Complex protein targeting to dinoflagellate plastids

Protein trafficking pathways to plastids are directed by N-terminal targeting peptides. In plants this consists of a relatively simple transit peptide, while in organisms with secondary plastids (which reside within the endomembrane system) a signal peptide is appended to the transit peptide. Despite amino acid compositional differences between organisms, often due to nucleotide biases, the features of plastid targeting sequences are generally consistent within species. Dinoflagellate algae deviate from this trend. We have conducted an expressed sequence tag (EST) survey of the peridinin-plastid containing dinoflagellate Heterocapsa triquetra to identify and characterize numerous targeting presequences of plastid proteins encoded in the nucleus. Consistent with targeting systems present in other secondary plastid-containing organisms, these all possess a canonical signal peptide at their N termini, however two major classes of transit peptides occur. Both classes possess a common N-terminal portion of the transit peptide, but one class of transit peptides contains a hydrophobic domain that has been reported to act as a stop-transfer membrane anchor, temporarily arresting protein insertion into the endoplasmic reticulum. A second class of transit peptide lacks this feature. These two classes are represented approximately equally, and for any given protein the class is conserved across all dinoflagellate taxa surveyed to date. This dichotomy suggests that two mechanisms, perhaps even trafficking routes, may direct proteins to dinoflagellate plastids. A four-residue phenylalanine-based motif is also a consistent feature of H. triquetra transit peptides, which is an ancient feature predating red algae and galucophytes that was lost in green plastids.     

Patterns of green fluorescent protein expression in transgenic plants

Modified forms of genes encoding green fluorescent protein (GFP) can be macroscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP visualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing transgenic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice.     

The potato granule bound starch synthase chloroplast transit peptide directs recombinant proteins to plastids

The chloroplast targeting transit sequence from potato granule bound starch synthase (gbss) was used to direct the accumulation of recombinant proteins to the plastid stroma. The potato gbss transit sequence was fused to the N-terminus of the green fluorescent protein (GFP) and the Catharanthus roseus strictosidine synthase (Str1) enzyme. Fluorescence microscopy confirmed that the recombinant gbss-GFP fusion protein was exclusively targeted to the plastid stroma in tobacco suspension cells, demonstrating that the transit sequence was functional in vivo. The Str1 fusion protein accumulated to high levels in plastids isolated from transgenic plants. We conclude that the potato gbss transit sequence is functional and directs import of recombinant proteins into the chloroplast stroma.     

Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny

Several modifications of a wild-type green fluorescent protein (GFP) gene were combined into a single construct, driven by the ubi-1 promoter and intron region, and transformed into maize. Green fluorescence, indicative of GFP expression, was observed in stably transformed callus as well as in leaves and roots of regenerated plants and their progeny. Cell wall autofluorescence made GFP expression difficult to observe in sections of leaves and roots. However, staining sections with toluidine blue allowed detection of GFP in transgenic tissue. Bright GFP fluorescence was observed in approximately 50% of the pollen of transgenic plants. These results suggest that GFP can be used as a reporter gene in transgenic maize; however, further modification, i.e., to alter the emission spectra, would increase its utility. Received: 17 December 1997 / Revision received: 6 March 1998 / Accepted: 20 March 1998     

Evidence for transit peptide acquisition through duplication and subsequent frameshift mutation of a preexisting protein gene in rice

Many proteins synthesized in the cytosol are delivered to their appropriate compartments in the cell by specific targeting signals. Here, we provide new insight into the generation of the chloroplast-targeting signal (called the transit peptide) in rice. First, we identified the mitochondrial ribosomal protein L13 (mt rpl13) gene on chromosome 5. Downstream of the gene, we identified a DNA fragment of 266 bp: a segment within a duplication of mt rpl13. The duplicated region was transcribed and found to encode an open reading frame (ORF) of 160 amino acids (aa) (orf160). The orf160 gene comprises C-terminal 60 aa derived from the mt rpl13 gene and N-terminal 100 aa derived from another duplicated fragment of a pentatricopeptide repeat (ppr)564 gene that encodes 564 aa with ppr motifs on chromosome 1. Examination of the localization of the ORF160 protein tagged with green fluorescent protein (GFP) showed that it is targeted to the chloroplasts. As such, ORF160 clearly contains a transit peptide. Interestingly, this was translated from the alternative reading frame of the duplicated fragment of ppr564. To confirm this, the reading frame of the ppr564 gene was shifted according to that of the orf160 gene, and the frameshifted ppr564 sequence was fused to the gene for GFP. The expressed GFP-fused protein was also located in the chloroplasts. These results provide clear evidence for the generation of the transit peptide through duplication and subsequent frameshifting of a reading frame of a preexisting protein gene. We also demonstrate the importance of sequence redundancy and frameshift mutation in this evolutionary process.     

绿色荧光蛋白GFP基因与新生隐球菌荚膜基因的融合表达系统

目的构建新生隐球菌荚膜基因与绿色荧光蛋白的融合表达系统。方法PCR法扩增CAP60基因片段,测序验证其准确性。将其与多个必需基因共同连人穿梭质粒。结果获得6150bps大小的质粒,该质粒含有荚膜基因启动子、终止子及荧光蛋白的基因。结论将新生隐球菌荚膜基因与荧光蛋白基因融合表达,将会有利于对荚膜的生化合成途径作进一步研究。     

绿色荧光蛋白——照亮生命科学的一盏明灯

绿色荧光蛋白的发现及应用具有划时代的重要意义,它不仅为当代生物学研究提供了极为实用的基本研究手段,并且在此基础上改造发展和发现了一系列荧光蛋白,拓展了应用范围。这使得对微观生物学的研究也可以进入一个时空结合,研究鲜活动态过程的新时代。本文主要回顾总结了绿色荧光蛋白的发现、优化改造及其应用。     

High yield recombinant silk-like protein production in transgenic plants through protein targeting

DP1B is a synthetic analogue of spider dragline silk protein. It can be spun to form silk fiber. Previously, it had been expressed in transgenic plants, showing the general feasibility of the plant-based DP1B production. However, success of such a plant-based platform requires a great increase of DP1B productivity in plant cells to reduce production cost. This report describes a protein targeting approach to accumulate DP1B in apoplast, ER lumen, and vacuole in Arabidopsis cells, by utilizing appropriate combinations of sporamin-targeting determinant peptides and ER retention peptide. The approach has dramatically enhanced DP1B accumulation, resulting in high production yield. The accumulation can be as high as 8.5 and 6.7% total soluble protein in leaf tissue by targeting to apoplast and ER lumen, respectively, or as high as 18 and 8.2% total soluble protein in seeds by targeting to ER lumen and vacuole, respectively. However, the vacuole targeting in leaves and the apoplast targeting in seeds have failed to accumulate full length DP1B molecules or any DP1B at all, respectively, suggesting that they may not be suitable for applications in leaf tissues and seeds. Data in this study recommend a combination of seed-specific expression and ER-targeting as one of the best strategies for yield enhancement of plant-based DP1B production.     

Light-dependent translocation of a phytochrome B-GFP fusion protein to the nucleus in transgenic Arabidopsis

Phytochrome is a ubiquitous photoreceptor of plants and is encoded by a small multigene family. We have shown recently that a functional nuclear localization signal may reside within the COOH-terminal region of a major member of the family, phytochrome B (phyB) (Sakamoto, K., and A. Nagatani. 1996. Plant J. 10:859-868). In the present study, a fusion protein consisting of full-length phyB and the green fluorescent protein (GFP) was overexpressed in the phyB mutant of Arabidopsis to examine subcellular localization of phyB in intact tissues. The resulting transgenic lines exhibited pleiotropic phenotypes reported previously for phyB overexpressing plants, suggesting that the fusion protein is biologically active. Immunoblot analysis with anti-phyB and anti-GFP monoclonal antibodies confirmed that the fusion protein accumulated to high levels in these lines. Fluorescence microscopy of the seedlings revealed that the phyB-GFP fusion protein was localized to the nucleus in light grown tissues. Interestingly, the fusion protein formed speckles in the nucleus. Analysis of confocal optical sections confirmed that the speckles were distributed within the nucleus. In contrast, phyB-GFP fluorescence was observed throughout the cell in dark-grown seedlings. Therefore, phyB translocates to specific sites within the nucleus upon photoreceptor activation.     

Amino terminal interaction in the prion protein identified using fusion to green fluorescent protein

In contrast to the well-characterized carboxyl domain, the amino terminal half of the mature cellular prion protein has no defined structure. Here, following fusion of mouse prion protein fragments to green fluorescence protein as a reporter of protein stability, we report extreme variability in fluorescence level that is dependent on the prion fragment expressed. In particular, exposure of the extreme amino terminus in the context of a truncated prion protein molecule led to rapid degradation, whereas the loss of only six amino terminal residues rescued high level fluorescence. Study of the precise endpoints and residue identity associated with high fluorescence suggested a domain within the amino terminal half of the molecule defined by a long-range intramolecular interaction between 23KKRPKP28 and 143DWED146 and dependent upon the anti-parallel beta-sheet ending at residue 169 and normally associated with the structurally defined carboxyl terminal domain. This previously unreported interaction may be significant for understanding prion bioactivity and for structural studies aimed at the complete prion structure.     

Binding of chimeric metal-binding green fluorescent protein to lipid monolayer

Membrane-based bioanalytical devices for metal determination using green fluorescent protein as the sensor molecule may be a useful future biomimetic material. However, in order to develop such a device, it is necessary first to understand the interaction of the protein with lipid membranes. Thus we have investigated the interaction between chimeric cadmium-binding green fluorescent proteins (CdBPGFPs) and lipid monolayers, using a film-balance technique complemented with epifluorescence microscopy. The binding avidity was monitored from the surface pressure vs. area isotherms or from the measured increase in the lateral pressure upon injection of the chimeric CdBPGFPs beneath the lipid monolayer. Increased fluidization as well as expansion of the surface area were shown to depend on the concentration of the CdBPGFPs. The kinetics of the protein-induced increase in lateral pressure was found to be biphasic. The chimeric CdBPGFPs possessed high affinity to the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer with a dissociation constant of K_d=10^–8M. Epifluorescence measurements showed that this affinity is due to the presence of the Cd-binding peptide, which caused the GFP to incorporate preferentially to the liquid phase and defect part of the rigid domain at low interfacial pressure. At high compression, the Cd-binding peptide could neither incorporate nor remain in the lipid core. However, specific orientation of the chimeric CdBPGFPs underneath the air–water interface was achieved, even under high surface pressure, when the proteins were applied to the metal-chelating lipid-containing surfaces. This specific binding could be controlled reversibly by the addition of metal ions or metal chelator. The reversible binding of the chimeric CdBPGFPs to metal-chelating lipids provided a potential approach for immobilization, orientation and lateral organization of a protein at the membrane interface. Furthermore, the feasibility of applying the chelator lipids for the codetermination of metal ions with specific ligands was also revealed. Our finding clearly demonstrates that a strong interaction, particularly with fluid lipid domains, could potentially be used for sensor development in the future.Abbreviations GFP green fluorescent protein - CdBPGFPs cadmium-binding green fluorescent protein - DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine - AAS atomic absorption spectrometry - Cd²⁺ cadmium (II) - Zn²⁺ zinc (II) - Cu²⁺ copper (II) - Ni²⁺ nickel (II) - E. coli Escherichia coli - NTA-DOGS 1,2-dioleoyl-sn-glycero-3-(N-(5-amino-1-carboxypentyl iminodiacetic acid) succinyl) - His6GFP hexahistidine green fluorescent protein - CdBP4GFP four-repeat cadmium-binding peptide green fluorescent protein - His6CdBP4GFP hexahistidine four-repeat cadmium-binding peptide green fluorescent protein - IMAC immobilized-metal-affinity chromatography - PBS phosphate-buffered saline - mN/m millinewton per metre - le liquid expanded - lc liquid condensed - PE phosphatidyl ethanolamine - PI phosphatidyl inositol - NTA nitrilotriacetic acid - EDTA ethylenediamine tetraacetic acid - RESA ring-infected erythrocyte surface antigen - CdBP cadmium-binding peptide     

异源基因α1,3半乳糖转移酶与增强型绿色荧光蛋白融合对荧光蛋白表达的影响

目的 研究异源(猪)基因α1,3半乳糖转移酶(3GT)与增强型绿色荧光蛋白(EGFP)基因形成的融合蛋白对其荧光表达量的影响.方法 BamHI,EcoRI酶切pcDNA3.1-α1,3GT重组载体后,回收含α1,3GT的片段,与BamHI、EcoRI酶切回收的pEGFP-N1载体连接,并酶切、测序鉴定重组真核表达载体p...     

Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2

The role of the actin cytoskeleton in plant development is intimately linked to its dynamic behavior. Therefore it is essential to continue refining methods for studying actin organization in living plant cells. The discovery of green fluorescent protein (GFP) has popularized the use of translational fusions of GFP with actin filament (F-actin) side-binding proteins to visualize in vivo actin organization in plants. The most recent of these live cell F-actin reporters are GFP fusions to the actin-binding domain 2 (ABD2) of Arabidopsis fimbrin 1 (ABD2-GFP). To improve ABD2-GFP fluorescence for enhanced in vivo F-actin imaging, transgenic Arabidopsis plants were generated expressing a construct with GFP fused to both the C- and N-termini of ABD2 under the control of the CaMV 35S promoter (35S::GFP-ABD2-GFP). The 35S::GFP-ABD2-GFP lines had significantly increased fluorescence compared with the original 35S::ABD2-GFP lines. The enhanced fluorescence of the 35S::GFP-ABD2-GFP-expressing lines allowed the acquisition of highly resolved images of F-actin in different plant organs and stages of development because of the reduced confocal microscope excitation settings needed for data collection. This simple modification to the ABD2-GFP construct presents an important tool for studying actin function during plant development.     

GFP在绿色荧光裸鼠不同组织中的表达及分析

目的研究外源绿色荧光蛋白（green fluorescent protein,简称GFP）基因在BALB/c绿色荧光裸鼠主要器官组织中的表达及其差异。方法小动物成像系统和RT-PCR方法检测GFP的组织分布以及荧光表达水平情况。结果经活体荧光影像系统观察及PCR方法检测发现GFP可以在裸鼠多个器官组织中表达,其中在胰腺、心脏、全脑、皮肤、睾丸中表达量较高。结论外源绿色荧光蛋白可以在模型动物体内成功表达且稳定遗传,其中在胰腺组织中高表达。