Immunocytochemical localization of chromatin regions UV-microirradiated in S phase or anaphase

Abstract

Chinese hamster cells (M3-1 line) in S phase were laser-UV-microirradiated (I, 257 nm) at a small site of the nucleus. Cells were fixed either immediately thereafter or in subsequent stages of the cell cycle, including prophase and metaphase. The microirradiated chromatin was visualized by indirect immunofluorescence microscopy using antibodies specific for UV-irradiated DNA. During the whole post-incubation period (4-15 h) immunofluorescent labelling was restricted to a small part of the nucleus (f, 4.5% of the total nuclear area). In mitotic cells segments of a few chromosomes only were labelled. Following microirradiation of chromosome segments in anaphase, immunofluorescent labelling was observed over a small part of the resulting interphase nucleus. A territorial organization of interphase chromosomes, i.e. interphase chromosomes occupying distinct domains, has previously been demonstrated by our group for the nucleus of Chinese hamster cells in Gl. Our present findings provide evidence that this organization pattern is maintained during the entire cell cycle.Evidence has recently been provided for a highly ordered arrangement of centromeres in metaphase plates of certain plant species [I]. Non-random arrangements of chromosomes have also been shown to a certain extent in metaphase plates of several mammalian species including man [2, 31. These findings have shed new light on the question of an ordered arrangement of interphase chromosomes as well, a question which still appears largely unsettled [3-71. Within this context it is important to know how the chromatin of individual chromosomes is distributed within the interphase nucleus at different stages of the cell cycle.In 1885, Rabl [8] suggested a model which predicts that interphase chromosomes maintain their anaphase-telophase orientation and occupy distinct nuclear

Specific staining of human chromosomes in Chinese hamster x man hybrid cell lines demonstrates interphase chromosome territories

“…In all cases label was found concentrated on a few chromosomes (Zorn et al 1979;Cremer et al 1982). The same result was obtained when a small site of the interphase nucleus was microirradiated at S-phase and the microirradiated chromatin was visualized in the subsequent metaphase by indirect immunofluorescence microscopy using antibodies specific for UV-irradiated DNA (Hens et al 1983). Recently it has been demonstrated that the number of chromosomes in which sister chromatid exchanges (SCEs) can be induced by laser-UV-microirradiation depends on the size of the nuclear area subjected to the laser-UV-microbeam (Raith et al 1984).…”

“…Thereafter slides were rinsed in 20 triM Tris/ HC1 (pH 7.5) containing 5 mM EDTA and air dried. Finally, cells and metaphase spreads were stained with Giemsa or DAPI (Hens et al 1983).…”

Chromatin Dynamics in Interphase Nuclei and Its Implications for Nuclear Structure

“…For example, a nuclear membrane–apposed/peripheral distribution has been observed for inactivated X chromosomes in fibroblasts, centromeres in some types of cells, and centromeres and telomeres of polytene chromosomes in Drosophila salivary gland nuclei ( Moroi et al, 1981 ; Mathog et al, 1984 ; Manuelidis and Borden, 1988 ; Dyer et al, 1989 ). Similarly, chromosomal confinement to domains has been observed or deduced from in situ hybridization studies and DNA irradiation studies ( Cremer et al, 1982 ; Hens et al, 1983 ; Schardin et al, 1985 ; Cremer et al, 1988 ; Lichter et al, 1988 ; Manuelidis and Borden, 1988 ; Pinkel et al, 1988 ; Leitch et al, 1990 ; Popp et al, 1990 ). Finally, active genes can exhibit nonrandom organization, concentrating in the nuclear periphery of mouse L and P19 embryonal carcinoma cells and near the borders of condensed chromatin in nucleated newt erythrocytes ( Hutchison and Weintraub, 1985 ; de Graaf et al, 1990).…”

Differences of size and shape of active and inactive X‐chromosome domains in human amniotic fluid cell nuclei

“…First, laser-UV-microbeam studies were applied to follow microirradiated chromatin in living Chinese hamster cells cultured in vitro throughout the cell cycle. Microirradiated chromatin was detected either by autoradiography (following incorporation of 3H-thymidine during unscheduled DNA synthesis) (Zorn et al, 1979;Cremer et al, 1982) or by indirect immunofluorescence with antibodies specific for UVirradiated DNA (Cremer et al, 1984;Hens et al, 1983). These experiments demonstrated that chromatin microirradiated at any small part of the interphase nucleus was composed of a few chromosome segments only.…”