Conformation of replicated segments of chromosome fibres in human S-phase nucleus

Recent statistical analysis of the folding of G(0)/G(1) chromosomes using f luorescence in situ hybridization (FISH) allowed development of a random wa lk/giant loop model of chromosome structure. According to this model there are two levels of organization of G(0)/G(1) chromosome fibres. On the first level, the fibres are arranged in giant loops several Mbp in size, and wit hin 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 visual ized at high resolution in S-phase nuclei using immunofluorescent methods a nd appear as worm-like fibres. In our earlier study, we analysed conformati on of the fibres in human cells blocked for 16 h at the G(1)/S boundary wit h 5-fluorodeoxyuridine (FdU) and then released into S-phase by the addition of a DNA precursor. However, long treatment of cells with FdU induces very short replicons and may promote apoptosis. In this study we analysed confo rmation of the fibres in normally proliferating human cells that had not be en blocked with FdU for a long time. It has been found that replicated chro mosome fibres visualized just after 2 h of incubation of the cells with a n on-radioactively labelled DNA precursor behave as flexible polymer chains w ithout major constraints, and that their local conformation in the range of several microns of their contour length may be considered as random. Confo cal analysis of human X chromosomes visualized in HeLa cells using FISH wit h a specific painting probe shows that in S-phase the chromosomes occupy di stinct nuclear territories and their apparent size does not differ from tha t in non-S-phase cells. This observation indicates that the second revel of chromosome organization also exists in S-phase chromosomes. It appears, th erefore, that the random walk/giant loop model developed earlier for G(0)/G (1) chromosomes is also valid for S-phase chromosomes.