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GFP Fusion Proteins (gfp + fusion_protein)
Selected AbstractsTsr,GFP accumulates linearly with time at cell poles, and can be used to differentiate ,old' versus ,new' poles, in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 6 2008Liyan Ping Summary In Escherichia coli, the chemotaxis receptor protein Tsr localizes abundantly to cell poles. The current study, utilizing a Tsr,GFP fusion protein and time-lapse fluorescence microscopy of individual cell lineages, demonstrates that Tsr accumulates approximately linearly with time at the cell poles and that, in consequence, more Tsr is present at the old pole of each cell than at its newborn pole. The rate of pole-localized Tsr accumulation is large enough that old and new poles can always be reliably distinguished, even for cells whose old poles have had only one generation to accumulate signal. Correspondingly, Tsr,GFP can be reliably used to assign new and old poles to any cell without use of information regarding pole heritage, thus providing a useful tool to analyse cells whose prior history is not available. The absolute level of Tsr,GFP at the old pole of a cell also provides a rough estimate of pole (and thus cell) age. [source] Cellular location and temperature-dependent assembly of IncHI1 plasmid R27-encoded TrhC-associated conjugative transfer protein complexesMOLECULAR MICROBIOLOGY, Issue 3 2001Matthew W. Gilmour Conjugal transfer of IncHI plasmid DNA between Gram-negative bacteria is temperature sensitive, as mating is optimal between 22°C and 30°C but is inhibited at 37°C. R27, isolated from Salmonella enterica serovar Typhi, is an IncHI1 plasmid of 180 kbp that has been sequenced completely. The gene encoding green fluorescent protein (GFP) was inserted into R27 in frame with trhC. TrhC is a mating pair formation (Mpf) protein that is essential for plasmid transfer and H-pilus production. Fluorescence microscopy allowed visualization of the TrhC,GFP fusion protein, and Escherichia coli cells were examined for the subcellular localization and temperature-dependent production of TrhC,GFP. At 27°C, TrhC,GFP was found at the periphery of cells as discrete foci, indicating an association of TrhC within protein complexes in the bacterial cell membrane, whereas at 37°C, little fluorescence was detected. These foci probably represent the intracellular position of protein complexes involved in conjugative transfer, as the formation of foci was dependent upon the presence of other Mpf proteins. During temperature shift experiments from 37°C to 27°C, a long lag period was required for generation of GFP foci. Conversely, during short shifts from 27°C to 37°C, the GFP foci remained stable. These results suggest that the expression of transfer genes in the Tra2 region of R27 is temperature dependent. Subcellular localization of TrhC was verified by cellular fractionation. Expression patterns of TrhC,GFP were confirmed with immunoblot analysis and reverse transcriptase,polymerase chain reaction (RT,PCR). These results allow us to propose mechanisms to explain the temperature-sensitive transfer of R27. [source] Presentation of functional organophosphorus hydrolase fusions on the surface of Escherichia coli by the AIDA-I autotransporter pathwayBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2008Chaokun Li Abstract We report, the surface presentation of organophosphorus hydrolase (OPH) and green fluorescent protein (GFP) fusions by employing the adhesin-involved-in-diffuse-adherence (AIDA-I) translocator domain as a transporter and anchoring motif. The surface location of the OPH,GFP fusion protein was confirmed by immunofluorescence microscopy, and protease accessibility, followed by Western blotting analysis. The investigation of growth kinetics and stability of resting cultures showed that the presence of the AIDA-I translocator domain in the outer membrane neither inhibits cell growth nor affects cell viability. Furthermore, the surface-exposed OPH,GFP was shown to have enzymatic activity and a functional fluorescence moiety. These results suggest that AIDA-I autotransporter is a useful tool to present heterologous macromolecule passenger proteins on the bacterial surface. Our strategy of linking GFP to OPH and the possibility to employ various bacterial species as host has enormous potential for enhancing field use. Biotechnol. Bioeng. 2008;99: 485,490. © 2007 Wiley Periodicals, Inc. [source] Identification of the CAB2/hCOS16 Gene Required for the Repair of DNA Double-strand Breaks on a Core Amplified Region of the 17ql2 Locus in Breast and Gastric CancersCANCER SCIENCE, Issue 11 2002Masahiko Nezu We previously reported that CAB1 and c -ERBB-2 genes were found to be located in a core amplified region of the 17q12 locus, which is frequently amplified in various cancers. During identification of this core region, CAB2, a human homologue of the yeast COS16 required for the repair of DNA double-strand breaks was cloned. Autofluorescence analysis of cells transfected with its GFP fusion protein demonstrated that CAB2 translocates into vesicles, suggesting that overexpression of CAB2 may decrease intercellular Mn2+ by accumulating it in the vesicles, in the same way as yeast COS16. This is the first report identifying all of the genes on the core amplified region of the 17q12 locus in breast and gastric cancers. [source] Yeast Saccharomyces cerevisiae has two cis -prenyltransferases with different properties and localizations.GENES TO CELLS, Issue 6 2001Implication for their distinct physiological roles in dolichol synthesis Background Dolichol is a family of long-chain polyprenols, which is utilized as a sugar carrier in protein glycosylation in the endoplasmic reticulum (ER). We have identified a key enzyme of the dolichol synthesis, cis -prenyltransferase, as Rer2p from Saccharomyces cerevisiae. We have also isolated a multicopy suppressor of an rer2 mutant and named it SRT1. It encodes a protein similar to Rer2p but its function has not been established. Results The cis -prenyltransferase activity of Srt1p has been proved biochemically in the lysate of yeast cells lacking Rer2p. The polyprenol product of Srt1p is longer in chain length than that of Rer2p and is not sufficiently converted to dolichol and dolichyl phosphate, unlike that of Rer2p. The subcellular localization of these two isozymes has been examined by immunofluorescence microscopy and by the use of GFP fusion proteins. Whereas GFP-Rer2p is localized to the continuous ER and some dots associated with the ER, GFP-Srt1p shows only punctate localization patterns. Immunofluorescence double staining with Erg6p, a marker of lipid particles in yeast, indicates that Srt1p is mainly localized to lipid particles (lipid bodies). RER2 is mainly expressed in the early logarithmic phase, while the expression of SRT1 is induced in the stationary phase. Conclusions We have shown that yeast has two active cis -prenyltransferases with different properties. This result implies that the two isozymes have different physiological roles during the life cycle of the yeast. [source] Changing transcriptional initiation sites and alternative 5,- and 3,-splice site selection of the first intron deploys Arabidopsis PROTEIN ISOASPARTYL METHYLTRANSFERASE2 variants to different subcellular compartmentsTHE PLANT JOURNAL, Issue 1 2008Randy D. Dinkins Summary Arabidopsis thaliana (L.) Heynh. possesses two PROTEIN-L-ISOASPARTATE METHYLTRANSFERASE (PIMT) genes encoding enzymes (EC 2.1.1.77) capable of converting uncoded l -isoaspartyl residues, arising spontaneously at l -asparaginyl and l -aspartyl sites in proteins, to l -aspartate. PIMT2 produces at least eight transcripts by using four transcriptional initiation sites (TIS; resulting in three different initiating methionines) and both 5,- and 3,-alternative splice site selection of the first intron. The transcripts produce mature proteins capable of converting l -isoaspartate to l -aspartate in small peptide substrates. PIMT:GFP fusion proteins generated a detectable signal in the nucleus. However, whether the protein was also detectable in the cytoplasm, endo-membrane system, chloroplasts, and/or mitochondria, depended on the transcript from which it was produced. On-blot-methylation of proteins, prior to the completion of germination, indicated that cruciferin subunits contain isoaspartate. The implications of using transcriptional mechanisms to expand a single gene's repertoire to protein variants capable of entry into the cell's various compartments are discussed in light of PIMT's presumed role in repairing the proteome. [source] Function of plastidial pyruvate kinases in seeds of Arabidopsis thaliana,THE PLANT JOURNAL, Issue 3 2007Sébastien Baud Summary Pyruvate kinase (PK) catalyses the irreversible synthesis of pyruvate and ATP, which are both used in multiple biochemical pathways. These compounds are essential for sustained fatty acid production in the plastids of maturing Arabidopsis embryos. Using a real-time quantitative reverse transcriptase (RT)-PCR approach, the three genes encoding putative plastidial PKs (PKps) in Arabidopsis, namely PKp1 (At3g22960), PKp2 (At5g52920) and PKp3 (At1g32440), were shown to be ubiquitously expressed. However, only PKp1 and PKp2 exhibited significant expression in maturing seeds. The activity of PKp1 and PKp2 promoters was consistent with this pattern, and the study of the PKp1:GFP and PKp2:GFP fusion proteins confirmed the plastidial localization of these enzymes. To further investigate the function of these two PKp isoforms in seeds comprehensive functional analyses were carried out, including the cytological, biochemical and molecular characterization of two pkp1 and two pkp2 alleles, together with a pkp1pkp2 double mutant. The results obtained outlined the importance of these PKps for fatty acid synthesis and embryo development. Mutant seeds were depleted of oil, their fatty acid content was drastically modified, embryo elongation was retarded and, finally, seed germination was also affected. Together, these results provide interesting insights concerning the carbon fluxes leading to oil synthesis in maturing Arabidopsis seeds. The regulation of this metabolic network by the WRINKLED1 transcription factor is discussed, and emphasizes the role of plastidial metabolism and the importance of its tight regulation. [source] Identification and characterization of a new conserved motif within the presequence of proteins targeted into complex diatom plastidsTHE PLANT JOURNAL, Issue 2 2005Oliver Kilian Summary Several groups of algae evolved by secondary endocytobiosis, which is defined as the uptake of a eukaryotic alga into a eukaryotic host cell and the subsequent transformation of the endosymbiont into an organelle. Due to this explicit evolutionary history such algae possess plastids that are surrounded by either three or four membranes. Protein targeting into plastids of these organisms depends on N-terminal bipartite presequences consisting of a signal and a transit peptide domain. This suggests that different protein targeting systems may have been combined during establishment of secondary endocytobiosis to enable the transport of proteins into the plastids. Here we demonstrate the presence of an apparently new type of transport into diatom plastids. We analyzed protein targeting into the plastids of diatoms and identified a conserved amino acid sequence motif within plastid preprotein targeting sequences. We expressed several diatom plastid presequence:GFP fusion proteins with or without modifications within that motif in the diatom Phaeodactylum tricornutum and found that a single conserved phenylalanine is crucial for protein transport into the diatom plastids in vivo, thus indicating the presence of a so far unknown new type of targeting signal. We also provide experimental data about the minimal requirements of a diatom plastid targeting presequence and demonstrate that the signal peptides of plastid preproteins and of endoplasmic reticulum-targeted preproteins in diatoms are functionally equivalent. Furthermore we show that treatment of the cells with Brefeldin A arrests protein transport into the diatom plastids suggesting that a vesicular transport step within the plastid membranes may occur. [source] Differential regulation of TGA transcription factors by post-transcriptional controlTHE PLANT JOURNAL, Issue 5 2002Dominique Pontier Summary Transcription factors often belong to multigene families and their individual contribution in a particular regulatory network remains difficult to assess. We show here that specific members from a family of conserved Arabidopsis bZIP transcription factors, the TGA proteins, are regulated in their protein stability by developmental stage-specific proteolysis. Using GFP fusions of three different Arabidopsis TGA factors that represent members of distinct subclasses of the TGA factor family, we demonstrate that two of these TGA proteins are specifically targeted for proteolysis in mature leaf cells. Using a supershift gel mobility assay, we found evidence for similar regulation of the cognate proteins as compared to the GFP fusion proteins expressed under the cauliflower mosaic virus (CaMV) 35S promoter. Using various inhibitors, we showed that the expression of at least one of these three TGA factors could be stabilized by inhibition of proteasome-mediated proteolysis. This study indicates that TGA transcription factors may be regulated by distinct pathways of targeted proteolysis that can serve to modulate the contribution of specific members of a multigene family in complex regulatory pathways. [source] | ||||||||||