Analysis of high-resolution R-bands, obtained by heat-denaturation and Giemsa staining, on human prophase chromosomes

RHG-bands (heat-denatured Giemsa R-bands) of human prophase chromosomes were analyzed at high resolution, and the banding patterns at prophase and metaphase are presented. The bands were compared with those of the International Standard Cytogenetic Nomenclature idiograms and of the G-band idiograms proposed by J. J. Yunis. The number, size, and position of the RHG-bands correspond rather well with their equivalent G-negative bands, but some differences were noted in the zones of preferential stretching, the juxtacentromeric regions, and the telomeres. Variations in the centromere index and the banding pattern in heterochromatin were also discussed.     

Karyotype analysis utilizing differentially stained constitutive heterochromatin of human and murine chromosomes

Constitutive heterochromatin of chromosomes can be visualized utilizing a new differential staining technique which was originally developed by Gall and Pardue (1971). The method facilitates the more certain identification of specific chromosomes within and between cell populations of different origins. Marker chromosomes can be identified in established cell lines over many months of serial passage. Chromosomes of similar morphology within karyotypes of man and mouse can be distinguished in a number of instances. For example, the Y chromosomes of both mouse and man can now be easily detected. The hetero-chromatic staining method also permits discrimination between mouse and human chromosomes in somatic cell hybrids, thus facilitating the assignment of gene markers to chromosomes in somatic cell genetics systems. Instances of translocation of centric heterochromatin to other parts of chromosomes in established tissue culture cell lines are described. An instance of the inheritance of a polymorphic variation in autosomal heterochromatin in man is reported. It is postulated that polymorphisms in the centric heterochromatin may account largely for small heritable chromosome length variations previously described in human populations and termed minor chromosome variants.     

Characterization of constitutive heterochromatin in Cebus apella (Cebidae, Primates) and Pan troglodytes (Hominidae, Primates): comparison to human chromosomes.

Using G bands, some homologies between the chromosomes of Cebus apella (CAP) and human chromosomes are difficult to establish. To solve this problem, we analyzed these homologies by fluorescence in situ hybridization using human whole chromosome probes (ZOO-FISH). The results indicated that 1) the human probe for chromosome 2 partially hybridizes with CAP chromosomes 13 and 5, 2) the human probe for chromosome 3 partially hybridizes with CAP chromosomes 18 and 20, 3) the human probe for chromosome 9 partially hybridizes with CAP chromosome 19, and 4) the human probe for chromosome 14 hybridizes with the p-terminal and q-terminal regions of CAP chromosome 6. However, none of the human probes employed hybridized with the heterochromatic regions of CAP chromosomes. For this reason, we characterized the heterochromatic regions of CAP chromosomes and of the chromosomes of Pan troglodytes (PTR), to allow comparison between CAP, PTR, and human chromosomes using in situ digestion of fixed chromosomes with the restriction enzymes AluI, HaeIII, and RsaI and by fluorescent staining with DA/DAPI. The results show that 1) centromeric heterochromatin is heterogeneous in the three species studied and 2) noncentromeric heterochromatin is homogeneous within each of the three species, but is different for each species. Thus, centromeric heterochromatin undergoes a higher degree of variability than noncentromeric heterochromatin.     

C- and Q-chromatin of the experimentally condensed human interphase chromosomes]

Condensed interphase chromosomes of the cultured human lymphocytes obtained by the fusion of interphase and metaphase cells were studied using C- and Q-bands techniques. The appearance and localization of the constitutive heterochromatin blocks on condensed chromosomes at G1-period were the same as on the metaphase ones. These characters were used for a group and individual identification of some chromosomes condensed at G1-period and for a study of the association of the constitutive heterochromatin blocks in the interphase nuclei. The fluorescent analysis of the chromosomes condensed at G1-period detected some bright fluorescent blocks of the constitutive heterochromatin.     

Counterstaining human chromosomes for flow karyology

Isolated human metaphase chromosomes stained with the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and chromomycin A3(CA3), and counterstained with nonfluorescent netropsin (NTR), have been analyzed by dual-laser flow cytometry. Counterstaining with NTR reduces DAPI fluorescence except at regions on chromosomes 1,9,15,16, and Y, corresponding to C-band heterochromatin. Bivariate flow karyology of human chromosomes treated with this triple-stain combination resolves chromosomes 1,9, and Y distinctly from the remaining chromosomes and resolves variations between chromosome homologues not detected by staining with propidium iodide (PI) or with the double stain combination Hoechst 33258(HO) and CA3.     

Entropic organization of interphase chromosomes

Chromosomes are not distributed randomly in nuclei. Appropriate positioning can activate (or repress) genes by bringing them closer to active (or inactive) compartments like euchromatin (or heterochromatin), and this is usually assumed to be driven by specific local forces (e.g., involving H bonds between nucleosomes or between nucleosomes and the lamina). Using Monte Carlo simulations, we demonstrate that nonspecific (entropic) forces acting alone are sufficient to position and shape self-avoiding polymers within a confining sphere in the ways seen in nuclei. We suggest that they can drive long flexible polymers (representing gene-rich chromosomes) to the interior, compact/thick ones (and heterochromatin) to the periphery, looped (but not linear) ones into appropriately shaped (ellipsoidal) territories, and polymers with large terminal beads (representing centromeric heterochromatin) into peripheral chromocenters. Flexible polymers tend to intermingle less than others, which is in accord with observations that gene-dense (and so flexible) chromosomes make poor translocation partners. Thus, entropic forces probably participate in the self-organization of chromosomes within nuclei.     

Assembly and characterization of heterochromatin and euchromatin on human artificial chromosomes

Background

Human centromere regions are characterized by the presence of alpha-satellite DNA, replication late in S phase and a heterochromatic appearance. Recent models propose that the centromere is organized into conserved chromatin domains in which chromatin containing CenH3 (centromere-specific H3 variant) at the functional centromere (kinetochore) forms within regions of heterochromatin. To address these models, we assayed formation of heterochromatin and euchromatin on de novo human artificial chromosomes containing alpha-satellite DNA. We also examined the relationship between chromatin composition and replication timing of artificial chromosomes.

Results

Heterochromatin factors (histone H3 lysine 9 methylation and HP1α) were enriched on artificial chromosomes estimated to be larger than 3 Mb in size but depleted on those smaller than 3 Mb. All artificial chromosomes assembled markers of euchromatin (histone H3 lysine 4 methylation), which may partly reflect marker-gene expression. Replication timing studies revealed that the replication timing of artificial chromosomes was heterogeneous. Heterochromatin-depleted artificial chromosomes replicated in early S phase whereas heterochromatin-enriched artificial chromosomes replicated in mid to late S phase.

Conclusions

Centromere regions on human artificial chromosomes and host chromosomes have similar amounts of CenH3 but exhibit highly varying degrees of heterochromatin, suggesting that only a small amount of heterochromatin may be required for centromere function. The formation of euchromatin on all artificial chromosomes demonstrates that they can provide a chromosome context suitable for gene expression. The earlier replication of the heterochromatin-depleted artificial chromosomes suggests that replication late in S phase is not a requirement for centromere function.

     

Attraction between centric heterochromatin of human chromosomes.

An analysis of the pattern of association of acrocentric chromosomes with nonacrocentric chromosomes in human lymphocyte metaphases was performed. This pattern in nonrandom with respect to chromosome length and intrachromosomal distribution. There is a general preference for the centric regions, most pronounced at the proximal segments of the long arms of chromosomes 1, 9, and 16, which is interpreted to reflect heterochromatin attraction during interphase. Comparison of the association patterns of homologous chromosome 1's differing with regard to the size of their heterochromatic regions corroborates this interpretation. The possible significance of heterochromatin attraction for the formation of spontaneous and induced chromosome anomalies is discused.     

Length of human prematurely condensed chromosomes during G0 and G1 phase

Length measurements on C-banded prematurely condensed no. 1 human chromosomes of G0 and G1 lymphocytes, as well as of synchronized G1 HEp cells revealed that (i) no length difference exists between mitotic chromosomes and G0 chromosomes; (ii) 1 h after PHA stimulation a clear increase in length is detectable; (iii) in isolated cases an increase by the factor 5 can be observed during G1; (iv) the increase is significantly less for constitutive heterochromatin than for euchromatin. The possibility is discussed that these conformational changes of chromatin reflect physiological differences, i.e. the rate of RNA synthesis during interphase.     

Experimental condensation inhibition in constitutive and facultative heterochromatin of mammalian chromosomes

What drives the dramatic changes in chromosome structure during the cell cycle is one of the oldest questions in genetics. During mitosis, all chromosomes become highly condensed and, as the cell completes mitosis, most of the chromatin decondenses again. Only chromosome regions containing constitutive or facultative heterochromatin remain in a more condensed state throughout interphase. One approach to understanding chromosome condensation is to experimentally induce condensation defects. 5-Azacytidine (5-aza-C) and 5-azadeoxycytidine (5-aza-dC) drastically inhibit condensation in mammalian constitutive heterochromatin, in particular in human chromosomes 1, 9, 15, 16, and Y, as well as in facultative heterochromatin (inactive X chromosome), when incorporated into late-replicating DNA during the last hours of cell culture. The decondensing effects of 5-aza-C analogs, which do not interfere with normal base pairing in substituted duplex DNA, have been correlated with global DNA hypomethylation. In contrast, decondensation of constitutive heterochromatin by incorporation of 5-iododeoxyuridine (IdU) or other non-demethylating base analogs, or binding of AT-specific DNA ligands, such as berenil and Hoechst 33258, may reflect an altered steric configuration of substituted or minor-groove-bound duplex DNA. Consequently, these compounds exert relatively specific effects on certain subsets of AT-rich constitutive heterochromatin, i.e. IdU on human chromosome 9, berenil on human Y, and Hoechst 33258 on mouse chromosomes, which provide high local concentrations of IdU incorporation sites or DNA-ligand-binding sites. None of these non-demethylating compounds affect the inactive X chromosome condensation. Structural features of chromosomes are largely determined by chromosome-associated proteins. In this light, we propose that both DNA hypomethylation and steric alterations in chromosomal DNA may interfere with the binding of specific proteins or multi-protein complexes that are required for chromosome condensation. The association between chromosome condensation defects, genomic instability, and epigenetic reprogramming is discussed. Chromosome condensation may represent a key ancestral mechanism for modulating chromatin structure that has since been realloted to other nuclear processes.     

Comparison of two measuring methods for the evaluation of C-heterochromatin in human chromosomes

Summary In this study two different methods for evaluating the size of the C heterochromatin blocks of human chromosomes 1, 9, 16, and Y were compared. The first method measured the lengths of both the euchromatin and the C heterochromatin parts of the p and q arms of chromosomes 1, 9, 16, and Y. The second method analyzed the same chromosome segments, but by measuring the areas.In the comparison, the relative C heterochromatin value (length or surface) of each chromosome, the mean for each individual, the standard deviation, and the coefficient of variation were taken into account. It is proposed that the best estimation for the size of a C heterochromatin segment is the ratio of its length to the total length of the chromosome; accurate estimation requires at least 20 metaphases.     

Why plant chromosomes do not show G-bands

Summary Giemsa techniques have refused to reveal G-banding patterns in plant chromosomes. Whatever has been differentially stained so far in plant chromosomes by various techniques represents constitutive heterochromatin (redefined in this paper). Patterns of this type must not be confused with the G-banding patterns of higher vertebrates which reveal an additional chromosome segmentation beyond that due to constitutive heterochromatin. The absence of G-bands in plants is explained as follows: 1) Plant chromosomes in metaphase contain much more DNA than G-banding vertebrate chromosomes of comparable length. At such a high degree of contraction vertebrate chromosomes too would not show G-bands, simply for optical reasons. 2) The striking correspondence of pachytene chromomeres and mitotic G-bands in higher vertebrates suggests that pachytene chromomeres are G-band equivalents, and that this may also be the case in plants. G-banded vertebrate chromosomes are on the average only 2.3 times shorter in mitosis than in pachytene; the chromomeric pattern therefore still can be shown. In contrast, plant chromosomes are approximately 10 times shorter at mitotic metaphase; their pachytene-like arrangement of chromomeres is therefore no longer demonstrable.     

Alterations of DNA methylation patterns in germ cells and Sertoli cells from developing mouse testis

In situ alterations of DNA methylation were studied between 14 d postcoitum and 4 d postpartum in Sertoli cells and germ cells from mouse testis, using anti-5-methylcytosine antibodies. Compared to cultured fibroblasts, Sertoli cells display strongly methylated juxtacentromeric heterochromatin, but hypomethylated chromatids. Germ cells always possess hypomethylated heterochromatin, whereas their euchromatin passes from a demethylated to a strongly methylated status between days 16 and 17 postcoitum. This hypermethylation occurs in the absence of DNA replication, germ cells being blocked in the G(0)-G(1) phase from day 15 postcoitum to birth. The DNA hypermethylation of germ cells is maintained until birth and could be visualized on both chromatids of metaphase chromosomes at the first postpartum cell division. Subsequently, the DNA hypermethylation is lost semiconservatively, being replaced by a methylation pattern recalling the typical fibroblast pattern. These alterations of DNA methylation follow a strict chronology, are chromosome structure and cell-type dependent, and may underlie profound changes of genome function.     

Heterochromatin is not an adequate explanation for close proximity of interphase chromosomes 1--Y, 9--Y, and 16--Y in human spermatozoa

Analysis of human spermatozoa and lymphocytes using C-banding techniques and in situ hybridization has shown a higher order packaging of the human genome. Chromosomes are not distributed entirely at random within the nucleus. In particular, chromosomes 1, 9, and 16, carrying large blocks of pericentromeric heterochromatin, and the Y chromosome, carrying heterochromatin in Yq12, are in close proximity to each other within the nucleus and are involved in somatic pairing with nonhomologous chromosomes. In order to determine whether the close proximity of these chromosomes in any way is attributable to the distribution of heterochromatin, double in situ hybridization was performed on chromosomes 1--Y, 9--Y, and 16--Y as well as on 1--X, 9--X, and 16--X-with chromosome X as the other gonosome carrying less heterochromatin-in human spermatozoa. Each pair was found to have a nonrandom spatial distribution. However, comparison of the arrangement of chromosomes 1--Y versus 1--X and 9--Y versus 9--X revealed that heterochromatin cannot be the only cause for the tendency of chromosome fusion, because only the results of the chromosome pair 1--Y/1--X could support this proposition. In conclusion, the heterochromatin effect cannot be, in itself, an adequate explanation for chromosome association, implicating as well other mechanisms.     

Scanning electron microscopy of mammalian chromosomes from prophase to telophase

Changes in the morphology of human and murine chromosomes during the different stages of mitosis have been examined by scanning electron microscopy. Two important findings have emerged from this study. The first is that prophase chromosomes do not become split into pairs of chromatids until late prophase or early metaphase. This entails two distinct processes of condensation, the earlier one starting as condensations of chromosomes into chromomeres which then fuse to form a cylindrical body. After this cylindrical body has split in two longitudinally, further condensation occurs by mechanisms that probably include coiling of the chromatids as well as other processes. The second finding is that the centromeric heterochromatin does not split in two at the same time as the rest of the chromosome, but remains undivided until anaphase. It is proposed that the function of centromeric heterochromatin is to hold the chromatids together until anaphase, when they are separated by the concerted action of topoisomerase II acting on numerous similar sites provided by the repetitive nature of the satellite DNA in the heterochromatin. A lower limit to the size of blocks of centromeric heterochromatin is placed by the need for adequate mechanical strength to hold the chromatids together, and a higher limit by the necessity for rapid splitting of the heterochromatin at anaphase. Beyond these limits malsegregation will occur, leading to aneuploidy. Because the centromere remains undivided until anaphase, it cannot undergo the later stage of condensation found in the chromosome arms after separation into chromatids, and therefore the centromere remains as a constriction.by U. Scheer     

Molecular cytogenetic research on the polymorphism of segments of the constitutive heterochromatin in human chromosomes

Cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes of high specificity to individual chromosomes (chromosomes 3, 11, 17, 18 and X) were hybridized in situ to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in definite heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The significant interindividual differences in relative copy number of alpha-satellite DNA have been detected. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms, as shown by intensity of hybridization with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences gives evidence for a high resolution power of in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes is variable for amount of alpha-satellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as novel general approach to analysis of chromosome heteromorphisms in man.     

Characterisation of repeated sequences from microdissected B chromosomes of Crepis capillaris

The B chromosome of Crepis capillaris was isolated from the standard chromosomes by microdissection, and the chromosomal DNA amplified using the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The PCR product was cloned and a Bspecific library created and characterised. Southern and in situ hybridisation analyses of the DOP-PCR product from microdissected B chromosomes confirmed that the B chromosome is composed mainly of sequences also present in the A chromosomes but lacks the main repeated DNA families located in the A-chromosomal heterochromatin. From 100 clones analysed, 12% of the generated B-chromosomal library was shown to be composed of dispersed repeats located in both the A and B chromosomes. No B-specific repeated sequence was detected. One of the most abundant repeated DNAs within the library, the family B134, was further characterised. Repeating units show a sequence similarity range from 69% to 90% and are characterised by their richness in (CA)n repeats. In situ hybridisation revealed that members of this family are dispersed throughout the A and B chromosomes but are more concentrated in the pericentromeric heterochromatin of the B, indicating that the molecular organization of B heterochromatin is different from that of the A chromosomes. Compared with the A chromosomes, the Bs contain about 20,000 copies per micron more of the B134 sequence. This indicates that B134 was amplified on the B chromosome after its origin. The B134 sequences in the B chromosomes have also diverged from those on the A chromosomes. Although the DNA composition of A and B chromosomes is similar, Bs are evolving separately from A chromosomes at the molecular level.     

Constitutive heterochromatin polymorphisms in human chromosomes identified by whole comparative genomic hybridization

Whole comparative genomic hybridization (W-CGH) is a new technique that reveals cryptic differences in highly repetitive DNA sequences, when different genomes are compared using metaphase or interphase chromosomes. W-CGH provides a quick approach to identify differential expansion of these DNA sequences at the single-chromosome level in the whole genome. In this study, we have determined the frequency of constitutive chromatin polymorphisms in the centromeric regions of human chromosomes using a whole-genome in situ cross-hybridization method to compare the whole genome of five different unrelated individuals. Results showed that the pericentromeric constitutive heterochromatin of chromosome 6 exhibited a high incidence of polymorphisms in repetitive DNA families located in pericentromeric regions. The constitutive heterochromatin of chromosomes 5 and 9 was also identified as highly polymorphic. Although further studies are necessary to corroborate and assess the overall incidence of these polymorphisms in human populations, the use of W-CGH could be pertinent and of clinical relevance to assess rapidly, from a chromosomal viewpoint, genome similarities and differences in closely related genomes such as those of relatives, or in more specific situations such as bone marrow transplantation where chimerism is produced in the recipient.     

Preferential somatic pairing between homologous heterochromatic regions of human chromosomes.

The cytidine analog 5-azacytidine (5-azaC) induces an undercondensation of the heterochromatin in human chromosomes 1, 9, 15, 16, and Y when it is added in low concentrations to the late S-phase of growing lymphocyte cultures. In interphase nuclei, these heterochromatic regions are frequently somatically paired. The somatic pairing configurations are preserved up to metaphase stage in the 5-azaC-treated cultures and are thus susceptible to a direct microscopical examination. The statistical analysis of 1,000 somatic pairing configurations from 5-azaC-treated cells showed that the somatic pairing between the heterochromatic regions of homologous chromosomes is preferred over that between nonhomologous chromosomes.     

New distinctions between regions of centromeric heterochromatin in human chromosomes by treatments with the isoschizomers NdeII-Sau3AI

The isoschizomers NdeII-Sau3AI (decreases GATC) have been used to characterize heterochromatic regions in human chromosomes. The findings with NdeII are identical with those previously published with MboI, but the results with Sau3AI provide evidence for new distinctions of centromeric heterochromatin in chromosomes 5 and 6. The results are discussed in relation to the chromatin organization at these regions an the mechanisms of the action of restriction endonucleases.