Improved green fluorescent protein as a fast reporter of gene expression in plant cells

An improved green fluorescent protein (GFP), S65TGFP, has new properties that make itself more suitable as a reporter of gene expression. The coding sequence for S65TGFP was placed under the control of the rice actin1 (Act1) promoter in pAct1-S65TGFP reconstruction. We transformed pAct1-S65TGFP into rice callus cells by particle bombardment and bright green fluorescent dots could be seen after 6-8 hours.     

Jellyfish green fluorescent protein as a reporter for virus infections

The gene encoding green fluorescent protein (GFP) of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X (PVX). Host plants of PVX inoculated with PVX.GFP became systemically infected. Production of GFP in these plants was detected initially between 1 and 2 days postinoculation by the presence of regions on the inoculated leaf that fluoresced bright green under UV light. Subsequently, this green fluorescence was evident in systemically infected tissue. The fluorescence could be detected by several methods. The simplest of these was by looking at the UV-illuminated plants in a darkened room. The PVX.GFP-infected tissue has been analysed either by epifluorescence or confocal laser scanning microscopy. These microscopical methods allow the presence of the virus to be localized to individual infected cells. It was also possible to detect the green fluorescence by spectroscopy or by electrophoresis of extracts from infected plants. To illustrate the potential application of this reporter gene in virological studies a derivative of PVX.GFP was constructed in which the coat protein gene of PVX was replaced by GFP. Confocal laser scanning microscopy of the inoculated tissue showed that the virus was restricted to the inoculated cells thereby confirming earlier speculation that the PVX coat protein is essential for cell-to-cell movement. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus-infected tissue.     

Use of a green fluorescent protein gene as a reporter in Zymomonas mobilis and Halomonas elongata

The mtl operon of Klebsiella pneumoniae KAY2026 (formerly Aerobacter aerogenes 1033-5P14) was shown to contain as the promoter-proximal gene mtlA, encoding a D-mannitol-specific enzyme II transporter (IICBA(Mtl)). This gene is followed by mtlD, coding for a mannitol-1-phosphate dehydrogenase (MtlD, 382 amino acid residues), and mtlR (MtlR, 195 amino acid residues) coding for a putative repressor, gene mtlR overlaps the termination codon of mtlD. The DNA and protein sequences are highly similar to the corresponding genes (81% identical bp) and proteins (79-85% identical amino acids) of Escherichia coli K-12. A truncated form of MtlD lacking the 162 C-terminal amino acid residues still shows 10% dehydrogenase activity which may explain the controversy in the literature concerning the properties of mannitol-phosphate and other medium-length dehydrogenases.     

Androgen receptor transactivation assay using green fluorescent protein as a reporter

For screening of a large number of samples for androgenic activity, a robust system with minimal handling is required. The coding sequence for human androgen receptor (AR) was inserted into expression plasmid YEpBUbi-FLAG1, resulting in the plasmid YEpBUbiFLAG-AR, and the estrogen response element (ERE) on the reporter vector YRpE2 was replaced by an androgen response element (ARE), resulting in the plasmid YRpE2-ARE. Thus, a fully functional transactivation assay system with beta-galactosidase as a reporter gene could be created. Furthermore, green fluorescent protein (GFP) was introduced as an alternative reporter gene that resulted in a simplification of the whole assay procedure. For evaluation of both reporter systems, seven steroidal compounds with known AR agonistic properties (5 alpha-dihydrotestosterone, testosterone, androstenedione, 17 alpha-methyltestosterone, progesterone, epitestosterone, and d-norgestrel) were tested, and their potencies obtained in the different assays were compared. Furthermore, potencies from the transactivation assays were compared with IC(50) values obtained in radioligand binding assays. The newly developed androgen receptor transactivation assay is a useful tool for characterizing compounds with androgenic activity.     

Green fluorescent protein functions as a reporter for protein localization in Escherichia coli

The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfp optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the sec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported beta-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.     

Assessment of membrane protein expression and stability using a split green fluorescent protein reporter

Membrane proteins are challenging targets for structural biologists. Finding optimal candidates for such studies requires extensive and laborious screening of protein expression and/or stability in detergent. The use of green fluorescent protein (GFP) as a reporter has enormously facilitated these studies; however, its 238 residues can potentially alter the intrinsic properties of the target (e.g., expression or stability). With the aim of minimizing undesired effects of full-length GFP, here we describe the utility of a split GFP reporter during precrystallization studies of membrane proteins. GFP fluorescence appeared by complementation of the first 15 residues of GFP (GFP(11)) (fused to the C terminus of a membrane protein target) with the remaining nonfluorescent GFP (GFP(1-10)). The signal obtained after sequential expression of SteT (l-serine/l-threonine exchanger of Bacillus subtilis) fused to GFP(11) followed by GFP(1-10) specifically measured the protein fraction inserted into the Escherichia coli cytoplasmic membrane, thereby discarding protein aggregates confined as inclusion bodies. Furthermore, in vitro complementation of purified SteT-GFP(11) with purified GFP(1-10) was exploited to rapidly assess the stability of wild-type and G294V mutant versions of SteT-GFP(11) following detergent solubilization and purification. This method can be applied in a medium- to high-throughput manner with multiple samples.     

Green fluorescent protein as a reporter for gene expression in the mucoralean fungus Absidia glauca

Mucoralean fungi (Zygomycota) are used for many industrial processes and also as important model organisms for investigating basic biological problems. Their genetic analysis is severely hampered by low transformation frequencies, by their strong tendency towards autonomous replication of plasmids instead of stable integration, and by the lack of reliable genetic reporter systems. We constructed plasmids for transforming the model zygomycete Absidia glauca that carry the versatile reporter gene coding for green fluorescent protein (GFP). gfp expression is controlled either by the homologous actin promoter or the promoter for the elongation factor of translation, EF1alpha. These plasmids also confer neomycin resistance and carry one of two genetic elements (rag1, seg1) that improve mitotic stability of the plasmid. The gfp constructs were replicated extrachromosomally and could be recovered from retransformed Escherichia coli cells. gfp expression was monitored by epifluorescence microscopy. The gfp reporter gene plasmids presented here for the model zygomycete A. glauca constitute the first reliable system that allows the monitoring of gene expression in this important group of fungi.     

Effect of mutations in a simian virus 40 PolyA signal enhancer on green fluorescent protein reporter gene expression

Our previous studies have shown that tandem Alu repeats inhibit green fluorescent protein (GFP) gene expression when inserted downstream of the GFP gene in the pEGFP-C1 vector. We found that the 22R sequence (5'-GTGAAAAAAATGCTTTATTTGT-3') from the antisense PolyA (240 bp polyadenylation signal) of simian virus 40, eliminated repression of GFP gene expression when inserted between the GFP gene and the Alu repeats. The 22R sequence contains an imperfect palindrome; based on RNA structure software prediction, it forms an unstable stem-loop structure, including a loop, a first stem, a bulge, and a second stem. Analysis of mutations of the loop length of the 22R sequence showed that the three-nucleotide loop (wild-type, 22R) induced much stronger GFP expression than did other loop lengths. Two mutations, 4TMI (A7→T, A17→T) and 5AMI (A6→T, T18→A), which caused the base type changes in the bulge and in the second stem in the 22R sequence, induced stronger GFP gene expression than 22R itself. Mutation of the bulge base (A17→T), leading to complete complementation of the stem, caused weaker GFP gene expression. Sequences without a palindrome (7pieA, 5'-GTGAAAAAAATG CAAAAAAAGT-3', 7pieT, 5'-GTGTTTTTTTTGCTTTTTTTGT-3') did not activate GFP gene expression. We conclude that an imperfect palindrome affects and can increase GFP gene expression.     

Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker

Background

The purpose of this study was to compare the effects of 0.5 fraction of inspired oxygen (FiO₂) and >0.95 FiO₂ on pulmonary gas exchange, shunt fraction and oxygen delivery (DO₂) in dorsally recumbent horses during inhalant anesthesia. The use of 0.5 FiO₂ has the potential to reduce absorption atelectasis (compared to maximal FiO₂) and augment alveolar oxygen (O₂) tensions (compared to ambient air) thereby improving gas exchange and DO₂. Our hypothesis was that 0.5 FiO₂ would reduce ventilation-perfusion mismatching and increase the fraction of pulmonary blood flow that is oxygenated, thus improving arterial oxygen content and DO₂.

Results

Arterial partial pressures of O₂ were significantly higher than preanesthetic levels at all times during anesthesia in the >0.95 FiO₂ group. Arterial partial pressures of O₂ did not change from preanesthetic levels in the 0.5 FiO₂ group but were significantly lower than in the >0.95 FiO₂ group from 15 to 90 min of anesthesia. Alveolar to arterial O₂ tension difference was increased significantly in both groups during anesthesia compared to preanesthetic values. The alveolar to arterial O₂ tension difference was significantly higher at all times in the >0.95 FiO₂ group compared to the 0.5 FiO₂ group. Oxygen delivery did not change from preanesthetic values in either group during anesthesia but was significantly lower than preanesthetic values 10 min after anesthesia in the 0.5 FiO₂ group. Shunt fraction increased in both groups during anesthesia attaining statistical significance at varying times. Shunt fraction was significantly increased in both groups 10 min after anesthesia but was not different between groups. Alveolar dead space ventilation increased after 3 hr of anesthesia in both groups.

Conclusions

Reducing FiO₂ did not change alveolar dead space ventilation or shunt fraction in dorsally recumbent, mechanically ventilated horses during 3 hr of isoflurane anesthesia. Reducing FiO₂ in dorsally recumbent isoflurane anesthetized horses does not improve oxygenation or oxygen delivery.     

Secretory fluorescent protein,a secretion green fluorescent fusion protein with alkaline phosphatase activity as a sensitive and traceable reporter in baculovirus expression system

The advantages of using traceable fluorescent protein (enhanced green fluorescent protein; EGFP) and a secretory alkaline phosphatase (SEAP) have been used to generate a reporter gene: the secretory fluorescent protein (SEFP). Sf21 cells, infected with the recombinant baculovirus containing the SEFP gene, revealed both traceable fluorescence and easily detectable alkaline phosphatase activity in the culture medium. The distribution of SEFP within the cells revealed that it was excluded from the nucleus, implying that the accumulation of SEFP in a secretory pathway, similar to that of the secretion signal-tagged FPs. Furthermore, the time- and dose-dependent release from the blockage of brefeldin A (BFA) confirmed that the secretion of SEFP was mediated by the secretion pathway and excluded leakage from viral infection. This SEFP reporter gene with traceable fluorescence and alkaline phosphatase activity may become a useful tool for studies on secretory protein production.     

Detection of mutation in transgenic CHO cells using green fluorescent protein as a reporter

A novel approach was developed for rapidly estimating the frequency of specific mutations in genetically engineered Chinese hamster ovary (CHO) cells. We designed double-transgenic CHO cell lines that contain a transgene consisting of the sequence coding for green fluorescent protein under the control of a tetracycline (Tet) responsive promoter and a second transgene coding for the constitutively expressed Tet repressor. Cultures of these CHO cells were treated with gamma-radiation, N-methyl-N-nitrosourea or methyl methanesulfonate, and the fluorescence of individual cells from both control and treated cultures was measured by flow cytometry. The treatments increased the number of highly fluorescent cells, those with presumed mutations in the Tet-repressor gene. Mutant cells from gamma-radiation-exposed cultures were isolated by fluorescence-activated cell sorting, cultured, and individual clones expanded. A PCR-based analysis indicated that the highly fluorescent expanded cells had lost the transgene coding for the Tet repressor, suggesting that the system mainly detects large genetic alterations. A similar approach may be useful for making high-throughput in vivo models for mutation detection.     

Predictive and interpretive simulation of green fluorescent protein expression in reporter bacteria.

We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P(A1/O4/O3) in response to increasing concentrations of the inducer IPTG (isopropyl-beta-D-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P(A1/O4/O3). Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfp fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.     

Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae.

A manganese peroxidase gene (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3' untranslated region of the glucoamylase gene of Aspergillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies.