In situ multi-wavelength fluorescence spectroscopy as effective tool to simultaneously monitor spore germination, metabolic activity and quantitative protein production in recombinant Aspergillus niger fed-batch cultures

The production of a mutant green fluorescent protein (S65TGFP), controlled by the maltose inducible glucoamylase promoter, was followed in situ in fed-batch cultures of recombinant Aspergillus niger using multi-wavelength fluorescence spectroscopy. Disturbance of quantitative product analysis by interfering fluorescence signals was resolved by using a set of defined combinations of excitation and emission wavelengths (lambda(ex)/lambda(em)). This technique resulted in excellent linearity between on-line signal and off-line determined S65TGFP concentrations. Spore germination was detectable in situ by monitoring the back scattered light intensity. Moreover, flavin-like fluorophores were identified as the dominating fungal host fluorophores. The time-dependent intensity of this fluorophore, potentially fungal flavin-containing oxidoreductase(s), did not correlate with the biomass concentration but correlated well with the fungal metabolic activity (e.g. respiratory activity). Other fluorophores commonly found in microbial cultures such NADH, pyridoxine and the aromatic amino acids, tryptophan, phenylalanine and tyrosine did not contribute significantly to the culture fluorescence of A. niger. Thus, multi-wavelength fluorescence spectroscopy has proven to be an effective tool for simultaneous on-line monitoring of the most relevant process variables in fungal cultures, e.g. spore germination, metabolic activity, and quantitative product formation.     

Endosperm-specific expression of green fluorescent protein driven by the hordein promoter is stably inherited in transgenic barley (Hordeum vulgare) plants

The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic barley (Hordeum vulgare L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by either a rice actin promoter or a barley endosperm-specific d-hordein promoter. The gene encoding phosphinothricin acetyltransferase (bar), driven by the maize ubiquitin promoter and intron, was used as a selectable marker to identify transgenic tissues. Strong GFP expression driven by the rice actin promoter was observed in callus cells and in a variety of tissues of T0 plants transformed with the sgfp(S65T)-containing construct. GFP expression, driven by the rice actin promoter, was observed in 14 out of 17 independent regenerable transgenic callus lines; however, expression was gradually lost in T0 and later generation progeny of diploid lines. Stable GFP expression was observed in T2 progeny from only 6 out of the 14 (43%) independent GFP-expressing callus lines. Four of the 8 lines not expressing GFP in T2 progeny, lost GFP expression during T0 plant regeneration from calli; one lost GFP expression in the transition from the T0 to T1 generations and three lines were sterile. Similarly, expression of bar driven by the maize ubiquitin promoter was lost in T1 progeny; only 21 out of 26 (81%) independent lines were Basta-resistant. In contrast to actin-driven expression, GFP expression driven by the d-hordein promoter exhibited endosperm-specificity. All seven lines transformed with d-hordein-driven GFP (100%) expressed GFP in the T1 and T2 generations, regardless of ploidy levels, and expression segregated in a Mendelian fashion. We conclude that the sgfp(S65T) gene was successfully transformed into barley and that GFP expression driven by the d-hordein promoter was more stable in its inheritance pattern in T1 and T2 progeny than that driven by the rice actin promoter or the bar gene driven by the maize ubiquitin promoter.     

A gfp reporter plasmid to visualize Azorhizobium caulinodans during nodulation of Sesbania rostrata

Compared with other labeling techniques, the use of the green fluorescent protein (GFP) is advantageous to visualize bacteria because observations can be performed in real time. This feature is particularly interesting to study invasion events of rhizobia during nodule development on their legume host plant. To investigate the symbiotic interaction between Azorhizobium caulinodans ORS571 and Sesbania rostrata, we constructed two plasmids, pMP220-hem-gfp5 and pBBR5-hem-gfp5-S65T, that carry a modified gfp gene, the expression of which is controlled by the constitutive hem promoter. Introduction of either of these plasmids into A. caulinodans allowed the visualization of single bacteria. Determination of the plasmid stability in cultured bacteria and in nodules demonstrated that pBBR5-hem-gfp5-S65T is more stable than pMP220-hem-gfp5. The plasmid pBBR5-hem-gfp5-S65T can be used to study early invasion events during nodule development on hydroponic roots of S. rostrata.     

Expression of green fluorescent protein and its inheritance in transgenic oat plants generated from shoot meristematic cultures

The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic oat ( Avena sativa L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by a rice actin promoter. In vitro shoot meristematic cultures (SMCs) induced from shoot apices of germinating mature seeds of a commercial oat cultivar, Garry, were used as a transformation target. Proliferating SMCs were bombarded with a mixture of plasmids containing the sgfp(S65T) gene and one of three selectable marker genes, phosphinothricin acetyltransferase (bar), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase (nptII). Cultures were selected with bialaphos, hygromycin B and geneticin (G418), respectively, to identify transgenic tissues. From 289 individual explants bombarded with the sgfp(S65T) gene and one of the three selectable marker genes, 23 independent transgenic events were obtained, giving a 8.0% transformation frequency. All 23 transgenic events were regenerable, and 64% produced fertile plants. Strong GFP expression driven by the rice actin promoter was observed in a variety of tissues of the T(0) plants and their progeny in 13 out of 23 independent transgenic lines. Stable GFP expression was observed in T(2) progeny from five independent GFP-expressing lines tested, and homozygous plants from two lines were obtained. Transgene silencing was observed in T(0) plants and their progeny of some transgenic lines.     

Real-time flow cytometric quantification of GFP expression and Gfp-fluorescence generation in Saccharomyces cerevisiae

A genetic and analytical methodology was developed based on a green fluorescent mutant protein (Gfp(S65T)) that allows the real-time quantification of gene expression in Saccharomyces cerevisiae. Using the UAS(GAL)(1-10)/CYC1 promoter and plasmids that are maintained in different copy numbers per cell, wild-type GFP and mutant GFP(S65T) were expressed in low to high concentration. Flow cytometric analysis was then applied to directly quantify Gfp((S65T)) (both wild type and mutant protein) expression at the single-cell level, and to indirectly measure the concentrations of non-fluorescent apoGfp((S65T)) and fluorescent Gfp((S65T)), which is autocatalytically formed from the apoprotein. Kinetics of apoGfp((S65T))/Gfp((S65T)) conversion during aerobic growth showed that the time required for complete apoGfp((S65T)) conversion is limited only by the amount of apoprotein that is expressed. When GFP(S65T) was expressed in single copy, the apoprotein did not accumulate and was instantly converted into its fluorescent form. The data indicate that an instant quantification of gene expression in S. cerevisiae is achievable based on Gfp(S65T), even if the gene is transcribed from a very strong promoter.     

Expression and analysis of green fluorescent proteins in human embryonic kidney cells by capillary electrophoresis

The green fluorescent protein (GFP) has attracted much interest as a reporter for gene expression. In this paper, application of capillary electrophoresis with laser-induced fluorescent (CE-LIF) for quantitation of green fluorescence protein in cellular extracts and single cells is investigated. The S65T mutant form of GFP protein was successfully expressed in human embryonic kidney (HEK293) cells, and its production was confirmed by fluorescence microscopy and CE-LIF. The mass limit of detection for the mutant S65T was 5.3 x 10(-20) mol, which was better than that for the wild-type GFP by a factor of six. Detection of a small amount of GFP is difficult by conventional techniques such as fluorescent microscopy due to interference from cell autofluorescence at low GFP concentrations. The HEK293 cells were transfected with the GFP plasmid that produced S65T-GFP. Transient production of S65T protein was detected 2 h after the transfection and reached a maximum after 48 h. The protein concentration began to decrease significantly after 96 h. Single cell analysis of HEK293 cells after transfection with GFP plasmid indicate a nonuniform production of S65T-GFP protein among cells.     

Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.)

Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate “escapes” and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained at frequencies of 3–11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic calli were regularly monitored under the microscope with blue light when they were transferred to media without selective agents. Green fluorescent shoots were obtained at frequencies of 2–5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has potential for the development of selectable marker-free transgenic sugar beet.     

GFP与微管结合蛋白和肌动蛋白结合区融合蛋白载体构建与表达

以pCAMBIA1 30 0、pBA0 0 5、pActl D和pMECA等 4个质粒载体为基础 ,根据不同需要 ,选择特定的限制性内切酶作完全或部分酶切 ,通过多步骤的酶切、连接、转化等过程 ,构建完成 2个可通过农杆菌介导转化的植物表达载体。分别命名为 :pActl GFPTALIN和pActl GFP2 MAP4BD。在洋葱表皮细胞中的瞬间表达结果显示 ,两个载体均可正常工作。     

Assessment of Transient Gene Expression in Plant Tissues Using the Green Fluorescent Protein as a Reference

Four different promoters (35S and enhanced 35S of the cauliflower mosaic virus, polyubiquitin of maize and actin1 of rice) were compared in a transient assay using maize leaves and particle bombardment. A gene encoding the jellyfish green fluorescent protein (GFP) driven by the 35S promoter was used as an internal standard to monitor the effectiveness of each bombardment. Normalisation of the transient expression assay using the GFP reference significantly reduced the variability between separate bombardments and allowed for a rapid and accurate evaluation of different promoters in microprojectile-bombarded leaves.     

Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species.

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and (15)N(2) gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.     

A novel plant nuclear gene encoding chloroplast ribosomal protein S9 has a transit peptide related to that of rice chloroplast ribosomal protein L12

We have cloned a novel nuclear gene for a ribosomal protein of rice and Arabidopsis that is like the bacterial ribosomal protein S9. To determine the subcellular localization of the gene product, we fused the N-terminal region and green fluorescent protein and expressed it transiently in rice seedlings. Localized fluorescence was detectable only in chloroplasts, indicating that this nuclear gene encodes chloroplast ribosomal protein S9. The N-terminal region of rice ribosomal protein S9 was found to have a high sequence similarity to the transit peptide region of the rice chloroplast ribosomal protein L12, suggesting that these transit peptides have a common lineage.     

A novel MaxiK splice variant exhibits dominant-negative properties for surface expression

We identified a novel MaxiK alpha subunit splice variant (SV1) from rat myometrium that is also present in brain. SV1 has a 33-amino acid insert in the S1 transmembrane domain that does not alter S1 overall hydrophobicity, but makes the S0-S1 linker longer. SV1 was transfected in HEK293T cells and studied using immunocytochemistry and electrophysiology. In non-permeabilized cells, N-terminal c-Myc- or C-terminal green fluorescent protein-tagged SV1 displayed no surface labeling or currents. The lack of SV1 functional expression was due to endoplasmic reticulum (ER) retention as determined by colabeling experiments with a specific ER marker. To explore the functional role of SV1, we coexpressed SV1 with the alpha (human SLO) and beta1 (KCNMB1) subunits of the MaxiK channel. Coexpression of SV1 inhibited surface expression of alpha and beta1 subunits approximately 80% by trapping them in the ER. This inhibition seems to be specific for MaxiK channel subunits since SV1 was unable to prevent surface expression of the Kv4.3 channel or to interact with green fluorescent protein. These results indicate a dominant-negative role of SV1 in MaxiK channel expression. Moreover, they reveal down-regulation by splice variants as a new mechanism that may contribute to the diverse levels of MaxiK channel expression in non-excitable and excitable cells.     

Functional correlation between the nuclear localization of Fht1p and its flocculation and heat tolerance activities in budding yeast Saccharomyces cerevisiae

Fht1p is involved in the flocculation and heat tolerance machinery of budding yeast Saccharomyces cerevisiae. Despite knowledge of its involvement in those phenotypes, a precise mechanism has yet to be discovered. To this end, we monitored the relationship between subcellular localization of Fht1p and its flocculation or heat tolerance function using newly developed expression vectors with a recombinant green fluorescent protein (GFP; S65T/S147P) of Aequorea victoria added at both the N- and C-terminus of Fht1p. The main fluorescent signal of the GFP tagged with either a wild-type Fht1p or mutants which preserve their flocculation function was detected in the nucleus, whereas signals of functionless mutants were dispersed to the cytoplasm.     

Structural and spectral response of green fluorescent protein variants to changes in pH

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become a useful tool in molecular and cell biology. Recently, it has been found that the fluorescence spectra of most mutants of GFP respond rapidly and reversibly to pH variations, making them useful as probes of intracellular pH. To explore the structural basis for the titration behavior of the popular GFP S65T variant, we determined high-resolution crystal structures at pH 8.0 and 4.6. The structures revealed changes in the hydrogen bond pattern with the chromophore, suggesting that the pH sensitivity derives from protonation of the chromophore phenolate. Mutations were designed in yellow fluorescent protein (S65G/V68L/S72A/T203Y) to change the solvent accessibility (H148G) and to modify polar groups (H148Q, E222Q) near the chromophore. pH titrations of these variants indicate that the chromophore pKa can be modulated over a broad range from 6 to 8, allowing for pH determination from pH 5 to pH 9. Finally, mutagenesis was used to raise the pKa from 6.0 (S65T) to 7.8 (S65T/H148D). Unlike other variants, S65T/H148D exhibits two pH-dependent excitation peaks for green fluorescence with a clean isosbestic point. This raises the interesting possibility of using fluorescence at this isosbestic point as an internal reference. Practical real time in vivo applications in cell and developmental biology are proposed.     

Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 1. Mutagenesis and structural studies

Wild type green fluorescent protein (wt-GFP) and the variant S65T/H148D each exhibit two absorption bands, A and B, which are associated with the protonated and deprotonated chromophores, respectively. Excitation of either band leads to green emission. In wt-GFP, excitation of band A ( approximately 395 nm) leads to green emission with a rise time of 10-15 ps, due to excited-state proton transfer (ESPT) from the chromophore hydroxyl group to an acceptor. This process produces an anionic excited-state intermediate I* that subsequently emits a green photon. In the variant S65T/H148D, the A band absorbance maximum is red-shifted to approximately 415 nm, and as detailed in the accompanying papers, when the A band is excited, green fluorescence appears with a rise time shorter than the instrument time resolution ( approximately 170 fs). On the basis of the steady-state spectroscopy and high-resolution crystal structures of several variants described herein, it is proposed that in S65T/H148D, the red shift of absorption band A and the ultrafast appearance of green fluorescence upon excitation of band A are due to a very short (相似文献   

Computational study of the absorption spectra of green fluorescent protein mutants

In this work, we present a theoretical study of the relationship between molecular structure and the red-shift in absorption spectra of S65G and S65T green fluorescent protein (GFP) mutants. To identify the effects of the protein environment, we combined results from molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics calculations to obtain structural properties, and applied time-dependent density functional theory to calculate the excitation energies. By using results from the MD simulations, we were able to provide a systematic analysis of the structural details that may effect the red-shift in the absorption spectra when taking into account temperature effects. Furthermore, a detailed study of hydrogen bonding during the MD simulations demonstrated differences between S65G and S65T, for example, regarding hydrogen bonding with Glu222. An analysis of the absorption spectra for different forms of the chromophore emphasized the dominance of the anionic forms in solution for the S65G and S65T GFP mutants.     

Construction of a Sinorhizobium meliloti strain carrying a stable and non-transmissible chromosomal single copy of the green fluorescent protein GFP-P64L/S65T

A single copy of the green fluorescent protein (GFP)-encoding gene gfp-P64L/S65T under the control of the constitutive nptII promoter was introduced in a neutral region of the Sinorhizobium meliloti chromosome, between the genes recA and alaS. Within the same chromosomal region downstream of gfp-P64L/S65T a tetracycline (Tc) resistant cassette was also inserted. Both markers were very stable during at least 40 bacterial generations without any selective pressure. Similarly, the gfp-Tc cassette was stable and functional in all rhizobia that were recovered from alfalfa nodules. The GFP-associated fluorescence derived from the (single copy) chromosomal gfp-P64L/S65T allowed detection of rhizobia during the colonisation of the root, infection thread formation, and nodule development. The gfp-Tc rhizobia showed indistinguishable phenotypes for nodulation, competitiveness, and nitrogen-fixation from the parental strain. The labelling system described here can be used for the stable fluorescent tagging of S. meliloti strains allowing their detection in biologically complex soil environments.