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Cytologická studie modelů DNA metylace a metylace histonů u lidských buněčných linií
Skalníková, M. ; Bártová, Eva ; Kozubek, Stanislav ; Kozubek, Michal
Epigenetic processes are defined as heritable changes in genome function that occur without a change in DNA sequence. Gene expression, chromosome segregation, DNA replication, repair, and recombination all act, not on DNA alone, but on the chromatin template. DNA methylation, along with histone lysine methylation, establishes the framework for long-term epigenetic maintenance. The discovery that enzymes can (re)organise chromatin into accessible and inaccessible configurations revealed epigenetic mechanisms that considerably extend the information potential of the genetic code. In mammals, heterochromatin is characterised by DNA methylation at CpG dinucleotides and methylation at lysine 9 of histone 3 (H3-K9), whereas euchromatin is associated with methylation at lysine 4 of histone 3 (H3-K4).
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Klastrování genů s podobným regulačním mechanismem během monocytární a garanulocytární diferenciace buněk HL-60
Koutná, I. ; Faltýsková, E. ; Krontorád, P. ; Svoboda, Z. ; Pavlík, T. ; Bártová, Eva ; Kozubek, Michal ; Kozubek, Stanislav
In the present study, a high density-microarrays of human cDNA containing 19,000 genetic elements were used to search for differences in gene expression of human promyelic leukaemia cell line HL-60 during monocyte (MD) and granulocyte differentiation (GD). For differentiation to monocytes (granulocytes), the TPA, 12-O-tetradecanoylphorbol-D-acetate (DMSO) were used, respectively. A total number of 4625 (4760) genes were found to be regulated during MD (GD). The genes have been divided into groups according to their kinetics of regulation (R-groups). The genes of the same group are not distributed randomly throughout the genome but form clusters on chromosomes (R-clusters). Thus, functionally interconnected genes are frequently localized near to each other on the DNA molecule.
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Analýza genové exprese kolorektálního karcinomu detekované cDNA mikročipy pomocí kombinace různých vyhodnocovacích přístupů
Jansová, E. ; Krontorád, P. ; Svoboda, Z. ; Koutná, I. ; Pavlík, T. ; Kozubek, Michal ; Jarošová, M. ; Žaloudík, J. ; Kozubek, Stanislav
We have compared colorectal carcinoma tissues with parallel samples of epithelial tissue and identified, by means of Human 1.7K cDNA microarrays, a set of genes with significantly altered expression in 12 patients. We used a combination of several approaches of microarray data analysis to be sure that the results were reliable. Although we have found differences between the results obtained by various image analysis software packages and using different statistical tools, we have identified the group of 22 genes with significantly altered expression in colorectal carcinoma tissue as compared with epithelial tissue using all evaluation approaches. Hierarchical clustering showed the possibility of dividing the patients into two groups according to the presence of metastases in regional lymph nodes. We have proposed 6 genes as potential markers for the detection of the presence of regional metastases in patients.
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Dynamika pohybu a umlčování transgenních lokusů HP1 v živých buňkách
Ondřej, Vladan ; Kozubek, Stanislav ; Lukášová, Emilie ; Matula, Pa. ; Matula, Pe. ; Kozubek, Michal
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.
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Využití vysokorozlišovací cytometrie ke studiu živých buněk
Vařecha, M. ; Amrichová, J. ; Ondřej, Vladan ; Lukášová, Emilie ; Kozubek, Stanislav ; Matula, Pa. ; Matula, Pe. ; Kozubek, Michal
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.
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Rychlé snímání obrazu živých buněk a jeho limity
Matula, Pa. ; Kozubek, Michal ; Kozubek, Stanislav ; Ondřej, Vladan
Technique of high-resolution cytometry (HRCM) developed in our laboratory is capable of automated (2D as well as 3D) acquisition and analysis of fluorescent stained cells. The cytometer can process large number of cells with high resolution. The acquisition can be repeated in time and that enables live cell studies. If very short events in living cells are studied then the sampling frequency must be high. The highest sampling frequency depends on the technical parameters of the motorized parts of cytometer (especially on the camera frame rate, the speed of filter exchange and the speed of axial movement) and on the control software, which must be appropriately optimized. The limits of the current setup are discussed and available sampling frequencies for different acquisition modes are listed.
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