Sofie Bekaert1, S Koll, O Thas, P Van Oostveldt. 1. Laboratory for Biochemistry and Molecular Cytology, Dept. of Molecular Biotechnology, Faculty for Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. sofie.bekaert@rug.ac.be
Abstract
BACKGROUND: The length of the terminal sequences of linear chromosomes changes dynamically during cellular proliferation. A crucial element in the study of telomere-related regulation mechanisms is the ability to measure telomere lengths of individual chromosomes. Individual telomere lengths can be measured using digital imaging fluorescence microscopy-based techniques. We extended this method using confocal microscopy for the acquisition of three-dimensional (3D) images. Consequently, variations in measured signal intensities due to erroneous focusing are avoided. METHODS: We employed our 3D telomere sizing method to compare telomere lengths of sister chromatids within metaphase preparations from human lymphocytes. The samples were treated following a quantitative fluorescence in situ hybridization (Q-FISH) protocol using fluorescein isothiocyanate (FITC)-labeled telomeric peptidic nucleic acid (PNA) probes and propidium iodide (PI) counterstain. RESULTS: We demonstrated that the telomere lengths of two sister chromatids are not necessarily equal in human lymphocytes. Profound statistical analysis demonstrated significant differences in the distribution of the sister chromatid telomere lengths, but we were not able to prove a discrete distribution of telomere sister ratios. These telomere length differences were more apparent in older individuals. CONCLUSION: Whereas the majority of sister telomere pairs have equal lengths, surprisingly, a minority was significantly different in each individual studied. We are convinced that these observations are not linked to the methodology or the protocol applied. We suggest that a biological phenomenon might be involved. Copyright 2002 Wiley-Liss, Inc.
BACKGROUND: The length of the terminal sequences of linear chromosomes changes dynamically during cellular proliferation. A crucial element in the study of telomere-related regulation mechanisms is the ability to measure telomere lengths of individual chromosomes. Individual telomere lengths can be measured using digital imaging fluorescence microscopy-based techniques. We extended this method using confocal microscopy for the acquisition of three-dimensional (3D) images. Consequently, variations in measured signal intensities due to erroneous focusing are avoided. METHODS: We employed our 3D telomere sizing method to compare telomere lengths of sister chromatids within metaphase preparations from human lymphocytes. The samples were treated following a quantitative fluorescence in situ hybridization (Q-FISH) protocol using fluorescein isothiocyanate (FITC)-labeled telomeric peptidic nucleic acid (PNA) probes and propidium iodide (PI) counterstain. RESULTS: We demonstrated that the telomere lengths of two sister chromatids are not necessarily equal in human lymphocytes. Profound statistical analysis demonstrated significant differences in the distribution of the sister chromatid telomere lengths, but we were not able to prove a discrete distribution of telomere sister ratios. These telomere length differences were more apparent in older individuals. CONCLUSION: Whereas the majority of sister telomere pairs have equal lengths, surprisingly, a minority was significantly different in each individual studied. We are convinced that these observations are not linked to the methodology or the protocol applied. We suggest that a biological phenomenon might be involved. Copyright 2002 Wiley-Liss, Inc.
Authors: Sven Perner; Silke Brüderlein; Cornelia Hasel; Irena Waibel; Alexandra Holdenried; Neslisah Ciloglu; Heiko Chopurian; Kirsten Vang Nielsen; Andreas Plesch; Josef Högel; Peter Möller Journal: Am J Pathol Date: 2003-11 Impact factor: 4.307
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