Non-random radial arrangements of interphase chromosome territories: evolutionary considerations and functional implications

In the nucleus of animal and plant cells individual chromosomes maintain a compartmentalized structure. Chromosome territories (CTs), as these structures were named by Theodor Boveri, are essential components of the higher-order chromatin architecture. Recent studies in mammals and non-mammalian vertebrates indicate that the radial position of a given CT (or segments thereof) is correlated with its size, its gene-density and its replication timing. As a representative case, chicken cell nuclei show highly consistent radial chromatin arrangements: gene-rich, early replicating microchromosomes are clustered within the nuclear interior, while gene-poor, later replicating macrochromosomes are preferentially located at the nuclear periphery. In humans, chromosomes 18 and 19 (HSA18 and 19) territories that are of similar size show a distinctly different position in the cell nuclei of lymphocytes and lymphoblastoid cells: the gene-rich and early replicating HSA19 CTs are typically found close to the nuclear center, while the gene-poor and later replicating HSA18 CTs are preferentially located at the nuclear periphery. Recent comparative maps between human and chicken chromosomes revealed that the chicken macrochromosomes 2 and Z contain the genes homologous to HSA18, while the genes on HSA19 are located onto the chicken microchromosomes. These data lend tentative support to the hypothesis that differences in the radial nuclear positions of gene-rich, early replicating and gene-poor, later replicating chromatin have been evolutionarily conserved during a period of more than 300 million years irrespective of the evolution of highly divergent karyotypes between humans and chicken.     

Micromethod for chromosome preparations from Chinese hamster lymphocytes.

Chromosome preparations from Chinese hamster lymphocytes were made by culturing for 2 to 6 days 50,000 to 800,000 lymphocytes in flat-bottomed Cooke microtiter plates in 0.1 or 0.2 ml Roswell Park Memorial Institute-1640 medium (RPMI-1640) supplemented with 5--40 microliters PHA/ml, 20% fetal calf serum (FCS), and 40 microns 2-mercaptoethanol. Depending on blood volume and cell concentrations used, it was possible to obtain up to 1400 well-spread metaphases from one venepuncture.     

Architecture of the Chinese hamster metaphase chromosome

The development of procedures for the isolation of unfixed metaphase chromosomes has made feasible a direct analysis of their morphology. Wholemount stereo electron microscopy was used to examine intact and partially disrupted chromosomes produced by physical shearing and extraction with salt and urea solutions. A model of chromosome architecture was developed to accommodate evidence from studies using both light and electron microscopy. In the proposed model the chromatid (anaphase chromosome) consists of two half-chromatids; each half-chromatid contains two deoxyribonucleoprotein ribbons wound into a single fiber (termed the core), with many loops of chromatin (termed epichromatin) attached along its length. The core ribbons are each about 50 Å thick by 4000 Å wide and are composed of many parallel deoxyribonucleoprotein strands. The epichromatin loops appear to be 250 Å supercoiled fibers containing about 75 per cent of the chromosomal DNA. The epichromatin can be selectively removed from the core fibers by extraction with 2.0 M NaCl or 6.0 M urea solutions.     

Homologous somatic association in radial metaphases of Crepis species

The chromosome arrangement in radial metaphases has been analyzed in root tips of Crepis capillaris (2n=6), C. taraxicifolia (2n=8) and C. rubra (2n=10) by using two statistical approaches: 1) measuring the distances between both members of each pair of homologues as the number of intervening chromosomes on the equatorial ring, and 2) applying a new statistical approach developed by Lacadena et al. (1977) which permits to analyze homologous somatic association considering as a whole the n chromosome pairs of the complement. The occurrence of somatic association of homologous chromosomes is clearly demonstrated with both approaches. Previous results obtained by other authors in different materials and with different statistical methods are discussed. The karyotypes of C. taraxifolia and C. rubra are described with numerical data.     

Non-random X chromosome inactivation in Aicardi syndrome

Most females have random X-chromosome inactivation (XCI), defined as an equal likelihood for inactivation of the maternally- or paternally-derived X chromosome in each cell. Several X-linked disorders have been associated with a higher prevalence of non-random XCI patterns, but previous studies on XCI patterns in Aicardi syndrome were limited by small numbers and older methodologies, and have yielded conflicting results. We studied XCI patterns in DNA extracted from peripheral blood leukocytes of 35 girls with typical Aicardi syndrome (AIC) from 0.25 to 16.42 years of age, using the human androgen receptor assay. Data on 33 informative samples showed non-random XCI in 11 (33%), defined as a >80:20% skewed ratio of one versus the other X chromosome being active. In six (18%) of these, there was a >95:5% extremely skewed ratio of one versus the other X chromosome being active. XCI patterns on maternal samples were not excessively skewed. The prevalence of non-random XCI in Aicardi syndrome is significantly different from that in the general population (p < 0.0001) and provides additional support for the hypothesis that Aicardi syndrome is an X-linked disorder. We also investigated the correlation between X-inactivation patterns and clinical severity and found that non-random XCI is associated with a high neurological composite severity score. Conversely, a statistically significant association was found between random XCI and the skeletal composite score. Correlations between X-inactivation patterns and individual features were made and we found a significant association between vertebral anomalies and random XCI.     

Non-random chromosome arrangement in triploid endosperm nuclei

The endosperm is at the center of successful seed formation in flowering plants. Being itself a product of fertilization, it is devoted to nourish the developing embryo and typically possesses a triploid genome consisting of two maternal and one paternal genome complement. Interestingly, endosperm development is controlled by epigenetic mechanisms conferring parent-of-origin-dependent effects that influence seed development. In the model plant Arabidopsis thaliana, we have previously described an endosperm-specific heterochromatin fraction, which increases with higher maternal, but not paternal, genome dosage. Here, we report a detailed analysis of chromosomal arrangement and association frequency in endosperm nuclei. We found that centromeric FISH signals in isolated nuclei show a planar alignment that may results from a semi-rigid, connective structure between chromosomes. Importantly, we found frequent pairwise association of centromeres, chromosomal segments, and entire arms of chromosomes in 3C endosperm nuclei. These associations deviate from random expectations predicted by numerical simulations. Therefore, we suggest a non-random chromosomal organization in the triploid nuclei of Arabidopsis endosperm. This contrasts with the prevailing random arrangement of chromosome territories in somatic nuclei. Based on observations on a series of nuclei with varying parental genome ratios, we propose a model where chromosomes associate pairwise involving one maternal and one paternal complement. The functional implications of this predicted chromosomal arrangement are discussed.     

On the selection for the modal chromosome number in Chinese hamster cells

Chinese hamster cell lines have a strong tendency to maintain as modal chromosome number the euploid one characteristic of the species. By following the evolution of karyotypes in cloned subpopulation it has been possible to show that these pseudodiploid constitutions are not simply established due to a selective advantage but they are actually generated in populations where pseudodiploid classes had been absent. The possible role of the mitotic apparatus in keeping and restoring the euploid number is discussed.     

Chromosome polymorphism involving heterochromatic blocks in Chinese hamster chromosome 9

A chromosome polymorphism was detected between two early passage euploid Chinese hamster cell strains when a fluorescence shift of the small metacentric No. 9 chromosome was resolved by flow cytometry. The characteristics of the polymorphism were studied using cultures established from ear clippings taken from 16 additional hamsters from our breeding colony. Additional variants of chromosome 9 were detected using flow cytometry, and a subset of these variants were analyzed by G- and C-banding. An increase of fluorescence recorded by flow cytometry correlated with an increase of centromeric heterochromatin. Autosomal normalization of the flow karyotype from 18 different animals indicated three distinct peak positions for chromosome 9. The results indicate that a discrete block of constitutive heterochromatin may be present in one or two extra copies within the small inbred colony of hamsters studied. To determine the inheritance patterns, hamsters with known polymorphic No. 9 chromosomes were bred. The flow karyotypes derived from the offspring of these matings provide strong evidence that chromosomal polymorphisms are inherited in Mendelian fashion.     

Non-random distribution of Alu-family repeats in human chromosomes

A phage lambda recombinant clone containing at least 8 Alu-family repeats (AFRs) has been isolated from a human genomic library, and DNA from the phage was used as a probe for in situ hybridization on G-banded human metaphase chromosomes of healthy donors and leukemic patients. Some chromosome bands show prominent clusters of silver grains in all individuals examined: 1p34, 1q23, 2q21–22, 10p14, 11p14, 10q21 and 11q14. The data suggest non-random distribution of AFRs in the human genome.     

Lack of specificity of chromosome breaks resulting from radiation-induced genomic instability in Chinese hamster cells

In V79 Chinese hamster cells, radiation-induced genomic instability results in a persistently increased frequency of micronuclei, dicentric chromosomes and apoptosis and in decreased colony-forming ability. These manifestations of radiation-induced genomic instability may be attributed to an increased rate of chromosome breakage events many generations after irradiation. This chromosomal instability does not seem to be a property which has been inflicted on individual chromosomes at the time of irradiation. Rather, it appears to be secondary to an increased level of non-specific clastogenic factors in the progeny of most if not all irradiated cells. This conclusion is drawn from the observations presented here, that all the chromosomes in surviving V79 cells are involved in the formation of dicentric chromosome aberrations 1 or 2 weeks after irradiation with about equal probability if corrections are made for chromosome length. Received: 5 March 1998 / Accepted in revised form: 1 July 1998     

Rate of spontaneous chromosome aberration occurrence in cultured Chinese hamster cells

The spontaneous chromosome mutation rate was studied in cultured aneuploid Chinese hamster cells (clone 237(1)) using the method of slowing down the rate of cell division in a limiting medium containing 0.1% of serum. It was shown that during one cell generation (which lasted 14 days in limiting medium) the accumulation of chromosome aberrations with time took place. The data obtained are in keeping with the assumption of a linear dependence of this accumulation on time. The spontaneous chromosome rearrangement rate was 1.2 X 10(-2) mutations per cell per 24 hours. Proceeding from this value the spontaneous chromosome aberration rate in cells with a normal duration of the cell cycle was 0.6 X 10(-2) per cell per generation.     

Non-random distribution of ring recoveries from trans-Saharan migrants indicates species-specific stopover areas

Many long-distance migrant birds regularly have to pass ecological barriers, like the Saharan desert, where fuelling is very difficult, and large fuel loads have to be stored in advance. In this paper, we have investigated how seven species of birds are distributed in autumn close to the Saharan desert in the eastern Mediterranean area by using ring recoveries from northern Europe. The result clearly shows that the species included are not randomly distributed at this point, about 3,000 km from the breeding area. Birds from rather large breeding areas were shown to converge in confined areas, which in several cases completely differ between species. This means that birds of the same species have to follow different migratory directions depending on the location of their starting point. The observed pattern support earlier findings indicating that birds, in combination with a clock-and-compass orientation procedure, must use some external cues in order to find confined species-specific areas. The possibility for birds to use information from the Earth's magnetic field as an external cue in this area is discussed.     

Chronology of chromosome reduplication in Chinese hamster cells incubated at low temperature

Experiments have been carried out on Chinese hamster fibroblasts. Cultures at the log-stage of growth were incubated at 15 or 25° C for 24 hrs. In two groups of experiments the cells were labeled with H³-TdR for 6 hrs at the respective temperature, washed and further incubated at 37° C. In each group of experiments cultures labeled with H³-TdR at 37° C for 20 min were used as a control. It was found: 1. the delay in the onset of cell passage through the mitotic cycle at 37° in cultures exposed to 15 or 25° C was about equal to 1,5-1 hr resp. and cells proceeded through the life cycle without blockages at any phase of the cycle; 2. the patterns of chromosome reproduction during the second half of the S-phase were the same after labeling at 15, 25 and 37° C. — In the third group the cells were labelled with HP-TdR for 10–60 min and 6 hrs resp. at 25° C. The patterns of reproduction of chromosome pairs 1–4 and small metacentrics were found to be the same in cells labeled briefly and those labeled for 6 hrs. After brief labeling asynchronous reduplication of different segments in many chromosomes became evident. It was masked because of heavy labeling after 6 hrs treatment.     

Identification of a chromosome that controls malignancy in Chinese hamster cells.

A chromosome that controls malignancy in Chinese hamster cells has been identified by analysis of the Giemsa banding pattern of a malignant cell line transformed by simian virus 40 (SV40), non-malignant revertants from this line, segregants from the revertants that were again malignant and a cell line transformed by methylcholanthrene. The malignant cell line transformed by SV40 was near diploid and had gained additional material of chromosome 3. Revertants with a suppression of malignancy and malignant revertants from which they were derived. Malignancy of these cells was associated with the ability to form colonies in agar. Cells of a line transformed by methylcholanthrene were malignant, formed almost no colonies in agar and the only chromosome change from the normal diploid chromosome banding complement was the addition of a long arm of chromosome 3. The results indicate that chromosome 3 carriers gene(s) that control malignancy in Chinese hamster cells in cell lines transformed by a viral or a chemical carcinogen and that malignancy was induced in both cell types by an increase of these genes.     

Pancreatic polypeptide and glucagon: Non-random distribution in pancreatic islets

When the technique of immunofluorescence is applied to rat pancreas to detect insulin, glucagon, somatostatin and pancreatic polypeptide (PP), two populations of islets having distinct cellular content and topographical distribution can be recognized. Islets from the lower part of the head show a well-defined rim of PP-containing cells, but very few or no glucagon-containing cells. Islets from the body and tail display the familiar rim of glucagon-containing cells and possess very few or no PP-containing cells. This inverse relationship between glucagon and PP-cells in different parts of the pancreas means that caution must be exercised when interpreting functional or morphological observations using different pancreatic fractions.     

Non-random distribution of chiasmata in rye,Crotalaria and coffee

Summary The two arms of a bivalent can be bound (contain at least one chiasma) or not-bound (contain no chiasma). Formulae are presented to test the randomness of distribution of the bound arms over the chromosome complement, under the assumption that the probability of being bound is related to the arm length.Testing meiotic configurations in Secale cereale (rye), Crotalaria intermedia and several species of Coffea, non-randomness was found in all cases, resulting in an excess of bivalents with only one arm bound. The effect is slightly different in the different plants. The basis is considered to be localization of chiasmata.In rye plants, homozygous for a rather large translocation, an excess of univalents was observed to more than compensate the localization effect. This is attributed to the limited probability of the newly formed short arm of the small translocation chromosome to be bound.The section on rye was started at the Laboratory of Genetics, director Prof. Dr. R. Prakken, Agricultural University, Wageningen, Netherlands. It was completed, with the addition of the sections on Crotalaria and coffee, at the Inter-American Institute of Agricultural Sciences, Turrialba, C. R.Cytogeneticist, Nuclear Energy Program, Inter-American Institute of Agricultural Sciences, Turrialba, Costa Rica, AEC Contract AT (30-1) 2043.     

Non-random distribution of abnormal mitoses in heteroploid cell lines.

We have performed absorption-cytometric DNA measurements of the DNA contents of the G0/G1, G2, metaphase, and telophase cells of the heteroploid MCa-11 and HL-60 lines, as well as the WCHE-5 line which has a narrowly restricted number of chromosomes. We found that morphologically unbalanced mitoses occurred much more frequently in telophase-cell pairs of the heteroploid MCa-11 and HL-60 lines than in those of the chromosomally stable WCHE-5 line. Furthermore, the morphologically unbalanced mitoses represented unequal segregation of DNA into each of the daughter telophase nuclei. Such mitotic segregation errors (MSE) occurred almost exclusively in telophase cells with DNA contents which were above, or below, the DNA content of the modal telophase population. The net effect of these non-random, unblanced divisions of heteroploid cells with non-modal DNA contents is to produce one daughter cell with a DNA content that tends to return to the modal DNA content peak.