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1.
Transgenic carnations obtained byAgrobacterium tumefciens-mediated transformation of leaf explants 总被引:1,自引:0,他引:1
Anne-Claire van Altvorst Tjitske Riksen Herma Koehorst Hans J. M. Dons 《Transgenic research》1995,4(2):105-113
For the development of anAgrobacterium-mediated transformation procedure of carnation (Dianthus caryophyllus L.), an intron-containing -glucuronidase (gus) gene was used to monitor the frequency of transformation events soon after infection of leaf explants. The efficiency of gene transfer was dependent on the carnation genotype, explant age and cocultivation time. Leaf explants from the youngest leaves showed the highest number of GUS-positive spots. After selection on a kanamycin-containing medium, transgenic shoots were generated among a relatively high number of untransformed shoots. The selection procedure was modified in such a way that the contact between explant and medium was more intense. This improved the selection and decreased the number of escapes. Kanamycin-resistant and GUS-positive plants were obtained from five cultivars after infection of leaf explants with the supervirulentAgrobacterium strain AGLO. A higher transformation frequency was observed with the binary vector pCGN7001 than with the p35SGUSint vector. Integration of the genes into the carnation genome was demonstrated by Southern blot hybridization. The number of incorporated T-DNA insertions varied between independent transformants from one to eight. Transformants were morphologically identical to untransformed plants. Segregation of the genes occurred in a Mendelian way. 相似文献
2.
Anne-Claire van Altvorst Herma Koehorst Jan de Jong Hans J. M. Dons 《Plant Cell, Tissue and Organ Culture》1996,45(2):169-173
Transgenic carnation plants were obtained after infection of petal explants with the supervirulent Agrobacterium tumefaciens strain AGLO. Southern blot techniques confirmed the transgenic nature of four transformed plants. The expression of the gus gene was verified in these plants by histochemical assays on selected shoots. It was very difficult to transfer the transgenic plants to the greenhouse due to vitrification and premature flowering.Abbreviations BA
6-benzyladenine
- GA3
gibberellic acid
-
gus-
glucuronidase gene
- MS
Murashige and Skoog (1962)
- NAA
naphthaleneacetic acid
-
nptII
neomycin phosphotransferase II gene
- PCR
polymerase chain reaction 相似文献
3.
4.
Karen?van Eunen Catharina?M.?L.?Volker-Touw Albert?Gerding Aycha?Bleeker Justina?C.?Wolters Willemijn?J.?van Rijt Anne-Claire?M.?F.?Martines Klary?E.?Niezen-Koning Rebecca?M.?Heiner Hjalmar?Permentier Albert?K.?Groen Terry?G.?J.?Derks Barbara?M.?BakkerEmail author 《BMC biology》2016,14(1):107
Background
Defects in genes involved in mitochondrial fatty-acid oxidation (mFAO) reduce the ability of patients to cope with metabolic challenges. mFAO enzymes accept multiple substrates of different chain length, leading to molecular competition among the substrates. Here, we combined computational modeling with quantitative mouse and patient data to investigate whether substrate competition affects pathway robustness in mFAO disorders.Results
First, we used comprehensive biochemical analyses of wild-type mice and mice deficient for medium-chain acyl-CoA dehydrogenase (MCAD) to parameterize a detailed computational model of mFAO. Model simulations predicted that MCAD deficiency would have no effect on the pathway flux at low concentrations of the mFAO substrate palmitoyl-CoA. However, high concentrations of palmitoyl-CoA would induce a decline in flux and an accumulation of intermediate metabolites. We proved computationally that the predicted overload behavior was due to substrate competition in the pathway. Second, to study the clinical relevance of this mechanism, we used patients’ metabolite profiles and generated a humanized version of the computational model. While molecular competition did not affect the plasma metabolite profiles during MCAD deficiency, it was a key factor in explaining the characteristic acylcarnitine profiles of multiple acyl-CoA dehydrogenase deficient patients. The patient-specific computational models allowed us to predict the severity of the disease phenotype, providing a proof of principle for the systems medicine approach.Conclusion
We conclude that substrate competition is at the basis of the physiology seen in patients with mFAO disorders, a finding that may explain why these patients run a risk of a life-threatening metabolic catastrophe.5.
6.
Eric Girard Stéphane Marchal Stéphanie Finet Roger Fourme Anne-Claire Dhaussy Marion Giffard Françoise Bonneté Jacques H. Abraini Nathalie Colloc'h 《Biophysical journal》2010,98(10):2365-2373
Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms. 相似文献
7.
The THAP domain: a novel protein motif with similarity to the DNA-binding domain of P element transposase 总被引:5,自引:0,他引:5
Roussigne M Kossida S Lavigne AC Clouaire T Ecochard V Glories A Amalric F Girard JP 《Trends in biochemical sciences》2003,28(2):66-69
We have identified a novel evolutionarily conserved protein motif - designated the THAP domain - that defines a new family of cellular factors. We have found that the THAP domain presents striking similarities with the site-specific DNA-binding domain (DBD) of Drosophila P element transposase, including a similar size, N-terminal location, and conservation of the residues that define the THAP motif, such as the C2CH signature (Cys-Xaa(2-4)-Cys-Xaa(35-50)-Cys-Xaa(2)-His). Our results suggest that the THAP domain is a novel example of a DBD that is shared between cellular proteins and transposases from mobile genomic parasites. 相似文献
8.
Gaudin F Nasreddine S Donnadieu AC Emilie D Combadière C Prévot S Machelon V Balabanian K 《PloS one》2011,6(7):e21546
Background
Little is known about the molecules that contribute to the growth of epithelial ovarian carcinomas (EOC), which remain the most lethal gynecological cancer in women. The chemokine Fractalkine/CX3CL1 has been widely reported to play a biologically relevant role in tumor growth and spread. We report here the first investigation of the expression and role of CX3CL1 in EOC.Results
Epithelial cells from the surface of the ovary and the Fallopian tubes and from benign, borderline and malignant tumors all stained positive for CX3CL1. In tumor specimens from 54 women who underwent surgical treatment for EOC diagnosis, CX3CL1 immunoreactivity was unevenly distributed in epithelial tumor cells, and ranged from strong (33%) to absent (17%). This uneven distribution of CX3CL1 did not reflect the morphological heterogeneity of EOC. It was positively correlated with the proliferation index Ki-67 and with GILZ (glucocorticoid-induced leucine zipper), previously identified as an activator of the proliferation of malignant EOC cells. Hierarchical clustering analysis, including age at diagnosis, tumor grade, FIGO stage, Ki-67 index, CX3CL1, SDF-1/CXCL12 and GILZ immunostaining scores, distinguished two major clusters corresponding to low and high levels of proliferation and differing in terms of GILZ and CX3CL1 expression. GILZ overexpression in the carcinoma-derived BG1 cell line resulted in parallel changes in CX3CL1 products. Conversely, CX3CL1 promoted through its binding to CX3CR1 AKT activation and proliferation in BG1 cells. In a mouse subcutaneous xenograft model, the overexpression of GILZ was associated with higher expression of CX3CL1 and faster tumor growth.Conclusion
Our findings highlight the previously unappreciated constitutive expression of CX3CL1 preceding tumorigenesis in ovarian epithelial cells. Together with GILZ, this chemokine emerges as a regulator of cell proliferation, which may be of potential clinical relevance for the selection of the most appropriate treatment for EOC patients. 相似文献9.
Soulet F Bailly K Roga S Lavigne AC Amalric F Bouche G 《The Journal of biological chemistry》2005,280(27):25604-25610
Fibroblast growth factor 2 (FGF-2) has been detected in the nuclei of many tissues and cell lines. Here we demonstrate that FGF-2 added exogenously to NIH3T3 cells enters the nucleus and interacts with the nuclear active 90-kDa ribosomal S6 kinase 2 (RSK2) in a cell cycle-dependent manner. By using purified proteins, FGF-2 is shown to directly interact through two separate domains with two RSK2 domains on both sides of the hydrophobic motif, namely the NH2-terminal kinase domain (residues 360-381) by amino acid Ser-117 and the COOH-terminal kinase domain (residues 388-400) by amino acids Leu-127 and Lys-128. Moreover, this interaction leads to maintenance of the sustained activation of RSK2 in G1 phase of the cell cycle. FGF-2 mutants (FGF-2 S117A, FGF-2 L127A, and FGF-2 K128A) that fail to interact in vitro with RSK2 fail to maintain a sustained RSK2 activity in vivo. 相似文献