Distamycin A/DAPI bands and the effects of 5-azacytidine on the chromosomes of the chimpanzee, Pan troglodytes

The chromosomes of the chimpanzee were stained with distamycin A/DAPI, which labels specific C-bands. Bright distamycin A/DAPI fluorescence was found in the heterochromatic regions of chromosomes 6, 11, 14 to 16, 18 to 20, and 23 and the Y. Lymphocyte cultures from chimpanzees were treated with low doses of 5-azacytidine during the last hours of culture. This cytosine analog induces highly distinct undercondensations in 28 heterochromatic regions of 19 chromosomes. These 5-azacytidine-sensitive regions are predominantly located in the terminal C-bands of the chromosomes. In vitro treatment with 5-azacytidine also preserves into the metaphase stage somatic pairings between the 5-azacytidine-sensitive heterochromatic regions in interphase nuclei. The homologies and differences regarding the chromosomal localization of distamycin A/DAPI-bright C-bands, 5-azacytidine-sensitive heterochromatin, 5-methylcytosine-rich DNA sequences, and satellite DNAs in the chimpanzee and man are discussed.     

Chromosome heteromorphisms in the gorilla karyotype. Analyses with distamycin A/DAPI, quinacrine and 5-azacytidine

The chromosomes of one male and three female gorillas were extensively studied with various regional banding methods. The chromosomes were stained with the fluorescent dyes quinacrine mustard and distamycin A/DAPI (DA/DAPI), which label different subsets of heterochromatin in the chromosome complement. Furthermore, lymphocyte cultures were treated with the cytidine analog 5-azacytidine (5-azaC). The 5-azaC-induced undercondensations were found in most of the DA/DAPI-bands as well as in many telomeric C-bands. The karyotype of the gorilla exhibits a considerable number of heterochromatin variants. Of the different types of heteromorphisms noted, the most striking is that involving the short arm regions of chromosomes 12 to 16 and 23 (satellite stalk regions) and the paracentromeric heterochromatin of chromosomes 17 and 18. There also are numerous heteromorphic C-bands localized in the telomeric regions of homologous chromosome arms. In comparison, only few heteromorphisms occur between C-bands in the centromeric and pericentromeric regions of homologs. Finally, a variability in the fluorescence intensity of quinacrine-bright satellites in the short arms of chromosomes 12 to 16, 22, and 23 is observed.     

A direct demonstration of somatically paired heterochromatin of human chromosomes

Human lymphocyte cultures were treated with different concentrations of 5-azacytidine for various lengths of time. This cytosine analog induces very distinct undercondensation in the heterochromatin of chromosomes 1, 9, 15, 16, and Y if applied in low doses during the last hours of culture. These regions are further distinguished by their intense distamycin A/DAPI-staining and highly methylated DNA. In interphase nuclei, these heterochromatic regions are frequently somatically paired. These somatic pairings are preserved up to the metaphase stage in the 5-azacytidine-treated cultures and are thus susceptible to direct analysis. The specific effect of 5-azacytidine on the heterochromatin of these chromosomes, its conserving effect on somatic pairing, and some of the consequences of the somatic pairing on the development of human chromosome aberrations are discussed.     

Specific silver staining of experimentally undercondensed chromosome regions

Treatment of human and mouse cell cultures with DNA binding AT-specific compounds and with some base analogues induced distinct undercondensations in several heterochromatic chromosome regions. All those heterochromatic regions undercondensed by AT-specific DNA ligands (distamycin A, DAPI, Hoechst 33258) could be heavily labeled with the silver(Ag)-staining technique; but the heterochromatic regions undercondensed with the cytidine analogue 5-azacytidine were Ag-negative. In metaphase chromosomes from BrdU-treated human cell cultures, the bifilarly substituted chromatids, which show a slight undercondensation, were also Ag-negative. Cytochemical analyses of the Ag-stained undercondensed heterochromatic regions showed that the Ag-stainable material consisted of nonhistone proteins. The mechanism of Ag staining in the undercondensed heterochromatic regions was compared with Ag staining of the nucleolus organizer regions.     

Distamycin A/DAPI staining of heterochromatin in male meiosis of man

The sequential staining with distamycin A/DAPI provides an ideal method for studying the behaviour of heterochromatic regions in human male meiosis. The various meiotic and postmeiotic stages were found to have different staining qualities. Although all heterochromatic regions in human pachytene cells show specific DA/DAPI fluorescence, bright and clearly stained heterochromatic blocks can be distinguished from small DA/DAPI spots. Pachytene nuclei exhibit associations between heterochromatic regions of non-homologous bivalents. The heterochromatin of bivalent 9 generally presents as a cluster of small, discrete bodies. The heterochromatic regions of chromosomes 1, 9, 15, 16 and Y are preferentially stained at diakinesis and metaphase of the second meiotic division. The specific DA/DAPI staining disappears with the progressive volume reduction of middle and late spermatid nuclei. The heterochromatin of the chromatids fuses to form a large chromocenter during spermatid differentiation.     

5-Azacytidine-induced undercondensations in human chromosomes

Summary The cytosine analogue 5-azacytidine induces very distinct undercondensations in human chromosomes if applied to lymphocyte cultures. The number of induced undercondensations and their chromosomal localization can be varied by the 5-azacytidine dose and the treatment time. Pulverized chromosomes or undercondensations in the G-band-positive chromosome regions are produced with high doses and long treatment times. If applied in low doses during the last hours of culture, 5-azacytidine induces specific undercondensations in the heterochromatin of chromosomes 1, 9, 15, 16, and Y. Optimum conditions required for inducing the various types of undercondensation in the chromosomes were determined. Various examples of the use 5-azacytidine in the analysis of chromosome rearrangements involving heterochromatic regions are presented.     

Heterochromatin characterization of sex chromosomes in Triturus marmoratus (Urodela, Salamandridae).

The sex chromosomes of the Iberian marbled newt, Triturus marmoratus, were studied using various banding techniques, including restriction enzyme/nick translation (RE/NT) procedures. Four types of heterochromatin on the sex chromosomes could be distinguished: (1) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT negative, and HaeIII/NT and HinfI/NT positive; (2) distamycin A/DAPI and chromomycin A3/distamycin A positive, but RE/NT negative; (3) AT rich, but RE/NT negative; and (4) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT and HinfI/NT negative, but HaeIII/NT positive. These data suggest a common origin for the terminal heterochromatic domains of both the X and Y chromosomes in this species.     

Counterstained enhancement of TaqI resistant sites after distamycin A/diamidinophenylindole treatment

Numerous selective and differential staining techniques have been used to investigate the hierarchical organisation of the human genome. This investigation demonstrates the unique characteristics that are produced on fixed human chromosomes when sequential procedures involving restriction endonuclease TaqI, distamycin A (DA) and 4,6-diamidino-2-phenylindole (DAPI) are employed. TaqI produces extensive gaps in the heterochromatic regions associated with satellite II and III DNAs of human chromosomes 1, 9, 15, 16 and Y. DA/DAPI selectively highlights, as brightly fluorescent C-bands, the heterochromatin associated with the alpha, beta, satellite II and III DNAs of these chromosomes. When DA and DAPI are used on chromosomes before TaqI digestion, and then stained with Giemsa, the centromeric regions appear to be more resistant, producing a distinct C-banding pattern and gaps in the heterochromatin regions. Sequential use of the DA/DAPI technique after TaqI treatment produces a bright fluorescence on the remaining pericentromeric regions of chromosomes 1, 9, 16 and Y, which also displayed a cytochemically unique banding pattern. This approach has produced specific enhanced chromosomal bands, which may serve as tools to characterize genomic heterochromatin at a fundamental level.     

Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A

A DNA-binding AT-specific oligopeptide antibiotic, distamycin A, was used as non-fluorescent counterstain in conjunction with the DNA-binding AT-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) to investigate the effect of the antibiotic on DAPI fluorescent banding of human chromosomes. Distamycin A-pretreated metaphases and interphase nuclei exhibited a significantly lower overall fluorescence intensity than DAPI controls. Chromosome arms were pale and intercalary DAPI bands (Q bands) were obliterated, but some specific regions of constitutive heterochromatin remained brightly fluorescent. These were mainly the constrictions of chromosomes 1, 9 and 16, the short arm of chromosome 15, and the distal part of the Y. The distamycin A/DAPI banding pattern appears to be comparable to that reported for anti-5-methylcytosine binding [11]. The observations are discussed as they relate to the roles of chromosomal DNAs and proteins in chromosome banding.     

Comparative mapping of human alphoid satellite DNA repeat sequences in the great apes

Heterochromatic regions of chromosomes contain highly repetitive, tandemly arranged DNA sequences that undergo very rapid variation compared to unique DNA sequences that are predominantly conserved. In this study the chromosomal basis of speciation has been looked at in terms of repeat sequences. We have hybridized twenty-one chromosome-specific human alphoid satellite DNA probes to metaphase spreads of the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus) to investigate the evolutionary relationship of heterochromatic regions among such hominoid species. The majority of the probes did not hybridize to their corresponding equivalent chromosome but presented hybridization signals on non-corresponding chromosomes. Such observations suggest that rapid changes may have occurred in the ancestral alphoid satellite DNA sequence, resulting in divergence among the great ape species. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Metaphase chromosomes of four species of the Drosophila nasuta subgroup

Metaphase chromosomes of four species of the Drosophila nasuta subgroup, D. nasuta, D. kepulauana, D. kohkoa, and D. neveifrons, were analysed by the C banding method to clarify the chromosomal differentiation during speciation. Four species were different in heterochromatic regions of their chromosomes, especially microchromosomes of the fourth chromosomes and the Y chromosomes. Intraspecific variations of heterochromatic regions were also found among isofemale lines from various localities of D. nasuta, D. kepulauana, and D. kohkoa. Intraspecific variations of heterochromatic regions were not different from interspecific variations morphologically. From these results, the evolutionary process of heterochromatic regions was discussed.     

Evolutionary diversity of reverse (R) fluorescent chromosome bands in vertebrates

Mitotic chromosomes, interphase cell nuclei, and male meiosis of 41 species representing all vertebrate classes were analyzed with distamycin A/mithramycin counterstaining. The purpose of the study was to recognize differences and common characteristics in the reverse (R) fluorescent banding patterns in the chromosomes of vertebrate species at various stages of evolution. In contrast to the warm-blooded mammals and birds, the euchromatic segments in the chromosomes of most reptiles, amphibians, and fishes contain no multiple fluorescent R-bands. This is thought to be due to the absence of the long homogeneous regions (isochores) in the DNA of the cold-blooded vertebrates. Distamycin A/mithramycin banding specifically reveals the GC-rich constitutive heterochromatin in all vertebrates. In most of the vertebrate chromosomes examined, the heterochromatic regions have opposite staining properties with mithramycin and quinacrine. Mithramycin labels the nucleolus organizer regions very brightly in the karyotypes of fishes, amphibians, reptiles and birds, but not of mammals. The lack of mithramycin fluorescence at the nucleolus organizer regions of mammals is attributed to the relatively low level of redundancy of the GC-rich ribosomal DNA in their genomes. Studies on the various meiotic stages of the cold-blooded vertebrates show that the mithramycin labeling of the nucleolus organizers is independent of their state of activity. This can be confirmed by mithramycin fluorescence at the nucleoli of actinomycintreated cells.Dedicated to the memory of Professor Dr. Hans Bauer     

Binding of anti-nucleoside antibodies reveals different classes of DNA in the chromosomes of the kangaroo rat (Dipodomys ordii)

The binding of highly purified anti-nucleoside antibodies to fixed metaphase chromosomes of the kangaroo rat (Dipodomys ordii) revealed the presence of different classes of DNA in different regions of the chromosomes. To permit antibody binding, the chromosomal DNA was first made single-stranded by either ultraviolet irradiation, which denatures some classes of AT-rich DNA, or photo-oxidation, which denatures GC-rich DNA. The antibody binding patterns obtained matched the location of the different classes of satellite DNA in kangaroo rat chromosomes. After either denaturation method, anti-5-methylcytidine (anti-M) bound intensely only to the centromeric heterochromatic regions which are known to contain the GC rich, highly methylated HS-β satellite DNA of this species. The basic repeating unit of the HS-β sequence is 5′-ACACAGCGGG-3′ 3′-TGTGTCGCCC-5′ [4]. The binding of anti-M after UV irradiation is permitted by the production of pyrmidine (CC and TC) dimers in the right-hand portion of this repeating sequence, supporting the idea that the 5-methylcytosine residues are in this portion. After photo-oxidation, anti-cytidine (anti-C) and anti-adenosine (anti-A) also showed intense binding to the centromeric heterochromatin. In addition, these antibodies showed moderately intense binding to non-centromeric heterochromatic regions, which contain the relatively GC-rich HS-α and MS satellite DNAs. After UV irradiation, anti-A binding produced a banding pattern identical to the quinacrine (Q-band) pattern, with bright chromosome arms and very dull centromeric heterochromatic regions, while anti-C showed moderate binding in the centromeric regions and fairly even but weak binding elsewhere.The results have clarified the way in which anti-nucleoside antibodies react with chromosomal DNA. The reactivity of anti-A, anti-C and anti-M with the partially denatured HS-β satellite DNA supports the idea that antibody binding requires denaturation of a sequence perhaps no more than 5 base pairs long. In addition, it appears that it is not necessary to have more than one identical base in a row to permit antibody binding.     

Evolutionary conservation of fragile sites induced by 5-azacytidine and 5-azadeoxycytidine in man,gorilla, and chimpanzee

Summary Lymphocyte cultures from man, gorilla, and chimpanzee were treated with 5-azacytidine and 5-azadeoxycytidine. These cytidine analogues induce common fragile sites in the chromosome bands 1q42 and 19q13 of man. A rare fragile site is induced by 5-azadeoxycytidine in the band 1q24. The optimum conditions required for inducing these new fragile sites were determined by a series of experiments. The common fragile site in human chromosome 1q42 also exists in the gorilla and chimpanzee in the homologous band 1q32. The fragile site in human chromosome 19q13 was demonstrated in the gorilla in the homologous chromosome band 20q13. These are the first examples found of evolutionary highly conserved fragile sites in homologous chromosome bands in related primate species. The interaction between 5-azacytidine, 5-azadeoxycytidine, and chromosomal DNA; the evolutionary conservation of genes located within or closely adjacent to the fragile sites in the chromosome 1 of Hominoidea; and the phylogenetic origin of the two new common fragile sites are discussed.     

Heterochromatin regions of the chromosomes of the hen and Japanese quail in mitosis and at the lampbrush stage

The mitotic and lampbrush chromosomes of the domestic fowl and Japanese quail were analysed by fluorochrome staining technique. The lampbrush chromosomes of both the subjects displayed a typical "loop-chromomere" structure. Three distinct kinds of loops were distinguished in Gallus g. domesticus--normal, telomeric bows, and lumps. The former are distributed along the whole chromosome length. The latter and the bows were observed in subtelomeric and telomeric regions. By DNA/RNA specific acridine orange staining it was shown that each loop (especially, "lumpy" loops) contained a rich RNP matrix. A comparative analysis of the chromomycin A3/distamycin A banding pattern of mitotic and lampbrush chromosomes shows that the telomeric "bows" and "lumps" are special loops developed in telomeric heterochromatic bands. In Coturnix c. japonica, the CMA/DA-positive bands were not observed in telomeres of mitotic macrochromosomes, except a smallest band in the 2p-arm telomere. The absence of telomeric heterochromatic bands which can be visualized in the quail mitotic chromosomes coincides with the absence of "bow"-like loops. Only small lump-like structures were seen in some telomeres of macroautosomes. The biological significance of loop formation and RNA synthesis in heterochromatic band loops in growing oocytes is briefly discussed.     

Chromosome banding and DNA replication patterns in bird karyotypes

The karyotypes of the domestic chicken (Gallus domesticus), Japanese quail (Coturnix coturnix), and griffon vulture (Gyps fulvus) were studied with a variety of banding techniques. The DNA replication patterns of bird chromosomes, analyzed by incorporation of 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT), are presented here for the first time. In particular, the time sequence of replication of the ZZ/ZW sex chromosomes throughout the S-phase was meticulously analyzed. BrdU and dT incorporation are very useful methods to identify homoeologies between karyotypes, as well as rearrangements that occurred in the macroautosomes during speciation. The Z chromosomes of the three birds displayed the same replication patterns, indicating a high degree of evolutionary conservation. In the homogametic male, BrdU and dT incorporation revealed no evidence of asynchronous replication between euchromatic bands in the ZZ pair. The same was true of the three Z chromosomes in a triploid-diploid chimeric chicken embryo. Minor replication asynchronies between the homologous ZZ or ZZZ chromosomes were restricted to heterochromatic C-bands. These results confirm that, in the ZZ male/ZW female sex-determining system of birds, dosage compensation for Z-linked genes does not occur by inactivation of one of the two Z chromosomes in the homogametic male. The heterochromatic W chromosomes of the three species showed bright labeling with distamycin A/mithramycin counterstain-enhanced fluorescence and exhibited significantly delayed DNA replication. The nucleolus organizers of birds, frequently located in microchromosomes, were also distinguished by bright distamycin A/mithramycin fluorescence.     

Nucleolus organizer regions and heterochromatin in the zebu (Bos indicus L.)

Summary Ag-NOR staining and a counterstain enhanced fluorescence technique (chromomycin A₃/distamycin A/DAPI-staining = CDD-method) and G-banding, respectively, have been applied to the zebu (Bos indicus L.) chromosomes. The nucleolus organizer regions (NORs) were found in the telomeric regions of chromosomes nos. 2, 3, 4, 11, and 28. CDD staining led to a well-defined R-banding pattern along the chromosome arms and to the visualization of centric heterochromatic bands of variable sizes.     

The chromosomal distribution of microsatellite repeats in the genome of the wolf fish Hoplias malabaricus, focusing on the sex chromosomes

Distribution of 12 mono-, di- and tri-nucleotide microsatellites on the chromosomes of 2 karyomorphs with 2 distinct sex chromosome systems (a simple XX/XY - karyomorph B and a multiple X(1)X(1)X(2)X(2)/X(1)X(2)Y - karyomorph D) in Hoplias malabaricus, commonly referred to as wolf fish, was studied using their physical mapping with fluorescence in situ hybridization (FISH). The distribution patterns of different microsatellites along the chromosomes varied considerably. Strong hybridization signals were observed at subtelomeric and heterochromatic regions of several autosomes, with a different accumulation on the sex chromosomes. A massive accumulation was found in the heterochromatic region of the X chromosome of karyomorph B, whereas microsatellites were gathered at centromeres of both X chromosomes as well as in corresponding regions of the neo-Y chromosome in karyomorph D. Our findings are likely in agreement with models that predict the accumulation of repetitive DNA sequences in regions with very low recombination. This process is however in contrast with what was observed in multiple systems, where such a reduction might be facilitated by the chromosomal rearrangements that are directly associated with the origin of these systems.     

Nucleolus-organizer regions and heterochromatin in three species of Bovidae

Ag-NOR staining and a counterstain-enhanced fluorescence technique (chromomycin A₃/distamycin A/DAPI staining = CDD-method) have been applied to ibex (Capra ibex L.), chamois (Rupicapra rupicapra L.) and bison (Bison bison L.) chromosomes.Chromomycin A₃ visualization led to a well defined R-banding pattern along the chromosome arms and to a clear demonstration of centric heterochromatic bands of variable size. The nucleolus organizer regions (NORs) were found in the telomeric regions of the chromosomes 2, 3, 4, 5 and 28 of the ibex, of the chromosomes 1/3 (short arm), 2, 4, 5 and 28 of the chamois and of the chromosomes 2, 3, 4, 11 and 28 of the bison.