A5040436840- RNA and protein synthesis mechanisms
- RNA modifications and cancer
- RNA Research and Splicing
- Genomics and Chromatin Dynamics
- DNA and Nucleic Acid Chemistry
- DNA Repair Mechanisms
- Cell Image Analysis Techniques
- Advanced biosensing and bioanalysis techniques
- RNA Interference and Gene Delivery
- Advanced Fluorescence Microscopy Techniques
- Bacteriophages and microbial interactions
- Molecular Biology Techniques and Applications
- Microtubule and mitosis dynamics
- Ubiquitin and proteasome pathways
- Biochemical and Molecular Research
- Click Chemistry and Applications
- Cancer-related Molecular Pathways
- Gene Regulatory Network Analysis
- Genetics, Bioinformatics, and Biomedical Research
- Advanced Electron Microscopy Techniques and Applications
- Bacterial Genetics and Biotechnology
- bioluminescence and chemiluminescence research
- Parvovirus B19 Infection Studies
- Monoclonal and Polyclonal Antibodies Research
- RNA regulation and disease
Palacký University Olomouc
2013-2025
Institute of Molecular and Translational Medicine
2013-2025
Czech Academy of Sciences
1992-2012
Czech Academy of Sciences, Institute of Molecular Genetics
2012
Czech Academy of Sciences, Institute of Biophysics
2012
Czech Academy of Sciences, Institute of Experimental Medicine
1989-2011
Charles University
2000-2008
Czech Academy of Sciences, Institute of Physiology
2006-2007
Tokyo University of Agriculture and Technology
2006
Ministry of Education, Culture, Sports, Science and Technology
2006
Mismatched nucleobase uracil is commonly repaired through the base excision repair initiated by DNA glycosylases. The data presented in this study strongly indicate that nuclear uracil-N-glycosylase activity and protein content human cell lines highest S phase of cycle its distribution kinetics partially reflect replication foci. In respect, demonstrate structural changes focus related to several dozens minutes before end replication. analysis also showed very popular synchronisation...
T he organization of transcriptionally active ribosomal genes in animal cell nucleoli is investigated this study order to address the long-standing controversy with regard intranucleolar localization these genes. Detailed analyses HeLa include direct by situ hybridization and their indirect via nascent transcript mappings. On light microscopy (LM) level, map 10–40 fluorescence foci per nucleus, transcription activity associated most foci. We demonstrate that each nucleolar focus observed LM...
In the present study, spatial organization of intron-containing pre-mRNAs Epstein–Barr virus (EBV) genes relative to location splicing factors is investigated. The intranuclear position transcriptionally active EBV genes, as well nascent transcripts, found be random with respect speckled accumulations (SC35 domains) in Namalwa cells, arguing against concept locus-specific mRNA speckles. Microclusters are, however, frequently superimposed on transcript sites. environment a dynamic structure...
We have investigated the possible involvement of ubiquitin-proteasome system (UPS) in ribosome biogenesis. find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate immunoprecipitation complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show fluorescence microscopy nucleolar morphology disrupted for some but not all involved Interference degradation induces accumulation 90S preribosomes,...
5-Bromo-2′-deoxyuridine (BrdU) and 2′-deoxy-5-ethynyluridine (EdU) are widely used as markers of replicated DNA. While BrdU is detected using antibodies, the click reaction typically with fluorescent azido-dyes for EdU localisation. We have performed an analysis ten samples antibodies against respect to their reactivity EdU. Except one sample all others evinced A high level persists in nuclear DNA even after if common concentration dye used. Although a ten-time increase azido-dye resulted...
CDD plays a pivotal role within the pyrimidine salvage pathway. In this study, novel, rapid method for identification of cell lines lacking functional cytidine deaminase was developed. This innovative utilizes immunocytochemical detection product 5-fluorocytidine deamination, 5-fluorouridine in cellular RNA, enabling these cells two hours. The approach employs an anti-bromodeoxyuridine antibody that also specifically binds to and its subsequent by fluorescently labeled antibody. Our results...
Abstract Cell quantification is widely used in basic or applied research. The current sensitive methods of cell are exclusively based on the analysis non-fixed cells and do not allow simultaneous detection various cellular components. A fast, cheap method fixed adherent described here. It incubation DAPI- Hoechst 33342-stained a solution containing SDS. presence SDS results quick de-staining DNA simultaneously, an up-to-1,000-fold increase fluorescence intensity dyes. This can be attributed...
Abstract DNA replication sites (RS) in synchronized HeLa cells have been studied at the electron microscopic level. Using an improved method for detection following vivo incorporation of biotin‐16‐deoxyuridine triphosphate, discrete RS, or foci are observed throughout S‐phase. In particular, much larger RS typically by fluorescence approaches mid‐ and late‐S‐phase, found to be composed smaller that virtually identical size early‐S‐phase. Pulse‐chase experiments demonstrate early‐S‐phase...
It is widely accepted that chromosomes occupy more or less fixed positions in mammalian interphase nucleus. However, relation between large-scale order of chromosome positioning and gene activity remains unclear. We used the model human ribosomal genes to address specific aspects this problem. Ribosomal are organized at particular chromosomal sites clusters termed nucleolus organizer regions (NORs). Only some NORs, called competent generally be transcriptionally active during interphase....
2′-deoxy-5-ethynyluridine (EdU) has been previously shown to be a cell poison whose toxicity depends on the particular line. The reason is not known. Our data indicates that different efficiency of EdU incorporation plays an important role. EdU-mediated was elevated by inhibition 2′-deoxythymidine 5′-monophosphate synthesis. resulted in abnormalities cycle including slowdown S phase and decrease DNA but cessation first division after administration observed all tested lines. In HeLa cells,...
A set of fifteen triterpenoid pyrazines and pyridines was prepared from parent 3-oxoderivatives (betulonic acid, dihydrobetulonic oleanonic moronic ursonic heterobetulonic allobetulone). Cytotoxicity all compounds tested in eight cancer two non-cancer cell lines. Evaluation the structure-activity relationships revealed that core determined whether final molecule is active or not, while heterocycle able to increase activity modulate specificity. Five (1b, 1c, 2b, 2c, 8) were found be...
ABSTRACT Earlier studies have established that the average speed of a replication fork is two to three times slower in early S-phase than late and intracellular 2′-deoxyribonucleoside 5′-triphosphate pools grow during S-phase. In this study, effect exogenous (dNTP) supply on synchronised population human HeLa cells was tested. The movement measured extended DNA fibers labelled with 2′-deoxythymidine analogues 5-chloro-2′-deoxyuridine 5-iodo-2′-deoxyuridine. We show introduction dNTPs...
A new method of the light microscopy detection BrdU-labeled DNA in situ is described. It based on oxidative attack at deoxyribose moiety by copper(I) presence oxygen, which leads to abstraction hydrogen atom from culminating elimination nucleobase, scission nucleic-acid strand and formation frequent gaps. The gaps allow reaction antibodies with commonly used markers replication (e.g. 5-bromo-2′-deoxyuridine), are otherwise masked. developed makes it possible detect nuclear mitochondrial...
5-Ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed a marker more frequently than EdC, its cytotoxicity commonly much higher the toxicity EdC. To reveal reason lower we performed DNA analysis five EdC-treated human cell lines. Surprisingly, not single one tested lines contained detectable amount EdC in their DNA. Instead, all EdU. The content incorporated differed particular cells...
Contamination of cell cultures by mycoplasmas is a very common phenomenon. As they can substantially alter metabolism and potentially spread to all in laboratory, their early detection necessary. One the fastest cheapest methods mycoplasma relies on direct staining mycoplasmas’ DNA DAPI or Hoechst dyes. Although this method easy fast perform, it suffers from low signal provided these dyes compared nuclear DNA. Therefore, reporter lines are used for cultivation before step. In study...
Abstract We have investigated the in situ organization of ribosomal gene (rDNA) transcription and replication HeLa cells. Fluorescence hybridization (FISH) revealed numerous rDNA foci nucleolus. Each focus corresponds to a higher order chromatin domain containing multiple genes. Multi‐channel labeling experiments indicated that, majority cells, all were active as demonstrated by co‐localization with signals fibrillarin, protein involved RNA processing. In some however, small portion did not...