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.
KeywordsChinese Hamster Ovary Metaphase Chromosome Interphase Nucleus Enlarge Nucleus Newborn Bovine Serum
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