Christian Zorn

Rabl's model of the interphase chromosome arrangement tested in Chinise hamster cells by premature chromosome condensation and laser-UV-microbeam experiments

SummaryIn 1885 Carl Rabl published his theory on the internal structure of the interphase nucleus. We have tested two predictions of this theory in fibroblasts grown in vitro from a female Chinese hamster, namely (1) the Rabl-orientation of interphase chromosomes and (2) the stability of the chromosome arrangement established in telophase throughout the subsequent interphase. Tests were carried out by premature chromosome condensation (PCC) and laser-UV-microirradiation of the interphase nucleus. Rabl-orientation of chromosomes was observed in G1 PCCs and G2 PCCs. The cell nucleus was microirradiated in G1 at one or two sites and pulse-labelled with 3H-thymidine for 2h. Cells were processed for autoradiography either immediately thereafter or after an additional growth period of 10 to 60h. Autoradiographs show unscheduled DNA synthesis (UDS) in the microirradiated nuclear part(s). The distribution of labelled chromatin was evaluated in autoradiographs from 1035 cells after microirradiation of a single nuclear site and from 253 cells after microirradiation of two sites. After 30 to 60h postincubation the labelled regions still appeared coherent although the average size of the labelled nuclear area fr increased from 14.2% (0h) to 26.5% (60h). The relative distance dr, i.e. the distance between two microirradiated sites divided by the diameter of the whole nucleus, showed a slight decrease with increasing incubation time. Nine metaphase figures were evaluated for UDS-label after microirradiation of the nuclear edge in G1. An average of 4.3 chromosomes per cell were labelled. Several chromosomes showed joint labelling of both distal chromosome arms including the telomeres, while the centromeric region was free from label. This label pattern is interpreted as the result of a V-shaped orientation of these particular chromosomes in the interphase nucleus with their telomeric regions close to each other at the nuclear edge. Our data support the tested predictions of the Rabl-model. Small time-dependent changes of the nuclear space occupied by single chromosomes and of their relative positions in the interphase nucleus seem possible, while the territorial organization of interphase chromosomes and their arrangement in general is maintained during interphase. The present limitations of the methods used for this study are discussed.

Unscheduled DNA synthesis after partial UV irradiation of the cell nucleus: Distribution in interphase and metaphase

Cells of an euploid strain of the Chinese hamster synchronized in the G1 phase were microirradiated in the nucleus with a laser UV microbeam (λ = 257 nm) and pulse-labelled with [3H]thymidine. In autoradiographs of cells fixed immediately after the pulse unscheduled DNA synthesis (UDS) was found restricted to the microirradiated part of the nucleus. The rate of UDS varied with the UV energy applied and the post-irradiation incubation time. In other experiments chromosome preparations were established after an additional chase and a subsequent growth period. In 28 mitotic cells autoradiographic label was found concentrated on a few chromosomes which lay adjacent to each other in one part of the metaphase plate. The distribution of label on the chromosomes could clearly be distinguished from patterns which originate from semi-conservative DNA synthesis within S phase. The label on chromosomes of microirradiated cells thus represents UDS. Our findings support the following ideas on the arrangement of interphase chromosomes: (1) Decondensed interphase chromosomes may occupy rather compact territories. (2) Chromosomes do not necessarily exhibit a close and permanent association with their respective homologues.

Laser UV Microirradiation of Interphase Nuclei and Post-Treatment with Caffeine A New Approach to Establish the Arrangement of Interphase Chromosomes

SummaryLaser UV microirradiation of Chinese hamster interphase cells combined with caffeine post-treatment produced different patterns of chromosome damage in mitosis following irradiation of a small area of the nucleus that may be classified in three categories: I) intact metaphase figures, II) chromosome damage confined to a small area of the metaphase spread, III) mitotic figures with damage on all chromosomes. Category III might be the consequence of a non-localized distortion of nuclear metabolism. By contrast, category II may reflect localized DNA damage induced by microirradiation, which could not be efficiently repaired due to the effect of caffeine. If this interpretation is right, in metaphase figures of category II chromosome damage should occur only at the irradiation site. The effect might then be used to investigate neighbourhood relationships of individual chromosomes in the interphase nucleus.

Localized ultraviolet laser microbeam irradiation of early Drosophila embryos: Fate maps based on location and frequency of adult defects

Drosophila embryos were locally irradiated with a 257-nm laser microbeam during blastoderm and germ band stages. Depending on stage and beam diameter (10–30 μm), from 0 to 45 nuclei were exposed to the uv radiation. The doses used, 5 or 10 erg, did not eliminate nuclei or cells at once, but up to 50% of the adult survivors from irradiated eggs carried defects in the thorax. These were scored with reference to the imaginal discs from which the affected structures derive. For each thoracic disc a “target center” was calculated as the weighted mean value of all beam locations affecting the respective adult derivatives. The target centers for the germ band stage map within the respective germ band segments. The pattern of target centers for the blastoderm stage is comparable to the thoracic region of published fate maps, and the distances between adjacent leg centers (approximately three cell diameters) agree with recent evidence based on mosaic flies. We discuss the question whether the target centers mark the position of the respective disc progenitor cells at the stages of irradiation and conclude that these positions are rendered rather correctly at least with reference to the longitudinal egg axis.

Effects of laser uv-microirradiation (lambda = 2573 A) on proliferation of Chinese hamster cells.

CREMER, C., CREMER, T., ZORN, C., AND SCHOELLER, L. Effects of Laser UV-Microirradiation (X = 2573 A) on Proliferation of Chinese Hamster Cells. Radiat. Res. 66, 106-121 (1976). A laser uv-microbeam with a wavelength of 2573 A* having a minimum spot diameter of approx 0.5 sm was used to microirradiate interphase cells of a V-79 subline of Chinese hamster cells. The incident energy necessary to induce a significant decrease of proliferation was 30 to 60 times larger after microirradiation of cytoplasm as compared with microirradiation of nucleoplasm. The mean value of relative cell numbers 40 hr after irradiation as a function of incident energy did not differ whether the cells were microirradiated lying singly or together in small groups. Analysis of individual growth curves of singly lying cells microirradiated in the nucleoplasm with the same energy showed heterogeneous reactions. The incident energy per cell compatible with proliferation of about 50% of the cells after microirradiation of nucleoplasm was approx. 2 X 10-3 ergs. From this value it is suggested that the energy density within the focus was in the region of several thousand ergs per square millimeter. Photochemical effects are thought to be the cause of growth disturbance,while thermal effects are excluded.

Formation of viable cell fragments by treatment with colchicine

Time-lapse cinematography of human fibroblasts revealed that mitotic cells separated into numerous cell fragments containing varying amounts of chromatin and cytoplasm when treated with colchicine. As cell fragments were very loosely attached to the surface of the culture vessel during their formation, they could be easily detached like mitotic cells by gently shaking the vessel and thus separated from normal interphase cells. Fragments obtained by this procedure were able to exclude trypan blue indicating, therefore, an intact cell membrane. When placed into Petri dishes many of them attached to and even spread out on the surface. Five hours later the majority of the attached fragments incorporated [3H]leucine. Time-lapse films showed that fragments were able to extend and retract pseudopodia at least for several hours after their formation. Although the fragments degenerated within a few days, in the present experiments the possibility was not excluded that fragments which had lost only a very small amount of chromatin and cytoplasm survived for longer periods of time. The observations clearly indicate viability of many newly formed fragments.