scholarly journals Green fluorescent protein fused to the C terminus of RAD51 specifically interferes with secondary DNA binding by the RAD51-ssDNA complex

2014 ◽  
Vol 89 (4) ◽  
pp. 169-179 ◽  
Author(s):  
Wataru Kobayashi ◽  
Satoshi Sekine ◽  
Shinichi Machida ◽  
Hitoshi Kurumizaka
Keyword(s):  
Green Fluorescent Protein ◽  
Dna Binding ◽  
Fluorescent Protein ◽  
C Terminus ◽  
Green Fluorescent

scholarly journals Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein

2000 ◽  
Vol 74 (23) ◽  
pp. 11339-11346 ◽  
Author(s):  
Vitaly Boyko ◽  
Jessica van der Laak ◽  
Jacqueline Ferralli ◽  
Elena Suslova ◽  
Myoung-Ok Kwon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Inclusion Bodies ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Leading Edge ◽  
Intercellular Transport ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

scholarly journals C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal

1998 ◽  
Vol 336 (2) ◽  
pp. 367-371 ◽  
Author(s):  
Leen AMERY ◽  
Chantal BREES ◽  
Myriam BAES ◽  
Chiaki SETOYAMA ◽  
Retsu MIURA ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Consensus Sequence ◽  
C Terminus ◽  
Targeting Signal ◽  
Fluorescent Pattern ◽  
Green Fluorescent ◽  
Positively Charged

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivoand in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP–PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP–PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.

161 EFFECTS OF LIPOSOMES ON SPERM MOTILITY AND DNA-BINDING EFFICIENCY

2017 ◽  
Vol 29 (1) ◽  
pp. 189
Author(s):  
S. N. Lotti ◽  
M. Rubessa ◽  
R. V. Knox ◽  
M. B. Wheeler
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Dna Binding ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Selection Method ◽  
Computer Assisted ◽  
Exogenous Dna ◽  
Liposome Preparation ◽  
Green Fluorescent

In mice, microinjection is the most common gene transfer method used. Unfortunately, this strategy does not translate as well to livestock. Another potential method is sperm-mediated gene transfer, which takes advantage of sperm’s natural ability to bind to naked DNA. Gene transfer using sperm-mediated gene transfer has been shown in pigs (Gandolfi et al. 1989 J. Reprod. Fert. Abstr. Ser. 4) and cattle (Perez et al. 1991 Biotecnol. Apl. 8, 90–94). Based on these observations, we examined the efficiency of exogenous DNA binding to sperm using liposomes. In this experiment, we analysed methods to select thawed bovine sperm for DNA binding and evaluated the binding of exogenous DNA to those sperm. To determine the optimal sperm-selection method, the sperm were analysed using a computer-assisted sperm analyzer (CASA), the parameters selected were: total motility, rapid motility, and progressive motility. To measure the binding of DNA we used an indirect analysis using NanoDrop technology (Thermo Scientific, Wilmington, DE, USA) to compare the different DNA concentrations among groups. Liposome preparation was done using a cationic lipid, 3-(trimethyl ammonium iodide) 1,2 dimystryl-propanediate and a neutral lipid, l-a Dioleoyl phosphatidyl-ethanolamine prepared according to the protocol of Russell (1997). Percoll or swim-up methods were used to select sperm after thawing (Rubessa et al. 2016), followed by incubation (3 h) with the liposome-DNA complexes according to liposome preparation protocol (Russell, 1997). We used enhanced green fluorescent protein in combination with the liposomes as a marker for exogenous DNA binding. Five treatments per selection method were analysed: 1) immediately after processing (Control), 2) After 3 h of incubation with no liposomes, 3) incubation with liposomes and no DNA, 4) incubation with 1 ng of DNA, and 5) incubation with 10 ng of DNA. This was repeated five times. The CASA results for total motility and rapid motility showed a greater amount of significant differences (P < 0.01) between the control and the other treatments in the Percol group as opposed to swim-up. These results confirm that the sperm selected with swim-up is more stable. Following CASA analysis, sperm was washed with PBS twice and collected in tubes. The DNA from all samples was extracted to determine the quantity of attaching varying amounts of DNA to sperm. The results showed a general increase in DNA concentrations with the increase of DNA added for both methods, but the statistical variation was too large to draw any definite conclusion. In future studies, real-time PCR will be used to determine the quantity of enhanced green fluorescent protein bound to the sperm. Table 1. Results of the computer-assisted sperm analyzer (CASA)

scholarly journals A Truncated Progesterone Receptor (PR-M) Localizes to the Mitochondrion and Controls Cellular Respiration

2013 ◽  
Vol 27 (5) ◽  
pp. 741-753 ◽  
Author(s):  
Qunsheng Dai ◽  
Anish A. Shah ◽  
Rachana V. Garde ◽  
Bryan A. Yonish ◽  
Li Zhang ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Dna Binding ◽  
Progesterone Receptor ◽  
Western Blot ◽  
Mitochondrial Membrane ◽  
Fluorescent Protein ◽  
Cellular Respiration ◽  
Mitochondrial Localization ◽  
Localization Signal ◽  
Green Fluorescent

Abstract The cDNA for a novel truncated progesterone receptor (PR-M) was previously cloned from human adipose and aortic cDNA libraries. The predicted protein sequence contains 16 unique N-terminal amino acids, encoded by a sequence in the distal third intron of the progesterone receptor PR gene, followed by the same amino acid sequence encoded by exons 4 through 8 of the nuclear PR. Thus, PR-M lacks the N terminus A/B domains and the C domain for DNA binding, whereas containing the hinge and hormone-binding domains. In this report, we have localized PR-M to mitochondria using immunofluorescent localization of a PR-M-green fluorescent protein (GFP) fusion protein and in Western blot analyses of purified human heart mitochondrial protein. Removal of the putative N-terminal mitochondrial localization signal obviated association of PR-M with mitochondria, whereas addition of the mitochondrial localization signal to green fluorescent protein resulted in mitochondrial localization. Immunoelectron microscopy and Western blot analysis after mitochondrial fractionation identified PR-M in the outer mitochondrial membrane. Antibody specificity was shown by mass spectrometry identification of a PR peptide in a mitochondrial membrane protein isolation. Cell models of overexpression and gene silencing of PR-M demonstrated a progestin-induced increase in mitochondrial membrane potential and an increase in oxygen consumption consistent with an increase in cellular respiration. This is the first example of a truncated steroid receptor, lacking a DNA-binding domain that localizes to the mitochondrion and initiates direct non-nuclear progesterone action. We hypothesize that progesterone may directly affect cellular energy production to meet the increased metabolic demands of pregnancy.

scholarly journals Sexual Pheromone Induces Diffusion of the Pheromone-Homologous Polypeptide in the Extracellular Matrix of Volvox carteri

2007 ◽  
Vol 6 (11) ◽  
pp. 2157-2162 ◽  
Author(s):  
Koichi Ishida
Keyword(s):  
Extracellular Matrix ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Sexual Pheromone ◽  
Volvox Carteri ◽  
Terminal Domain ◽  
Induction Signal ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT The C-terminal domain of pherophorin II is homologous to the sexual pheromone of Volvox carteri and is released from other domains during sexual induction. Green fluorescent protein fused to the C terminus of pherophorin II was located at the extracellular matrix directly surrounding the gonidium, the final target of the sexual-induction signal.

scholarly journals Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae

2002 ◽  
Vol 363 (3) ◽  
pp. 737-744 ◽  
Author(s):  
Sandra PAIVA ◽  
Arthur L. KRUCKEBERG ◽  
Margarida CASAL
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Reporter Protein ◽  
C Terminus ◽  
Lactate Transporter ◽  
Green Fluorescent ◽  
Endocytic Pathways

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123s−1. It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p—GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p—GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Δdoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.

scholarly journals Functional Regions of the Pseudomonas aeruginosa Cytotoxin ExoU

2005 ◽  
Vol 73 (1) ◽  
pp. 573-582 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Alan R. Hauser
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Phospholipase Activity ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a potent patatin-like phospholipase, causes rapid cell death following its injection into host cells by the Pseudomonas aeruginosa type III secretion system. To better define regions of ExoU required for cytotoxicity, transposon-based linker insertion mutagenesis followed by site-directed mutagenesis of individual residues was employed by using a Saccharomyces cerevisiae model system. Random insertion of five amino acids identified multiple regions within ExoU that are required for cell killing. Five regions were chosen for further characterization: three corresponded to the oxyanion hole, hydrolase motif, and catalytic aspartate motif of the patatin-like domain within the N-terminal half of ExoU; one corresponded to an uncharacterized part of the patatin-like domain; and one corresponded to a region near the C terminus. Specific individual amino acid substitutions in each of the four N-terminal regions prevented killing of yeast and significantly reduced phospholipase activity. Whereas five amino acid insertions in the fifth region near the C terminus markedly reduced cytotoxicity and phospholipase activity, substitution of individual amino acids did not abolish either activity. To determine whether each of the five identified regions of ExoU was also essential for cytotoxicity in human cells, representative mutant forms of ExoU fused to green fluorescent protein were expressed in HeLa cells. These variants of ExoU were readily visualized and caused minimal cytotoxicity to HeLa cells, while wild-type ExoU fused to green fluorescent protein induced significant cell lysis and no detectable fluorescence. Thus, a minimum of five regions, including one which is well removed from the patatin-like domain, are required for the cytotoxicity and phospholipase activity of ExoU.

scholarly journals The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells

2003 ◽  
Vol 372 (2) ◽  
pp. 335-345 ◽  
Author(s):  
Martin E. LIDELL ◽  
Malin E. V. JOHANSSON ◽  
Matthias MÖRGELIN ◽  
Noomi ASKER ◽  
James R. GUM ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Chinese Hamster Ovary ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Chinese Hamster ◽  
Full Length ◽  
Globular Domain ◽  
Wild Type ◽  
C Terminus ◽  
Green Fluorescent

The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine- and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin κ-chain signal sequence to direct the protein to the secretory pathway. Pulse–chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bond-stabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The C-terminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2.

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