Functional expression of green fluorescent protein derivatives in Halobacterium salinarum

Protocols with demonstrated reliability have been established for the diagnosis of numerous movement disorders. whereas in the essential tremor (ET) literature, there is no discussion about the reliability of diagnostic protocols. Lack of knowledge of the reliability of diagnostic protocols in ET limits the use of these protocols because reliability is an essential requirement for scientific quality in data management. The objective of this study was to determine the reliability of a protocol for diagnosing ET. The protocol consists of a Tremor Interview, a videotaped Tremor Examination, and a diagnostic algorithm. Eighty-three subjects with ET, identified in a community-based health study in Washington Heights-Inwood, New York, were matched with 83 control subjects from the same community. These subjects and their relatives are being recruited to participate in the Washington Heights-Inwood Genetic Study of ET. Two hundred twenty-six subjects have been evaluated to date (35 ET cases, 40 controls, 151 relatives). All 226 underwent an 84-item Tremor Interview and 26-item videotaped Tremor Examination. Diagnoses (normal, possible ET, probable ET, definite ET) were independently assigned by two blinded neurologists specializing in movement disorders. The kappa statistic, k, was used to determine diagnostic agreement between these two neurologists. The concordance rate between two raters using diagnostic categories definite ET, probable ET. possible ET, and normal was 80%; kw = 0.84 (near perfect to perfect agreement). The concordance rate between two raters using two diagnostic categories (definite ET and normal) was 100%; k = 1.00 (perfect agreement). There was high correlation between the two raters' total tremor scores (r = 0.89, p < 0.00001). This diagnostic protocol is highly reliable. Research in ET would greatly benefit from diagnostic protocols with demonstrated reliability.     

Constitutive and inducible green fluorescent protein expression in Bartonella henselae

The green fluorescent protein (GFP) gene was expressed on a plasmid in B. henselae, and GFP-expressing bacteria were visualized by fluorescence microscopy. HEp-2 cells infected with GFP-expressing bacteria were separated from uninfected cells with a fluorescence activated cell sorter. Promoter fusions of B. henselae chromosomal DNA to gfp were examined by flow cytometry, and a B. henselae groEL promoter fusion which induced expression at 37 degreesC was isolated.     

Use of green fluorescent protein to tag and investigate gene expression in marine bacteria

Two broad-host-range vectors previously constructed for use in soil bacteria (A. G. Matthysse, S. Stretton, C. Dandie, N. C. McClure, and A. E. Goodman, FEMS Microbiol. Lett. 145:87-94, 1996) were assessed by epifluorescence microscopy for use in tagging three marine bacterial species. Expression of gfp could be visualized in Vibrio sp. strain S141 cells at uniform levels of intensity from either the lac or the npt-2 promoter, whereas expression of gfp could be visualized in Psychrobacter sp. strain SW5H cells at various levels of intensity only from the npt-2 promoter. Green fluorescent protein (GFP) fluorescence was not detected in the third species, Pseudoalteromonas sp. strain S91, when the gfp gene was expressed from either promoter. A new mini-Tn10-kan-gfp transposon was constructed to investigate further the possibilities of fluorescence tagging of marine bacteria. Insertion of mini-Tn10-kan-gfp generated random stable mutants at high frequencies with all three marine species. With this transposon, strongly and weakly expressed S91 promoters were isolated. Visualization of GFP by epifluorescence microscopy was markedly reduced when S91 (mini-Tn10-kan-gfp) cells were grown in rich medium compared to that when cells were grown in minimal medium. Mini-Tn10-kan-gfp was used to create an S91 chitinase-negative, GFP-positive mutant. Expression of the chi-gfp fusion was induced in cells exposed to N'-acetylglucosamine or attached to chitin particles. By laser scanning confocal microscopy, biofilms consisting of microcolonies of chi-negative, GFP+ S91 cells were found to be localized several microns from a natural chitin substratum. Tagging bacterial strains with GFP enables visualization of, as well as monitoring of gene expression in, living single cells in situ and in real time.     

Overexpression of human DNA topoisomerase II alpha by fusion to enhanced green fluorescent protein

DNA topoisomerase (topo) II alpha is a major target for many anticancer agents. However, progress towards understanding how these agents interact with this enzyme in human cells and how resistance to these agents arises is greatly impeded by difficulties in expressing this gene. Here, we report on achieving a high level of expression of a full-length human topo II alpha gene in human cells. We started with the topo II alpha cDNA driven by a strong cytomegalovirus promoter and transiently transfected HeLa cells. Although topo II alpha mRNA was consistently detected in transfected cells, no exogenous topo II alpha protein was detected. By contrast, when the same cDNA was fused to an enhanced green fluorescent protein (EGFP), we detected a high level of expression at both mRNA and protein levels. The exogenous topo II alpha was localized to cell nuclei as expected, indicating that the fusion protein is properly folded. Furthermore, overexpression of the EGFP-topo II alpha fusion protein increased the sensitivity of the transfected cells to teniposide, suggesting that it functions as the endogenous counterpart. Thus, in addition to being used as a gene tag, the GFP fusion approach may be generally applicable for expressing genes, such as topo II alpha, that are difficult to express by conventional methods.     

Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells

Two mutants of the green fluorescent protein (GFP), RSGFP4 and GFPS65T, have been recently created which differ from the wildtype GFP of A. victoria in their excitation maxima. Here we show that human fibroblasts transfected with either of the two mutant GFP genes emit a green fluorescence that is 18-fold brighter than the cells transfected with the wildtype GFP gene. Retroviral vectors expressing the improved GFP gene were also constructed to determine their suitability for stable gene transduction into mammalian cells. The inclusion of the RSGFP4 gene in a retroviral vector did not reduce the viral titer and resulted in a fluorescent signal in viable transduced cells detectable by both fluorescence microscopy and fluorescence-activated cell sorter (FACS) analysis. Therefore, the improved mutant GFP provides a vital marker for monitoring gene transfer and expression in mammalian cells.     

The molecular structure of green fluorescent protein

The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 A by multiwavelength anomalous dispersion phasing methods. The protein is in the shape of a cylinder, comprising 11 strands of beta-sheet with an alpha-helix inside and short helical segments on the ends of the cylinder. This motif, with beta-structure on the outside and alpha-helix on the inside, represents a new protein fold, which we have named the beta-can. Two protomers pack closely together to form a dimer in the crystal. The fluorophores are protected inside the cylinders, and their structures are consistent with the formation of aromatic systems made up of Tyr66 with reduction of its C alpha-C beta bond coupled with cyclization of the neighboring glycine and serine residues. The environment inside the cylinder explains the effects of many existing mutants of GFP and suggests specific side chains that could be modified to change the spectral properties of GFP. Furthermore, the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.     

Recombinant Bombyx mori nucleopolyhedrovirus harboring green fluorescent protein

The gene encoding the green fluorescent protein (gfp) under the control of the highly expressed Autographa californica nucleopolyhedrovirus (AcMNPV)-polyhedrin promoter has been introduced into the polyhedrin (polh) locus of Bombyx mori nucleopolyhedrovirus (BmNPV) by homologous recombination. The insect host larvae and the cultured cells infected with this recombinant virus (vBmGFP) showed high levels of expression of gfp. The larval tissues permissive to virus multiplication could be readily visualized using the tagged recombinant virus, thus providing a direct approach to study the progress of virus infection or its control in the animal host. The highly expressed recombinant protein, GFP, could be easily solubilized from fat bodies. Thus, the caterpillar-based expression could serve as an economic alternative method for the large-scale production of recombinant proteins, even when they are nonsecretory in nature. Further, if the recombinant vBmGFP is used as a parent in generating other recombinants, conversion of the fluorescent plaques to colorless plaques serves as an easy means for screening recombinants. Such a method is especially helpful for BmNPV-recombinant selections in the absence of the other simplified techniques as are available for the prototype baculovirus AcMNPV system.     

Using green fluorescent protein to understand the mechanisms of G-protein-coupled receptor regulation

G protein-coupled receptor (GPCR) activation is followed rapidly by adaptive changes that serve to diminish the responsiveness of a cell to further stimulation. This process, termed desensitization, is the consequence of receptor phosphorylation, arrestin binding, sequestration and down-regulation. GPCR phosphorylation is initiated within seconds to minutes of receptor activation and is mediated by both second messenger-dependent protein kinases and receptor-specific G protein-coupled receptor kinases (GRKs). Desensitization in response to GRK-mediated phosphorylation involves the binding of arrestin proteins that serve to sterically uncouple the receptor from its G protein. GPCR sequestration, the endocytosis of receptors to endosomes, not only contributes to the temporal desensitization of GPCRs, but plays a critical role in GPCR resensitization. GPCR down-regulation, a loss of the total cellular complement of receptors, is the consequence of both increased lysosomal degradation and decreased mRNA synthesis of GPCRs. While each of these agonist-mediated desensitization processes are initiated within a temporally dissociable time frame, recent data suggest that they are intimately related to one another. The use of green fluorescent protein from the jellyfish Aqueora victoria as an epitope tag with intrinsic fluorescence has facilitated our understanding of the relative relationship between GRK phosphorylation, arrestin binding, receptor sequestration and down-regulation.     

Development and applications of enhanced green fluorescent protein mutants

The introduction of several mutations resulted in the generation of improved mutants of the green fluorescent protein (GFP). A strong green (GFPsg25) and blue (BFPsg50) fluorescent protein, gave 50-fold-100-fold brighter fluorescence compared to wild-type GFP and BFP (Tyr66His), respectively, upon expression in mammalian cells. GFPsg25 and BFPsg50 have different excitation and emission maxima. This allows their use as an efficient dual-color tagging system and their independent detection in living cells.     

Detection of brain tumor invasion and micrometastasis in vivo by expression of enhanced green fluorescent protein

OBJECTIVE: To determine whether fluorescence from human brain tumor cells transfected with the enhanced green fluorescent protein (EGFP) gene in vitro and xenotransplanted into the brain of nude mice would permit the detection of brain tumor invasion and metastasis in vivo. METHODS: Daoy medulloblastoma cells were transfected with a long terminal repeat-based retroviral vector containing the EGFP gene. Stable EGFP-expressing clones were isolated and stereotactically injected into the frontal cortex of nude mice. Four weeks later, whole brain sections were examined using fluorescence microscopy, immunohistochemistry, and routine hematoxylin and eosin staining for the visualization and detection of tumor cell invasion and metastasis. RESULTS: We demonstrate that EGFP-transduced Daoy cells maintain stable high-level EGFP expression in the central nervous system during their growth in vivo. EGFP fluorescence clearly demarcated the primary tumor margins and readily allowed for the visualization of distant micrometastases and local invasion on the single-cell level. Small metastatic and locally invasive foci, including those immediately adjacent to the tumor's leading invasive edge, were virtually undetectable by routine hematoxylin and eosin staining and immunohistochemistry. EGFP expression also persisted in vitro after cell reculture from brain tissue extracts. CONCLUSION: We show, for the first time, that EGFP-transduced human brain tumor cells can be visualized by fluorescence microscopy after intracerebral implantation. This method is superior to routine hematoxylin and eosin staining and immunohistochemistry for the detection and study of physiologically relevant patterns of brain tumor invasion and metastasis in vivo.     

Infectious simian varicella virus expressing the green fluorescent protein

PURPOSE: To determine if vascular occlusion and nonperfusion is associated with the outer retinal atrophy, retinopathy, and choroidopathy (chorioretinopathy) that occurs in the alpha H beta S[beta MDD] and alpha H beta S [alpha MD beta MDD] transgenic mouse models of sickle cell disease. METHODS: Mice from the alpha H beta S[beta MDD] and alpha H beta S[alpha MD beta MDD] transgenic mouse lines that express high levels of human beta S globin were anesthetized and administered horseradish peroxidase (HRP) intracardially. After 1 min, the animals were sacrificed, and the retina from one eye was excised, fixed, and developed in diaminobenzidine (DAB). The contralateral eye was fixed, embedded whole in glycol methacrylate, and HRP developed in 2.5 microns sections. RESULTS: HRP reaction product (HRP-RP) and stained erythrocytes (RBCs) (due to endogenous peroxidase) were diffusely distributed within all vascular lumens in flatmount retinas from control animals (littermates homozygous for the mouse Beta Major deletion not expressing the beta S transgene). In 42.5% of the transgenic mice expressing beta S without any proliferative retinopathy, many blood vessels contained RBC plugs and lacked lumenal HRP-RP. In addition to packed RBCs, fibrin was sometimes present at sites of occlusion. In sections from whole eyes of the same animals, foci of photoreceptor degeneration were associated with areas of choriocapillaris nonperfusion (lumen that lacked HRP-PR). In areas with normal photoreceptors, the choriocapillaris appeared perfused (HRP-RP was present). In animals with proliferative chorioretinopathy, some neovascular formations lacked luminal HRP-RP, suggesting autoinfarction. CONCLUSIONS: Nonperfused retinal and choroidal vessels were observed in mice from the alpha H beta S[beta MDD] and alpha H beta S[alpha MD beta MDD] lines without retinal and choroidal neovascularization, whereas, all mice with neovascularization had nonperfused areas. Furthermore, small foci of PR loss were associated with areas of nonperfused choriocapillaris. These results suggest that sickle cell-mediated vaso-occlusions are an initial event in the chorioretinopathy and outer retinal atrophy that occurs in these models.     

Functional characteristics of ES cell-derived cardiac precursor cells identified by tissue-specific expression of the green fluorescent protein

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions. In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac alpha-actin promoter. First, fluorescent areas in ES cell-derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2+ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments proved the functional expression of the L-type Ca2+ current (ICa) starting from day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, ICa was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of ICa proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, ICa was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP3-sensitive intracellular Ca2+ stores and Ca2+-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca2+ stores, detected by caffeine-induced Ca2+ release, appeared in most GFP-positive cells 1-2 d after ICa. Coexpression of both ICa and ryanodine-sensitive Ca2+ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs. Thus, the functional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca2+-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.     

Use of a green fluorescent protein as a marker for human immunodeficiency virus type 1 infection

We constructed a recombinant human immunodeficiency virus type 1 (HIV-1) provirus called R7-GFP that expresses a modified form of a green fluorescent protein (GFP) from the jellyfish Aequorea victoria by substituting GFP-coding sequences for Nef-coding sequences. Alanine was substituted for serine at amino acid position 65 in the modified GFP, resulting in markedly increased fluorescence at an excitation wavelength of 488 nm as compared to wild-type GFP. The replication kinetics of R7-GFP were identical to that measured with an isogenic, nef-negative strain lacking GFP. Expression of GFP by replication-competent HIV-1 allowed simultaneous quantitation of viral infection and cell surface CD4 levels, revealing rapid and nearly complete CD4 downregulation on R7-GFP-infected PBMCs.     

Selective neuronal expression of green fluorescent protein with cytomegalovirus promoter reveals entire neuronal arbor in transgenic mice

In simple nervous systems, identified groups of neurons can be studied in depth. To allow the same advantage in the mammalian brain, we have generated green fluorescent protein (GFP) transgenic mice in which only a few types of neurons are strongly labeled with a fluorescent molecule, which the neurons synthesize internally, allowing the cells, their dendrites, filopodia, and axons to be identified in both living and fixed CNS, in slices and culture. The same neurons, with GFP expression controlled by part of the major immediate early promoter of human cytomegalovirus (CMV), show GFP beginning early in development, from one generation to the next, allowing cellular and physiological studies of axonal and dendritic growth, fate mapping, anatomical connections, and synapse formation in identified neurons. The human CMV promoter sequence we used was different from that used in previous work with other reporter genes and gave a dramatically different pattern of expression. Two transgenic lines with the same CMV promoter show similar anatomical patterns of expression in the present study. Strong GFP labeling was found in a subpopulation of mossy fibers that innervated parasagittal bands in the cerebellar cortex and olfactory axons that projected into the olfactory bulb, subsets of motoneurons and dorsal root ganglion cells, granule but not mitral cells of the olfactory bulb, and a group of neurons in the hypothalamic suprachiasmatic nucleus. A novel type of neuron was strongly labeled in the olfactory bulb external plexiform layer. In normal brains, CMV does not constitute a threat, but in the developing brain, CMV can cause debilitating neurodegeneration and death; studies using the CMV promoter aid in understanding the affinity of CMV that has been suggested for specific brain regions.     

Characterization of the photoconversion of green fluorescent protein with FTIR spectroscopy

Green Fluorescent Protein (GFP) is a bioluminescence protein from the jelly fish Aequorea victoria. It can exist in at least two spectroscopically distinct states: GFP395 and GFP480, with peak absorption at 395 and 480 nm, respectively, presumably resulting from a change in the protonation state of the phenolic ring of its chromophore. When GFP is formed upon heterologous expression in Escherichia coli, its chromophore is mainly present as the neutral species. UV and visible light convert (the chromophore of) GFP quantitatively from this neutral- into the anionic form. On the basis of X-ray diffraction, it was recently proposed (Brejc, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2306-2311; Palm, G. J. et al. (1997) Nat. Struct. Biol. 4, 361-365) that the carboxylic group of Glu222 functions as the proton acceptor of the chromophore of GFP, during the transition from the neutral form (i.e., GFP395) to the ionized form (GFP480). However, X-ray crystallography cannot detect protons directly. The results of FTIR difference spectroscopy, in contrast, are highly sensitive to changes in the protonation state between two conformations of a protein. Here we report the first characterization of GFP, and its photoconversion, with FTIR spectroscopy. Our results clearly show the change in protonation state of the chromophore upon photoconversion. However, they do not provide indications for a change of the protonation state of a glutamate side chain between the states GFP395 and GFP480, nor for an isomerization of the double bond that forms part of the link between the two rings of the chromophore.