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21.
A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells 总被引:17,自引:12,他引:5 下载免费PDF全文
Epinat JC Arnould S Chames P Rochaix P Desfontaines D Puzin C Patin A Zanghellini A Pâques F Lacroix E 《Nucleic acids research》2003,31(11):2952-2962
Homologous gene targeting is the ultimate tool for reverse genetics, but its use is often limited by low efficiency. In a number of recent studies, site- specific DNA double-strand breaks (DSBs) have been used to induce efficient gene targeting. Engineering highly specific, dedicated DNA endonucleases is the key to a wider usage of this technology. In this study, we present two novel, chimeric meganucleases, derived from homing endonucleases. The first one is able to induce recombination in yeast and mammalian cells, whereas the second cleaves a novel (chosen) DNA target site. These results are a first step toward the generation of custom endonucleases for the purpose of targeted genome engineering. 相似文献
22.
Morin A Huysveld N Braun F Dimova D Sakanyan V Charlier D 《Journal of molecular biology》2003,332(3):537-553
The degree of sequence conservation of arginine repressor proteins (ArgR) and of the cognate operators (tandem pairs of 18 bp imperfect palindromes, ARG boxes) in evolutionarily distant bacteria is unusually high, and the global mechanism of ArgR-mediated regulation appears to be similar. However, here we demonstrate that the arginine repressor from the hyperthermophilic bacterium Thermotoga neapolitana (ArgR(Tn)) exhibits characteristics that clearly distinguish this regulator from the well-studied homologues from Escherichia coli, Bacillus subtilis and B.stearothermophilus. A high-resolution contact map of ArgR(Tn) binding to the operator of the biosynthetic argGHCJBD operon of Thermotoga maritima indicates that ArgR(Tn) establishes all of its strong contacts with a single ARG box-like sequence of the operator only. Protein array and electrophoretic mobility-shift data demonstrate that ArgR(Tn) has a remarkable capacity to bind to arginine operators from Gram-negative and Gram-positive bacteria, and to single ARG box-bearing targets. Moreover, the overall effect of L-arginine on the apparent K(d) of ArgR(Tn) binding to various cognate and heterologous operator fragments was minor with respect to that observed with diverse bacterial arginine repressors. We demonstrate that this unusual behaviour for an ArgR protein can, to a large extent, be ascribed to the presence of a serine residue at position 107 of ArgR(Tn), instead of the highly conserved glutamine that is involved in arginine binding in the E.coli repressor. Consistent with these results, ArR(Tn) was found to behave as a superrepressor in E.coli, inhibiting growth in minimal medium, even supplemented with arginine, whereas similar constructs bearing the S107Q mutant allele did not inhibit growth. We assume that ArgR(Tn), owing to its broad target specificity and its ability to bind single ARG box sequences, might play a more general regulatory role in Thermotoga 相似文献
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24.
X-ray diffraction characterization of the dense phases formed by nucleosome core particles 下载免费PDF全文
Multiple dense phases of nucleosome core particles (NCPs) were formed in controlled ionic conditions (15-160 mM monovalent salt, no divalent ions), under osmotic pressures ranging from 4.7 x 10(5) to 2.35 x 10(6) Pa. We present here the x-ray diffraction analysis of these phases. In the lamello-columnar phase obtained at low salt concentration (<25 mM), NCPs stack into columns that align to form bilayers, kept separated from one another by a layer of solvent. NCPs form a monoclinic lattice in the plane of the bilayer. For high salt concentration (>50 mM), NCPs order into either a two-dimensional columnar hexagonal phase or into three-dimensional orthorhombic (quasi-hexagonal) crystals. The lamellar and hexagonal (or quasi-hexagonal) organizations coexist in the intermediate salt range; their demixing requires a long time. For an applied pressure P = 4.7 10(5) Pa, the calculated NCPs concentration ranges from approximately 280 to 320 mg/ml in the lamello-columnar phase to 495 to 585 mg/ml in the three-dimensional orthorhombic phase. These concentrations cover the concentration of the living cell. 相似文献
25.
Several investigators, including ourselves, have reported lower yield of GM-CSF bone marrow-derived dendritic cells (DC) with altered MHC class II and co-stimulatory molecules expression in the non-obese diabetic (NOD) mice. However, whether this defect was intrinsic to the DC lineage and/or related to abnormal expansion of other cell types responding to GM-CSF remained an opened issue. We performed phenotypical and morphological analysis of cells from GM-CSF-supplemented-bone marrow-cultures and of freshly isolated bone marrow and blood cells from unmanipulated prediabetic NOD mice. The results show a heretofore undescribed bias towards generation of granulocytes in NOD mice, concomitant with quantitative and qualitative alterations of the DC lineage in both the bone marrow and the blood of this mouse strain. We propose that increased generation of granulocytes in NOD mice might contribute to autoimmunity. First, high numbers of granulocytes per se might favor inflammatory environment. Second, granulocytes, by interfering with DC development, might favor unbalanced antigen presenting cell function leading to T cell autoimmunity. 相似文献
26.
Fuentes AM Díaz-Mejía JJ Maldonado-Rodríguez R Amábile-Cuevas CF 《FEMS microbiology letters》2001,205(2):271-275
When Escherichia coli cells are under superoxide stress, proteins SoxR and SoxS, acting sequentially, control the expression of a set of repair and defense genes. One of these genes, fumC, encoding fumarase C, was reported to be also activated by iron deprivation in a soxRS-dependent manner. However, the same condition failed to induce the expression of a soxS'::lacZ fusion. The expression of acnA (aconitase A) is also activated by SoxR alone when under iron deprivation, but not of sodA (Mn-superoxide-dismutase). SoxR completely inhibited the migration of a DNA fragment containing the promoter region of fumC, in gel-shift experiments. SoxR might bind to a different region than SoxS within the fumC promoter, or an unknown intermediate other than SoxS might be acting. It is possible that the regulatory role of SoxR is more complex than previously considered. 相似文献
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28.
Two new iminodiacetyl-hydroxamate derivatives, the N-benzyl-N-carboxymethyl-iminoacetohydroxamic acid (H(2)L(1)) and the N-benzyl-N'-hydroxypiperazine-2,6-dione (HL(2)), have been recently reported as very effective inhibitors against a set of zinc-containing matrix metalloproteinases (MMPs). Herein, aimed at understanding that inhibitory function, these compounds are studied in their complex formation equilibria with three biologically relevant first-row transition M(2+) metal ions (M=Cu, Zn, Ni) by using potentiometric and spectroscopic techniques. At physiological conditions, complexation of these metal ions by H(2)L(1) mostly occurs with formation of 1:1 species by tridentate co-ordination (O,N,N) (carboxylate-amino-hydroxamate), whereas complexation with HL(2) mainly involves the formation of 1:2 (M:L) species with normal (O,O) hydroxamate coordination. Moreover, at higher pH, H(2)L(1) is able to form a pentanuclear tetrameric copper complex with an interesting 12-metallacrown-4 structure. 相似文献
29.
The aim of this work was to design a biodegradable delivery system for oligonucleotides providing both a sustained release and an improved intracellular penetration. To this purpose oligonucleotide/polyethylenimine (ON/PEI) complexes at nitrogen to phosphate (N/P) molar ratios of about 15 or 40 were encapsulated into poly(lactide-co-glycolide) microspheres by the multiple emulsion-solvent evaporation technique. ON/PEI complexes were efficiently entrapped inside microspheres. The introduction of salts within the external aqueous phase allowed an improvement of microsphere characteristics. In particular, the use of sodium chloride led to a reduced microsphere porosity and a more homogeneous ON distribution inside the polymeric matrix. These effects were attributed to the reduced flux of water from the external aqueous phase toward the internal aqueous droplets, due to the osmotic effect of sodium chloride. Both, the reduced porosity and the improved ON distribution inside the matrix, were considered responsible for the lower burst effect and the slower ON release rate from microsphere prepared with sodium chloride. ON/PEI complexes encapsulated inside microspheres were also protected toward enzymatic degradation in fetal calf serum. Interestingly, ON/PEI complexes slowly released from microspheres efficiently penetrated inside HeLa cells and oligonucleotides were preferentially located in the nucleus. 相似文献
30.
Santos MA Marques S Gil M Tegoni M Scozzafava A Supuran CT 《Journal of enzyme inhibition and medicinal chemistry》2003,18(3):233-242
A series of succinyl hydroxamates/bishydroxamates as well as a new structural type of matrix metalloproteinase (MMP)/bacterial protease (BP) inhibitors, incorporating iminodiacetic (IDA) hydroxamate/bishydroxamate moieties, has been synthesized and tested for interaction with four vertebrate proteases, MMP-1, MMP-2, MMP-8 and MMP-9, and a BP, the collagenase isolated from Clostridium histolyticum (ChC). The new derivatives generally showed inhibition constants in the range of 8-62 nM against the five proteases mentioned above. 相似文献