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The Nuclear Position of Pericentromeric DNA of Chromosome 11 Appears to Be Random in G<Subscript>O</Subscript> and N

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The Nuclear Position of Pericentromeric DNA of Chromosome 11 Appears to Be Random in G<Subscript>O</Subscript> and N UvA-DARE (Digital Academic Repository) The nuclear position of pericentromeric DNA of chromosome 11 appears to be random in G0 and non-random in G1 human lymphocytes Hulspas, R.; Houtsmuller, A.; Krijtenburg, P.-J.; Bauman, J.G.J.; Nanninga, N. DOI 10.1007/BF00352253 Publication date 1994 Published in Chromosoma Link to publication Citation for published version (APA): Hulspas, R., Houtsmuller, A., Krijtenburg, P-J., Bauman, J. G. J., & Nanninga, N. (1994). The nuclear position of pericentromeric DNA of chromosome 11 appears to be random in G0 and non-random in G1 human lymphocytes. Chromosoma, 103, 286-292. https://doi.org/10.1007/BF00352253 General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:01 Oct 2021 Chromosoma (1994) 103:286-292 CHROMOSOMA Springer-Verlag 1994 The nuclear position of pericentromeric DNA of chromosome 11 appears to be random in G Oand non-random in G 1 human lymphocytes R. Hulspas 1,*, A.B. Houtsmuller 2, P.-J. Krijtenburg 1, J.G.J. Bauman 1, N. Nanninga 2 1Institute for Applied Radiobiologyand ImmunologyTNO, Department of Molecular Pathology, P.O. Box 5815, NL-2280 HV Rijswijk, The Netherlands 2 Section of Molecular Cytology,Institute for Molecular Cell Biology, BioCentrumAmsterdam, Universityof Amsterdam, Plantage Muidergracht 14, NL 1018 TV Amsterdam, The Netherlands Received: 29 December 1993; in revised form: 30 March 1994 / Accepted: 7 April 1994 Abstract. The nuclear topography of pericentromeric the (decondensed) chromosome position is more compli- DNA of chromosome 11 was analyzed in G O (nonstimu- cated. Using [3H]thymidine labeling and electron micro- lated) and G 1 [phytohemagglutinin (PHA) stimulated] scope autoradiography in synchronized human amnion human lymphocytes by confocal microscopy. In addition cells, Comings (1968) proposed a model of the organiza- to the nuclear center, the centrosome was used as a sec- tion of the interphase nucleus in which the chromosomes ond point of reference in the three-dimensional (3D) are attached to the nuclear membrane at the site of initia- analysis. Pericentromeric DNA of chromosome 11 and tion of DNA replication. Also, it was suggested that ho- the centrosome were labeled using a combination of flu- mologous chromosomes might be attached closer to orescent in situ hybridization (FISH) and immunofluo- each other than to nonhomologous chromosomes. The rescence. To preserve the 3D morphology of the cells, techniques to visualize and analyze (parts of) interphase these techniques were performed on whole cells in sus- chromosomes have evolved rapidly, revealing more ac- pension. Three-dimensional images of the cells were an- curate data on the position of chromosomes during inter- alyzed with a recently developed 3D software program phase (for reviews see Manuelidis 1990; Jackson 1991; (Interactive Measurement of Axes and Positioning in 3 Lichter et al. 1991). Dimensions). The distribution of the chromosome 11 Many studies on the localization of centromeres of centromeres appeared to be random during the G O stage human chromosomes have been done by means of anti- but clearly non-random during the G 1 stage, when the bodies present in serum of patients with CREST syn- nuclear center was used as a reference point. Further sta- drome. These antibodies are specific for kinetochore tistical analysis of the G1 cells revealed that the centro- proteins enabling the detection of the centromeric re- meres were randomly distributed in a shell underlying gions of all chromosomes. Pair-wise arrangements of the nuclear membrane. A topographical relationship be- centromeres have been reported in interphase nuclei of tween the centrosome and the centromeres appeared to rat-kangaroo and Indian muntjac cells, using such be absent during the G Oand G I stages of the cell cycle. CREST serum antibodies (Hadlaczky et al. 1986). In murine cells, clustered and peripheral centromere distri- butions have been found in meiotic prophase I (Brinkley et al. 1986) and cell cycle specific distributions of cen- Introduction tromeres have been found in human fibroblasts and lym- phocytes (Bartholdi 1991; Weimer et al. 1992). The use The position of chromosomes has been well defined for of CREST serum antibodies, however, provides no infor- cells in mitosis. In subsequent phases they move toward mation about the centromere distributions of specific the center of the cell and align at the equatorial plate, chromosomes. followed by a relocation of the separated chromatids at The centromere region of a chromosome contains opposite sides of the cell (Alberts et al. 1989). The high- specific, alpha satellite DNA sequences (Willard 1985; ly condensed state of the chromosomes during this phase Jorgensen et al. 1986; Schulman and Bloom 1991). To- of the cell cycle, makes it comparatively easy to analyze day, specific DNA probes for almost every individual their locations. During interphase, however, the study of human chromsome are available. Using (fluorescent) in situ hybridization [(F)ISH], the analysis of the centro- *Present address: UMMC Cancer Center, Two Biotech, suite 202, mere distribution of individual chromosomes has been 373 Plantation St., Worcester, MA 01605, USA reported in several studies. Experiments performed on Edited by: U. Scheer isolated nuclei centrifuged onto glass slides, revealed Correspondence to: R. Hulspas that the centromeres of chromosome 1 appeared to be 287 randomly distributed in interphase nuclei of human cere- lying the nuclear membrane. No significant relation- brum, whereas a pair-wise arrangement was found in hu- ship between the position of the centromeres and the man cerebellum nuclei (Arnoldus et al. 1989). A mainly centrosome was found in either the G o or the G 1 lym- peripheral location of the centromeres of chromosome 1 phocytes. was seen in isolated interphase nuclei of human lympho- cytes (Van Dekken et al. 1990). In this study, the nuclei were kept in suspension to preserve the three-dimension- Materials and methods al (3D) morphology. Random distributions of chromosome 7 centromeres Isolation, growth and fixation of human lymphocytes. Lympho- have been reported in both human cerebrum and cerebel- cytes were isolated from peripheral blood with Lymphocyte Sepa- lum interphase nuclei (Arnoldus et al. 1989). In isolated ration Medium (Organon Teknika; Durham, N.C.). They were ei- G0/G 1 nuclei of human lymphocytes, however, the distri- ther directly fixed in phosphate buffered saline (PBS) with 2% butions of centromeres of chromosomes 1 and 7 were (w/v) paraformaldehyde, pH 7.4 (20 min, room temperature) and shown to be located exclusively at the nuclear periphery. stored in 70% ethanol at -20 ~ C, or cultured in Alpha Modification of Eagle's Medium (Flow Laboratories, Irvine, Scotland) with Similarly, the centromeres of chromosomes 11 and 17 10% (v/v) fetal calf serum. Cells were stimulated to enter the cell were found to be preferentially at the nuclear membrane. cycle by addition of 1% (v/v) phytohemagglutinin (PHA, Welcome During the G 2 stage, a more interior location of these Diagnostics; Dartford, UK). After 65 h of stimulation, 10 ~tg/ml centromeres was reported, indicating a relocation of bisbenzimide no. 33342 (Hoechst 42, Riedel-de Haen; Hannover, these centromeres during interphase (Ferguson and Ward Germany) was added and the culture was incubated for 1 h at 37 ~ 1992). C. Next, the cells were washed twice in PBS containing 10 gg/ml Hoechst 42. Finally, 1.2 gg/ml propidium iodide (PI) was added From these findings it appears that during interphase for flow cytometric analysis. the localization of the centromeres of each individual chromosome might be cell type specific. Also, the spe- Flow cytometric analysis and sorting. Cells were analyzed on the cific interphase stage (G 0, G~, S, G2) seems to be rele- RELACS-2 flow cytometer (Van den Engh and Stokdijk 1989), vant for the centromere distribution. Furthermore, the equipped with two lasers for dual beam flow cytometry. The first centromere distribution might be influenced by whether laser (Coherent Innova 90; Palo Alto, Calif.) was set at 488 nm (300 roW) to excite PI. PI fluorescence was detected through a cells are flattened on glass slides or kept in suspension, BP630P10 emission filter (Corion; Holliston, Mass.). The second or whether isolated nuclei are used. laser (Spectra Physics, Series 2000; Mountain View, Calif.) was set An important aspect in 3D analysis, is the choice of a at ultraviolet wavelengths (351 nm and 364 nm; 150 mW) to excite reference point. Usually, the position of centromeres is Hoechst 42 that was detected through a 408 long pass plus 450 described in relationship to the nuclear center or relative short pass filter (Schott Glaswerke; Mainz, Germany). The same to each other. The position of a centromere relative to a setup was used to sort the PI negative lymphocytes in the G 1 stage of the cell cycle. The sorted cells were fixed and stored as de- nuclear or a cellular structure might give more informa- scribed above. tion about its biological role. During mitosis a clear to- pological relationship between centromeres and the cen- Preparation of the biotinylated DNA probe.
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