We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority.

• MeSH
• Bromodeoxyuridine metabolism   MeSH
• Clone Cells MeSH
• DNA metabolism   MeSH
• HCT116 Cells MeSH
• HeLa Cells MeSH
• Idoxuridine analogs & derivatives   metabolism   MeSH
• Humans MeSH
• Antibodies, Monoclonal metabolism   MeSH
• Peptide Mapping * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5'-deoxy-5'-iodouridine MeSH Browser
• Bromodeoxyuridine MeSH
• DNA MeSH
• Idoxuridine MeSH
• Antibodies, Monoclonal MeSH

T he organization of transcriptionally active ribosomal genes in animal cell nucleoli is investigated in this study in order to address the long-standing controversy with regard to the intranucleolar localization of these genes. Detailed analyses of HeLa cell nucleoli include direct localization of ribosomal genes by in situ hybridization and their indirect localization via nascent ribosomal transcript mappings. On the light microscopy (LM) level, ribosomal genes map in 10-40 fluorescence foci per nucleus, and transcription activity is associated with most foci. We demonstrate that each nucleolar focus observed by LM corresponds, on the EM level, to an individual fibrillar center (FC) and surrounding dense fibrillar components (DFCs). The EM data identify the DFC as the nucleolar subcompartment in which rRNA synthesis takes place, consistent with detection of rDNA within the DFC. The highly sensitive method for mapping nascent transcripts in permeabilized cells on ultrastructural level provides intense and unambiguous clustered immunogold signal over the DFC, whereas very little to no label is detected over the FC. This signal is strongly indicative of nascent "Christmas trees" of rRNA associated with individual rDNA genes, sampled on the surface of thin sections. Stereological analysis of the clustered transcription signal further suggests that these Christmas trees may be contorted in space and exhibit a DNA compaction ratio on the order of 4-5.5.

• MeSH
• Bromouracil analogs & derivatives   MeSH
• Cell Nucleolus genetics   ultrastructure   MeSH
• Microscopy, Electron MeSH
• Transcription, Genetic physiology   MeSH
• Genes, rRNA genetics   MeSH
• HeLa Cells MeSH
• In Situ Hybridization MeSH
• Humans MeSH
• Permeability MeSH
• Uridine analogs & derivatives   pharmacokinetics   MeSH
• Gold MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5-bromouridine MeSH Browser
• Bromouracil MeSH
• Uridine MeSH
• Gold MeSH