A New Immunocytochemical Technique for Ultrastructural Analysis of DNA Replication in Proliferating Cells After Application of Two Halogenated Deoxyuridines

Jaunin, Françoise; Visser, Astrid E.; Cmarko, Dušan; Aten, Jacob A.; Fakan, Stanislav

doi:10.1177/002215549804601014

Cited by 20 publications

(34 citation statements)

References 24 publications

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“…In addition, we verified that only one size of gold particles was present following incubation of sections in the presence of only rat anti-BrdU (or mouse anti-BrdU) and then in the presence of a mixture of 6-nm gold labeled anti-mouse antibody and 10-nm gold labeled anti-rat antibody. Finally, in both optical and ultrastructural studies, we verified that the Harlan Sera-Lab rat anti-BrdU does not recognize IdUsubstituted DNA, while conversely mouse Becton Dickinson anti-BrdU does not recognize CldU-substituted DNA, as previously reported (Alten et al, 1992;Jaunin et al, 1998).…”

Section: Ultrastructural Studiessupporting

confidence: 83%

“…These data suggested that BCL6 bodies are associated with replicating DNA and/or that their close vicinity is a nuclear micro-environment prone to support DNA replication, and raised the possibility that they influence the replication foci (RF) assembly, positioning, or activity (Albagli et al, 2000). Moreover, together with the proposed spatial coincidence or close apposition between transcription and replication sites in nucleus (Fakan and Nobis, 1978;Jaunin et al, 1998Jaunin et al, , 2000Wei et al, 1998), these previous data led us here to also compare the localization of BCL6 bodies and RNA.…”

Section: Introductionmentioning

confidence: 81%

“…To induce BCL6 expression, UTA-L cells were plated in GM only supplemented with tetracycline at 200 ng/ml at day 1, then rinsed three times and fed with fresh GM at day 2, and exposed to 10 M (unless otherwise stated) of XdU (BrdU, CldU, and/or IdU, Sigma Aldrich) at day 3, i.e., when most of the cells express BCL6 as judged by immunofluorescence and electron microscopy (Albagli et al, 2000). Some cultures were fixed at the end of the pulse period whereas others were rapidly washed twice with prewarmed GM supplemented with thymidine (100 M), then twice in GM, before reincubation in GM for different times (see text) prior to fixation, as previously described (Jaunin et al, 1998). In double labeling experiments, cell cultures were pulse-labeled with IdU (or CldU), chased as described above, and, finally, incubated in the presence of CldU (or IdU) for the indicated time prior to fixation.…”

Section: Materials and Methods Cell Cultures And Nucleic Acid Labelinmentioning

confidence: 99%

“…Importantly, although the above results validated the use of short pulses of any XdU to visualize replicating DNA, they also showed that CldU (and BrdU) incorporation alters the fate of DNA while IdU-substituted DNA more closely reflects the fate of normal DNA. We thus assumed that, to approach the normal situation, IdU can be indifferently used to visualize either replicating or post-replicative DNA while CldU should be used only for replicating DNA: cells were thus exposed to IdU and CldU for the first and second pulse, respectively, as already described (Jaunin et al, 1998;Tashiro et al, 2000). From 4-and 8-hour chases, almost all 6-nm gold particles, which localized the DNA synthesized during the first pulse, constituted clusters or threads located in the nucleoplasm or in the perinuclear layer of condensed chromatin without any contact with the surface of the BCL6 bodies in agreement with the data of the above pulse-chase experiments (Fig.…”

Section: Concomitant Visualization Of Replicating Andmentioning

confidence: 99%

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The relationship between BCL6 bodies and nuclear sites of normal and halogenated DNA and RNA synthesis

Puvion‐Dutilleul¹,

Souquere-Besse²,

Albagli-Curiel

2003

Microscopy Res & Technique

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BCL6 is a POZ/BTB and zinc finger transcription factor that self-interacts and accumulates into discrete nuclear "bodies" of unknown function. We recently reported that BCL6 bodies associate with bromodeoxyuridine (BrdU)-substituted DNA, suggesting their implication in replication. To examine this possibility, we examine here by electron and confocal microscopy the relation between BCL6 bodies and replication foci (RF) using incorporation of various halogenated nucleotides (BrdU, chlorodeoxyuridine, CldU, and iododeoxyuridine, IdU) or PCNA (proliferating cell nuclear antigen) staining. We show that BCL6 bodies are found associated with RF, as revealed by PCNA staining. However, such association is markedly prolonged upon BrdU or CldU incorporation, but less, or not at all, upon IdU incorporation. Pulse-chase and double-labeling experiments indicate that IdU-substituted DNA leaves BCL6 bodies after a few tenths of minutes while BrdUor CldU-substituted DNA stalls in their vicinity for several hours, thereby giving the characteristic "crowns" of DNA entirely surrounding BCL6 bodies. In all cases, however, the halogenated DNA ends up undergoing a movement from BCL6 bodies toward nucleoplasm and nuclear periphery to reach euchromatin and heterochromatin, respectively. We propose that replicating DNA is prone to be bound by BCL6, while BrdU/CldU incorporation increases this propensity possibly because these two events have synergistic effects on the structure and chromatinisation of the newly synthesized DNA. Finally, despite the known proximity between nuclear sites of transcription and replication, we show via several approaches that BCL6 bodies do not appear to be involved either in RNA synthesis or storage.

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Section: Ultrastructural Studiessupporting

confidence: 83%

Section: Introductionmentioning

confidence: 81%

Section: Materials and Methods Cell Cultures And Nucleic Acid Labelinmentioning

confidence: 99%

Section: Concomitant Visualization Of Replicating Andmentioning

confidence: 99%

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The relationship between BCL6 bodies and nuclear sites of normal and halogenated DNA and RNA synthesis

Puvion‐Dutilleul¹,

Souquere-Besse²,

Albagli-Curiel

2003

Microscopy Res & Technique

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“…Cl-and Icontaining deoxyuridine has been used for DNA double labeling in pulse-chase experiments (Jaunin et al 1998). …”

mentioning

confidence: 99%

Use of Halogenated Precursors for Simultaneous DNA and RNA Detection by Means of Immunoelectron and Immunofluorescence Microscopy

Vecchio

Solimando

Biggiogera

et al. 2007

J Histochem Cytochem.

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(MB) S U M M A R Y We have developed a novel approach for in situ labeling and detection of nucleic acids in cultured cells. It is based on in vivo incorporation of chlorouridine (ClU) or iododeoxyuridine (IdU) into Chinese hamster ovary cells with the aim of labeling RNA and DNA, respectively. The halogenated nucleotides are immunolabeled on ultrathin sections with anti-bromodeoxyuridine (BrdU) monoclonal antibodies that specifically react with either IdU or ClU. Furthermore, we combined ClU and IdU incubation to label simultaneously RNA and DNA in the same cell. Both were visualized by means of anti-BrdU antibodies exhibiting strong affinity for one of the two halogenated epitopes. Confocal imaging of interphase nuclei and electron microscopic analysis showed evidence of a partial colocalization of newly synthesized DNA and RNA inside the cell nucleus. RNase and DNase digestion of ultrathin sections after formaldehyde fixation and acrylic resin embedding confirmed the specificity of incorporation. Consequently, this method allows us to differentially label DNA and RNA on the same section. Using short pulses with the precursors, we could show that newly synthesized DNA and RNA both preferentially occur within the perichromatin region at the border of condensed chromatin domains. (J Histochem Cytochem 56:45-55, 2008) K E Y W O R D S halogenated precursors immunocytochemistry electron microscopy RNA and DNA THE EUKARYOTIC CELL NUCLEUS is organized into functional domains where nuclear processes such as DNA replication and RNA transcription and processing take place (Spector 2003;Stein et al. 2003;Fakan 2004;van Driel and Fransz 2004;Cremer et al. 2006).Although structural characterization of these compartments has been extensively studied during the last four decades, especially thanks to ultrastructural cytochemical studies (Monneron and Bernhard 1969), we still look for high-resolution tools, allowing one to better approach the occurrence of different functions within the architecture of the cell nucleus.The application of halogenated precursors in ultrastructural in situ analyses of DNA replication and RNAtranscription is now well established. Cl-and Icontaining deoxyuridine has been used for DNA double labeling in pulse-chase experiments (Jaunin et al. 1998).Moreover, bromodeoxyuridine (BrdU) has been shown to be suitable for labeling of chromatin domains after a long incorporation period followed by several cell cycles, giving rise to multiple chromatid segregations (Visser et al. 2000). Bromouridine (BrU) and 5-bromouridine 5 ¶-triphosphate (Br-UTP) were especially used for short labeling of RNA and detection of transcription sites (Cmarko et al. 1999;Trentani et al. 2003).In this study, we analyzed the location of newly replicated and transcribed molecules with regard to chromatin structure. It was achieved in Chinese hamster ovary cells (CHO) by detecting newly synthesized DNA and RNA using incorporation of different halogenated precursors by means of immunoelectron cytochemistry. This high-resolut...

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Modulation of BACE1 Activity by Chemically Modified Aptamers

et al. 2018

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A modified DNA aptamer that binds BACE1, a therapeutic target involved in Alzheimer's disease has been developed. This ssXNA not only tightly binds to BACE1 but also inhibits its protease activity in vitro in the same range as a previously described unmodified aptamer. We report the in vitro selection of functional oligonucleotides incorporating two nucleobase modifications: 5-chlorouracil and 7-deazaadenine. The nucleoside analogue 5-chloro-2'-deoxyuridine has already been explored as a replacement for thymidine in a chemically modified genome of a bacterium. Thus, 5-chlorouracil modification is a good candidate to support genetic transfer in vivo as well as functional activity.

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A New Immunocytochemical Technique for Ultrastructural Analysis of DNA Replication in Proliferating Cells After Application of Two Halogenated Deoxyuridines

Cited by 20 publications

References 24 publications

The relationship between BCL6 bodies and nuclear sites of normal and halogenated DNA and RNA synthesis

The relationship between BCL6 bodies and nuclear sites of normal and halogenated DNA and RNA synthesis

Use of Halogenated Precursors for Simultaneous DNA and RNA Detection by Means of Immunoelectron and Immunofluorescence Microscopy

Modulation of BACE1 Activity by Chemically Modified Aptamers

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