Libraries of green fluorescent protein fusions generated by transposition in vitro

Two artificial transposons have been constructed that carry a gene encoding Green Fluorescent Protein and can be used for generating libraries of GFP fusions in a gene of interest. One such element, AT2GFP, can be used to generate GFP insertions in frame with the amino acid sequence of the protein of interest, with a stop codon at the end of the GFP coding sequence; AT2GFP also contains a selectable marker that confers trimethoprim resistance in bacteria. The second element, GS, can be used to generate tribrid GFP fusions because there is no stop codon in the GFP transposon, and the resulting fusion proteins contain the entire amino acid sequence encoded by the gene. The GS element consists of a gfp open reading frame and a supF amber suppressor tRNA gene; the supF portion of the GS transposon can be utilized as a selectable marker in bacteria. Its sequence contains a fortuitous open reading frame, and thus it can be translated continuously with the gfp amino acid sequence. As a target for GFP insertions, we used a plasmid carrying the native Ty1 retrotransposon of the yeast Sacharomyces cerevisiae. The resulting multiple GFP fusions to Ty1 capsid protein Gag and Ty1 integrase were useful in determining the cellular localization of these proteins. Libraries of GFP fusions generated by transposition in vitro represent a novel and potentially powerful method to study the cell distribution and cellular localization signals of proteins.     

Translational control and target recognition by Escherichia coli small RNAs in vivo

Small non-coding RNAs (sRNAs) are an emerging class of regulators of bacterial gene expression. Most of the regulatory Escherichia coli sRNAs known to date modulate translation of trans-encoded target mRNAs. We studied the specificity of sRNA target interactions using gene fusions to green fluorescent protein (GFP) as a novel reporter of translational control by bacterial sRNAs in vivo. Target sequences were selected from both monocistronic and polycistronic mRNAs. Upon expression of the cognate sRNA (DsrA, GcvB, MicA, MicC, MicF, RprA, RyhB, SgrS and Spot42), we observed highly specific translation repression/activation of target fusions under various growth conditions. Target regulation was also tested in mutants that lacked Hfq or RNase III, or which expressed a truncated RNase E (rne701). We found that translational regulation by these sRNAs was largely independent of full-length RNase E, e.g. despite the fact that ompA fusion mRNA decay could no longer be promoted by MicA. This is the first study in which multiple well-defined E.coli sRNA target pairs have been studied in a uniform manner in vivo. We expect our GFP fusion approach to be applicable to sRNA targets of other bacteria, and also demonstrate that Vibrio RyhB sRNA represses a Vibrio sodB fusion when co-expressed in E.coli.     

Green fluorescent protein (GFP) fusion constructs in gene therapy research

The history of green fluorescent protein (GFP) as a marker is less than 10 years old, but it has already made a major impact on many areas of natural sciences, especially on cell biology and histochemistry. GFP can be detected in living cells without selection or staining and it can be fused to other proteins to yield fluorescent chimeras. The potential of GFP has also been recognised by gene therapy researchers and various GFP-tagged therapeutic proteins have been constructed. These chimeric proteins have been used to determine the expression level, site and time course of the therapeutic gene, or the correlation between gene transfer rate and therapeutic outcome. This review summarises the status of the applications of GFP fusions in gene therapy research.     

Differential localisation of GFP fusions to cytoskeleton-binding proteins in animal,plant, and yeast cells. Green-fluorescent protein

The structure and functioning of the cytoskeleton is controlled and regulated by cytoskeleton-associated proteins. Fused to the green-fluorescent protein (GFP), these proteins can be used as tools to monitor changes in the organisation of the cytoskeleton in living cells and tissues in different organisms. Since the localisation of a specific cytoskeleton protein may indicate a particular function for the associated cytoskeletal element, studies of cytoskeleton-binding proteins fused to GFP may provide insight into the organisation and functioning of the cytoskeleton. In this article, we focused on two animal proteins, human T-plastin and bovine tau, and studied the distribution of their respective GFP fusions in animal COS cells, plant epidermal cells (Allium cepa), and yeast cells (Saccharomyces cerevisiae). Plastin-GFP localised preferentially to membrane ruffles, lamellipodia and focal adhesion points in COS cells, to the actin filament cytoskeleton within cytoplasmic strands in onion epidermal cells, and to cortical actin patches in yeast cells. Thus, in these 3 very different types of cells plastin-GFP associated with mobile structures in which there are high rates of actin turnover. Chemical fixation was found to drastically alter the distribution of plastin-GFP. Tau-GFP bound to microtubules in COS cells and onion epidermal cells but failed to bind to yeast microtubules. Thus, animal and plant microtubules appear to have a common tau binding site which is absent in yeast. We conclude that the study of the distribution patterns of microtubule- and actin-filament-binding proteins fused to GFP in heterologous systems should be a valuable tool in furthering our knowledge about cytoskeleton function in eukaryotic cells.     

High-throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata

A strategy was developed for the high-throughput localization of unknown expressed proteins in Nicotiana benthamiana. Libraries of random, partial cDNAs fused to the 5' or 3' end of the gene for green fluorescent protein (GFP) were expressed in planta using a vector based on Tobacco mosaic virus. Viral populations were screened en masse on inoculated leaves using a confocal microscope fitted with water-dipping lenses. Each viral infection site expressed a unique cDNA-GFP fusion, allowing several hundred cDNA-GFP fusions to be screened in a single day. More than half of the members of the library carrying cDNA fusions to the 5' end of gfp that expressed fluorescent fusion proteins displayed discrete, noncytosolic, subcellular localizations. Nucleotide sequence determination of recovered cDNA sequences and subsequent sequence searches showed that fusions of GFP to proteins that had a predicted subcellular "address" became localized with high fidelity. In a subsequent screen of >20,000 infection foci, 12 fusion proteins were identified that localized to plasmodesmata, a subcellular structure for which very few protein components have been identified. This virus-based system represents a method for high-throughput functional genomic study of plant cell organelles and allows the identification of unique proteins that associate with specific subcompartments within organelles.     

Reconstructing evolutionary graphs: 3D parsimony

The increasing recognition that symbioses have greatly altered evolution through genome fusions is creating a need for algorithms that can reliably detect and reconstruct fusions. Here, we generalize the bootstrappers gambit algorithm (a quartet method) in order to permit it to analyze both bifurcations and fusions under a single mathematical model, and thereby detect past genomic branchings and endosymbioses. This new method, 3-dimensional parsimony, can be applied to aligned sequences, such as gene, indel, or other genomic presence/absence sequences. It also provides a statistical measure of support for each possible graph. The usefulness of this method is demonstrated by applying it to the ring of life.     

Efficient PCR-based gene targeting with a recyclable marker for Aspergillus nidulans

The rapid accumulation of genomic sequences from a large number of eukaryotes, including numerous filamentous fungi, has created a tremendous scientific potential, which can only be realized if precise site-directed genome modifications, like gene deletions, promoter replacements, in-frame GFP fusions and specific point mutations can be made rapidly and reliably. The development of gene-targeting techniques in filamentous fungi and other higher eukaryotes has been hampered because foreign DNA is predominantly integrated randomly into the genome. For Aspergillus nidulans, we have developed a flexible method for gene-targeting employing a bipartite gene-targeting substrate. This substrate is made solely by PCR, which obviates the need for bacterial subcloning steps. The method reduces the number of false positives and can be used to produce virtually any genome alteration. A major advance of the method is that it allows multiple subsequent genome manipulations to be performed as the selectable marker is recycled.     

GFP为报告基因的酵母哺乳动物细胞穿梭载体的构建及其在人血管内皮细胞中的表达

为研究酵母作为载体在口服基因治疗及免疫中的作用 ,需要一种能够在酵母中复制而在哺乳动物细胞中表达的穿梭载体 .利用通用载体质粒融合系统 (UPS)构建了一种以GFP为报告基因的新载体 ,以常规的氯化锂法对酿酒酵母进行转化 ,证明该载体能够在酵母中复制 ;以脂质体介导向人血管内皮细胞进行了转染 ,有绿色荧光 ,证明该载体能够在哺乳动物细胞中表达 .所获得的新型的穿梭载体为口服酵母在基因治疗中的应用提供了物质准备     

New transposons to generate GFP protein fusions in Candida albicans

Transposon elements are important tools for gene function analysis, for example they can be used to easily create genome-wide collections of insertion mutants. Transposons may also carry sequences coding for an epitope or fluorescent marker useful for protein expression and localization analysis. We have developed three new Tn5-based transposons that incorporate a GFP (green fluorescent protein) coding sequence to generate fusion proteins in the important fungal pathogen Candida albicans. Each transposon also contains the URA3 and Kan(R) genes for yeast and bacterial selection, respectively. After in vitro transposition, the insertional allele is transferred to the chromosomal locus by homologous recombination. Transposons Tn5-CaGFP and Tn5-CaGFP-URA3::FLIP can generate C-terminal truncated GFP fusions. A URA3 flipper recycling cassette was incorporated into the transposon Tn5-CaGFP-URA3::FLIP. After the induction of Flip recombinase to excise the marker, the heterozygous strain is transformed again in order to obtain a GFP-tagged homozygous strains. In the Tn5-CaGFP-FL transposon the markers are flanked by a rare-cutting enzyme. After in vitro transposition into a plasmid-borne target gene, the markers are eliminated by restriction digestion and religation, resulting in a construct coding for full-length GFP-fusion proteins. This transposon can generate plasmid libraries of GFP insertions in proteins where N- or C-terminal tagging may alter localization. We tested our transposon system by mutagenizing the essential septin CDC3 gene. The results indicate that the Cdc3 C-terminal extension is important for correct septin filament assembly. The transposons described here provide a new system to obtain global gene expression and protein localization data in C. albicans.     

In vivo functional characterization of a yeast nucleotide sequence: construction of a mini-Mu derivative adapted to yeast

We have constructed a derivative of the bacteriophage Mu (called MudIIZZ1), which contains the lacZ gene coding for beta-galactosidase (beta Gal) and markers suited for yeast transformation (2 mu circle replication origin and LEU2). This new transposon is an efficient tool for studying the expression of cloned yeast nucleotide sequences through beta Gal-protein fusions. It is also adapted for one-step disruption experiments so that a functional map of the same sequence can be drawn. We have used this MudIIZZ1 transposon to study a 5-kb DNA fragment which had been cloned by complementation of a cold-sensitive respiration-deficient phenotype. By testing the expression of the beta Gal fusions and the disruption phenotype, we have confirmed the presence of a gene required for mitochondrial functions, and revealed another two open reading frames in the same fragment; one of these also interferes with mitochondrial biogenesis. The method is fast and reliable, and has potential for more general purposes which are discussed.     

Effect of an amyloidogenic sequence attached to yellow fluorescent protein

Green fluorescent protein (GFP) is often misfolded into nonfluorescent states when an aggregatable sequence is attached to its N-terminus. However, GFP fusions with highly aggregatable, prion-determining, and highly charged sequences from yeast prions, such as Sup35 and Ure2p, form green fibrils with properly folded GFP. To gain further insight into the general effect of an aggregatable sequence attached to fluorescent protein, we designed eight fusion proteins of a yellow variant of GFP (YFP) containing an aggregation-prone amyloidogenic sequence derived from human medin, attached via different lengths of linker sequence. Seven fusion proteins formed white fibrils lacking native YFP function. However, the fusion with an 18-residue medin sequence and a 50 amino acid linker formed fibrils with yellow color of folded YFP. Deconvolution analysis of infrared spectra also supports the presence of properly folded YFP in the fibrils formed by this protein. These results suggest that, the presence of an amyloidogenic sequence to a folded protein can promote the formation of fibrils and disrupt the native structures whereas the structure of the folded region is retained by optimizing sequences of amyloidogenic and linker regions.     

Secretion and surface display of green fluorescent protein using the yeast Saccharomyces cerevisiae

Green fluorescent protein (GFP) continues to be a very useful tool in biotechnology, but soluble production of GFP and GFP-protein fusions has been difficult. In this study, we have produced yeast-enhanced green fluorescent protein (yEGFP) in Saccharomyces cerevisiae as a soluble, secreted product with a purified level of 6 mg/L. Expression was directed by the inducible GAL1-10 promoter and synthetic prepro leader sequence. The secretion of yEGFP by yeast was strongly dependent on temperature, with 20 degrees C induction being optimal. Use of 2 micro multicopy expression constructs elevated yields over a low-copy CEN-based system by approximately 2-fold. Yeast-enhanced GFP was also expressed as a fusion to the Aga2p mating agglutinin in order to test the secretory processing fidelity of yEGFP-protein fusions. When the cell surface anchoring protein, Aga1p, was co-overexpressed with the Aga2p-yEGFP fusion, the Aga2p-yEGFP protein was tethered to the yeast cell surface. Flow cytometry and fluorescence microscopy analysis indicated that the fusion was displayed on the yeast cell surface at high levels. In the absence of high level Aga1p expression, the Aga2p-yEGFP fusion protein was instead secreted in its entirety with no detectable surface display. These findings reveal that yeast is a suitable host for secretion of GFP and GFP-protein fusions and thus could enable a wide range of biochemistry and biotechnology applications.     

Differential localisation of GFP fusions to cytoskeleton-binding proteins in animal, plant, and yeast cells

Summary.  The structure and functioning of the cytoskeleton is controlled and regulated by cytoskeleton-associated proteins. Fused to the green-fluorescent protein (GFP), these proteins can be used as tools to monitor changes in the organisation of the cytoskeleton in living cells and tissues in different organisms. Since the localisation of a specific cytoskeleton protein may indicate a particular function for the associated cytoskeletal element, studies of cytoskeleton-binding proteins fused to GFP may provide insight into the organisation and functioning of the cytoskeleton. In this article, we focused on two animal proteins, human T-plastin and bovine tau, and studied the distribution of their respective GFP fusions in animal COS cells, plant epidermal cells (Allium cepa), and yeast cells (Saccharomyces cerevisiae). Plastin-GFP localised preferentially to membrane ruffles, lamellipodia and focal adhesion points in COS cells, to the actin filament cytoskeleton within cytoplasmic strands in onion epidermal cells, and to cortical actin patches in yeast cells. Thus, in these 3 very different types of cells plastin-GFP associated with mobile structures in which there are high rates of actin turnover. Chemical fixation was found to drastically alter the distribution of plastin-GFP. Tau-GFP bound to microtubules in COS cells and onion epidermal cells but failed to bind to yeast microtubules. Thus, animal and plant microtubules appear to have a common tau binding site which is absent in yeast. We conclude that the study of the distribution patterns of microtubule- and actin-filament-binding proteins fused to GFP in heterologous systems should be a valuable tool in furthering our knowledge about cytoskeleton function in eukaryotic cells. Received January 12, 2002; accepted March 7, 2002; published online June 24, 2002 RID="*" ID="*" Correspondence and reprints (present address): Institute of Botany, University of Bonn, Kirschallee 1, 53115 Bonn, Federal Republic of Germany. Abbreviation: smRS-GFP soluble modified red-shifted GFP.     

Functional analysis of mitochondrial protein import in yeast

In order to facilitate studies on protein localization to and sorting within yeast mitochondria, we have designed an experimental system that utilizes a new vector and a functional assay. The vector, which we call an LPS plasmid (for leader peptide substitution), employs a yeast COX5a gene (the structural gene for subunit Va of the inner membrane protein complex cytochrome c oxidase) as a convenient reporter for correct mitochondrial localization. Using in vitro mutagenesis, we have modified COX5a so that the DNA sequences encoding the wild-type subunit Va leader peptide can be precisely deleted and replaced with a given test sequence. The substituted leader peptide can then be analyzed for its ability to direct subunit Va to the inner mitochondrial membrane (to target and sort) by complementation or other in vivo assays. In this study we have tested the ability of several heterologous sequences to function in this system. The results of these experiments indicate that a functional leader peptide is required to target subunit Va to mitochondria. In addition, leader peptides, or portions thereof, derived from proteins located in other mitochondrial compartments can also be used to properly localize this polypeptide. The results presented here also indicate that the information necessary to sort subunit Va to the inner mitochondrial membrane does not reside in the leader peptide but rather in the mature subunit Va sequence.     

Cellular localization of choline-utilization proteins in Streptococcus pneumoniae using novel fluorescent reporter systems

The molecular mechanisms underlying cell growth, cell division and pathogenesis in Streptococcus pneumoniae are still not fully understood. Single-cell methodologies are potentially of great value to investigate S. pneumoniae cell biology. Here, we report the construction of novel plasmids for single and double cross-over integration of functional fusions to the gene encoding a fast folding variant of the green fluorescent protein (GFP) into the S. pneumoniae chromosome. We have also established a zinc-inducible system for the fine control of gfp -fusion gene expression and for protein depletion experiments in S. pneumoniae . Using this novel single cell toolkit, we have examined the cellular localization of the proteins involved in the essential process of choline decoration of S. pneumoniae teichoic acid. GFP fusions to LicA and LicC, enzymes involved in the activation of choline, showed a cytoplasmic distribution, as predicted from their primary sequences. A GFP fusion to the choline importer protein LicB showed clear membrane localization. GFP fusions to LicD1 and LicD2, enzymes responsible for loading of teichoic acid subunits with choline, are also membrane-associated, even though both proteins lack any obvious membrane spanning domain. These results indicate that the decoration of teichoic acid by the LicD enzymes is a membrane-associated process presumably occurring at lipid-linked teichoic acid precursors.     

Insights into ATP synthase assembly and function through the molecular genetic manipulation of subunits of the yeast mitochondrial enzyme complex

Development of an increasingly detailed understanding of the eucaryotic mitochondrial ATP synthase requires a detailed knowledge of the stoichiometry, structure and function of F(0) sector subunits in the contexts of the proton channel and the stator stalk. Still to be resolved are the precise locations and roles of other supernumerary subunits present in mitochondrial ATP synthase complexes, but not found in the bacterial or chloroplast enzymes. The highly developed system of molecular genetic manipulation available in the yeast Saccharomyces cerevisiae, a unicellular eucaryote, permits testing for gene function based on the effects of gene disruption or deletion. In addition, the genes encoding ATP synthase subunits can be manipulated to introduce specific amino acids at desired positions within a subunit, or to add epitope or affinity tags at the C-terminus, enabling questions of stoichiometry, structure and function to be addressed. Newly emerging technologies, such as fusions of subunits with GFP are being applied to probe the dynamic interactions within mitochondrial ATP synthase, between ATP synthase complexes, and between ATP synthase and other mitochondrial enzyme complexes.     

Topology of membrane insertion in vitro and plasma membrane assembly in vivo of the yeast arginine permease

We have examined the topology of the yeast arginine permease, a plasma-membrane protein with multiple membrane-spanning domains. Using fusions of the permease with the glycosylatable secreted yeast protein, acid phosphatase, we identified membrane-spanning sequences that can translocate adjacent acid phosphatase across the membrane of the endoplasmic reticulum (ER), as measured by in vitro glycosylation. Examination for the presence or absence of glycosylation in a systematic series of such fusions gave an internally consistent model for the lumenal or cytoplasmic disposition of the acid phosphatase reporter, defining the topology of the permease. The phenotypes of a further set of arginine permease gene fusions with portions of the gene for the secreted protein, killer toxin, suggest that the pathways of export of membrane and secreted proteins need not be functionally distinct.     

The protein import pore Tom40 in the microsporidian Nosema bombycis

Microsporidia, an unusual group of unicellular parasites related to fungi, possess a highly reduced mitochondrion known as the mitosome. Since mitosomes lack an organellar genome, their proteins must be translated in the cytosol before being imported into the mitosome via translocases. We have identified a Tom40 gene (NbTom40), the main component of the translocase of the outer mitochondrial membrane, in the genome of the microsporidian Nosema bombycis. NbTom40 is reduced in size, but it is predicted to form a β-barrel structure composed of 19 β-strands. Phylogenetic analysis confirms that NbTom40 forms a clade with Tom40 sequences from other species, distinct from a related clade of voltage-dependent anion channels (VDACs). The NbTom40 contains a β-signal motif that the polar residue is substituted by glycine. Furthermore, we show that expression of NbTom40, as a GFP fusion protein within yeast cells, directs GFP to mitochondria of yeast. These findings suggest that NbTom40 may serve as an import channel of the microsporidian mitosome and facilitate protein translocation into this organelle.