Fluorescent heterochromatin staining in primate chromosomes

Recently, in addition to quinacrine staining, fluorochrome techniques have been developed which brilliantly stain other heterochromatic regions. Two of these staining techniques are Distamycin/DAPI (DA/DAPI) and D287/170. We stained the chromosomes of all species of great apes and 14 species of primates (48 individuals) using these three fluorochrome techniques. Only african apes and man show brilliant quinacrine staining while, man and all the great apes show brilliant DA/DAPI staining and only species belonging to the hominoidea (including the siamang) showed bright D287/170 staining. In the lower primates a medium level of DA/DAPI fluorescence was found in some species with large amount of pericentromeric heterochromatin. Brilliant DA/DAPI staining could represent a derived trait linking all great apes and humans, while D287/170 may link all hominoidea. Fluorochrome staining is believed to be correlated with some satellite DNA sequences. However, data available on the chromosome location of satellite DNAs in non-human primates were derived from buoyant density fractions resulting in cross hybridization and now are not considered reliable. Before making any correlation between fluorochrome staining and satellite DNAs in non human primates there is need of data onin situ hybridization with cloned DNA sequences on primate chromosomes. These data would help clarify the evolution and relationship of satellite DNAs and heterochromatin in primates.     

DA/DAPI-Fluorescent heteromorphism of human Y chromosome

Summary Variation of DA/DAPI intensity in the Yq12 band was observed in five amniotic cell specimens and one blood specimen from the father of one fetus. Three distinct classes of Yq heterochromatin were identified by distamycin A (DA) treatment of the cell cultures and various staining techniques. The heterochromatin in the Yq11.23 sub-band does not under-condense when exposed to DA, and shows pale fluorescence with quinacrine staining, positive C-banding, and bright fluorescence with DA/DAPI technique. This class of heterochromatin was consistently observed in all specimens studied. The other two classes of heterochromatin are in the Yq12 band. Both show undercondensation when exposed to DA, quinacrine-bright fluorescence, and positive C-banding; howover, one class of heterochromatin shows DA/DAPI-bright fluorescence and the other shows pale fluorescence. The size and banding intensity of the two classes of heterochromatin in Yq12 are variable. These results provide cytological evidence of heterogeneity within the Y heterochromatin region containing AT-rich DNA.     

Composition of constitutive heterochromatin of Pseudonannolene strinatii Mauriès, 1974 (Diplopoda, Spirostreptida) analyzed by AT/CG specific fluorochromes

Triple staining with fluorochromes (DA/DAPI/CMA) and C-banding were used to characterize the composition of Pseudonannolene strinatii heterochromatin. C-banding showed C+ bands of different labeling intensity on chromosomes 1 and 2 in some cells. Fluorochrome staining revealed DAPI+ regions corresponding to the C-banding pattern, indicating that the heterochromatin of this species is abundant in AT-rich sequences.     

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.     

Heterochromatin differentiation shows the pathways of karyotypic evolution in Israeli mole rats (Spalax, Spalacidae, Rodentia)

C-banding, base-specific fluorochrome staining (CMA3/DA/DAPI), and comparative genomic hybridization (CGH) were used to analyze the constitutive heterochromatin in two Israeli Spalax species, S. galili (2n = 52) and S. judai (2n = 60). It was shown that C-positive centromeric heterochromatin and some telomeric sites comprise GC-rich DNA sequences in both species. Comparative genomic in situ hybridization revealed slight qualitative differences in highly repetitive sequences in the two Spalax species. Eight acrocentric pairs in S. judai that are involved in Robertsonian rearrangements, possessed composite heterochromatin with a preference of S. judai highly repetitive sequences in the proximal region. Heterochromatin of the sex chromosomes, two biarmed homologous pairs (4 and 5) in both species, and acrocentric chromosomes from the group with a variable centromere position in S. judai was entirely species-specific. The high level of homology in the composition of heterochromatin may relate to the recent divergence of Israeli Spalax. Interspecies heterochromatin differences are discussed in the context of possible mechanisms in the Spalax chromosome evolution.     

Satellite DNA and cytological staining patterns in heterochromatic inversions of human chromosome 9

Summary A series of partial inversions of the heterochromatic C-band of chromosome 9 have been stained with distamycin A plus 4,6-diamidino-2-phenyl-indol-2 HCl (DA/DAPI) and found to consist of three classes: (a) those in which only the C-band in the long arm fluoresces with DA/DAPI (these are the most frequent), (b) those in which only the C-band in the short arm fluoresces with DA/DAPI, and (c) those in which the C-bands in both arms fluoresce with DA/DAPI.There are also differences in the satellite DNA content of each type of inversion as measured by hybridisation in situ. Types (a) and (b) have satellite DNA contents similar to those of their normal homologues, while type (c) has a satellite DNA content almost double that of the normal homologue.It appears that DA/DAPI specifically stains heterochromatin that contains satellite DNA.The ability to distinguish these three types of inversion may help to resolve the question of the clinical significance of such inversions.     

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.     

Cytogenetics of the darkling beetles Zophobas aff. confusus and Nyctobates gigas (Coleoptera, Tenebrionidae)

Males of Zophobas aff. confusus and Nyctobates gigas (Tenebrionidae) collected in the State of Pernambuco, Brazil, were studied through conventional staining, C-banding, silver nitrate impregnation (AgNO(3)), and the base specific fluorochromes CMA(3) and DAPI. Z. aff. confusus was found to have 2n = 20 (9+Xyp) while N. gigas exhibited 2n = 18 (8+neoXY). Large pericentromeric blocks of constitutive heterochromatin (CH) were detected throughout the autosomal complement of the two species, except in one autosomal pair of N. gigas in which no heterochromatic block was observed. The sex chromosomes of both species were almost totally heterochromatic. Double staining with CMA(3)/DA (distamycin) and DAPI/DA marked CH in Z. aff. confusus. However, DAPI staining was more intense. N. gigas was found to possess blocks of CH-positive CMA(3) and homogeneous DAPI. AgNO(3) staining also revealed differences between the two species. In Z. confusus an NOR was observed in the sexual bivalent Xyp and N. gigas was found to have an autosomal NOR.     

Heterochromatins and band karyotypes in three species of salmonids

Summary The heterochromatins of rainbow trout (Salmo gairdneri R.), brown trout (Salmo trutta fario L.) and brook trout (Salvelinus fontinalis M.) were characterized by sequential chromomycin A₃/distamycin A/DAPI (CDD) and DAPI/actinomycin D (DAPI/AmD) fluorescence. On most biarmed chromosomes, an equilocal localization of prominent DAPI/AmD positive, chromomycin A₃ negative, AT-rich blocks at the centromeres were observed in all three species. Band karyotypes of the three species were established. In rainbow trout, several DAPI/AmD positive heterochromatin blocks behaved positive in a silver-staining method. Mitotic and interphase studies proved the presence of inter-individual NOR variation in brown trout. The NORs of brook trout were localized on chromosomes 5, 10, 14, 15 and 29.     

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.     

Colocalization of repetitive DNAs and silencing of major rRNA genes. A case report of the fish Astyanax janeiroensis

The heterochromatin composition and loca- tion in the genome of the fish Astyanax janeiroensis was investigated using Chromomycin A(3) and DAPI fluorochromes and fluorescence in situ hybridization (FISH) with 18S rDNA and As51 satellite DNA probes, respectively. Distinct repetitive DNA classes were found, namely: (1) C-positive centromeric/telomeric heterochromatin, (2) NOR-associated GC-rich heterochromatin (18S(+)/GC(+)) and (3) As51(+)/18S(+) heterochromatin colocalized on 14 distinct heterochromatic domains with attenuated fluorescence of DAPI staining (As51(+)/18S(+)/DAPI attenuated signal). Besides these fourteen associated repetitive DNAs, another eight sites with only 18S rDNA were also found, comprising altogether 22 18S rDNA sites in the genome of the species under study. Up to seven 18S rDNA sites were found to be active, i.e., were characterized as positive after silver staining (Ag-NORs). It was noteworthy that in all As51(+)/18S(+) domains the 18S rDNA were not found to be active sites due to the silencing of these genes when associated with the As51 satellite DNA in the same heterochromatic domain. The dispersion of the As51 sites in the genome of the species is hypothesized to probably originate from a transposable element. Several chromosomal and karyotype markers are similar between A. janeiroensis and A. scabripinnis, indicating a close relationship between these species.     

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。     

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.     

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.     

Cytogenetic analysis of the neotropical spider Nephilengys cruentata (Araneomorphae, Tetragnathidae): standard staining nors, C-bands and base-specific fluorochromes.

The aim of this work is to characterize Nephilengys cruentata in relation to the diploid number, chromosome morphology, type of sex determination chromosome system, chromosomes bearing the Nucleolar Organizer Regions (NORs), C-banding pattern, and AT or GC repetitive sequences. The chromosome preparations were submitted to standard staining (Giemsa), NOR silver impregnation, C-banding technique, and base-specific fluorochrome staining. The analysis of the cells showed 2n = 24 and 2n = 26 chromosomes in the embryos, and 2n = 26 in the ovarian cells, being all the chromosomes acrocentric. The long arm of the pairs 1, 2 and 3 showed an extensive negative heteropycnotic area when the mitotic metaphases were stained with Giemsa. The sexual chromosomes did not show differential characteristics that allowed to distinguish them from the other chromosomes of the complement. Considering the diploid numbers found in N. cruentata and the prevalence of X1X2 sex determination chromosome system in Tetragnathidae, N. cruentata seems to possess 2n = 24 = 22 + X1X2 in the males, and 2n = 26 = 22 + X1X1X2X2 in the females. The pairs 1, 2 and 3 showed NORs which are coincident with the negative heteropycnotic patterns. Using the C-banding technique, the pericentromeric region of the chromosomes revealed small quantity or even absence of constitutive heterochromatin, differing of the C-banding pattern described in other species of spiders. In N. cruentata the fluorochromes DAPI/DA, DAPI/MM and CMA3/DA revealed that the constitutive heterochromatin is rich in AT bases and the NORs possess repetitive sequences of GC bases.     

Cold-sensitive chromosome regions and heterochromatin inCestrum aurantiacum (Solanaceae)

The karyotype ofCestrum aurantiacum was analyzed for the presence of coldsensitive regions (CSRs) and other types of constitutive heterochromatin. A range of techniques was employed including the fluorescent DAPI, chromomycin/DAPI double staining and actinomycin D/DAPI counter-staining, and the non-fluorescent C-banding applied as single or sequential staining, sequential N-banding and silver impregnation. Four classes of constitutive heterochromatin were recognized: CSRs, nucleolar organizers, non-nucleolar chromomycin-positive bands, and indifferently fluorescent bands. The banded karyotype ofC. aurantiacum is compared with those of otherCestrum species. The sectionsHabrothamnus andCestrum are not karyologically distinct.     

Chromosome CPD(PI/DAPI)- and CMA/DAPI-Banding Patterns in Allium cepa L.

Chromosome banding patterns of Allium cepa L. were obtained by using fluorescent dye combinations chromomycin A₃ (CMA) + 4",6-diamidino-2-phenylindole (DAPI), DAPI + actinomycin D (AMD) and propidium iodide (PI) + DAPI. In A. cepa,telomeric heterochromatin displayed dull fluorescence after staining with DAPI and DAPI/AMD. After joint staining with the GC-specific CMA and AT-specific DAPI, the CMA-positive fluorescence of the NOR region and the telomeric bands of C-heterochromatin was observed. In combination with DAPI, PI, a dye with low AT/GC specificity, produced almost uniform fluorescence of chromosomal arms and heterochromatin, whereas the NOR-adjoining regions displayed bright fluorescence. Denaturation of chromosomal DNA (2 × SSC, 95°C for 1–3 min) followed by renaturation (2 × SSC, 37°C, 12 h) altered the chromosome fluorescence patterns: specific PI-positive bands appeared and the contrast of CMA-banding increased. Bright fluorescence of NOR and adjoining regions was also observed in the case. Three-minute denaturation led also to a bright PI-positive fluorescence of telomeric heterochromatin. The denaturation of chromosomal DNA before staining results in changes of the DAPI fluorescence pattern and in the appearance of bright DAPI fluorescence in GC-rich NOP regions. The mechanisms underlying the effects of denaturation/renaturation procedures on chromosome banding patterns obtained with different fluorochromes are discussed.     

Heterochromatin characterization in five species of Heteroptera

The amount, composition and location of heterochromatin in Athaumastus haematicus (Stål, 1859), Leptoglossus impictus (Stål, 1859), Phthia picta (Drury, 1770) (Coreidae), Largus rufipennis Laporte, 1832 (Largidae) and Jadera sanguinolenta (Fabricius, 1775) (Rhopalidae) are analyzed by C-banding and DAPI/CMA fluorescent banding. As the rule for Heteroptera the possession of holokinetic chromosomes and a pre-reductional type of meiosis cytogenetically characterize these five species. Besides, all of them (except L. rufipennis) present a pair of m chromosomes. C-banding technique reveals the absence of constitutive heterochromatin in A. haematicus, scarce C-positive blocks in L. impictus and J. sanguinolenta, and C-positive heterochromatin terminally located in P. picta and L. rufipennis. All C-bands are DAPI bright, except for a DAPI dull/CMA bright band at one telomeric end of the X chromosome in L. rufipennis, which probably corresponds to a nucleolar organizing region. The results of the banding techniques are analyzed in relation to the chiasma frequency and distribution in the five species, and it is concluded that there should exist some constraints to the acquisition and/ or accumulation of heterochromatin in their karyotypes.     

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.