Green Fluorescent Protein

Green fluorescent protein (GFP) is an excellent biosensor as a result of its ability to be easily monitored in a wide variety of applications. Enzymes and proteins have been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. The purpose of this work was to study GFP stability in chlorinated water for injection (WFI) and chlorinated buffered solutions at various pH ranges, with and without agitation, to evaluate the exposure time required for chlorine to decrease 90% of its fluorescence intensity (decimal reduction time, D-value, min, 25°C). Fluorescence intensity (Ex/Em max) 394/509 nm) was measured immediately after the addition of GFP (8.0-9.0 µg/mL) into buffered or unbuffered chlorine solutions with or without constant stirring. With solutions constantly stirred, GFP fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH) 7.15 (0.08), GFP retained its structure between 52 and 94 ppm, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. The recovery of GFP fluorescence intensity due to renaturation was observed between 30 and 100 ppm chlorine in WFI (final pH) 11.01 (0.23) without stirring. Stirring enhanced the contact between GFP and chlorine throughout the assay and provided a more accurate D-value evaluation. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness.

Inducible expression of tetracycline responsive element (TRE)-regulated genes in nearly all cells in a stable clone has generally been problematic, especially in long-term culture. Heterogeneity of tet-inducible expression is generally attributed to the instability of the original tet-transactivators tTA and rtTA. These transactivators have cryptic splice sites, prokaryotic codons and full VP16 domains, all of which contribute to their instability. Moreover, they also require high concentrations of Doxycycline (Dox). The 5 amino acid substitutions in the rtTA variant rtTA2S-M2 confer exquisite sensitivity to Dox. Moreover, humanized codons, removal of cryptic splice sites and minimal VP16 domains in rtTA2S-M2 results in its being better tolerated within cells. However, the ability of this modified transactivator to maintain homogeneous inducibility in long-term culture has not been examined. We demonstrate that rtTA2S-M2 expressing clones exhibit functional transactivator activity for over 7 months in culture. Furthermore, rtTA2S-M2 expressing clones with chromosomally integrated copies of a TRE -green fluorescent protein (GFP) reporter also exhibited homogeneous inducibility in long-term culture. Importantly, the inherent reduced toxicity and improved stability of rtTA2S-M2 obviates the need to continuously select for its message, once clones with functional transactivator are isolated. The use of rtTA2S-M2 did not, however, preclude clones with stably integrated TRE-reporter from exhibiting leakiness. However, inclusion of flanking double copies of a 'minimal core element' of the chicken h-globin gene insulator, instead of the 1.4 kb region, in the TRE-reporter was sufficient to markedly reduce the frequency of clones with high basal expression. Inclusion of the insulator core also did not affect the maximal expression levels of the inducible gene, which typically equaled or exceeded that observed with the strong constitutive CMV promoter. Finally, with this system homogeneous inducibility was observed rapidly and with low doses of Dox. D

nature materials | VOL 3 | OCTOBER 2004 | www.nature.com/naturematerials I n the last decade we have witnessed a fast growth in the research, development and marketing of biosensors, although no clear decisions have been made in favour of specific materials 1 . An ideal biosensor should combine nature's sensitivity and specificity with the advantages of modern microelectronics. Because of its technological advancement, silicon has been used in most of the past and current developments 2-5 . However, its poor biocompatibility and chemical instability prevent silicon from becoming the ideal material for biosensor applications. In contrast, diamond is known as a biocompatible material in itself 6 , consisting of just carbon atoms. Efforts were therefore made in the past to functionalize diamond surfaces 7-9 . Nevertheless, economic considerations and a lack of information have prevented a clear judgement of the potential of diamond in this particular field.The advent of a new diamond thin-film deposition technology 10 and recent breakthroughs in substitutional doping of diamond 11,12 have generated renewed interest. Indeed, the possibility of depositing large-area thin films of nanocrystalline diamond (NCD) on diverse substrates at a relatively low cost, and the fact that NCD has nearly the same combination of exceptional properties as single-crystal diamond-such as biocompatibility, chemical inertness and superb electrochemical properties 13 -have quickly led to considerable progress 15 . For instance, Yang et al. have demonstrated the modification of NCD surfaces with DNA oligonucleotides, and shown that NCD as a substrate for DNA immobilization has superior properties to those of other materials such as glass, gold or silicon 15 .

Objective
Maize (Zea mays L.) is a key cereal crop throughout the world and important model for plant genetics and biology. Establishment of an efficient transient gene expression system in maize facilitates plant functional genomics projects using gene silencing or heterologous protein overexpression strategies. The present study was aimed at optimizing an Agroinjection-mediated SCMV-based systemic heterologous gene expression system in maize.
Materials and methods
Recombinant DNA encoding sugarcane mosaic virus (SCMV) containing the coding sequence of green fluorescent protein between the protein 1 (P1) and helper component‐proteinase (HC‐Pro) cistrons, in‐frame with the viral open reading frame, was introduced into the meristematic tissue, above the coleoptilar node, of maize seedlings via a direct Agroinoculation procedure. The efficiency of Agrobacterium tumefaciens strains EHA105 and GV3101 in delivering the recombinant vector into three and seven-day old seedlings of three maize varieties, including sweet corn (Iochief and Golden Bantam varieties) and dent corn (inbred line B73), was examined. Expression of GFP transgene in symptomatic Agroinoculated plants was assessed by confocal fluorescent microscopy and RT-PCR in comparison with controls.
Results
Results of RT-PCR and confocal fluorescent microscopy revealed that: 1) A. tumefaciens GV3101 is significantly more successful in delivering the recombinant SCMV-based vector into maize plants than EHA105, 2) the percentage of GFP-expressing Golden Bantam plants is significantly higher than two other maize varieties, and 3) The effectiveness of growth stage of maize seedlings on the percentage of GFP expressing Agroinjected plants depends upon the interactions between Agrobacterium strain and maize genotype.
Conclusions
In conclusion, a combination of the Golden Bantam maize variety and A. tumefaciens strain GV3101 for direct Agroinjection of the SCMV-based vector into seedlings at the early two-leaf stage could be a fast and efficient system for investigating gene functions in maize.

Coiled bodies are nuclear organelles that contain components of at least three RNA-processing pathways: pre-mRNA splicing, histone mRNA 3′- maturation, and pre-rRNA processing. Their function remains unknown. However, it has been speculated that coiled bodies may be sites of splicing factor assembly and/or recycling, play a role in histone mRNA 3′-processing, or act as nuclear transport or sorting structures. To study the dynamics of coiled bodies in living cells, we have stably expressed a U2B"–green fluorescent protein fusion in tobacco BY-2 cells and in Arabidopsis plants. Time-lapse confocal microscopy has shown that coiled bodies are mobile organelles in plant cells. We have observed movements of coiled bodies in the nucleolus, in the nucleoplasm, and from the periphery of the nucleus into the nucleolus, which suggests a transport function for coiled bodies. Furthermore, we have observed coalescence of coiled bodies, which suggests a mechanism for the decrease in coiled bo...

Fluorescence resonance energy transfer (FRET) is a widely utilized optical technique for measuring small distances of 1-10 nm in live cells. In recent years, its application has been greatly popularized by the discovery of green fluorescent protein (GFP) and many improved variants which make good donor-acceptor fluorophore pairs. GFP-based proteins are structurally stable, relatively inert, and can be reliably attached to points of interest. The combination of easy access to the GFP-based FRET technique and its obvious usefulness in many applications can lead to complacency. Potential problems such as light contaminants, e.g., bleed-through and cross-talk, and inconsistent donor and acceptor concentrations are easily overlooked and can lead to errors in FRET calculation and data interpretation. In this article, we outline possible pitfalls of GFP-based FRET and approaches that address these issues, including a "Spectra FRET" technique that can be easily applied to live cell studies.

The Arabidopsis Nucleolar Protein Database (http://bioinf.scri.sari.ac.uk/cgi-bin/atnopdb/home) provides information on 217 proteins identified in a proteomic analysis of nucleoli isolated from Arabidopsis cell culture. The database is organized on the basis of the Arabidopsis gene identifier number. The information provided includes protein description, protein class, whether or not the plant protein has a homologue in the most recent human nucleolar proteome and the results of reciprocal BLAST analysis of the human proteome. In addition, for one-third of the 217 Arabidopsis nucleolar proteins, localization images are available from analysis of full-length cDNA–green fluorescent protein (GFP) fusions and the strength of signal in different parts of the cell—nucleolus, nucleolus-associated structures, nucleoplasm, nuclear bodies and extra-nuclear—is provided. For each protein, the most likely human and yeast orthologues, where identifiable through BLASTX analysis, are given with links to relevant information sources.

Herpes simplex virus type1 VP22 (HSV-1 VP22) Green fluorescent protein (GFP) a b s t r a c t An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.

The VPS4 gene is a member of the AAA-family; it codes for an ATPase which is involved in lysosomal/endosomal membrane trafficking. VPS4 genes are present in virtually all eukaryotes. Exhaustive data mining of all available genomic databases from completely or partially sequenced organisms revealed the existence of up to three paralogues, VPS4a, -b, and -c. Whereas in the genome of lower eukaryotes like yeast only one VPS4 representative is present, we found that mammals harbour two paralogues, VPS4a and VPS4b. Most interestingly, the Fugu fish contains a third VPS4 paralogue (VPS4c ). Sequence comparison of the three VPS4 paralogues indicates that the Fugu VPS4c displays sequence features intermediate between VPS4a and VPS4b. Using complete mammalian VPS4a and VPS4b cDNA clones as probes, genomic clones of both VPS4 paralogues in human and mouse were identified and sequenced. The chromosomal loci of all four VPS4 genes were determined by independent methods. A BLAST search of the human genome database with the human VPS4A sequence yielded a double match, most likely due to a faulty assembly of sequence contigs in the human draft sequence. Fluorescent in situ hybridization and radiation hybrid analyses demonstrated that human and mouse VPS4A/a and VPS4B/b are located on syntenic chromosomal regions. Northern blot and semi-quantitative reverse transcription analyses showed that mouse VPS4a and VPS4b are differentially expressed in different organs, suggesting that the two paralogues have developed different functional properties since their divergence. To investigate the subcellular distribution of the murine VPS4 paralogues, we transiently expressed various fluorescent VPS4 fusion proteins in mouse 3T3 cells. All tested VPS4 fusion proteins were found in the cytosol. Expression of dominant-negative mutant VPS4 fusion proteins led to their concentration in the perinuclear region. Co-expression of VPS4a-GFP and VPS4b-dsRed fusion proteins revealed a partial co-localization that was most prominent with mutant VPS4a and VPS4b proteins. A physical interaction between the mouse paralogues was also supported by two-hybrid analyses. q

Two cDNAs, isolated from a Trypanosoma cruzi amastigote library immunoscreened with sera from patients with Chagas disease, encode proteins with sequence homology to eukaryotic components of the cellular sorting and recycling machinery. These proteins, denominated TcAGL, present an N-terminal lectin domain and a C-terminal region containing repetitive amino acids and a poly-glutamine tract. They are products of polymorphic alleles of a single copy gene constitutively expressed during the parasite life cycle. Polyclonal antibodies obtained from mice immunized with the recombinant antigen recognize proteins with apparent molecular weight ranging from 95 to 120 kDa in cell lysates from all three life stages and in various strains of the parasite. Sera from Chagas disease patients recognize the recombinant antigen in ELISA and immunoprecipitation assays but not in Western blot assays under denaturing conditions. Consistent with its proposed role in the glycoprotein secreting pathway, immunofluorescence analyses and expression of a green fluorescent protein-tagged TcAGL protein indicate a sub-cellular localization in the vicinity of the flagellar pocket membrane and the Golgi complex of the parasite. Ó

Here we show that codon optimization of the SARS-CoV S-protein gene substantially enhanced S-protein expression. We also found that two retroviruses, simian immunodeficiency virus (SIV) and murine leukemia virus, both expressing green fluorescent protein and pseudotyped with SARS-CoV S protein or S-protein variants, efficiently infected HEK293T cells stably expressing ACE2. Infection mediated by an S-protein variant whose cytoplasmic domain had been truncated and altered to include a fragment of the cytoplasmic tail of the human immunodeficiency virus type 1 envelope glycoprotein was, in both cases, substantially more efficient than that mediated by wild-type S protein.

Multiple-probe fluorescence imaging applications demand an ever-increasing number of resolvable probes, and the use of fluorophores with resolvable fluorescence lifetimes can help meet this demand. Green fluorescent protein (GFP) and its variants have been widely used in spectrally resolved multiprobe imaging, but as yet, there has not been a systematic set of mutants generated with resolvable lifetimes. Therefore, to generate such mutants, we have utilized error-prone PCR and fluorescence lifetime imaging to screen for mutants of UV-excited green fluorescent protein (GFPuv) that exhibit altered fluorescence decay lifetimes. This has resulted in the isolation of GFPuv mutants displaying at least three distinctly different lifetimes in the range of 1.9-2.8 ns. Mutation of Y145 to either histidine or cysteine was found to shift the fluorescence lifetime of GFPuv from 3.03 ( 0.03 to 2.78 ( 0.05 ns for the Y145H mutant and to 2.74 ( 0.05 ns for Y145C. Some of the shorter-lifetime mutants exhibited excitation peaks that were red-shifted relative to their maximal absorption, indicating that the mutations allowed the adoption of additional conformations relative to wtGFPuv. The utility of these mutants for applications in simultaneous imaging and quantification is shown by the ability to quantify the composition of binary mixtures in time-resolved images using a single detector channel. The application of the screening method for generating lifetime mutants of other fluorescent proteins is also discussed.

Previous studies demonstrated that the rat neuronspecific enolase (NSE) promoter is effective for transgene expression in the brain in a variety of adeno-associated virus-2 vectors. This study evaluated the dose response and longer time course of this promoter and compared it to two cytomegalovirus/chicken ␤-actin hybrid (CBA) promoter-based systems. NSE promoterdriven green fluorescent protein (GFP)-expressing neurons were found at doses as low as 10 7 particles, with expression increasing in a dose-dependent manner over a 3.3-log range. Bicistronic expression of GFP via an internal ribosome entry site coupled to the NSE promoter was also dose dependent, although the potency was decreased by 3.4-fold. The number of GFP-expressing neurons was stable for at least 25 months. The CBA promoter increased the numbers of GFP-expressing cells versus the NSE promoter, although the expression pattern remained neuronal and persisted for at least 18 months. The CBA promoter permitted detection of cells distal to the injection site that had retrogradely transported the vector from their terminal areas. Incorporating the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) into a CBA promoter vector induced greater expression levels in the hippocampus, as measured by stereological estimates of cell numbers and by Western blots, which demonstrated an 11-fold increase. Incorporation of the WPRE also improved transgene expression in primary neuronal cultures. The increased efficiency obtained with vector elements such as the CBA promoter and the WPRE may enhance the ability to genetically modify larger portions of the brain while requiring smaller doses and volumes. © 2002 Elsevier Science (USA)

We have developed a simple correlative photooxidation method that allows for the direct ultrastructural visualization of the green fluorescent protein (GFP) upon illumination. The method, termed GRAB for GFP recognition after bleaching, uses oxygen radicals generated during the GFP bleaching process to photooxidize 3,3¢-diaminobenzidine (DAB) into an electron-dense precipitate that can be visualized by routine electron microscopy and electron tomography. The amount of DAB product produced by the GRAB method appears to be linear with the initial fluorescence, and the resulting images are of sufficient quality to reveal detailed spatial information. This is exemplified by the observed intra-Golgi stack and intracisternal distribution of a human Golgi resident glycosylation enzyme, N-acetylgalactosaminyltransferase-2 fused either to enhanced GFP or CFP.

Detailed evaluation of all steps in tumor cell metastasis is critical for evaluating the cell mechanisms controlling metastasis. Using green fluo- rescent protein transfectants of metastatic (MTLn3) and nonmetastatic (MTC) cell lines derived from the rat mammary adenocarcinoma 13762 NF, we have measured tumor cell density in the blood, individual tumor cells in the lungs, and lung metastases. Correlation of

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the carboxy-terminus of the Pho84 phosphate permease of Saccharomyces cerevisiae. Both components of the fusion protein displayed their native functions and revealed a cellular localization and degradation of the Pho84-GFP chimera consistent with the behavior of the wild-type Pho84 protein. The GFP-tagged chimera allowed for a detection of conditions under which the Pho84 transporter is localized to its functional environment, i.e. the plasma membrane, and conditions linked to relocation of the protein to the vacuole for degradation. By use of the methodology described, GFP should be useful in studies of localization and degradation also of other membrane proteins in vivo.

Background The isolation of green fluorescent protein (GFP) and the development of spectral variants over the past decade have begun to reveal the dynamic nature of protein trafficking and organelle motility. In planta analyses of this dynamic process have typically been limited to only two organelles or proteins at a time in only a few cell types. Results We generated a transgenic Arabidopsis plant that contains four spectrally different fluorescent proteins. Nuclei, plastids, mitochondria and plasma membranes were genetically tagged with cyan, red, yellow and green fluorescent proteins, respectively. In addition, methods to track nuclei, mitochondria and chloroplasts and quantify the interaction between these organelles at a submicron resolution were developed. These analyzes revealed that N-ethylmaleimide disrupts nuclear-mitochondrial but not nuclear-plastids interactions in root epidermal cells of live Arabidopsis seedlings. Conclusion We developed a tool and associated methods for analyzing the complex dynamic of organelle-organelle interactions in real time in planta. Homozygous transgenic Arabidopsis (Kaleidocell) is available through Arabidopsis Biological Resource Center.

We have generated transgenic zebraWsh that express green Xuorescent protein (GFP) in glial cells driven by the zebraWsh glial Wbrillary acidic protein (GFAP) regulatory elements. Transgenic lines Tg(gfap:GFP) were generated from three founders; the results presented here are from the mi2001 line. GFP expression was Wrst visible in the living embryo at the tail bud-stage, then in the developing brain by the 5-somite-stage (»12 h post-fertilization, hpf) and then spreading posteriorly along the developing spinal cord by the 12-somite stage (»15 hpf). At 24 hpf GFP-expressing cells were in the retina and lens. By 72 hpf GFP expression levels were strong and localized to the glia of the brain, neural retina, spinal cord, and ventral spinal nerves, with moderate expression in the enteric nervous system and weaker levels in the olfactory sensory placode and otic capsule. GFP expression in glia co-localized with anti-GFAP antibodies, but did not colocalize with the neuronal antibodies HuC/D or calretinin in mature neurons.

Reporter genes have proved to be an excellent tool for studying disease progression. Recently, the green fluorescent protein (GFP) ability to quantitatively monitor gene expression has been demonstrated in different organisms. This report describes the use of Leishmania tarentolae (L. tarentolae) expression system (LEXSY) for high and stable levels of GFP production in different Leishmania species including L. tarentolae, L. major and L. infantum. The DNA expression cassette (pLEXSY-EGFP) was integrated into the chromosomal ssu locus of Leishmania strains through homologous recombination. Fluorescent microscopic image showed that GFP transgenes can be abundantly and stably expressed in promastigote and amastigote stages of parasites. Furthermore, flow cytometry analysis indicated a clear quantitative distinction between wild type and transgenic Leishmania strains at both promastigote and amastigote forms. Our data showed that the footpad lesions with GFP-transfected L. major are progressive over time by using fluorescence small-animal imaging system. Consequently, the utilization of stable GFP-transfected Leishmania species will be appropriate for in vitro and in vivo screening of anti-leishmanial drugs and vaccine development as well as understanding the biology of the host-parasite interactions at the cellular level.

The recombinant mouse brain serotonin receptor (5HT1c) was used to study the response of plant cells and oocytes to a stress signal activated by the serotonin-serotonin receptor interaction and associated Ca 2+ flux. Basing on plant expression vectors, recombinant constructs were obtained to direct production of 5HT1c fused with the green fluorescent protein in plant cells. The mRNAs for hybrid proteins were synthesized in an in vitro transcription system. The expression and function of the hybrid protein and the function of the associated ion channels were electrophysiologically studied in Xenopus laevis oocytes injected with the hybrid mRNA. The hybrid protein was functional and changed the operation of the Ca 2+ channel in oocytes. To study the expression of the hybrid constructs in plant cells, the in vitro transcription product was inoculated in tobacco leaves, which fluoresced thereupon.

Luminous proteins include primary light producers, such as aequorin, and secondary photoproteins that in some organisms red-shift light emission for better penetration in space. When expressed in heterologous systems, both types of proteins may act as versatile reporters capable of monitoring phenomena as diverse as calcium homoeostasis, protein sorting, gene expression, and so on. The Ca# + -sensitive photoprotein aequorin was targeted to defined intracellular locations (organelles, such as mitochondria, endoplasmic reticulum, sarcoplasmic reticulum, Golgi apparatus and nucleus, and cytoplasmic regions, such as the bulk cytosol and the subplasmalemmal rim), and was used to analyse Ca# + homoeostasis at the subcellular level. We will discuss this application, reviewing its advantages and disadvantages and the experimental procedure. The applications of green fluorescent protein (GFP) are even broader. Indeed, the ability to molecularly engineer and recombinantly express a strongly fluorescent probe Abbreviations used : BFP, blue flourescent protein ; BRET, bioluminescence resonance energy transfer ; [Ca 2 + ] c , cytoplasmic Ca 2 + concentration ; ER, endoplasmic reticulum ; FRET, fluorescence resonance energy transfer ; GFP, green fluorescent protein ; eGFP (etc.), ' humanized ' mutant of wild-type GFP (etc.) ; HA1, short epitope tag derived from haemagglutinin ; KRB, Krebs-Ringer buffer ; PKC, protein kinase C. ).

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 Â 10 À 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. D

Brachypodium distachyon is a novel model system for structural and functional genomics studies of temperate grasses because of its biological and genetic attributes. Recently, the genome sequence of the community standard line Bd21 has been released and the availability of an efficient transformation system is critical for the discovery and validation of the function of Brachypodium genes. Here, we provide an improved procedure for the facile and efficient Agrobacterium-mediated transformation of line Bd21. The protocol relies on the transformation of compact embryogenic calli derived from immature embryos using visual and chemical screening of transformed tissues and plants. The combination of green fluorescent protein expression and hygromycin resistance enables early identification of transformation events and drastically reduces the quantity of tissue to be handled throughout the selection process. Approximately eight independent fully developed transgenic Bd21 plants can be produced from each immature embryo, enabling the generation of thousands of T-DNA lines. The process-from wild-type seeds to transgenic T 1 seeds-takes B8 months to complete.

The dissection and subsequent reassembly of a protein from peptidic fragments provides an avenue for controlling its tertiary structure and hence its function. Here, we describe a general method for the reassembly of protein fragments mediated by the noncovalent association of antiparallel leucine zippers. 1 Although a majority of leucine zippers associate in a parallel fashion, recent examples of both naturally occurring and designed antiparallel leucine zippers have appeared in the literature. 1,2 We report here a strategy for the noncovalent reconnection of the N-and C-termini of a dissected surface loop of a protein by means of antiparallel leucine zippers ( ). 3 We have successfully applied this oligomerization strategy, both in vitro and in vivo, to the 238 residue green fluorescent protein (GFP) from Aequorea Victoria. 4 GFP provides an easily testable system for correct reassembly by virtue of its autocatalytically generated fluorescence, which is intimately linked to its properly folded structure. 5 Moreover, the current interest in utilizing GFP as a biosensor provides further motivation for generating new tools for biotechnological applications based on the strategy we describe. 5 The unassisted reconstitution of proteins from peptide fragments has been demonstrated for several proteins; including ribonuclease, 6a chymotrypsin inhibitor-2, 6b tRNA synthetases, 6c and inteins. 6d Protein reassembly has thus become an important avenue for understanding enzyme catalysis, 6a protein folding, 6b and protein evolution. 6c Recently, assisted protein reassembly or "fragment complementation" has been applied to the in vivo detection of protein-protein interactions in such systems as dihydrofolate reductase (DHFR), 7a-c ubiquitin, 7d,e and -galactosidase. 7f These reassembly processes are contingent upon the proper choice of a dissection site within a protein and can be aided by techniques such as limited proteolysis, circular permutation 8 and loop insertions. 9 In particular, recent circular permutation 8c and protein insertion 8b,9 strategies have provided convincing evidence that GFP can fold, fluoresce, and serve as a biosensor despite the rearrangement of the natural coding sequence.

In the present study, new applications of the transgenic technology in developing novel varieties of ornamental fish and bioreactor fish were explored in a model fish, the zebrafish (Danio rerio). Three "living color" fluorescent proteins, green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP or dsRed), were expressed under a strong muscle-specific mylz2 promoter in stable lines of transgenic zebrafish. These transgenic zebrafish display vivid fluorescent colors (green, red, yellow, or orange) visible to unaided eyes under both daylight and ultraviolet light in the dark. The level of foreign protein expression is estimated between 3% and 17% of total muscle proteins, equivalent to 4.8-27.2 mg/g wet muscle tissue. Thus, the fish muscle may be explored as another useful bioreactor system for production of recombinant proteins. In spite of the high level of foreign protein expression, the expression of endogenous mylz2 mRNAs was not negatively affected. Furthermore, compared to the wild-type fish, these fluorescent transgenic fish have no advantage in survival and reproduction.

Controlled proteolysis of regulatory or aberrant proteins by the ubiquitin/proteasome system is indispensable for cell viability. Conformational diseases such as Alzheimer's, Parkinson's and Huntington's disease are characterised by the accumulation of misfolded or aggregation-prone proteins. Since these proteins are typical substrates of the ubiquitin/proteasome system, it is not surprising that various models propose impairment of this system as a contributing factor to the pathology of conformational disorders. The complex nature of the ubiquitin/proteasome system and its universal role in cell physiology however turns evaluation of these attractive hypotheses into a major challenge. Several reporter substrates for the ubiquitin/proteasome system have recently been developed to facilitate functional studies of the system in living cells. In this review, we will discuss these new tools as well as the proteins associated with conformational disease that have been studied with these reporters.

Unlike that of mammals, the brain of teleost fish exhibits an intense aromatase activity due to the strong expression of one of two aromatase genes (aromatase A or cyp19a1a and aromatase B or cyp19a1b) that arose from a gene duplication event. In situ hybridization, immunohistochemistry and expression of GFP (green fluorescent protein) in transgenic tg(cyp19a1b-GFP) fish demonstrate that aromatase B is only expressed in radial glial cells (RGC) of adult fish. These cells persist throughout life and act as progenitors in the brain of both developing and adult fish. Although aromatase B-positive radial glial cells are most abundant in the preoptic area and the hypothalamus, they are observed throughout the central nervous system and spinal cord. In agreement with the fact that brain aromatase activity is correlated to sex steroid levels, the high expression of cyp19a1b is due to an auto-regulatory loop through which estrogens and aromatizable androgens up-regulate aromatase expression.

Background: Articular cartilage has very limited intrinsic regenerative capacity, making cell-based therapy a tempting approach for cartilage repair. Cell tracking can be a major step towards unraveling and improving the repair process of these therapies. We studied superparamagnetic iron oxides (SPIO) for labeling human bone marrow-derived mesenchymal stem cells (hBMSCs) regarding effectivity, cell viability, long term metabolic cell activity, chondrogenic differentiation and hBMSC secretion profile. We additionally examined the capacity of synovial cells to endocytose SPIO from dead, labeled cells, together with the use of magnetic resonance imaging (MRI) for intra-articular visualization and quantification of SPIO labeled cells.

The tapetum, the innermost of four sporophytic layers in the anther wall, comes in direct contact with the developing male gametophyte and is thought to play a crucial role in the development and maturation of microspores. Here, we report the identification of rice (Oryza sativa) Undeveloped Tapetum1 (Udt1), which is required for the differentiation of secondary parietal cells to mature tapetal cells. T-DNA or retrotransposon Tos17 insertions in the Udt1 gene caused male sterility. The anther walls and meiocytes of the mutants were normal during the early premeiosis stage, but their tapeta failed to differentiate and became vacuolated during the meiotic stage. In addition, meiocytes did not develop to microspores, and middle layer degeneration was inhibited. Consequently, the anther locules contained no pollen. The UDT1:green fluorescent protein fusion protein was localized to the nucleus. This, together with its homology with other basic helix-loop-helix proteins, suggests that UDT1 is a transcription factor. DNA microarray analysis identified 958 downregulated and 267 upregulated genes in the udt1-1 anthers, suggesting that Udt1 plays a major role in maintaining tapetum development, starting in early meiosis.

Cf proteins are receptor-like proteins (RLPs) that mediate resistance of tomato (Solanum lycopersicum) to the foliar pathogen Cladosporium fulvum. These transmembrane immune receptors, which carry extracellular leucine-rich repeats that are subjected to posttranslational glycosylation, perceive effectors of the pathogen and trigger a defense response that results in plant resistance. To identify proteins required for the functionality of these RLPs, we performed immunopurification of a functional Cf-4-enhanced green fluorescent protein fusion protein transiently expressed in Nicotiana benthamiana, followed by mass spectrometry. The endoplasmic reticulum (ER) heat shock protein70 binding proteins (BiPs) and lectin-type calreticulins (CRTs), which are chaperones involved in ER-quality control, were copurifying with Cf-4-enhanced green fluorescent protein.

Two transcriptional reporter shuttle vectors were constructed for the filamentous cyanobacterium Nostoc punctiforme using the green fluorescence protein (GFP) reporter. Both the ampicillin-and kanamycin-resistant versions of the plasmid allow promoters to be directionally cloned into a multiple cloning site preceding a promoterless gfp gene using an Escherichia coli host. The ability of the self-replicating shuttle plasmids to report cell-type-specific gene expression in N. punctiforme was tested by cloning promoters expressed in normal vegetative cells, nitrogen-fixing heterocysts and spore-like akinetes. A P psaC reporter gene fusion was expressed in vegetative cells and not in heterocysts, whereas GFP driven from P hetR was found highly expressed in heterocysts. GFP expression driven by the promoter for the N. punctiforme homologue of the akinete-specific gene avaK was expressed in developing akinetes. Decreased expression of GFP from the P psaC reporter in hormogonia was also observed. The results demonstrate the utility of these GFP vectors to study cell-type-specific gene expression in differentiating filamentous cyanobacteria. D

Fluorescent imaging microscopy has been an essential tool for biologists over many years, especially after the discovery of the green fluorescent protein and the possibility of tagging virtually every protein with it. In recent years dramatic enhancement of the level of detail at which a fluorescing structure of interest can be imaged have been achieved. We review classical and new developments in high-resolution microscopy, and describe how these methods have been used in biological research. Classical methods include widefield and confocal microscopy whereas novel approaches range from linear methods such as 4Pi, I(5) and structured illumination microscopy to non-linear schemes such as stimulated emission depletion and saturated structured illumination. Localization based approaches (e.g. PALM and STORM), near-field methods and total internal refraction microscopy are also discussed. As the terms 'resolution', 'sensitivity', 'sampling' and 'precision&#3...

As part of our analysis of branched-chain amino acid metabolism in plants, we analyzed the function of Arabidopsis thaliana BRANCHED-CHAIN AMINOTRANSFERASE4 (BCAT4). Recombinant BCAT4 showed high efficiency with Met and its derivatives and the corresponding 2-oxo acids, suggesting its participation in the chain elongation pathway of Met-derived glucosinolate biosynthesis. This was substantiated by in vivo analysis of two BCAT4 T-DNA knockout mutants, in which Met-derived aliphatic glucosinolate accumulation is reduced by ;50%. The increase in free Met and S-methylmethionine levels in these mutants, together with in vitro substrate specificity, strongly implicate BCAT4 in catalysis of the initial deamination of Met to 4-methylthio-2-oxobutyrate. BCAT4 transcription is induced by wounding and is predominantly observed in the phloem. BCAT4 transcript accumulation also follows a diurnal rhythm, and green fluorescent protein tagging experiments and subcellular protein fractions show that BCAT4 is located in the cytosol. The assignment of BCAT4 to the Met chain elongation pathway documents the close evolutionary relationship of this pathway to Leu biosynthesis. In addition to BCAT4, the enzyme methylthioalkylmalate synthase 1 has been recruited for the Met chain elongation pathway from a gene family involved in Leu formation. This suggests that the two pathways have a common evolutionary origin.

Mitochondria trigger apoptosis by releasing caspase activators, including cytochrome c (cytC). Here we show, using a pH-sensitive green fluorescent protein (GFP), that mitochondria-dependent apoptotic stimuli (such as Bax, staurosporine and ultraviolet irradiation) induce rapid, Bcl-2-inhibitable mitochondrial alkalinization and cytosol acidification, followed by cytC release, caspase activation and mitochondrial swelling and depolarization. These events are not induced by mitochondria-independent apoptotic stimuli,

An assay capable of simultaneously measuring both general toxicity and more subtle genotoxicity, in aqueous environmental samples, is described. The assay uses eukaryotic (yeast) cells, genetically modified to express a green fluorescent protein (GFP) whenever DNA damage, as a result of exposure to genotoxic agents, is repaired. A measure of the reduction in cell proliferation is used to characterise general toxicity producing familiar EC 50 and LOEC data. The assay protocol has been developed for proposed use in the field and hence employs dedicated, portable instrumentation, the development of which is described. A range of environmentally relevant substances has been evaluated using the assay, including solutions of metal ions, solvents and pesticides. Preliminary data comparing the yeast assay's response to that of a standard Daphnia test in the analysis of the toxicity of 34 varied industrial waste effluents are also presented. The sensitivity to a wide range of substances and effluents suggests the assay should be useful for environmental toxicity monitoring.

In this study, we explored the capacity of the naturally occurring compound solasodine to promote neurogenesis in vitro and in vivo. Mouse embryonic teratocarcinoma P19 cells exposed to solasodine for 2 days followed by a 5-day washout differentiated into cholinergic neurons that expressed specific neuronal markers and displayed important axonal formation that continued growing even 30 days after treatment. In vivo, a 2-week infusion of solasodine into the left ventricle of the rat brain followed by a 3-week washout resulted in a significant increase in bromodeoxyuridine uptake by cells of the ependymal layer, subventricular zone, and cortex that co-localized with doublecortin immunostaining, demonstrating the proliferative and differentiating properties of solasodine on neuronal progenitors. In addition, these data demonstrate that under our experimental conditions adult ependymal cells retrieved their proliferative and differentiating abilities. The GAP-43/HuD pathway was activated both in vitro and in vivo, suggesting a role in the differentiating process triggered by solasodine. Solasodine treatment in rats resulted in a dramatic increase in expression of the cholesterol-and drug-binding translocator protein in ependymal cells, suggesting a possible role played by neurosteroid production in solasodine-induced neurogenesis. In GAD65-GFP mice that express the green fluorescent protein under the control of the glutamic acid decarboxylase 65-kDa promoter, solasodine treatment increased the number of GABAergic progenitors and neuroblasts generated in the subventricular zone and present in the olfactory migratory tract. Taken together, these results suggest that solasodine offers an interesting approach to stimulate in situ neurogenesis from resident neuronal progenitors as part of neuron replacement therapy.