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.     

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.     

Preferential fluorescent staining of heterochromatic regions in human chromosomes 9, 15, and the Y by D 287/170

Summary The utility of a newly synthesized chemical variation of DAPI (4-6-diamidino-2-phenyl-indole), D 287/170, for differential staining of constitutive heterochromatin in man is demonstrated. Direct staining of human chromosomes with D 287/170 results in brilliant fluorescence of the paracentromeric C-band of chromosome 9, of a proximal short-arm segment of chromosome 15 and of certain heterochromatic regions in the Y. Bright, but less conspicuous fluorescence is occassionally seen at the centromeres of other chromosomes. The staining differentiation obtained by D 287/170 is very distinct, and the intensity of the fluorescent light is unusually high. The new fluorochrome should prove particularly useful for detecting and analyzing human chromosome 9 heterochromatin at various stages of the cell cycle in normal and structurally altered chromosomes.     

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.     

Expression of heterochromatin by restriction endonuclease treatment and distamycin A/DAPI staining of Indian muntjac (Muntiacus muntjak) chromosomes

The constitutive heterochromatin of the Indian muntjac (Muntiacus muntjak) was examined following digestion with various restriction endonucleases (AluI, HaeIII, HinfI, and MboI), as well as by selective fluorescence staining with distamycin A plus 4'-6-diamidino-2-phenylindole. Distinct areas within the C-bands were found to have characteristic staining patterns which were more conspicuous in the sex chromosomes. Two dot-like structures resistant to AluI were found in the X and Y1 chromosomes in the same position as the nucleolus organizer regions.     

Cytogenetics of bisexual/unisexual species of Poecilia. II. Analysis of heterochromatin and nucleolar organizer regions in Poecilia mexicana mexicana by C-banding and DAPI, quinacrine, chromomycin A3, and silver staining.

Chromosomes of Poecilia mexicana mexicana, one of the bisexual species involved in the hybrid origin of the unisexual teleost fish species P. formosa, were analyzed by several staining techniques. Sex-specific, differential heterochromatin, found in other congeneric species, was not observed in P. m. mexicana. Nucleolar organizer regions were polymorphic among individual specimens within a given population sample. A single specimen exhibiting intraindividual variability of chromosome pair 1 and a specimen with a triploid karyotype are also described.     

Cytofluorometric determination of the DNA base content in human chromosomes with quinacrine mustard, Hoechst 33′258, DAPI, and mithramycin

The human chromosomes 1, 9, 16, 21, and Y were analysed cytofluorometrically with the AT-specific DNA ligands quinacrine mustard (QM), Hoechst 33′258, and DAPI, and the GC-specific DNA ligand mithramycin. All three AT dyes give similar results, though QM produces more distinct banding than DAPI or Hoechst. The sum of AT and GC fluorescence is very well correlated to the amount of DNA estimated densitometrically. The AT/GC ratios of chromosomes 16, 22, and Y differ clearly from that of whole nuclei, and accord fairly well with the results obtained by flow cytometry. For the Y a significant difference in calculated base content between donors was found with all three AT dyes even though differences in the karyotypes were not distinguishable by the eye.     

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 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.     

Fluorescent chromosome banding in inbred chicken: quinacrine bands,sequential chromomycin and Dapi bands

Summary Highly inbred White Leghorn chickens were used for an investigation of the banding pattern of the macrochromosomes. A standard was set for the Q-bands. GC-specific fluorochrome chromomycin and the AT-specific Dapi were used in a sequential stain. The comparison of these two stains disclosed quantitative differences in the base distribution of the DNA. Factors responsibe for the binding mechanism and the appearance of the bands are discussed.     

Characterization of normal and rearranged human chromosomes by simultaneous Q- and R-banding with chromomycin A3

Simultaneous Q- and R-type banding patterns in human chromosomes have been achieved by staining with chromomycin A3. Some peculiarities of these patterns as compared to the patterns induced by other fluorochromes are described. The resolution power of this technique in analyzing structural rearrangements of human chromosomes is discussed.     

5-Methylcytosine in heterochromatic regions of chromosomes: chimpanzee and gorilla compared to the human

Fixed metaphase chromosomes of gorilla and chimpanzee were UV-irradiated to produce regions of single-stranded DNA and then treated with antibodies specific for the minor DNA base 5-methylcytosine (5 MeC). An indirect immunofluorescence technique was used to visualize sites of antibody binding. In the gorilla six pairs of autosomes contained major fluorescent regions, indicating localized regions of highly methylated DNA. These corresponded, with the exception of chromosome 19, to the major regions of constitutive heterochromatin as seen by C-banding. The Y chromosome also contained a highly fluorescent region which was located just proximal to the intense Q-band region. In the chimpanzee no comparable concentrations of highly methylated DNA were seen. Smaller regions of intense 5 MeC binding were present on perhaps six chimpanzee chromosomes, including the Y. Five of these corresponded to chromosomes which were highly methylated in the gorilla.--There is diversity among the human, gorilla and chimpanzee in both the size and location of concentrations of 5 MeC, supporting the idea that satellite DNA evolves more rapidly than DNA in the remainder of the chromosome.     

Longitudinal differentiation of metaphase chromosomes of Indian muntjac as studied by restriction enzyme digestion,in situ hybridization with cloned DNA probes and distamycin A plus DAPI fluorescence staining

The longitudinal differentiation of metaphase chromosomes of the Indian muntjac was studied by digestion with restriction enzymes, in situ hybridization with cloned DNA probes and distamycin A plus DAPI (4-6-diamidino-2-phenylindole) fluorescence staining. The centromeric regions of chromosomes 3 and 3 + X of a male Indian muntjac cell line were distinct from each other and different from those of other chromosomes. Digestion with a combination of EcoRI^* and Sau3A revealed a pattern corresponding to that of C-banding. Digestion with AluI, EcoRII or RsaI yielded a band specific to the centromeric region only in chromosomes 3 and 3 + X. Furthermore, HinfI digestion yielded only a band at the centromeric region of chromosome 3, whereas DA-DAPI staining revealed a single band limited to the extreme end of the C-band heterochromatin of the short arm of 3 + X. These results suggest that centromeres of Indian muntjac chromosomes contain at least four different types of repetitive DNA. Such diversity in heterochromatin was also confirmed by in situ hybridization using specific DNA probes isolated and cloned from highly repetitive DNA families. Heterozygosity between chromosome homologs was revealed by restriction enzyme banding. Evidence is presented for the presence of nucleolus organizer regions (NORs) on the long arm of chromosome 1 as well as on the secondary constrictions of 3 and 3 + X.Abbreviations DA distamycin A - DAPI 4-6-diamidino-2-phenylindole - NOR(s) nucleolus organizer region(s) - PBS phosphate-buffered saline - PI propidium iodide     

DA/DAPI fluorescent bands in the chromosomes of Pan paniscus

The fluorochrome pattern produced by DA/DAPI double staining in Pan paniscus chromosomes is reported. The location of DA/DAPI prominent bands differs from that reported for all other hominoid species. However, the pattern in the pygmy chimpanzee is most similar to that seen in Pan troglodytes. Comparison of the DA/DAPI pattern of the other hominoid species allows the construction of a proposed hominoid ancestral karyotype and a preliminary phylogenetic reconstruction of DA/DAPI bands for the great apes and man.     

A comparison between quinacrine fluorescence banding and 3H-thymidine incorporation patterns in human chromosomes

Summary Late ³H-thymidine incorporation patterns and quinacrine fluorescence banding patterns were analyzed in metaphase chromosomes prepared from human blood cultures. In general, late-labeling regions correspond to the strongly fluorescent bands in the chromosomes. However, the dully fluorescent secondary constrictions of the chromosomes Nos. 1, 9 and 16 may show late replication in some instances. In contrast, the brilliantly fluorescent distal part of the Y chromosome is not labeled during the latest phase of the DNA replication. In the male X and in the isopycnotic X of the female the labeling pattern also agrees with the quinacrine fluorescence banding. The heteropycnotic X of the female is still more strikingly late-labeling. However, the pattern of its late replication agrees also with the quinacrine fluorescence bands.

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Zusammenfassung An aus menschlichen Blutkulturen gewonnenen Chromosomenpräparaten wurden vergleichende Untersuchungen über die späten Replikationsmuster (mittels ³H-Thymidin-Autoradiographie) und die Quinacrin-Fluorescenz-Bänderungsmuster durchgeführt. Im allgemeinen stimmen die spät replizierenden Abschnitte mit den intensiv fluorescierenden Regionen der Chromosomen überein. Allerdings können die nur schwach fluorescierenden sekundären Constrictionen der Chromosomen Nr. 1, 9 und 16 manchmal auch eine späte Replikation zeigen. Auf der anderen Seite wird der stark fluorescierende Abschnitt des Y-Chromosoms in den letzten Phasen der DNS-Replikation nicht mehr markiert. Beim X-Chromosom des Mannes und beim isopyknotischen X der Frau wird ebenfalls eine Übereinstimmung des Spät-Replikationsmusters und der Quinacrin-Bänderung gefunden. Das heteropyknotische X der Frau zeigt eine noch deutlichere Spät-Replikation; das Muster der Silberkörner stimmt aber ebenfalls mit den Quinacrine-Bändern überein.

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This study was supported by the österreichischen Fonds zur Förderung der Wissenschaftlichen Forschung.     

限制酶AluI显带和CA_3/DA/DAPI染色表达的家猪染色体结构异染色质

采用限制酶AluI显带、CA_(?)/DA/DAPI荧光染色和常规C带技术研究了家猪染色体着丝粒结构异染色质,结果表明:着丝粒结构异染色质至少可被区分为3类,并且在染色体组内各有其特异的染色体分布。将家猪染色体DA/DAPI荧光带和限制酶AluI显带与人类染色体比较,发现家猪13—18号端着丝粒染色体显带特征与人染色体1,9、16、Y一致。提示家猪13—18号端着丝粒区结构异染色质存在与人类随体DNA相似的DNA组成。     

Heterogeneity of human chromosome 9 constitutive heterochromatin as revealed by sequential distamycin A/DAPI staining and C-banding

Summary Distamycin A/DAPI staining and sequential C-banding of human lymphocyte chromosomes reveals the regular occurrence of differentially staining subfractions of chromosome 9 constitutive heterochromatin. These subfractions are regionally organized as two subsegments: a distal one, which fluoresces brightly with DAPI after preincubation with distamycin A and a proximal one, which stains intensely with Giemsa after sequential C-banding. Observations are presented that indicate an occasionally independent genetic behavior of these heterochromatin subfractions.