Conformation of Replicated Segments of Chromosome Fibres in Human S-phase Nucleus

Recent statistical analysis of the folding of G0/G1 chromosomes using fluorescence in situ hybridization (FISH) allowed development of a random walk/giant loop model of chromosome structure. According to this model there are two levels of organization of G0/G1 chromosome fibres. On the first level, the fibres are arranged in giant loops several Mbp in size, and within each loop the fibres are randomly folded. On the second level, the loop attachment sites form a chromosome backbone that also shows random folding. Newly replicated segments of mammalian chromosomes may be directly visualized at high resolution in S-phase nuclei using immunofluorescent methods and appear as worm-like fibres. In our earlier study, we analysed conformation of the fibres in human cells blocked for 16 h at the G1/S boundary with 5- fluorodeoxyuridine (FdU) and then released into S-phase by the addition of a DNA prec ursor. However, long treatment of cells with FdU induces very short replicons and may promote apoptosis. In this study we analysed conformation of the fibres in normally proliferating human cells that had not been blocked with FdU for a long time. It has been found that replicated chromosome fibres visualized just after 2 h of incubation of the cells with a non-radioactively labelled DNA precursor behave as flexible polymer chains without major constraints, and that their local conformation in the range of several microns of their contour length may be considered as random. Confocal analysis of human X chromosomes visualized in HeLa cells using FISH with a specific painting probe shows that in S-phase the chromosomes occupy distinct nuclear territories and their apparent size does not differ from that in non-S-phase cells. This observation indicates that the second level of chromosome organization also exists in S-phase chromosomes. It appears, theref ore, that the random walk/giant loop model developed earlier for G0/G1 chromosomes is also valid for S-phase chromosomes.