Higher-order chromatin structure of human granulocytes.

The structural organisation of chromatin in eukaryotes plays an important role in a number of biological processes. Our results provide a comprehensive insight into the nuclear topography of human peripheral blood granulocytes, mainly neutrophils. The nuclei of granulocytes are characterised by a segmented shape consisting of two to five lobes that are in many cases connected by a thin DNA-containing filament. The segregation of chromosomes into the nuclear lobes was studied using fluorescence in situ hybridisation (FISH). We were able to distinguish different topographic types of granulocytes on the basis of the pattern of segregation. Five topographic types were detected using dual-colour FISH in two-lobed nuclei. The segregation of four sets of genetic structures could be studied with the aid of repeated FISH and a large number of topographic types were observed. In all these experiments a non-random distribution of chromosomes into nuclear lobes was found. The painting of a single type of chromosome in two-lobed nuclei showed the prevalence of symmetric topographic types (on average in 65.5% of cases) with significant variations among individual chromosomes. The results of analysis of five topographic types (defined by two chromosomes in two-lobed nuclei) showed that the symmetric topographic types for both chromosomes are significantly more frequent than predicted. Repeated hybridisation experiments confirmed that the occurrence of certain patterns of chromosome segregation is much higher than that predicted from the combination of probabilities. The frequency of symmetric topographic types for chromosome domains was systematically higher than for genes located on these chromosomes. It appears that the prevalence of symmetric segregation patterns is more probable for large objects such as chromosome domains than for genes located on chromatin loops extending outwards from the surface of the domain defined by specific chromosome paints. This means that one chromosome domain may occur in different lobes of granulocytic nuclei. This observation is supported by the fact that both genes and centromeres were observed on filaments joining different lobes. For all chromosomes, the distances between the membrane and fluorescence gravity centre of the chromosome were measured and correlated with the segregation patterns. A higher percentage of symmetric topographic types was found in those chromosomes that were located closer to the nuclear membrane. Nuclear positioning of all genetic elements in granulocytic nuclei was studied in two-dimensional projection; however, the results were verified using three-dimensional analysis.

Exchange aberrations among 11 chromosomes of human lymphocytes induced by gamma-rays.

PURPOSE: To detect the frequencies of interchanges among 11 chromosomes in lymphocytes irradiated with gamma-rays and to find out whether these frequencies reflect the proximity of some of these chromosomes within the interphase nucleus. MATERIAL AND METHODS: Exchange aberrations were detected in the first mitosis after irradiation of human lymphocytes with 3 and 5 Gy gamma-rays of 60Co. Two-colour repeated FISH with two differently chemically modified probes in each hybridization was applied. The microscope stage positions of each mitosis were recorded after the first hybridization and used for the automatic scanning of images after all successive experiments. Five images were obtained for each mitosis differing in visualized pairs of chromosomes. Comparing these images, exchanges among 10 chromosomes could be detected. Painting of the p arm of chromosome 21 with the painting probe for chromosome 22 also made it possible to detect exchanges of this chromosome with other chromosomes of the selected group. RESULTS: Frequencies of exchange aberrations induced in chromosomes of the selected group as well as interchanges between many pairs of chromosomes of this group were roughly proportional to the DNA content of chromosomes. Higher frequencies of interchanges than expected according to the model of linear proportionality were found between several chromosomes involved in translocations frequent in different subtypes of leukaemia. CONCLUSIONS: Frequencies of interchanges among 11 chromosomes of human lymphocytes induced by gamma-rays do not indicate as clearly as fast neutrons the non-random arrangement of chromosomes in the cell nucleus. The interaction of a large number of chromosomes in exchange aberrations suggests that the chromatin in the territory of one chromosome is accessible for several other chromosomes.