRET) studies, in addition to other recent “two-color” advances including photoswitching and bimolecular fluorescence complementation (BiFC). It will be seen that green and red fluorescent proteins now exist with nearly ideal properties for dual-color microscopy and FRET.     

A green fluorescent protein kinase substrate allowing detection and localization of intracellular ERK/MAP kinase activity

We describe a versatile intracellular reporter of ERK/MAP kinase activity: a cDNA construct, pGFP.MBP, encoding amino acids 85-144 of the human myelin basic protein fused to the C-terminus of an enhanced green fluorescent protein (GFP). The fused fragment of myelin basic protein contains a single consensus ERK/MAP kinase phosphorylation motif (PRTP, where the threonine is phosphorylated). Phosphorylation of the specific motif can be detected via immunoblotting or immunofluorescence with a commercially available phospho-specific monoclonal antibody. When expressed in mammalian cells by either transient or stable transfection, the fusion protein acts as a bona fide kinase substrate, as demonstrated by rapid serum-induced phosphorylation that is blocked by a specific MEK inhibitor. Moreover, the localization of the total substrate pool is easily visualized by GFP autofluorescence and the extent of its phosphorylation simultaneously detected within intact fixed cells by immunofluorescence using the commercially available phospho-specific antibody. The approach described should be generally applicable to the intracellular analysis of many specific protein kinase substrates for which phospho-specific antibodies have been produced.     

In vivo oxygen imaging using green fluorescent protein

In vivo oxygen measurement is the key to understanding how biological systems dynamically adapt to reductions in oxygen supply. High spatial resolution oxygen imaging is of particular importance because recent studies address the significance of within-tissue and within-cell heterogeneities in oxygen concentration in health and disease. Here, we report a new technique for in vivo molecular imaging of oxygen in organs using green fluorescent protein (GFP). GFP-expressing COS-7 cells were briefly photoactivated with a strong blue light while lowering the oxygen concentration from 10% to <0.001%. Red fluorescence (excitation 520–550 nm, emission >580 nm) appeared after photoactivation at <2% oxygen (the red shift of GFP fluorescence). The red shift disappeared after reoxygenation of the cell, indicating that the red shift is stable as long as the cell is hypoxic. The red shift of GFP fluorescence was also demonstrated in single cardiomyocytes isolated from the GFP knock-in mouse (green mouse) heart. Then, we tried in vivo molecular imaging of hypoxia in organs. The red shift could be imaged in the ischemic liver and kidney in the green mouse using macroscopic optics provided that oxygen diffusion from the atmospheric air was prevented. In crystalloid-perfused beating heart isolated from the green mouse, significant spatial heterogeneities in the red shift were demonstrated in the epicardium distal to the coronary artery ligation. We conclude that the present technique using GFP as an oxygen indicator may allow in vivo molecular imaging of oxygen in organs. heart; ischemia; hypoxia; molecular imaging     

Differential localization of protein kinase C delta by phorbol esters and related compounds using a fusion protein with green fluorescent protein

Enzyme localization often plays a controlling role in determining its activity and specificity. Protein kinase C (PKC) has long been known to translocate in response to physiological stimuli as well as to exogenous ligands such as the phorbol esters. We report here that different phorbol derivatives and related ligands, selected for differences in chemical structure and profile of biological activity, induce distinct patterns of redistribution of PKC delta. Localization of a PKC delta-green fluorescent protein (GFP) fusion construct was monitored in living Chinese hamster ovary cells as a function of ligand, concentration, and time using confocal laser scanning microscopy. delta-PKC-GFP was expressed predominantly in the cytoplasm, with some in the nucleus and perinuclear region. Phorbol 12-myristate 13-acetate (PMA) induced plasma membrane translocation followed by slower nuclear membrane translocation. As the concentration of PMA increased, the proportion of nuclear to plasma membrane localization increased markedly. In contrast to PMA, bryostatin 1, a unique activator of PKC that induces a subset of PMA-mediated responses while antagonizing others, at all doses induced almost exclusively nuclear membrane translocation. Like PMA, the complete tumor promoter 12-deoxyphorbol 13-tetradecanoate induced plasma membrane and slower nuclear membrane translocation, whereas the inhibitor of tumor promotion 12-deoxyphorbol 13-phenylacetate, which differs only in its side chain, induced a distinctive distribution of PKC delta-GFP. Finally, the novel constrained diacylglycerol derivative B8-DL-B8 induced a slow Golgi localization. We speculate that differential control of PKC delta localization may provide an interesting strategy for producing ligands with differential biological consequences.