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.     

Morphologic variability of human chromosomes: Polymorphism of constitutive heterochromatin

Summary Polymorphism of constitutive heterochromatin has been studied in a series of 30 normal individuals. A high frequency of C-band variants were observed. Twenty-six of the 30 individuals studied had at least one polymorphic variant of the C band. A total of 42 variants were recorded which were predominately localized near the centromeric heterochromatin block of chromosome 9 (26.19%), chromosome 16 (19.05%), and chromosome 1 (16.66%). These results are discussed together with the findings revealed by different studies.Aided by U.G.C. grant No. 9-32/75 X (RF).     

Distribution of constitutive heterochromatin in mammalian chromosomes

Using a special staining technique, a survey of the chromosomes of many mammalian species showed that constitutive heterochromatin is present in all cases and that the heterochromatin pattern appears to be specific and consistent or each chromosome and each taxon. Usually heavy heterochromatin is found in the centromeric areas, but terminal heterochromatin is not uncommon. Occasionally interstitial heterochromatin bands occur. In some species, such as the Syrian hamster and Peromyscus, many chromosome arms are completely heterochromatic.Supported in part by Research Grant GB-13661 from the National Science Foundation.     

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.     

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.     

Heterogeneity of constitutive heterochromatin in somatic Syrian hamster chromosomes.

Syrian hamster constitutive heterochromatin was analyzed for C-band distribution and for BrU late-replication pattern. Characteristic for this species is relatively large amounts of sex-chromosome and autosomal heterochromatin. The distribution of constitutive heterochromatin was determined. The long term of the X chromosome, the whole Y, the short arms of 8 autosomal pairs, the long arm of the smallest metacentric pair, and the centromeric regions of 12 pairs stained intensely dark on C-band preparations. In contrast to the heterochromatin in the centromeric regions, the autosomal short-arm heterochromatin has an increased susceptibility to the denaturation process, as indicated by prolonged exposure to NaOH or Ba(OH)2. Such further exposure to denaturing agents results in an intense dark stain only on the sex-chromosome heterochromatin and centromeric regions of the autosomes. The BrdU late-replication pattern demonstrated that the late-replicating regions correspond to C-bands. Centromeric regions replicate late in the S phase; however, no centromeric region is among the latest replicating segments of the complement. Centromeric and noncentromeric heterochromatin are two distinct categories of constitutive heterochromatin.     

Differential sensitivity of constitutive and facultative heterochromatin in orthopteran chromosomes to digestion by DNaseI

In situ pancreatic DNaseI digestions were used as probes to study the structural organization of facultative and constitutive heterochromatin during both mitotic and meiotic divisions. Three different types of heterochromatic regions from three insect species were chosen for this study. These regions had been previously characterized by in situ treatments with restriction endonucleases (AT and GC rich DNA sequences). Progressive increase in DNaseI concentration (from 10 to 200 ng/ml) or in incubation time (from 5 to 30 min) revealed a specific pattern of sequential digestion of the constitutive heterochromatic regions, the centromeric ones (AT-rich DNA) being the most resistant to DNaseI action. The interstitial C-bands (with AT or GC-rich DNA) were more sensitive to DNaseI, and the band 4.4 from Baetica ustalata was the most resistant of the non-centromeric bands. Similar results were obtained during meiosis, but increased accessibility to DNAseI was observed compared to mitosis. DNA methylation in the non-centromeric band 4.4 of B. ustulata could be responsible for its differential digestion with respect to the remaining intercalar heterochromatin. Facultatively heterochromatic regions (X chromosomes) were found to exhibit a differential response to DNaseI attack from mitosis to meiosis. While they behaved as cuchromatin during mitosis, they were the most resistant together with centromeric heterochromatin regions, during metaphase I and II. The different responses to digestion of the X chromosome and X-derived regions between somatic and meiotic divisions are probably a consequence of the changes in the organization of this chromosome during the facultative heterochromatinization process.     

A comparison of constitutive heterochromatin staining methods in two cases of familial heterochromatin deficiencies

Summary Using DAPI staining after pretreatment with distamycin A we detected a familial deficiency of chromosome 16 heterochromatin. A distinct positively staining band, however, was seen after C-banding. Thus, by using these different heterochromatin staining methods, heterogeneity of the constitutive heterochromatin in the centromeric region of human chromosome 16 was indicated. The same C-banding procedure was also applied to a previously described familial deficiency of chromosome 9 heterochromatin evidenced using distamycin A/DAPI staining and G 11 staining (Buys et al., 1979). In this case a C-band appeared to be virtually absent on the relevant chromosome. These staining methods may be valuable tools in the study of chromosome polymorphisms.     

Characterization of human chromosomal constitutive heterochromatin

The constitutive heterochromatin of human chromosomes is evaluated by various selective staining techniques, i.e., CBG, G-11, distamycin A plus 4,6-diamidino-2-phenylindole-2-HCl (DA/DAPI), the fluorochrome D287/170, and Giemsa staining following the treatments with restriction endonucleases AluI and HaeIII. It is suggested that the constitutive heterochromatin could be arbitrarily divided into at least seven types depending on the staining profiles expressed by different regions of C-bands. The pericentromeric C-bands of chromosomes 1, 5, 7, 9, 13-18, and 20-22 consist of more than one type of chromatin, of which chromosome 1 presents the highest degree of heterogeneity. Chromosomes 3 and 4 show relatively less consistent heterogeneous fractions in their C-bands. The C-bands of chromosomes 10, 19, and the Y do not have much heterogeneity but have characteristic patterns with other methods using restriction endonucleases. Chromosomes 2, 6, 8, 11, 12, and X have homogeneous bands stained by the CBG technique only. Among the chromosomes with smaller pericentric C-bands, chromosome 18 shows frequent heteromorphic variants for the size and position (inversions) of the AluI resistant fraction of C-band. The analysis of various types of heterochromatin with respect to specific satellite and nonsatellite DNA sequences suggest that the staining profiles are probably related to sequence diversity.     

Two types of constitutive heterochromatin in the chromosomes of some Fritillaria species

A Giemsa banding technique has been used to study C-banding in mitotic chromosomes in root tips of Fritillaria graeca, F. crassifolia and F. rhodocanakis, all diploids (2n=24) belonging to the graeca group. In the first two the C-bands were of two types, diverging in respect of staining regularly and specifically within chromosomes. In one type it was weak, being intermediate between that of intensely stained ones, representing the other class, and the euchromatin. In F. graeca the pale bands were proximally localized and confined to 5 pairs, whereas in F. crassifolia they occurred only in the 4 M chromosomes, in each within the centromeric constriction as a large inclusion. The interphase nuclei of both species contained pale and heavily stained chromocentres. No pale ones occurred in such nuclei of F. rhodocanakis. The probability is discussed that the two classes of C-band represent distinct types of heterochromatin, differing both in respect of condensation throughout the whole mitotic cycle and in the repetitive DNA sequences they most likely contain. In all 3 species pairs of Giemsa-positive centromeric dots, representing the centromeres, were masked both by proximally or centromerically localized bands, irrespective of the class of heterochromatin they represented.     

The constitutive heterochromatin in chromosomes of Fritillaria Sp., as revealed by Giemsa banding.

The incidence of C-bands (constitutive heterochromatin), as determined by differential Giemsa staining, was studied in the chromosomes of 56 species, varietal forms and subgenera of Fritillaria and 30 of them are illustrated. With the exception of the subgenera Korolkowi, a supposed link between lilies and fritillaries, and chromsome complements of all plants contained bands. There were wide differences in the size and number of these bands among species both within and between groups. In those with the largest and most abundant bands, there was a pronounced tendency for centromeric localization, both in Old and New World species. The Giemsa positive centromeres were masked when this occurred. Heteromorphy in respect of banding occurred in most species. The relation of repetitive DNA sequences with heterochromatin is discussed, as is also the problem of evolution in Fritillaria.     

Image analysis as a simple and useful tool for evaluating the constitutive heterochromatin (C-banding) in human chromosomes

Image analysis can contribute to those fields of cytogenetics that are influenced most by subjectivity, especially evaluation of chromosome regions that are histochemically polymorphic. C-banding of human or primate chromosomes may be used as a typical example of this concept. In addition, using it quantitatively it is useful in studies of population cytogenetics of man and Primates. Therefore, the aim of this study was to determine the best conditions for measurement by image analysis of C-positive regions in a sample of 29 normal subjects. We looked for relationships of chromosomal C-positive areas with the dimensions of the corresponding metaphases. Finally, we suggest some criteria for potential use of C-banding in population and comparative studies.     

Asynchronous replication of constitutive heterochromatin on X chromosomes in female Mus dunni

Euchromatin DNA of one X chromosome in mammalian females, which becomes facultatively heterochromatinized, is known to replicate asynchronously late in S phase compared to its active homologue. In the females of a pygmy mouse species Mus dunni, which has prominent segment of constitutive heterochromatin as the short arm of its submetacentric X chromosome, we have observed asynchronous replication of c-heterochromatin arm as well, predominant number of cells showing the segment associated with the facultatively heterochromatic X to be terminating later. The preferential later termination of replication of the c-heterochromatic arm on the lyonized X appears to be due to the influence of facultative heterochromatin on the adjacent constitutive heterochromatin.     

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.     

Robertsonian polymorphism and constitutive heterochromatin distribution in chromosomes of the rainbow trout (Salmo gairdneri).

White-blood-cell culture was used to examine the chromosomes of 53 rainbow trout (Salmo gairdneri) from three locations in the Pacific Northwest of the United States. A Robertsonian chromosome polymorphism is present, resulting in diploid numbers of 60, 59, or 58 in different individuals with 104 chromosome arms. The low level of intraindividual Robertsonian variation, differences in the number of subtelocentric chromosomes between individuals with different chromosome numbers, and frequencies of fish with different chromosome numbers in one population suggest that the interindividual differences are inherited and not somatic. C-banding shows that constitutive heterochromatin is localized near the centromeres and near the secondary constriction one chromosome pair.