No (200)
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Image Analysis of Gene Locus Positions Within Chromosome Territories in Human Lymphocytes
Stepka, K.; Falk, Martin
2014 - English
One of the important areas of current cellular research with substantial impacts on medicine is analyzing the spatial organization of genetic material within the cell nuclei. Higher-order chromatin structure has been shown to play essential roles in regulating fundamental cellular processes, like DNA transcription, replication, and repair. In this paper, we present an image analysis method for the localization of gene loci with regard to chromosomal territories they occupy in 3D confocal microscopy images. We show that the segmentation of the territories to obtain a precise position of the gene relative to a hard territory boundary may lead to undesirable bias in the results, instead, we propose an approach based on the evaluation of the relative chromatin density at the site of the gene loci. This method yields softer, fuzzier ´boundaries´, characterized by progressively decreasing chromatin density. The method therefore focuses on the extent to which the signals are located inside the territories, rather than a hard yes/no classification.
Keywords:
selection method; deletion
Available at various institutes of the ASCR
Image Analysis of Gene Locus Positions Within Chromosome Territories in Human Lymphocytes
One of the important areas of current cellular research with substantial impacts on medicine is analyzing the spatial organization of genetic material within the cell nuclei. Higher-order chromatin ...
P36 VOLTAMETRIC DETECTION OF DNA DELETION ON PENCIL ELECTRODE
Haroniková, Lucia; Špaček, Jan; Fojta, Miroslav
2014 - English
In this work, we present a new qualitative approach of detection of PCR products using electrochemistry on pencil electrodes. PCR products with an incorporated biotin-labeled dNTP (dCTP in this study) are detected via conjugated streptavidinealkaline phospatase. 1-Naphthyl phosphate (1-NP), which is dephosphorylated by alkaline phosphatase to release 1-naphthol, was used as a substrate in electrochemical detection of the PCR product. Voltammetric measurement on disposable pencil electrode is a very cheap and easy to use method. The system was optimized for plasmid DNA PCR product with potential to detect human genome DNA products and possible application in gene deletion monitoring.
Keywords:
DNA
Available at various institutes of the ASCR
P36 VOLTAMETRIC DETECTION OF DNA DELETION ON PENCIL ELECTRODE
In this work, we present a new qualitative approach of detection of PCR products using electrochemistry on pencil electrodes. PCR products with an incorporated biotin-labeled dNTP (dCTP in this study) ...
Detection of Single Nucleotide Polymorphisms Using Selective Incorporation of Biotin in DNA Strand and Subsequent Enzymatic Detection at Pencil Electrode
Plucnara, Medard; Ecsin, E.; Erdem, A.; Fojta, Miroslav
2014 - English
Enzyme-linked electrochemical assay using DNA labeling by biotin followed by binding of enzyme-streptavidin conjugates has been found to be a potent tool for DNA diagnostic. This approach brings a special advantage of signal amplification due to the fact, that only very small number of enzymatic labels can produce a number of molecules of an electrochemically detectable product from an inactive substrate to obtain sufficiently strong signal. A new way of using of this technique combined with selective primer extension reaction designed for the detection of single nucleotide polymorphism is presented here. The assay was combined with measurements at a pencil graphite electrode, which is a very practical tool for potential clinical applications due to its cheapness and disposability.
Keywords:
Single nucleotide polymorphism; Enzymatic labeling; Pencil graphite electrode
Available at various institutes of the ASCR
Detection of Single Nucleotide Polymorphisms Using Selective Incorporation of Biotin in DNA Strand and Subsequent Enzymatic Detection at Pencil Electrode
Enzyme-linked electrochemical assay using DNA labeling by biotin followed by binding of enzyme-streptavidin conjugates has been found to be a potent tool for DNA diagnostic. This approach brings a ...
TAIL-LABELED OLIGONUCLEOTIDE PROBES FOR A DUAL ELECTROCHEMICAL MAGNETIC IMMUNOPRECIPITATION ASSAY OF DNA-PROTEIN BINDING
Hermanová, Monika; Špaček, Jan; Orság, Petr; Fojta, Miroslav
2014 - English
A novel assay for detection of DNA-protein binding has been developed. Oligonucleotides bearing or lacking specific binding site of the p53 protein were tail-labeled by two different modified deoxynucleotide triphosphates using terminal deoxynucleotidyl transferase. Electrochemical detection enabled to discriminate between sequence-specific and non-specific p53-DNA binding in a competition assay.
Keywords:
DNA-PROTEIN
Available at various institutes of the ASCR
TAIL-LABELED OLIGONUCLEOTIDE PROBES FOR A DUAL ELECTROCHEMICAL MAGNETIC IMMUNOPRECIPITATION ASSAY OF DNA-PROTEIN BINDING
A novel assay for detection of DNA-protein binding has been developed. Oligonucleotides bearing or lacking specific binding site of the p53 protein were tail-labeled by two different modified ...
A Preliminary Study of Modification of 7-Deazaadenine with a Complex of Osmium Tetroxide with 2,2 '-Bipyridine
Vítová, Lada; Havran, Luděk; Fojta, Miroslav; Šedo, O.; Zdráhal, Z.; Vespalec, Radim
2014 - English
Reactivity of a purine nucleic base analogue 7-deazaadenine (A*) in short oligodeoxynucleotide (ODN) towards an osmium tetroxide complex with 2,2'-bipyridine (Os, bipy) was investigated by means of electrochemistry, capillary electrophoresis and MALDI-TOF mass spectrometry. Electrochemical measurement, electrophoretic analyses and mass spectrometric analyses of reaction mixture proved that ODN with an A* residue yields an osmium adduct with properties similar to those exhibited by the well-known adduct of thymine.
Keywords:
dna; bases; Electrochemical analysis
Available at various institutes of the ASCR
A Preliminary Study of Modification of 7-Deazaadenine with a Complex of Osmium Tetroxide with 2,2 '-Bipyridine
Reactivity of a purine nucleic base analogue 7-deazaadenine (A*) in short oligodeoxynucleotide (ODN) towards an osmium tetroxide complex with 2,2'-bipyridine (Os, bipy) was investigated by means of ...
Magnetic Beads-Based Electrochemical Techniques for DNA-Protein Interaction Monitoring
Fojta, Miroslav; Pivoňková, Hana; Němcová, Kateřina; Horáková Brázdilová, Petra; Havran, Luděk; Orság, Petr; Vidláková, Pavlína; Macíčková-Cahová, Hana; Balintová, Jana; Hocek, Michal
2011 - English
Electrochemical techniques, in connection with separation of nucleoprotein complexes at magnetic beads, are suitable for the monitoring of DNA-protein interactions. For the detection of complexes captured at the beads it is possible to utilize intrinsic electrochemical activity of the protein, intrinsic structure-selective signals of the DNA, or indicator DNA substrates tail-labeled with electroactive moieties.
Keywords:
DNA-protein interaction; Electrochemistry; Magnetic beads
Available at various institutes of the ASCR
Magnetic Beads-Based Electrochemical Techniques for DNA-Protein Interaction Monitoring
Electrochemical techniques, in connection with separation of nucleoprotein complexes at magnetic beads, are suitable for the monitoring of DNA-protein interactions. For the detection of complexes ...
Using Anthraquinone Labeled Nucleotide Triphosphates for Electrochemical Analysis of DNA
Vidláková, Pavlína; Balintová, Jana; Horáková Brázdilová, Petra; Havran, Luděk; Orság, Petr; Fojta, Miroslav; Hocek, Michal
2011 - English
Base-modified anthraquinone-nucleotide triphosphates were used for DNA labeling. Anthraquinone modified nucleotides were incorporated into DNA by primer extension with KOD polymerase. In cyclic and square wave voltammetry at HMDE or PGE antraquinone gave a well developed characteristic signal suitable for differentiation of the labeled DNA from unlabeled one.
Keywords:
DNA; Anthraquinone; Electrochemical analysis
Available at various institutes of the ASCR
Using Anthraquinone Labeled Nucleotide Triphosphates for Electrochemical Analysis of DNA
Base-modified anthraquinone-nucleotide triphosphates were used for DNA labeling. Anthraquinone modified nucleotides were incorporated into DNA by primer extension with KOD polymerase. In cyclic and ...
Colocalization of PML bodies and PML/RARalpha microspeckles with up- and down-regulated loci and changes of chromatin structure in APL leukemia cells
Falk, Martin; Lukášová, Emilie; Faretta, M.; Dellino, I.; Kozubek, Stanislav; Pellici, G. I.; Kozubek, Michal; Rochi, M.
2004 - English
Acute promyelocytic leukemia (APL) is associated with the reciprocal translocation between PML and RARa genes; however, the mechanism of its pathogenesis is still not well understood. In this article we demonstrate that PML/RARa fusion protein colocalizes with particular chromosomal loci containing clusters of genes downregulated by this protein. Binding of PML/RARa to those loci is consequently followed by local chromatin contraction. Treatment of leukemic cells with retinoic acid (ATRA) leads to reconstruction of PML bodies and reverts chromatin condensation to original value in healthy cells. Therefore we propose and discuss the mechanistic model of downregulation in APL based on the strong attraction of histone deacethylases (HDAC) to downregulated loci by the PML/RARa fusion chimera. Akutní promyelocytární leukémie (APL) je asociována s reciprokou translokací mezi geny PML a RARa; mechanismus patogeneze této choroby není nicméně doposud objasněn. V tomto článku ukazujeme, že fúzní protein PML/RARa kolokalizuje v jádře s lokusy obsahujícími klastry genů, umlčenými tímto proteinem, přičemž vazba PML/RARa k těmto lokusům je následně doprovázena lokální kontrakcí chromatinu. Léčba leukemických buněk pomocí all-trans kyseliny retinové (ATRA) vede naopak k rekonstituci PML tělísek a dekondenzaci chromatinu na původní hodnoty pozorované u zdravých buněk. Na základě těchto výsledků navrhujeme a diskutujeme mechanistický model utlumení genové exprese u APL, spočívající v silné vazbě histonových deacethylas (HDAC) k umlčovaným lokusům prostřednictvím represivního komplexu stabilizovaného fúzní chimérou PML/RARa.
Keywords:
acute promyelocytic leukemia; PML bodies; higher order chromatin structure
Available at various institutes of the ASCR
Colocalization of PML bodies and PML/RARalpha microspeckles with up- and down-regulated loci and changes of chromatin structure in APL leukemia cells
Acute promyelocytic leukemia (APL) is associated with the reciprocal translocation between PML and RARa genes; however, the mechanism of its pathogenesis is still not well understood. In this article ...
In vivo imaging of cell interior using automated high-resolution cytometry
Vařecha, M.; Amrichová, J.; Ondřej, Vladan; Lukášová, Emilie; Kozubek, Stanislav; Matula, Pa.; Matula, Pe.; Kozubek, Michal
2004 - English
The fluorescent proteins are important innovation in the field of cell biology and in vivo experiments that use fluorescent proteins bring new interesting results from a different point of view than in vitro and in situ experiments. This presentation will introduce our automated 2D/3D high-resolution time-lapse image acquisition and analysis of living cells transfected with plasmids encoding fusion proteins or dyed with fluorescence probes. One of our in vivo projects, we will mention, is focused on the study of mitochondrial and nuclear apoptotic processes in living cells. Another in vivo project concentrates on the study of topography of telomeres and incidence of telomere-association phenomenon in various types of human tumor and healthy cells. In our experiments, cells are stably or transiently transfected with plasmid DNAs coding mitochondrial apoptogenic or telomere-binding proteins with fluorescent proteins. Použití fluorescenčních proteinů v buněčné biologii přináší nové poznatky často odlišné od experimentů dosud prováděných pouze in situ. Tato práce představuje metodu studia živých buněk v čase ve 2D i 3D pomocí vysokorozlišovací cytometrie, popisuje také snímání obrazových dat i jejich analýzu. Jako vzorky byly použity živé buňky, jak geneticky obohacené o geny kódující fúzní fluorescenční proteiny, tak obarvené fluorescenčními sondami vhodnými pro živé buňky. Jeden z našich projektů se soustřeďuje na studium mitochondriálních a jaderných apoptotických procesů v živých buňkách a druhý projekt se zabývá studiem topographie telomer a četností telomerických asociací v různých typech lidských nádorových a zdravých buněk. V našich experimentech používáme buňky, které jsou stabilně nebo tranzientně transfekovány plazmidovou DNA kódující mitochondriální apoptotické nebo na telomery se vázající proteiny s připojenými fluorescenčními proteiny.
Keywords:
high-resolution cytometry; fluorescent proteins; in vivo experiments
Available at various institutes of the ASCR
In vivo imaging of cell interior using automated high-resolution cytometry
The fluorescent proteins are important innovation in the field of cell biology and in vivo experiments that use fluorescent proteins bring new interesting results from a different point of view than ...
Dynamics of HP1 transgene loci movement and silencing in living cells
Ondřej, Vladan; Kozubek, Stanislav; Lukášová, Emilie; Matula, Pa.; Matula, Pe.; Kozubek, Michal
2004 - English
Intranuclear localization and mobility of Cy3 labelled transgene loci were studied in living cells. Observations showed occurrence of several transgene copies in each cell nucleus. The majority of copies were localized in the centromeric heterochromatin defined by HPlbeta-GFP fusion protein, the minority of copies in euchromatin. The tracking of loci showed restricted diffusive motion of these in the short-time range. During long-time observation of HPlbeta domains and transgene loci, we have found that in most cases the proximity of these objects decreased within time. The changes of positions of HPl domains were very small but transgene loci displayed directional movement towards the relevant HPl domain. Our data records support the idea that the cell nucleus consists of several separated higher-order compartments. The genes relocate within these compartments, which is finally connected with changes of their expression status. Na živých buňkách byla studována jaderná lokalizace a pohyblivost transgenních lokusů barvených Cy3. Pozorování ukázala výskyt několika kopií transgenů v každém buněčném jádře. Většina kopií byla lokalizována v centrometrickém heterochromatinu definovaném HPlbeta-GFP fúzním proteinem, menšina kopií v euchromatinu. Sledování lokusů prokázalo jejich omezený difúzní pohyb v krátkém časovém rozmezí. Během dlouhodobého sledování HP1beta domén a transgenních lokusů jsme zjistili, že ve většině případů proximita těchto objektů klesá v čase. Změny v pozici HP1 domén byly velmi malé, ale transgenní lokusy vykazovaly pohyb směrem k relevantní HP1 doméně. Naše data podporují myšlenku, že buněčná jádra se skládají z několika oddělených výše organizovaných kompartmentů. Geny se uvnitř těchto kompartmentů přemísťují, což je v konečném důsledku spojeno se změnami jejich exprese.
Keywords:
silencing in living cells; HP1beta; GFP fusion protein
Available at various institutes of the ASCR
Dynamics of HP1 transgene loci movement and silencing in living cells
Intranuclear localization and mobility of Cy3 labelled transgene loci were studied in living cells. Observations showed occurrence of several transgene copies in each cell nucleus. The majority of ...
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