Restrictive distribution of asymmetric C-bands in the chromosome complement of the rhesus (Macaca mulatto)

Lymphocyte chromosomes from a cercopithecoid species, Macaca mulatta, were studied for the occurrence of lateral asymmetry in constitutive heterochromatin. The technique consisted of growing the lymphocytes for one cell cycle in BrdUrd, staining with 33258 Hoechst, exposing them to UV light, treating them with 2 SSC and staining with Giemsa. This procedure revealed asymmetric staining in the region of constitutive heterochromatin of the nucleolar organizer marker chromosome (no. 13 of the complement). In these chromosomes, the darkly staining region was confined at any given point to a single chromatid, while the corresponding region on the sister chromatid was lightly stained. This pattern of asymmetric staining in the constitutive heterochromatic region was not observed in any other chromosome of Macaca mulatta. The lateral asymmetry of constitutive heterochromatin in this species is presumed to reflect the strand bias in the distribution of thymine in the alphoid DNA fractions.     

Lateral asymmetry in human constitutive heterochromatin

Human lymphocytes were grown for one replication cycle in BrdU, stained with 33258 Hoechst, exposed to UV light and subsequently treated with 2 x SSC and stained with Giemsa. This technique differentially stains the constitutive heterochromatin of chromosomes 1, 9, 15, 16, and the Y. In the heterochromatin of chromosome 9 both sister chromatids stained darkly and symmetrically but in the other four chromosomes the heterochromatin showed lateral asymmetry, one chromatid being darkly stained while its sister chromatid was as pale or paler than the rest of the chromosome. The lateral asymmetry is presumed to reflect an underlying asymmetry in distribution of thymine between the two strands of the DNA duplex in the satellite DNA component of the chromosomes. In some number 1 chromosomes compound lateral asymmetry was seen; darkly staining material was present on both sister chromatids although at any given point lateral asymmetry was maintained so that if one chromatid stained darkly the corresponding point on the sister chromatid was very pale. The pattern of compound lateral asymmetry varied among the number 1 chromosomes studied but was constant for any one homologue from one individual. This technique reveals a previously unsuspected type of polymorphism within the constitutive heterochromatin of man.     

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.     

Cytological dissection of sex chromosome heterochromatin of Drosophila hydei

Prophase chromosomes of Drosophila hydei were stained with 0.5 g/ml Hoechst 33258 and examined under a fluorescence microscope. While autosomal and X chromosome heterochromatin are homogeneously fluorescent, the entirely heterochromatic Y chromosome exhibits an extremely fine longitudinal differentiation, being subdivided into 18 different regions defined by the degree of fluorescence and the presence of constrictions. Thus high resolution Hoechst banding of prophase chromosomes provides a tool comparable to polytene chromosomes for the cytogenetic analysis of the Y chromosome of D. hydei. — D. hydei heterochromatin was further characterized by Hoechst staining of chromosomes exposed to 5-bromodeoxyuridine for one round of DNA replication. After this treatment the pericentromeric autosomal heterochromatin, the X heterochromatin and the Y chromosome exhibit numerous regions of lateral asymmetry. Moreover, while the heterochromatic short arms of the major autosomes show simple lateral asymmetry, the X and the Y heterochromatin exhibit complex patterns of contralateral asymmetry. These observations, coupled with the data on the molecular content of D. hydei heterochromatin, give some insight into the chromosomal organization of highly and moderately repetitive heterochromatic DNA.     

G-banding in the barbary macaque (Macaca sylvanus, Linnaeus, 1758) of Morocco

G-banding was performed on the lymphocytes of six barbary macaques (Macaca sylvanus). Four chromosomes appear to have polymorphic variants: number 1, number 4, number 16, and the “marker” chromosome, number 9. These polymorphic forms differe from the G-banding reported for Macaca mulatta, Macaca fascicularis, and Macaca arctoides. It may be that evolutionary relationships among various macaque species can be ascertained by using such chromosomal polymorphisms.     

Normal and BrdC-substituted chromosomes during spermatogenesis with an endomeiotic chromosome reduplication in Carausius morosus Br.

Spermatogenesis involving an additional chromosome reduplication during zygotene in sporadic males and intersexes of the thelytokous phasmid Carausius morosus Br. has been examined using differential staining of chromatids after 5-bromodeoxycytidine incorporation. After reduplication autobivalents are formed by synapsis between identical sister chromosomes. Chiasmata are only formed after reduplication; they do not occur in constitutive heterochromatin, but can be formed in facultative heterochromatin, dependent on heteropycnosis and sex. Quadrivalents and U-type exchanges occur. In spermatogonia and spermatocytes the number of differentially stained chromosomes varies considerably; sister chromatid exchanges hardly appear. Sex bivalents with differentially stained chromosomes have a lower chiasma frequency than normally stained sex bivalents. Bivalents show reduced staining of all four, two outer, or one inner chromatid. Autobivalents arise in the same way as diplochromosomes; chromatids with the oldest DNA sub-units remain together during reduplication and are thus involved in sister chromosome pairing. The additional reduplication begins 7 days after the premeiotic S-phase, first metaphase after 19 days. Spermatogenesis is abnormal from first anaphase onwards.     

NOR Sites Detected by Ag-DAPI Staining of an Unusual Autosome Chromosome of Bradysia Hygida (Diptera:Sciaridae) Colocalize with C-banded Heterochromatic Region

The study of chromosomes in insects is a good tool in mitotic process analysis, zoographic localization and evolution investigation. Among them, the Sciaridae offers a karyotype with a small number of chromosomes, where the heterochromatin and nucleolar organizer region, NOR, are easily analyzed in metaphase chromosomes obtained from cerebral ganglia squashes. In this work, the heterochromatic regions on Bradysia hygida mitotic chromosomes, revealed by C-banding, were identified as centromeric blocks on A and C chromosomes and as dark interstitial region in B and X chromosomes. By Ag-DAPI staining, active nucleolus organizer region, NOR, was revealed associated to the constitutive heterochromatin in the end of the C autosome chromosome. The C-band regions and the unusual ribosomal site localization are discussed.     

Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258

A benzimidazole derivative, Hoechst 33258 can induce decondensation of constitutive heterochromatin in the mouse derived L cell chromosomes when the compound is given in sufficiently high concentration (40 micrograms/ml) to the L cell culture. Hoechst 33258 at low concentration (1 micrograms/ml, 16 h) cannot produce this effect on L cell chromosomes. Bromodeoxyuridine (BUdR) incorporation for one cell cycle simultaneous with the Hoechst 33258 treatment at low concentration could decondense heterochromatin segments in metaphase chromosomes. The heterochromatin decondensation, however, was asymmetric; it was observed only on one chromatid and the other of a chromosome remained in condensed state. The observation of asymmetric decondensation of heterochromatin by Hoechst 33258 after BUdR incorporation for one cell cycle, the association of A-T rich satellite DNA to mouse heterochromatin, and available data on the specific binding of Hoechst 33258 to A-T base pairs of DNA and on the higher affinity of the compound to BUdR substituted DNA than to ordinary DNA implied that the binding of Hoechst 33258 molecules to A-T rich satellite DNA is the cause of heterochromatin decondensation.     

Differential silver staining of chromatin in metaphase chromosomes

The silver techniques used to demonstrate nucleolar organizer regions and cores in chromosomes can also differentially stain chromatin within chromosomes. Direct silver staining of mouse and human chromosomes resulted in preferential staining of centromeric regions and non-nucleolar secondary constrictions, both of which are composed of constitutive heterochromatin. After C-banding, these regions were no longer silver-stainable, suggesting that the biochemical constituents (presumably non-histone proteins) which contain the reaction sites for silver are extracted during the banding treatment. Light and electron microscopy of chromosomes G-banded with trypsin and then silver-stained revealed heavier deposits of silver over the condensed aggregates of chromatin within the band regions than over the more dispersed interband chromatin. At the ultrastructural level, chromatin fibres were covered with silver grains, indicating that there are many reaction sites for this metal along the fibres. These results suggest that the degree of silver staining in any region of the chromosome may be contingent upon the concentration of chromatin in that region. This finding may have important implications concerning the nature of the silver-stained core-like structure in chromosomes. If a preferential dispersion of chromatin fibres occurs at the periphery of the chromosome during slide preparation, leaving the central region of each chromatid relatively undispersed, this difference in the concentration of chromatin may account for the differential silver staining of these regions and the consequent appearance of a core-like structure.     

Variation in lateral asymmetry of human chromosome 1

Variations in lateral asymmetry of human chromosome 1 were studied in 17 amniotic cell samples and eight blood samples by the 5-bromodeoxyuridine (BrdU) quenching of 4'-6-diamidino-2-phenylindole (DAPI) fluorescence. The size and the relative proportion of the bright fluorescent spots on each chromatid in the heterochromatic region of chromosome 1 (1qh) are variable from different amniotic (or blood) samples after one cycle of BrdU incorporation. However, the particular pattern for a given chromosome 1 is consistent within the individual sample. Size variations were classified into three groups, and variations in the pattern (proportion) of bright fluorescence on each chromatid in the 1qh region were classified into four groups. A preliminary estimate of the type and frequency of lateral asymmetry variations was obtained. These results suggest a high frequency of variability of heterochromatin in the population. The BrdU-DAPI fluorescence technique was found to be very useful for characterizing variations in the 1qh region; variations in organization of heterochromatin DNA with the 1qh region can be detected, and a simple system of nomenclature is proposed for naming the variations in this region.     

Location,structure and behaviour of C-heterochromatin during meiosis in Dichroplus silveiraguidoi (Acrididae-Orthoptera)

The constitutive heterochromatin of Dichroplus silveiraguidoi, a species which shows an exceptionally low chromosome number (2n=8), was studied at meiosis with a staining technique on normal and hypotonically treated specimens. The results showed: 1) an unusual behaviour of the heterochromatic blocks located in the so-called synaptic region of the sex bivalent (Neo Y-Neo X), which remains paired from early prophase through metaphase I; 2) in normal or in hypotonically treated cells a heterogeneous configuration of the C-heterochromatic blocks was observed. This configuration is characterized by the existence of small positive granules interconnected by euchromatic filaments and is enhanced by treatment with a low ionic strength solution; 3) A weakly positive stained (intermediate) material was demonstrated in the Neo X chromosome; 4) A large amount of heterochromatin is distributed in the form of granular material along the length of the autosomes and as telomeric and centromeric blocks in all chromosomes. The possible evolutionary mechanisms involved and the significance of the C-band heterochromatin demonstrated in this species are discussed.     

Tritiated uridine induced chromosome aberrations in relation to heterochromatin and nucleolar organisation in Microtus agrestis L.

Microtus agrestis is characterised by long sex chromosomes, most of which are constitutively heterochromatic, and thus supposedly, genetically inactive. A method to assess the template activity of the chromosomes is to study the distribution of chromatid aberrations produced by H³UdR, among and within the chromosomes. In such a study, in female Microtus agrestis cells in culture, it was found that, a large number of localised chromatid aberrations was induced in the constitutively heterochromatic regions of both X chromosomes. The frequency distribution and types of aberrations were found to be cell cycle dependent. With differential staining it has been possible to demonstrate that the constitutive heterochromatin of the sex chromosomes are involved in the nucleolar organisation in this species, thus containing the ribosomal RNA cistrons.     

Sequence of Centromere Separation: Role of Centromeric Heterochromatin

The late metaphase-early anaphase cells from various tissues of male Mus musculus, M. poschiavinus, M. spretus, M. castaneus, female and male Bos taurus (cattle) and female Myopus schisticolor (wood lemming) were analyzed for centromeres that showed separation into two daughter centromeres and those that did not show such separation. In all strains and species of mouse the Y chromosome is the first one to separate, as is the X or Y in the cattle. These sex chromosomes are devoid of constitutive heterochromatin, whereas all autosomes in these species carry detectable quantities. In cattle, the late replicating X chromosome appears to separate later than the active X. In the wood lemming the three pairs of autosomes with the least amount of centromeric constitutive heterochromatin separate first. These are followed by the separation of seven pairs of autosomes carrying medium amounts of constitutive heterochromatin. Five pairs of autosomes with the largest amounts of constitutive heterochromatin are the last in the sequence of separation. The sex chromosomes with medium amounts of constitutive heterochromatin around the centromere, and a very large amount of distal heterochromatin, separate among the very late ones but are not the last. These observations assign a specific role to centromeric constitutive heterochromatin and also indicate that nonproximal heterochromatin does not exert control over the sequence in which the centromeres in the genome separate. It appears that qualitative differences among various types of constitutive heterochromatin are as important as quantitative differences in controlling the separation of centromeres.     

Nature and distribution of constitutive heterochromatin and NOR location in the grasshopper Phaeoparia megacephala (Romaleidae: Orthoptera)

The constitutive heterochromatin (CH) of Phaeoparia megacephala was studied using C-banding and fluorochrome staining (CMA3, DAPI and acridine orange). The nucleolar organizer regions (NOR) were identified with silver staining. The chromosome complement of this species was 2n = 23, XO in males, and 2n = 24, XX in females. The CH was pericentromeric in all chromosomes. L1, L2, L3 and X chromosomes showed large blocks of CH, while the medium and small chromosomes had small blocks. The staining procedure with acridine orange revealed the same pattern. All the pericentromeric regions showed small blocks of CMA3-positive constitutive heterochromatin (GC-rich regions), while only part of the large C-band positive chromosome segments (L1, L2, L3 and X) were CMA3 positive. This character demonstrates an uncommon heterogeneity of constitutive heterochromatin in P. megacephala. The fluorochrome DAPI did not reveal DAPI-positive regions (AT-rich regions). Silver staining revealed only one pair of medium chromosomes with NOR.     

Characterisation of a very complex constitutive heterochromatin in two Gerbillus species (Rodentia)

A large amount of heterochromatin is observed in two species of the genus Gerbillus, G. nigeriae and G. hesperinus. The C-band material represents about one-half of the total karyotype length in the former species, and about one-third in the latter. Several banding techniques and various 5-bromodeoxyuridine (BrdU) treatments were used to characterise these heterochromatic segments. After applying the R-banding technique, three different staining responses of the heterochromatin can be distinguished. In G. nigeriae, strongly stained segments (R-band positive) appear in most chromosomes and, in particular, constitute the short arms of all the larger chromosomes. Palely staining heterochromatic segments (R-band negative) are less abundant in G. nigeriae but predominate in G. hesperinus. In addition, in both species an intermediate staining of heterochromatin is observed near the centromere or in the heterochromatic short arms of some acrocentric and small submetacentric chromosomes. Very short BrdU treatment during the end of the last cell cycle results in asymmetrical staining of chromatids in heterochromatic segments after applying the acridine orange or FPG (fluorescence plus Giemsa) technique. The alternating location of strongly staining segments in one or the other chromatid simulates sister chromatid exchanges (pseudo-SCE). This pattern persists after longer BrdU treatment during different stages of the last cell cycle and is independent of the R-staining properties of the heterochromatin. The lateral asymmetric appearance of the large heterochromatic segments in Gerbillus is interpreted as reflecting an uneven distribution of adenine and thymidine between the two strands of DNA.     

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.     

The distribution of SCEs within and between the genomes of an interspecific hybrid

The distribution of sister chromatid exchanges has been examined in the chromosomes of a hybrid male wallaby (Macropus rufogriseus x Wallabia bicolor ), and in the X chromosomes of M. parryi and M. rufus. Comparisons were made of SCE frequency between the two genomes of the hybrid, only one of which has an appreciable amount of constitutive heterochromatin, and between the euchromatic and heterochromatic regions of the M. rufogriseus genome. The frequency of SCEs is closely correlated with the DNA content of the individual chromosomes. The distribution of the SCEs between the euchromatin and heterochromatin in the M. rufogriseus genome showed a deficiency of SCEs observed in the heterochromatin compared with the euchromatin. —A substantial excess of SCEs occurred at the nucleolar organiser region of the M. rufogriseus X chromosome. This excess was absent from the nucleolar organiser region of the X chromosome of the two other macropodine species studied and is accounted for by the presence of an adjacent euchromatin-heterochromatin junction.     

Preferential Breakpoints in the Recovery of Broken Dicentric Chromosomes in Drosophila melanogaster

We designed a system to determine whether dicentric chromosomes in Drosophila melanogaster break at random or at preferred sites. Sister chromatid exchange in a Ring-X chromosome produced dicentric chromosomes with two bridging arms connecting segregating centromeres as cells divide. This double bridge can break in mitosis. A genetic screen recovered chromosomes that were linearized by breakage in the male germline. Because the screen required viability of males with this X chromosome, the breakpoints in each arm of the double bridge must be closely matched to produce a nearly euploid chromosome. We expected that most linear chromosomes would be broken in heterochromatin because there are no vital genes in heterochromatin, and breakpoint distribution would be relatively unconstrained. Surprisingly, approximately half the breakpoints are found in euchromatin, and the breakpoints are clustered in just a few regions of the chromosome that closely match regions identified as intercalary heterochromatin. The results support the Laird hypothesis that intercalary heterochromatin can explain fragile sites in mitotic chromosomes, including fragile X. Opened rings also were recovered after male larvae were exposed to X-rays. This method was much less efficient and produced chromosomes with a strikingly different array of breakpoints, with almost all located in heterochromatin. A series of circularly permuted linear X chromosomes was generated that may be useful for investigating aspects of chromosome behavior, such as crossover distribution and interference in meiosis, or questions of nuclear organization and function.     

Qualitative analysis of constitutive heterochromatin and primate evolution

The results of qualitative heterochromatin analysis in 16 species of primates: Homo sapiens , Pan troglodytes and Gorilla gorilla (F. Hominidae), Hylobates syndactilus (F. Hylobatidae), Macaca fascicularis , M. tibetana , Mandrillus sphinx , M. leucophaeus , Cercopithecus aethiops , C. sabaeus and C. albogularis (F. Cercopithecidae), Cebus apella , Ateles belzebuth hybridus , Aotus azarae , Saimiri sciureus and Lagothrix lagothricha (F. Cebidae) are presented in this work. We characterized heterochromatin using: (a) in situ digestion with restriction enzymes AluI, HaeIII, RsaI and Sau3A, and (b) chromosome staining with DA/DAPI on unbanded chromosomes, on C-banded chromosomes and on sequentially G-C-banded chromosomes. The aim of this work was to relate the qualitative characteristics of constitutive heterochromatin observed with the cytogenetic evolutive processes in the primate group. Results obtained show that (1) in the family Cercopithecidae, Papionini species do not present chromosomal rearrangements when their karyotypes are compared and the heterochromatin characteristics are uniform, while Cercopithecini species show a high number of chromosomal reorganizations, but they have the same heterochromatic characteristics; (2) the Platyrrhini species analysed show variability in their karyological and heterochromatic characteristics; (3) the Hominoidea present two different situations: Pan , Gorilla and Homo with few chromosomal reorganizations among their karyotypes but with a high variability in their heterochromatin characteristics, and Hylobates with low heterochromatin variability and a highly derived karyotype. Speciation processes related to chromosome changes and heterochromatin variations in different groups of primates are discussed.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 107–124.     

Chromosome banding in Amphibia

The mitotic and meiotic chromosomes of the marsupial frog Gastrotheca riobambae were analysed with various banding techniques. The karyotype of this species is distinguished by considerable amounts of constitutive heterochromatin and unusual, heteromorphic XY sex chromosomes. The Y chromosome is considerably larger than the X chromosome and almost completely heterochromatic. The analysis of the banding patterns obtained with GC- and AT-base-pair-specific fluorochromes shows that the constitutive heterochromatin in the Y chromosome consists of at least three different structural categories. The only nucleolus organizer region (NOR) of the karyotype is localized in the short arm of the X chromosome. This causes a sex-specific difference in the number of NOR: female animals have two NORs in diploid cells, male animals one. No cytological indications were found for the inactivation of one of the two X chromosomes in the female cells. In male meiosis, the heteromorphic sex chromosomes form a characteristic sex-bivalent by pairing their telomeres in an end-to-end arrangement. The significance of the XY/XX sex chromosomes of G. riobambae for the study of X-linked genes in Amphibia, the evolution of sex chromosomes and their specific DNA sequences, and the significance of the meiotic process of sex chromosomes are discussed.