Fluorescent Analysis of Replication and Intermediates of Chromatin Folding in Nuclei of Mammalian Cells
The scarcity of evidence regarding chromatin folding is due to the fact that individual chromosomes cannot be seen during their development in the interphase nucleus. Consequently, chromosome structure has been studied primarily in metaphase preparations (Kirsch-Volders et al., 1980; Zang and Back, 1968; Zankl and Zang, 1974), while direct approaches to visualize the topography of chromatin in the nucleus has remained limited (Comings, 1968; Comings, 1980; Cremer et al., 1979; Sperling and Luedtke, 1981; Vogel and Schroeder, 1974) and indirect conclusions were drawn mainly from metaphase studies. This article describes a new approach to the study of chromosome condensation confirming the existence of a flexible folding pattern through a series of transient geometric forms of chromosomes in nuclei of Chinese hamster ovary (CHO) cells. Exponentially growing cells were permeabilized reversibly and nascent DNA was synthesized in the presence of the four dNTPs, dTTP being replaced by biotin-11-dUTP (Banfalvi et al., 1989). Biotinylated nucleotides do not perturb DNA replication (Banfalvi et al., 1989; Blow and Watson, 1987; Hiriyanna et al., 1988; Hunting et al., 1985; Langer et al., 1981; Nakayasu and Bereznay, 1989), but their interference with chromosome folding (Banfalvi et al., 1989, 1990) can be exploited to accumulate intermediates of the condensation process.
KeywordsSucrose Trypsin Hepes Dextran DAPI
Banfalvi, G., Sooki-Toth, A., Saikar, N., Csuzi, S., and Antoni, F. (1984) Nascent DNA chains synthesized in reversibly permeable cells of mouse thymocytes. Eur. J. Biochem.
139: 553–559.PubMedCrossRefGoogle Scholar
Banfalvi, G., Wiegant, J., Sarkar, N., and van Duijn, P. (1989) Immunofluorescent visualization of DNA replication sites within nuclei of CHO cells. Histochemistry
93: 81–86.PubMedCrossRefGoogle Scholar
Banfalvi, G., Tanke, H., Raap, A.K., Slats, J., and van der Ploegh, M. (1990) Early replication signals in nuclei of Chinese hamster ovary cells. Histochemistry
94: 435–440.PubMedCrossRefGoogle Scholar
Blow, J.J., and Watson, J.V. (1987) Nuclei act as independent units of replication in Xenopus
cell-free DNA replication system. EMBO J.
6: 1997–2002.PubMedGoogle Scholar
Comings, D.E. (1968) The rationale for an ordered arrangement of chromatin in the interphase nucleus. Am. J. Hum. Genet.
20: 440–460.PubMedGoogle Scholar
Comings, D.E. (1980) Arrangement of chromatin in the nucleus. Hum. Genet.
53: 131–143.PubMedCrossRefGoogle Scholar
Cremer, C, Cremer, T., Zorn, C, and Cioreanu, V. (1979) Partial irradiation of Chinese hamster cell nuclei and detection of unscheduled DNA synthesis in interphase and methaphase. A tool to investigate the arrangement of interphase chromosomes in mammalian cells. Hoppe Seyler’s Z. Physiol. Chem.
360: 244–245.Google Scholar
Hiriyanna, K.T., Varkey, J., Beer, M., and Benbow, M. (1988) Electron microscopic visualization of sites of nascent DNA synthesis by streptavidin-gold binding to biotinylated nucleotides incorporated in vivo
. J. Cell Biol.
107: 33–44.PubMedCrossRefGoogle Scholar
Hunting, DJ., Dresler, S.L., and Murcia, G. (1985). Incorporation of biotin-labelled deoxyuridine triphosphate into DNA during excision repair and electron microscopic visualization of repair patches. Biochemistry
24: 5729–5734.PubMedCrossRefGoogle Scholar
Kirsch-Volders, M., Hens, L., and Susanne, C. (1980) Telomere and centromere association tendencies in the human male metaphase complement. Hum. Genet.
54: 69–77.PubMedCrossRefGoogle Scholar
Langer, P.R., Waldrop, A.A., and Ward, D.C. (1981) Enzymatic synthesis of biotin-labelled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA
78: 6633–6637.PubMedCrossRefGoogle Scholar
Nakayasu, H., and Bereznay, R. (1989) Mapping replicational sites in the eukaryotic cell nucleus. J. Cell Biol.
108: 1–11.PubMedCrossRefGoogle Scholar
Sperling, K., and Luedtke, E-K. (1981) Arrangement of prematurely condensed chromosomes in cultured cells and lymphjocytes of the Indian muntjac. Chromosoma
83: 541–553.PubMedCrossRefGoogle Scholar
Vogel, F., and Schroeder, T.M. (1974) The internal order of the interphase nucleus. Hum. Genet.
25: 265–297.CrossRefGoogle Scholar
Zang, K.D., and Back, E. (1968) Quantitative studies of the arrangement of human chromosomes. I. Individual features in the association pattern of acricentric chromosomes of normal males and females. Cytogenetics
7: 455–470.PubMedCrossRefGoogle Scholar
Zankl, H., and Zang, K.D. (1974) Quantitative studies on the arrangement of human metaphase chromosomes. IV. The association frequency of human acrocentric marker chromosomes. Hum. Genet.
23: 259–265.CrossRefGoogle Scholar
© Springer Science+Business Media New York 1993