Improved in situ detection method for telomeric tandem repeats in metaphase spreads and interphase nuclei

Uhlmann, V; Prasad, Madhu; Silva, I; Luettich, Karsta; Grande, L; Alonso, Leonardo G.; Thisted, M; Pluzek, K J; Gorst, J; Ring, Martina; Sweeney, Marion; Kenny, C; Martín, C; Russell, J; Bermingham, Nessan A.; O’Donovan, Mike; Sheils, Orla; O’Leary, John

doi:10.1136/mp.53.1.48

Cited by 13 publications

(9 citation statements)

References 20 publications

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“…The cells were treated with KCl (0.56% w/v) for 20 min and then washed several times in Carnoýs fixative (MeOH:HAc (3:1)), as described by Uhlmann et al (2000). The nuclei suspension was then finally dropped onto SuperFrosts Plus slides.…”

Section: Telomere Length By Telomere Fluorescent In Situ Hybridizatiomentioning

confidence: 99%

Possibility of mixed progenitor cells in sea star arm regeneration

et al. 2010

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In contrast to most vertebrates, invertebrate deuterostome echinoderms, such as the sea star Asterias rubens, undergo regeneration of lost body parts. The current hypothesis suggests that differentiated cells are the main source for regenerating arm in sea stars, but there is little information regarding the origin and identity of these cells. Here, we show that several organs distant to the regenerating arm responded by proliferation, most significantly in the coelomic epithelium and larger cells of the pyloric caeca. Analyzing markers for proliferating cells and parameters indicating cell ageing, such as levels of DNA damage, pigment, and lipofuscin contents as well as telomere length and telomerase activity, we suggest that cells contributing to the new arm likely originate from progenitors rather than differentiated cells. This is the first study showing that cells of mixed origin may be recruited from more distant sources of stem/progenitor cells in a sea star, and the first described indication of a role for pyloric caeca in arm regeneration. Data on growth rate during arm regeneration further indicate that regeneration is at the expense of whole animal growth. We propose a new working hypothesis for arm regeneration in sea stars involving four phases: wound healing by coelomocytes, migration of distant progenitor cells of mixed origin including from pyloric caeca, proliferation in these organs to compensate for cell loss, and finally, local proliferation in the regenerating arm.

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Section: Telomere Length By Telomere Fluorescent In Situ Hybridizatiomentioning

confidence: 99%

Possibility of mixed progenitor cells in sea star arm regeneration

et al. 2010

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“…Sections (10 µm thick) of each specimen were mounted on ProbeOn slides (Fisher Scientific, Pittsburgh, Pa., USA) for ISH. The telomere repeat probe (TTAGGG) 4 [23, 24]was labeled with fluorescein isothiocyanate (FITC) on the 3′-tail (EspecOligo Service, Tsukuba, Japan). The probe was diluted to set to 20 µg/ml by probe hybridization solution [50% formamide (Sigma, St. Louis, Mo., USA), 0.5 M NaCl, 5% polyethylene glycol 8000 (Sigma)].…”

Section: Methodsmentioning

confidence: 99%

<i>Helicobactor pylori</i> Infection Is Closely Associated with Telomere Reduction in Gastric Mucosa

Kuniyasu¹,

Kitadai²,

Mieno³

et al. 2003

Oncology

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Objective: To investigate differential reduction in telomere DNA in tissue components of gastric mucosa with respect to Helicobactor pylori infection. Materials and Methods: The telomere content was examined by fluorescent in situ hybridization with the (TTAGGG)₄ probe. To compare the signal intensities from the probe (telomere volume) with telomere length, five gastric carcinoma cell lines were used. Telomere volumes were examined in 9 healthy persons, 124 non-cancer patients, and 86 gastric cancer patients. Results: Telomere volume showed a linear correlation with telomere length measured by Southern blotting in gastric carcinoma cells. In healthy persons without H. pylori infection, the telomere volumes of gastric epithelial tissues were 70–79% that of intramucosal lymphocytes (internal control). In 124 patients without gastric cancer, telomere volume of H.-pylori-infected mucosa was significantly less than that of H.-pylori-negative mucosa in both metaplastic and non- metaplastic tissues (p < 0.0001). In 86 gastric cancer patients, telomere volumes in intestinal metaplasia adjacent to cancer were 75% that of intestinal metaplasia of non-cancer patients (p = 0.0001). hTERT expression was detected in 6 cancer-associated and 2 cancer-negative intestinal metaplasia specimens, in which telomere volume was markedly reduced. Conclusion:H. pylori infection is closely associated with telomere reduction in gastric epithelium.

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“…The PNA probe resulting duplexes are more stable than the DNA/DNA or DNA/RNA duplexes formed by the conventional oligonucleotide probes, because the charged phosphate-deoxyribose backbone is replaced by uncharged repeating N-(2-amino ethyl)-glycine backbone linked by peptide bonds [51]. The digital images were recorded with a CCD camera on a fluorescence microscope, and analyzed quantitatively [29,49].…”

Section: Fluorescence In Situ Hybridization (Fish)mentioning

confidence: 99%

“…The modified oligonucleotide probes in the FISH detection of telomere which previously described are suggested to be applied to PRINS [51]. The high target affinity of 2'-OMe-ribose-and 5-(1-propynyl) pyrimidine-modified probes could be suit for PRINS in the detection of telomere [51].…”

Section: Primed In Situ (Prins)mentioning

confidence: 99%

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The telomere length dynamic and methods of its assessment

Lin

Yan

2005

J Cellular Mol Med

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Human telomeres are composed of long repeating sequences of TTAGGG, associated with a variety of telomere‐binding proteins. Its function as an end‐protector of chromosomes prevents the chromosome from end‐to‐end fusion, recombination and degradation. Telomerase acts as reverse transcriptase in the elongation of telomeres, which prevent the loss of telomeres due to the end replication problems. However, telomerase activity is detected at low level in somatic cells and high level in embryonic stem cells and tumor cells. It confers immortality to embryonic stem cells and tumor cells. In most tumor cells, telomeres are extremely short and stable. Telomere length is an important indicator of the telomerase activity in tumor cells and it may be used in the prognosis of malignancy. Thus, the assessment of telomeres length is of great experimental and clinical significance. This review describes the role of telomere and telomerase in cancer pathogenesis and the dynamics of the telomeres length in different cell types. The various methods of measurement of telomeres length, i.e. southern blot, hybridization protection assay, fluorescence in situ hybridization, primed in situ, quantitative PCR and single telomere length analysis are discussed. The principle and comparative evaluation of these methods are reviewed. The detection of G‐strand overhang by telomeric‐oligonucleotide ligation assay, primer extension/nick translation assay and electron microscopy are briefly discussed.

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Improved in situ detection method for telomeric tandem repeats in metaphase spreads and interphase nuclei

Cited by 13 publications

References 20 publications

Possibility of mixed progenitor cells in sea star arm regeneration

Possibility of mixed progenitor cells in sea star arm regeneration

<i>Helicobactor pylori</i> Infection Is Closely Associated with Telomere Reduction in Gastric Mucosa

The telomere length dynamic and methods of its assessment

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