Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes

Studies of higher-order chromatin arrangements are an essential part of ongoing attempts to explore changes in epigenome structure and their functional implications during development and cell differentiation. However, the extent and cell-type-specificity of three-dimensional (3D) chromosome arrangements has remained controversial. In order to overcome technical limitations of previous studies, we have developed tools that allow the quantitative 3D positional mapping of all chromosomes simultaneously. We present unequivocal evidence for a probabilistic 3D order of prometaphase chromosomes, as well as of chromosome territories (CTs) in nuclei of quiescent (G0) and cycling (early S-phase) human diploid fibroblasts (46, XY). Radial distance measurements showed a probabilistic, highly nonrandom correlation with chromosome size: small chromosomes—independently of their gene density—were distributed significantly closer to the center of the nucleus or prometaphase rosette, while large chromosomes were located closer to the nuclear or rosette rim. This arrangement was independently confirmed in both human fibroblast and amniotic fluid cell nuclei. Notably, these cell types exhibit flat-ellipsoidal cell nuclei, in contrast to the spherical nuclei of lymphocytes and several other human cell types, for which we and others previously demonstrated gene-density-correlated radial 3D CT arrangements. Modeling of 3D CT arrangements suggests that cell-type-specific differences in radial CT arrangements are not solely due to geometrical constraints that result from nuclear shape differences. We also found gene-density-correlated arrangements of higher-order chromatin shared by all human cell types studied so far. Chromatin domains, which are gene-poor, form a layer beneath the nuclear envelope, while gene-dense chromatin is enriched in the nuclear interior. We discuss the possible functional implications of this finding.     

Ploidy of bovine nuclear transfer blastocysts reconstructed using in vitro produced blastomere donors

The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro development. Because little is known about ploidy errors in nuclear transfer embryos, this was examined using embryos reconstructed from in vitro produced embryo donors. In vitro matured oocytes were enucleated and then activated using calcium ionophore A23187 followed by 6-dimethylaminopurine (6-DMAP). Subsequently, embryos were reconstructed using blastomeres from day 4-5 in vitro produced donors. The embryos were cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 +/- 20.2 (mean % +/- SD) nuclei/embryo were examined. Of these 16, 7 embryos (43.8%) were potentially abnormal because in these, 1.1%, 1.4%, 5.3%, 7.5%, 26.3%, 30.4%, and 66.2% % of the nuclei had a chromosome composition deviating from the diploid condition, indicating a wide degree of variation between embryos. These errors comprised mainly triploid (8.2 +/- 10.3 [0-26.3]: % +/- SD [range]) and tetraploid (10.6 +/- 19.9 [0-54.9]) nuclei with other ploidy combinations accounting for only 0.9 +/- 2.1 [0-2.1]% of deviant nuclei. The proportion of completely normal nuclear transfer embryos was no less than those produced by in vitro fertilization but the distribution of chromosome abnormalities was different (p = 0.0002). In conclusion, nuclear transfer embryos reconstructed using blastomere cells can produce over 50% blastocysts with a diploid chromosome complement. However, the contribution of chromosome abnormalities to embryonic loss in the remaining embryos deserves further investigation.     

Double in situ hybridization in combination with digital image analysis: a new approach to study interphase chromosome topography

Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a 2D-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the 2D-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far.     

Lateral asymmetry of constitutive heterochromatin in human chromosomes

Summary Variations in lateral asymmetry of constitutive heterochromatin were studied in 30 normal individuals with reference to the chromosomal regions 1q12, 9q12, 15p11, 16q12 and Yq12. The technique consisted of growing human lymphocytes for one cell cycle in BrdU, staining with 33258 Hoechst, exposing them to UV light, treating them with 2 x SSC, and staining with Giemsa. This procedure revealed asymmetric staining in the region of constitutive heterochromatin in these chromosomal regions. Chromosomes 15, 16, and Y showed simple lateral asymmetry, whereas chromosome 1 showed both simple and compound asymmetry. In 15 cases, compound lateral asymmetry was evident in both homologues of chromosome 1, 12 cases showed compound lateral asymmetry in one homologue and simple lateral asymmetry in the other, and the remaining three cases showed simple lateral asymmetry in both the homologues. The centromere region of chromosome 9 stained symmetrically with this technique. The lateral asymmetry is presumed to reflect the strand bias in the distribution of thymine in satellite DNA fractions.     

Localization of genetic elements of intact and derivative chromosome 11 and 22 territories in nuclei of Ewing sarcoma cells

Recurring chromosomal abnormalities are associated with specific tumour types. The EWSR1 and FLI1 genes are involved in balanced translocation t(11;22)(q24;q12), which is present in more than 85% of Ewing sarcomas. In our previous study, we have found that the fusion genes pertaining to both derivative chromosomes 11 and 22 in Ewing sarcoma cell nuclei are shifted to the midway nuclear position between the native EWSR1 and FLI1 genes. In this contribution we focused our attention at nuclear positioning of other genetic elements of chromosomes 11 and 22 in order to find if the whole derivative chromosomes or only their translocated parts change their nuclear positions in comparison with the native chromosomes. Using repeated fluorescence in situ hybridization and high-resolution cytometry, 2D radial positions of EWSR1, BCR, FLI1, BCL1 genes and fluorescence weight centres of chromosome territories were compared for intact and derivative chromosomes 11 and 22 in nuclei of three Ewing sarcoma samples. Significant radial shift was obtained for the derivative EWSR1, FLI1 and BCL1 genes and for the derivative chromosome 11 compared with the intact ones and not very significant for chromosome 22 and the BCR gene. Our results also suggest that the mean nuclear positions of fusion genes are determined by the final structure of the derivative chromosomes and do not depend on the location of the translocation event.     

Relationship between cell size and nuclear volume in nucleated red blood cells of developmentally matched diploid and tetraploid mouse embryos.

Analysis of control diploid and polyploid amphibia indicated that cell and nuclear volumes were closely related to their ploidy, so that an increase in ploidy was generally associated with an increase in cell size. This relationship is also believed to occur in mammalian polyploids. However, since the latter are only rarely encountered spontaneously, or only occasionally following experimental manipulation, no detailed morphometric studies have been carried out to date to confirm whether such a relationship exists. In this study, the cellular and nuclear volume of primitive red blood cells was analyzed in carefully developmentally matched control diploid mouse embryos and tetraploid embryos produced by the technique of electrofusion. All of the cells and/or nuclei studied had a characteristic spherical shape which greatly simplified the morphometric analysis. A defined and predictable relationship between ploidy and cellular and/or nuclear volume was observed in the red blood cells between 8.25 and 14.5 days of gestation. During this period the primitive red blood cells are gradually replaced by the definitive erythrocytes. The ratio of control values to tetraploid values was found to be close to the theoretical value of 1:2 when comparable cells and/or their nuclei were analyzed in carefully developmentally matched material.     

Mitosis is not the only distributor of mutated cells: Non‐mitotic endopolyploid cells produce reproductive genome‐reduced cells

Giant endopolyploid nuclei (>16n) can spontaneously fragment by endomitosis (nuclear internal division) into near‐diploid cells with reproductive capacity (depolyploidization), and endotetra/octopolyploidy can undergo chromosome‐visible meiotic‐like genome reductional divisions also to replicative subcells. These unconventional divisions are associated with production of aneuploidy, which led to the question in this study of whether endopolyploidy, in general, can contribute genetic variability to tumorigenic potential. For this purpose, non‐proliferative endopolyploid cells (range: 4n–32n) in near‐senescence of normal diploid cell strains were analysed for nuclear–morphogenic changes associated with the presence of diploid‐sized nuclei in the cytoplasm. A one‐by‐one nuclear‐cutoff process gave rise to reproducing genome‐reduced cells. It was concluded that these unconventional cell divisions are, indeed, suspects of originating genetic variability. Details of these irregular mitoses were compared to ‘mitotic–meiosis’ in primitive organisms, which suggested activation of an ancestral trait in the mammalian cells.     

Sex specific chromosome polymorphisms in the common Indian Krait,Bungarus caeruleus Schneider (Ophidia,Elapidae)

Chromosome analyses of common Indian Krait, B. caeuleus from three geographical regions of India have revealed variable diploid numbers of 43, 44 and 45 in different female individuals but a constant diploid number of 44 in the males. C-banding and in situ hybridization studies, using radio labelled W sex chromosome specific satellite DNA as a probe, have shown that C-banding and sex chromosome associated satellite DNA's are exclusively localised in the W chromosome. The W chromosome is involved in reciprocal translocations either with a medium sized macroautosome or with a microchromosome resulting in a multiple sex chromosome constitution of Z₁Z₁Z₂Z₂/Z₁Z₂W type. In some female individuals dissociation of the W has resulted in multiple W chromosomes, W₁ and W₂. These polymorphisms are uniquely confined to the female sex only. A predominance of polymorphic females, involving particularly the translocation of a medium sized macrochromosome, in all three geeographical regions and the restriction of the females having the original chromosome constitution (ZW) to one geographical region suggests that polymorphic individuals have adaptive flexibility and higher fecundity.     

Cytogenetic analysis of diploidy in cloned bovine embryos using an improved air-dry karyotyping method

Of the few published studies on the cytogenetic analyses of bovine nuclear transferred (NT) embryos, results differ between air-dry and fluorescent in situ hybridization (FISH) procedures. A modified air-dry procedure is reported in this study that provides more metaphase plates for analysis. Day 5 and Day 7 bovine NT embryos were cultured in colcemid-containing CR1aa for 10-12 or 16-18 h, then treated in hypotonic sodium citrate for 3-5 min. The standard procedure of 5h in colcemid and 15-20 min in hypotonic solution was the control. A much higher (P<0.01) percent of mitotic nuclei was observed in the experimental groups. The 33 and 41% mitotic nuclei were obtained from 10 to 12 h and 16 to 18 h-colcemid-treated Day 5 embryos, respectively, which was higher (P<0.001) than the control (15%). The mitotic nuclei in Day 7 NT embryos were 24% in 10-12 h- and 28% in 16-18 h-colcemid-treated groups, which also was higher (P<0.05) than the control (10%). The majority of analyzable embryos were diploid. Analyses of mixoploid embryos showed on average that 70% of the cells were diploid. Day 5 mixoploid embryos contained numerically higher polyploid cells than Day 7 embryos, although statistically there were no differences. We concluded that the modified air-dry method provided a larger source of mitotic nuclei for chromosome analyses of cloned bovine embryos.     

Genomics of type I diabetes mellitus and its late complications

In ethnic Russians, MHC (HLA) was shown to be the major locus determining the predisposition to type 1 diabetes mellitus (T1DM). To map the regions linked to T1DM, families with concordant or discordant sib pairs were selected from the Russian population of Moscow. With these families, linkage to T1DM was demonstrated for CTLA4 (IDDM12, 2q32.1-q33), which codes for a T-cell surface antigen, and PDCD2 (IDDM8, 6q25-q27), which is homologous to the mouse programmed cell death activator gene. With polymorphic microsatellites, regions 3q21-q25 (IDDM9) and 10p12.2 (IDDM10) were also linked to T1DM. Case/control and family studies of the polymorphic markers from region 11p13 revealed a new T1DM-associated locus in the vicinity of the catalase gene (CAT); linkage to this locus was not reported earlier for other populations. Diabetic polyneuropathy (DPN) proved to be associated with single-nucleotide polymorphisms Ala(-9)Val (SOD2), Arg213Gly (SOD3), and T(-262)C (CAT) and with a polymorphic microsatellite of the NOS2 promoter. Hence oxidative stress, which results from hyperglycemia, increased mitochondrial production of superoxide radicals, and insufficient activities of antioxidative enzymes, was assumed to play an important part in DPN development in T1DM. Diabetic nephropathy (DN) showed no association with the antioxidative enzyme genes. However, the association was observed for the insertion/deletion (I/D) polymorphism of ACE and the ecNOS34a/4b polymorphism of NOS3, two genes involved in controlling vascular tonicity, and for the I/D polymorphism of APOB and the epsilon 2/epsilon 3/epsilon 4 polymorphism of APOE, two genes involved in lipid transport. In addition, polymorphic microsatellites of chromosome 3q21-q25 proved to be closely associated with DN. The tightest association was established for D3S1550, carriers of allele 12 or genotype 12/14 having high risk of DN (OR = 4.85 and 6.25, respectively). Region 3q21-q25 was assumed to contain a major gene determining DN development, while the other DN-associated genes mostly affect the progression of DN.     

Distribution of chromosome 18 and X centric heterochromatin in the interphase nucleus of cultured human cells

In situ hybridization of human chromosome 18 and X-specific alphoid DNA-probes was performed in combination with three dimensional (3D) and two dimensional (2D) image analysis to study the interphase distribution of the centric heterochromatin (18c and Xc) of these chromosomes in cultured human cells. 3D analyses of 18c targets using confocal laser scanning microscopy indicated a nonrandom disposition in 73 amniotic fluid cell nuclei. The shape of these nuclei resembled rather flat cylinders or ellipsoids and targets were preferentially arranged in a domain around the nuclear center, but close to or associated with the nuclear envelope. Within this domain, however, positionings of the two targets occurred independently from each other, i.e., the two targets were observed with similar frequencies at the same (upper or lower) side of the nuclear envelope as those on opposite sides. This result strongly argues against any permanent homologous association of 18c. A 2D analytical approach was used for the rapid evaluation of 18c positions in over 4000 interphase nuclei from normal male and female individuals, as well as individuals with trisomy 18 and Bloom's syndrome. In addition to epithelially derived amniotic fluid cells, investigated cell types included in vitro cultivated fibroblastoid cells established from fetal lung tissue and skin-derived fibroblasts. In agreement with the above 3D observations 18c targets were found significantly closer (P less than 0.01) to the center of the 2D nuclear image (CNI) and to each other in all these cultures compared to a random distribution derived from corresponding ellipsoid or cylinder model nuclei. For comparison, a chromosome X-specific alphoid DNA probe was used to investigate the 2D distribution of chromosome X centric heterochromatin in the same cell types. Two dimensional Xc-Xc and Xc-CNI distances fit a random distribution in diploid normal and Bloom's syndrome nuclei, as well as in nuclei with trisomy X. The different distributions of 18c and Xc targets were confirmed by the simultaneous staining of these targets in different colors within individual nuclei using a double in situ hybridization approach.     

The changes in chromosome 6 spatial organization during chromatin polytenization in the Calliphora erythrocephala Mg. (Diptera: Calliphoridae) nurse cells

Localization of Calliphora erythrocephala chromosome 6 in a 3D nuclear space at different stages of nurse cell chromatin polytenization was analyzed by fluorescence in situ hybridization and 3D microscopy. The obtained results suggest a large-scale chromatin relocation in the C. erythrocephala nurse cell nuclei, which is accompanied by a change in the chromosome territory of chromosome 6 associated with the change in expression activity of the nucleus and formation of reticular chromatin structure. It was revealed that the relocation of chromosome 6 (nucleolus organizer chromosome) is accompanied by fragmentation of the single large nucleolus into micronucleoli, which are spread over the entire nuclear space being associated with their nucleolar organizer regions. Presumably, the chromosome 6 material during transition to a highly polytenized structure is redistributed in the nucleus so that the inactive pericentromeric regions are displaced to the nuclear periphery, while the chromosome regions carrying rDNA sequences loop out beyond the chromosome territory. Being dispersed over the entire nuclear space, rDNA sequences are likely to be amplified, thereby providing numerous small signals from the chromosome 6-specific DNA probe. Micronucleoli are formed around the actively transcribed nucleolar organizer regions.     

Different breakage-prone regions on chromosome 1 detected in t(11;14)-positive mantle cell lymphoma cell lines and multiple myeloma cell lines are associated with different tumor progression-related mechanisms

To better define secondary aberrations that occur in addition to translocation t(11;14)(q13;q32) in mantle cell lymphomas (MCL) and in multiple myelomas (MM), seven t(11;14)-positive MCL cell lines and four t(11;14)-positive MM cell lines were analysed by fluorescence R-banding and spectral karyotyping (SKY). Compared with published data obtained by G-banding, most chromosome aberrations were redefined or further specified. Furthermore, several additional chromosome aberrations were identified. Thus, these cytogenetically well defined t(11;14)-positive MCL and MM cell lines may be useful tools for the identification and characterization of genes that might be involved in the pathogenesis of MCL and MM, respectively. Since MCL and MM were found to have different alterations of chromosome 1, these were investigated in more detail by fluorescence in situ hybridization (FISH) and multicolor banding (MCB) analyses. The most frequently altered and deletion-prone loci in MCL cell lines were regions 1p31 and 1p21. In contrast, breakpoints in MM cell lines most often involved the heterochromatic regions 1p12-->p11, and the subcentromeric regions 1q12 and 1q21. These data are in accordance with previously published data of primary lymphomas. Our findings may indicate that different pathways of clonal evolution are involved in these morphologically distinct lymphomas harboring an identical primary chromosome aberration, t(11;14).     

Cytogenetics of neuroblastoma: a study using fine needle aspiration cultures.

Neuroblastoma is associated with chromosomal aberrations of 1p and 1q in a majority of cases. Some nonrandom secondary changes were observed in this study. The role of these changes in the development and progression of neuroblastoma is examined. Chromosomal analysis was performed on 33 children with neuroblastoma using fine needle aspiration cultures. Metaphases were observed in 57.5% of cases. 86.6% showed the involvement of chromosome 1. Double minutes and unidentifiable markers was also observed. The most frequent secondary changes included add(4)(q35), add(11)(p13), add(14)(q32), add(16)(q12). add(17)(p13), add(19)(q12) and add(21)(q22). Minority of cases showed deletions and translocations. Ploidy pattern ranged from diploid to hypotetraploid. The present study substantiates aberration of chromosome 1 in the form of deletions, additions, duplications and iso-chromosome with some notable secondary abnormalities.     

Internuclear transfer of genetic information in kar1-1/KAR1 heterokaryons in Saccharomyces cerevisiae.

Heterokaryons of Saccharomyces cerevisiae have been constructed utilizing the kar1-1 mutation, which prevents nuclear fusion during conjugation (J. Conde and G. Fink, Proc. Natl. Acad. Sci. U.S.A. 73:3651-3655, 1976). Each heterokaryon contained two haploid nuclei that were marked on several chromosomes. They segregated haploid progeny (cytoductants), most of which have the nuclear genotype of one or the other of the heterokaryon parents, but they occasionally segregated progeny having a recombinant genotype (exceptional cytoductants). Exceptional cytoductants receive the majority of their genome from one parent (the recipient) and a minority from the other (the donor). Transfer of two markers from the donor nucleus to the recipient is rarely coincident for markers located on different chromosomes but is nearly always coincident for those markers located on the same chromosome, suggesting that whole chromosomes are transferred from the donor nucleus to the recipient. In crosses of kar1-1 X KAR1 parents, either nucleus may act as a recipient or donor with equal probability. Recipient nuclei acquired 9 of the 10 chromosomes examined, with frequencies which were inversely correlated with the size of the chromosome. When a chromosome is acquired by the recipient nucleus, it either replaces its homolog or exists in a disomic condition. Haploid progeny emanating from kar1 X KAR1 crosses are frequently inviable. I tested whether this inviability might be the result of chromosome loss by donor nuclei. Viability of progeny from kar1 X KAR1 heterokaryons was improved when the parental nuclei were diploid to an extent consistent with the hypothesis, and diploid progeny which had become monosomic were recovered from these heterokaryons. The following sequence of events accounts for chromosome transfer in kar1 X KAR1 heterokaryons. After cell fusion, each nucleus in the heterokaryon has a probability of about 0.38 of losing one or more chromosomes. A nucleus sustaining such a loss can become a donor in a chromosome transfer event. If the other nucleus does not sustain a mortal chromosome loss, it can become a recipient in a transfer event. The chance of acquiring a chromosome lost by the donor is greater for smaller chromosomes than for larger ones and is about 0.05 for the average chromosome.     

The radial arrangement of the human chromosome 7 in the lymphocyte cell nucleus is associated with chromosomal band gene density

In the nuclei of human lymphocytes, chromosome territories are distributed according to the average gene density of each chromosome. However, chromosomes are very heterogeneous in size and base composition, and can contain both very gene-dense and very gene-poor regions. Thus, a precise analysis of chromosome organisation in the nuclei should consider also the distribution of DNA belonging to the chromosomal bands in each chromosome. To improve our understanding of the chromatin organisation, we localised chromosome 7 DNA regions, endowed with different gene densities, in the nuclei of human lymphocytes. Our results showed that this chromosome in cell nuclei is arranged radially with the gene-dense/GC-richest regions exposed towards the nuclear interior and the gene-poorest/GC-poorest ones located at the nuclear periphery. Moreover, we found that chromatin fibres from the 7p22.3 and the 7q22.1 bands are not confined to the territory of the bulk of this chromosome, protruding towards the inner part of the nucleus. Overall, our work demonstrates the radial arrangement of the territory of chromosome 7 in the lymphocyte nucleus and confirms that human genes occupy specific radial positions, presumably to enhance intra- and inter-chromosomal interaction among loci displaying a similar expression pattern, and/or similar replication timing.