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DNA double-strand breaks (DSBs) can efficiently kill cancer cells, but they can also produce unwanted chromosome rearrangements when DNA ends from different DSBs are erroneously joined. Movement of DSB-containing chromatin domains might facilitate these DSB interactions and promote the formation of chromosome rearrangements. Therefore, we analyzed the mobility of chromatin domains containing DSBs, marked by the fluorescently tagged DSB marker 53BP1, in living mammalian cells and compared it with the mobility of undamaged chromatin on a time-scale relevant for DSB repair. We found that chromatin domains containing DSBs are substantially more mobile than intact chromatin, and are capable of roaming a more than twofold larger area of the cell nucleus. Moreover, this increased DSB mobility, but not the mobility of undamaged chromatin, can be reduced by agents that affect higher-order chromatin organization.

Purpose: It is generally accepted that chromosome exchanges in irradiated cells are formed through interactions between separate DNA double-strand breaks (DSB). Here we tested whether non-irradiated DNA participates in the formation of chromosome aberrations when complex DNA DSB are induced elsewhere in the nucleus., Materials and Methods: Synchronized Chinese hamster cells containing an X chromosome with a late replicating q arm (X(q) domain) were labelled with 125I-iododeoxyuridine (125IdUrd) in a period of S-phase when the vast majority of the X(q) domain was not replicating. DNA damage from 125I decay was accumulated at the G1/S border while the cells were stored in liquid nitrogen. Decay of 125I induced DSB in the immediate vicinity of the 125I atom. Chromosome aberrations involving what is essentially the 125I-free X domain were scored at the first mitosis after cell thawing. As a positive control, cells were treated with 125IdUrd at a later period in S-phase when the X(q) domain replicates, yielding a labelled X(q) domain., Results: The 125I-free X(q) domain exhibited chromosome aberrations (exchanges and fragments). The frequency of these aberrations was linearly dependent on the number of 125I decays elsewhere in the cell nucleus. The efficiency of formation of chromosome aberrations by the 125I-free X(q) domain was approximately half of that observed in the 125I-labelled X(q) domain., Conclusions: The involvement of the 125I-free X(q) domain in chromosome aberrations suggests that DNA not damaged by the decay of incorporated 125I can interact with damaged DNA, indicating the existence of an alternative pathway for the formation of chromosome aberrations.

We developed a method for partial irradiation of cell nuclei and for highlighting the irradiated chromatin domain(s) in both interphase nuclei and metaphase chromosomes. The method involves the use of the replication program of chromosomes and consists of three major steps: I) selection of a suitable chromatin domain, II) damage induction by 125I, and III) visualization of the domain. Here, the first step of the method, applied to Chinese hamster HA-1 cells, is described. Using pulse labelling with the replication marker IUdR, it was shown that Xq does not replicate at early S-phase and that the replication timing of Xq can be highly effectively synchronized with hydroxyurea in a whole cell population. Thus, the replication timing of Xq may be used to exclude or to incorporate 125I into the Xq. Other chromatin can be selected and targeted with 125I in a similar way. Examples of possible applications of the method are given.

Transcription and replication are, like many other nuclear functions and components, concentrated in nuclear domains. Transcription domains and replication domains may play an important role in the coordination of gene expression and gene duplication in S-phase. We have investigated the spatial relationship between transcription and replication in S-phase nuclei after fluorescent labelling of nascent RNA and nascent DNA, using confocal immunofluorescence microscopy. Permeabilized human bladder carcinoma cells were labelled with 5-bromouridine 5'-triphosphate and digoxigenin-11-deoxyuridine 5'-triphosphate to visualize sites of RNA synthesis and DNA synthesis, respectively. Transcription by RNA polymerase II was localized in several hundreds of domains scattered throughout the nucleoplasm in all stages of S-phase. This distribution resembled that of nascent DNA in early S-phase. In contrast, replication patterns in late S-phase consisted of fewer, larger replication domains. In double-labelling experiments we found that transcription domains did not colocalize with replication domains in late S-phase nuclei. This is in agreement with the notion that late replicating DNA is generally not actively transcribed. Also in early S-phase nuclei, transcription domains and replication domains did not colocalize. We conclude that nuclear domains exist, large enough to be resolved by light microscopy, that are characterized by a high activity of either transcription or replication, but never both at the same time. This probably means that as soon as the DNA in a nuclear domain is being replicated, transcription of that DNA essentially stops until replication in the entire domain is completed.

Time-resolved measurements of the nuclear volume response of human peripheral T-lymphocytes, under aniosmotic conditions, are presented. In the experiments slit scanning FlowCytometry methods were used. We propose an extension to the standard solid viscoelastic model to interpret the observed dynamical behavior of the nucleus. It is shown that experimental and theoretical evidence indicates a passive nuclear response merely induced by a mechanical link (i.e., the cytoskeleton) between the cell membrane and the nuclear envelope. Implications of this work to the field of cellular mechanics and cytoskeletal rheology are surveyed.

An instrument is described which analyses shapes of fluorescence profiles generated by particles passing through the focussed laser beam of a flow cytometer. The output signal of this pulse-shape analyzer is used as input for the signal processing electronics of a commercial flow cytometer system. The instrument detects dips in pulse-profiles; a shape parameter named Pulse Dip Index (PDI) is defined as the ratio of the integrated signal from the beginning of the pulse until the first dip, relative to the integrated signal of the complete profile. This PDI is similar to the Centromeric Index of chromosomes. The composition of aggregates in mixtures of fluorescent particles of different sizes was evaluated by PDI analysis. In our experiments the PDI was determined within 30 microseconds from the onset of the pulse-profile and particles with a specified morphology of interest were selected for on-line registration of their profiles as digitized pulse-shapes. In a cell sorter system, the PDI can be used as a parameter for sorting.

Incubation of Bacillus subtilis after outgrowth from spores in the presence of four different antibiotics in two different concentrations, showed that septation can occur without termination of nuclear division. Septation is then only partially uncoupled from the normal division cycle. Observations on location and development of mesosomes in the presence of the antibiotics, made in three-dimensional cell reconstructions, suggest that the mesosome plays a role in the normal coordination between nuclear and cell division, and may explain the partial independence between these two processes in B. subtilis.