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DNA replication and chromatin: From 3D to function
Ligasová, Anna ; Koberna, Karel (advisor) ; Cmarko, Dušan (referee) ; Nedvídek, Josef (referee)
We characterized the spatio-temporal organization of replication sites. In agreement with the previous studies, five different replication patterns were observed during the S phase in HeLa cells at the level of light microscopy. Using the electron microscopy approach, we observed the close association of numerous silver grains at many sites in the early S phase. The labeled sites had discrete sizes in terms of both area and diameter. Moreover, the much larger heterochromatin domains that replicate in the late S phase, and to a lesser extent in the mid S phase, were actually composed of closely-associated, labeled RS with virtually identical diameters and areas as the early-S-labeled RS. The replication sites labeled in the early S phase were maintained as similarly labeled clusters of colloidal gold particles later in the S phase and in the next cell generation. The number of replication sites was similar in the early- and mid-S-phase cells with a slight decrease in the late S phase. The electron microscopy tomography revealed that most of the observed replication sites corresponded to individual replicons. We found that the replisomes operate as tightly-associated couples during replication. Our results concern only human HeLa cells, but considering that the findings on prokaryotic cells and budding yeast...
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Targeting DNA and DNA synthesis in situ
Strunin, Dmytro ; Koberna, Karel (advisor) ; Hocek, Michal (referee) ; Forstová, Jitka (referee)
The thesis is focused on the development and application of the new methods for targeting DNA and DNA synthesis in situ. Three methodical approaches were developed, which significantly improve detection of cellular DNA. Concurrently, a new insights in the mech- anism of 2'-deoxy-5-ethynyluridine cytotoxicity were described. The approaches developed in this work provide an important tool for study of DNA replication and repair mechanisms, the study of mito- chondrial DNA, as well as the analysis of cell proliferation. The first part of the thesis presents the new method for the detection of 5-bromo-2'-deoxyuridine incorporated into the newly synthesized DNA using monovalent copper ions in the presence of oxygen. This method is applicable for the detection of nuclear DNA synthesis as well as for the mitochondrial DNA synthesis. Next part of the work describes the approach enabling highly efficient detection of over- all nuclear and mitochondrial DNA. The third approach concerned the elimination of the unspecific binding of anti 5-bromo-2'-deoxyuridine antibodies to another modified nucleoside - 2'-deoxy-5-ethynyluridine. This method is useful in the studies where the necessity of two con- secutive labeling pulses is needed. The approach is based on the use of non-fluorescent azido-molecules. The...
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DNA replication and chromatin: From 3D to function
Ligasová, Anna ; Koberna, Karel (advisor) ; Cmarko, Dušan (referee) ; Nedvídek, Josef (referee)
We characterized the spatio-temporal organization of replication sites. In agreement with the previous studies, five different replication patterns were observed during the S phase in HeLa cells at the level of light microscopy. Using the electron microscopy approach, we observed the close association of numerous silver grains at many sites in the early S phase. The labeled sites had discrete sizes in terms of both area and diameter. Moreover, the much larger heterochromatin domains that replicate in the late S phase, and to a lesser extent in the mid S phase, were actually composed of closely-associated, labeled RS with virtually identical diameters and areas as the early-S-labeled RS. The replication sites labeled in the early S phase were maintained as similarly labeled clusters of colloidal gold particles later in the S phase and in the next cell generation. The number of replication sites was similar in the early- and mid-S-phase cells with a slight decrease in the late S phase. The electron microscopy tomography revealed that most of the observed replication sites corresponded to individual replicons. We found that the replisomes operate as tightly-associated couples during replication. Our results concern only human HeLa cells, but considering that the findings on prokaryotic cells and budding yeast...
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Transport of ribosomal RNA within the nucleolus
Staněk, David ; Koberna, Karel ; Pliss, Artem ; Čtrnáctá, Vlasta ; Malínský, Jan ; Mašata, Martin ; Večeřová, Jaromíra ; Raška, Ivan
In the present study, we have explored the fact that incorporation of BrU into prerRNA did not interfere with pre-rRNA processing (11) and we have investigated the transport of non - isotopically labeled RNA within the nucleolus by means of transmissiion electron microscopy and confocal laser scanning microscopy.Within 20 minutes, BrU - labeled nucleolar RNA moved from the transcription sites in nucleolar DFC to GC. Double localization of bromouridine labeled RNA and fibrillarin revealed that only a ppart of the fibrillarin rich domains were transcriptionally active.
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