Characterization of different types of condensed chromatin inCostus (Zingiberaceae)

The chromatin structure of six diploids species ofCostus was analysed using conventional Giemsa staining, C-banding and DAPI/CMA fluorochromes. The interphase nuclei in all the species show an areticulate structure and the prophase chromosomes show large blocks of proximal condensed chromatin. After banding procedures, each chromosome exhibits only centromeric dot-like DAPI⁺/CMA^– C-bands whereas the satellites (one pair at each karyotype) are weakly stained after C-banding and show a DAPI^–/CMA⁺ fluorescence. Two chromocentres show bright fluorescence with CMA and weak staining after C-banding whereas the others chromocentres show only a small fraction of DAPI⁺ heterochromatin. These results were interpreted to mean that the greater part of the condensed chromatin has an euchromatic nature whereas two types of well localized heterochromatin occur in a small proportion. The Z-stage analysis suggests that heterochromatin and condensed euchromatin decondense at different times. The chromosome number and morphology of all species are given and the implications of the condensed euchromatin are discussed.Dedicated to Prof.Elisabeth Tschermak-Woess on the occasion of her 70th birthday.     

X-linked heterochromatin distribution in the holocentric chromosomes of the green apple aphid <Emphasis Type="Italic">Aphis pomi</Emphasis>

Chromatin organization in the holocentric chromosomes of the green apple aphid Aphis pomi has been investigated at a cytological level after C-banding, NOR, Giemsa, fluorochrome staining and fluorescent in situ hybridization (FISH). C-banding technique showed that heterochromatic bands are exclusively located on X chromosomes. This data represents a peculiar feature that clearly contradicts the equilocal distribution of heterochromatin typical of monocentric chromosomes. Moreover, silver staining and FISH carried out with a 28S rDNA probe localized rDNA genes on one telomere of each X chromosome; CMA₃ staining reveals that these silver positive telomeres are the only GC-rich regions among A. pomi heterochromatin, whereas all other C-positive bands are DAPI positive thus containing AT-rich DNA.     

Chromatin differentiation between Vigna radiata (L.) R. Wilczek and V. unguiculata (L.) Walp. (Fabaceae)

A comparative chromosomal evaluation was carried out between Vigna unguiculata (cowpea) and V. radiata (mung bean) with chromomycin A₃ (CMA₃)/4’,6-diamidino-2-phenylindole (DAPI) banding and fluorescent in situ hybridization (FISH) using 5S/45S ribosomal DNA (rDNA) probes. Both species had symmetric karyotypes (2n = 22), with prevalence of centromeres in chromosomes at median (m) and submedian (sm) regions and chromosomes ranging in size from 2.1 to 1.25 μm (V. unguiculata) and 2.18 to 0.93 μm (V. radiata). Three different banding patterns were identified for V. unguiculata: CMA₃⁺/DAPI⁰, CMA₃⁺⁺/DAPI^–, and CMA₃⁺/DAPI^–. The CMA₃⁺/DAPI⁰ bands were observed in the pericentromeric regions of all chromosomes, while the CMA₃⁺⁺/DAPI^– and CMA₃⁺/DAPI^– bands were co-localized with the 45S rDNA in the subtelomeric position (chromosomes B, G, and D, J, respectively) and in the proximal position in chromosome F. Two pairs of chromosomes (D and I) bearing interstitial 5S rDNA have been also identified. Vigna radiata displayed CMA₃⁰/DAPI⁺ bands distributed in the centromeric region of chromosomes B, C, and F, while CMA₃⁺⁺/DAPI^– bands were co-localized with the 45S rDNA sites in the subtelomeric position of the short arm in the F and K chromosome pairs. Three pairs of 5S rDNA sites were identified, the first in the proximal region of the long arm in chromosome E and the two others in the proximal and subterminal positions in the long arm of chromosome J. These data highlight some divergences regarding the amount and composition of the heterochromatin in both species, allowing the identification of individual chromosomes in V. unguiculata and V. radiata, and a comparison with other members of the Phaseoloid clade.     

Heterochromatin and rDNA patterns in Solanum species of the Morelloid and Dulcamaroid clades (Solanaceae)

The heterochromatin distribution and the position of 18-5.8-26S, and 5S rDNA loci were determined in 13 species of Solanum of the Morelloid and Dulcamaroid clades. The CMA/DAPI staining and FISH were employed. Two types of constitutive heterochromatin were determined: CMA⁺/DAPI^? associated to NOR and CMA⁺/DAPI^? distributed as terminal bands. In the Morelloid clade, CMA⁺/DAPI^? bands were found in five species while in the Dulcamaroid clade, only S. angustifidum presented this feature. In the Morelloid clade, two to four 18-5.8-26S rDNA loci occupied terminal positions and two rDNA 5S loci were found with variable positions (terminal, intercalary, and centromeric). In the Dulcamaroid clade, two terminal 18-5.8-26S rDNA loci were detected with the exception of S. salicifolium which possessed four such loci and two to four 5S rDNA loci. Solanum crispum is the only species possessing the 5S in synteny with 18-5.8-26S rDNA loci. Karyotype features chromosome banding pattern as well as the location of ribosomal genes which varied among the species, reflecting the chromosome differentiation and evolutionary divergence. The findings obtained contributed to the development of tools that can be used for establishing chromosomic homeologies among species and hence to clarify their taxonomic relationships.     

Meiosis Aspects and Nucleolar Activity in Triatoma vitticeps (Triatominae, Heteroptera)

Some aspects of both the nucleolar organizer activity and meiosis were studied in the testes of Triatoma vitticeps (Heteroptera, Triatominae). The techniques used included squashing followed by lacto-acetic orcein staining, silver-ion impregnation, fluorescent banding (CMA₃, Quinacrine mustard and DAPI) and fluorescent in situ hybridization (FISH). A close relationship between heterochromatin and nucleolus in testicular cells was observed. During meiosis, the silver-ion impregnation pattern varied. At metaphase plate, a small body appeared apart from the chromosomes. In the spermatids this small body was seen in preparations stained with orcein and silver- ion impregnation but not with fluorochromes or FISH. These characteristics combined suggest that these corpuscles represent a source of ribonucleoproteins (RNP) – RNA and specific nucleolar proteins. Silver-ion impregnation and (FISH) revealed nucleolar organizer activity in two metaphase sex chromosomes (X). These results indicate that, in these species, nucleolar organizer regions (NORs) are located in the sex chromosomes, X chromosomes were CMA₃⁺ and Y chromosome was DAPI⁺.     

Molecular cytogenetics of <Emphasis Type="Italic">Oligosarcus hepsetus</Emphasis> (Teleostei,Characiformes) from two Brazilian locations

Karyotype and cytogenetic markers of Oligosarcus hepsetus from two Brazilian locations in the Paraíba do Sul River Basin (Brazil) were investigated using differential staining techniques (C-banding, silver (Ag)- and chromomycin A₃ (CMA₃)-staining) and fluorescent in situ hybridization (FISH) using 18 S rDNA and 5 S rDNA probes. The diploid chromosome number was invariably 2n = 50 with 3 pairs of metacentric, 5 pairs of submetacentric, 8 pairs of subtelocentric and 9 pairs of acrocentric chromosomes. No heteromorphic sex chromosomes were observed. The nucleolar organizer regions (NORs) were detected in the short arms of the largest acrocentric pair using Ag-, CMA₃- stainings and FISH with 18 S rDNA probe, the latter showing also positive labeling in the short arms of a small acrocentric pair, not visualized by the former methods. FISH with 5 S rDNA probe showed positive labeling in the two chromosome pairs. While the CMA₃-staining exhibited GC-rich heterochromatin segments in two pairs of chromosomes, including those coincided with Ag-NORs, the DAPI staining did not reveal any signal, indicating the absence of AT-rich heterochromatin. FISH with an As-51 satellite DNA probe derived from the closely related Astyanax scabripinnis did not reveal any positive signal, demonstrating the absence of this class of DNA in the genome of the specimens under study.     

Classical and molecular cytogenetics and DNA content in Maihuenia and Pereskia (Cactaceae)

We studied cacti species of the subfamilies Pereskioideae (five species of the southern clade) and both species of Maihuenioideae using molecular cytogenetic techniques and DNA content. Mitotic chromosomes were analyzed for Pereskia aculeata, P. bahiensis, P. grandifolia, P. nemorosa, P. sacharosa, Maihuenia poeppigii, and M. patagonica, using the Feulgen stain, CMA/DAPI fluorescent chromosome banding, fluorescence in situ hybridization (FISH, probes of 5S rDNA and pTa71 for 18-5.8-26S rDNA), and DNA content by flow cytometry technique. The karyotypes were highly symmetrical, most of the pairs being metacentric (m). CMA/DAPI banding revealed the presence of CMA⁺/DAPI^? bands associated with NORs in the first m pair of all species. The co-localization of 18-5.8-26S rDNA loci with CMA⁺/DAPI^?/NORs blocks allowed the identification of homeologous chromosome pairs between species of both subfamilies. FISH using probe 5S rDNA was applied for the first time in both subfamilies. Diploid species had always one m pair carrying 5S rDNA genes, with pericentromeric location in different chromosome pairs. In the tetraploid cytotype of M. patagonica, the 5S rDNA probe hybridized to two pairs. The 2C DNA content obtained by FC varied twofold (from 1.85 to 2.52 pg), with significant differences between species. Mean chromosome length, karyotype formula, percentage of heterochromatin position of 5S rDNA locus, and nuclear Cx DNA content vary among Maihuenia and Pereskia species and allowed to differentiate them. Both genera are closely related and that the differences found are not strong enough to separate Maihuenioideae from Pereskioideae.     

Multiple nucleolus organizer regions in Leptodactylus mystacinus (Amphibia, Anura) and comments on its systematic position in the L. fuscus group based on cytogenetic and molecular analyses

Specimens of Leptodactylus mystacinus from Brazil were karyotyped with conventional and differential staining. The 2n = 22 karyotype is similar to that found for the majority of the Leptodactylus, the karyotypic conservatism also confirmed by the similarity of the replication banding patterns with those previously described. L. mystacinus has a small amount of C-banded heterochromatin, located mainly at the centromeres, although telomeric or interstitial bands have also been noticed. With DA/CMA₃ some chromosome regions showed slightly bright fluorescence, and with DA/DAPI, no particular AT-rich repetitive region was observed. Silver staining showed an extensive inter- and intraindividual variation in the number and position of Ag-positive regions, in 1p, 4p, 8p, 8q, and 11p. Nevertheless, FISH using rDNA probes confirmed only the signals on the short arms of chromosomes 4 and 8 as true NORs. The remaining silver stained regions are probably due to the heterochromatin with some affinity to the Ag-staining. Phylogenetic analysis based on partial cytochrome b sequence revealed that L. mystacinus forms a basal branch, so that the presence of multiple NORs in pairs 4 and 8 in this species indicates an autapomorphy. Supported by FAPESP and CNPq.     

Analysis of heterochromatin by combination of C-banding and CMA3 and DAPI staining in two fish species (Pimelodidae,Siluriformes)

The chromosomes of Steindachneridion sp. (2n = 56) and Rhamdia quelen (2n = 58) were analyzed by C-banding (CB) and Chromomycin A₃ (CMA₃) and 4,6-diamidino-2-phenylindole (DAPI) staining, separately and consecutively, in order to understand the role of base-specific fluorochrome treatment after CB. Both species' chromosomes shared common staining profiles as follows. CB with Giemsa (CBG) revealed weak heterochromatic blocks in the telomeric regions of some chromosomes and conspicuous bands on the short arms of one chromosome pair, where nucleolar organizer regions (NORs) were evidenced by silver-staining. Without CB pretreatment, the NORs were stained conspicuously with CMA₃, but not with DAPI. The latter uniformly stained all chromosomes, but leaving the NORs pale. Combination of CMA₃ or DAPI staining with CB showed distinctive fluorescent blocks in the NOR-bearing short arms of the single chromosome pair along with several bright fluorescent signals on other chromosomes, which were not evidenced by single CMA₃ or DAPI staining. These results suggest a modification of chromatin structure by CB treatment, which may increase the stainability of CMA₃ and DAPI.     

Mitotic and meiotic analysis of a pericentric inversion associated with a tandem duplication in Eleutherine bulbosa

The mitotic and meiotic chromosomes of five populations of Eleutherine bulbosa were analysed after C-banding and fluorochrome (DAPI/CMA) staining. All individuals showed heterozygosity in pair number I for a pericentric inversion and a tandem duplication in the inverted region of one of the homologues. The duplication comprised the secondary constriction and ca. 40% of its associated CMA⁺ heterochromatin. All the secondary constrictions were nucleolar organizers, but the repeated one seemed to be the most active. The inverted segment constituted ca. 70% of the chromosome length and included, in addition to the duplication and the centromere, two DAPI⁺ C-bands. These markers, especially the CMA⁺ blocks which were unique in the complement, permitted a detailed analysis of meiotic pairing. In practically all examples of late zygotene the CMA blocks were paired, which seems to mean that non-homologous pairing never occurred. At this stage the duplicated CMA⁺ block was clearly unpaired. At pachytene-diplotene the inverted region formed a typical loop without chiasmata. Although male meiosis was normal, no indication of sexual reproduction was found. A causal relationship between duplication, inversion and asexual reproduction is proposed to explain the maintenance of the heterozygosity.     

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.     

Karyology of the Antarctic scallop Adamussium colbecki, with some comments on the karyological evolution of pectinids

Karyotype, location of the nucleolar organiser region (NOR) and heterochromatin presence and composition were studied in the Antarctic scallop Adamussium colbecki Smith, 1902. The karyotype exhibits 2n = 38 chromosomes with 11 pairs of metacentrics, 5 of submetacentrics, one subtelocentric and two telocentrics. Ag–NOR, CMA₃, DA/MM and NOR–FISH evidenced paracentromeric NORs on the short arm of 2nd pair chromosomes. Digestion with three restriction endonucleases followed by sequential staining with Giemsa, CMA₃ and DAPI evidenced on all chromosomes centromeric heterochromatin positive for both DAPI and CMA₃. In situ hybridisation analysis showed the presence of an AT-rich satellite DNA in the centromeric heterochromatin of several chromosomes. A mosaicism was detected in the germinal cell lines of one specimen, as in six of the 20 plates examined the set had 37 chromosomes with a missing pair of telocentrics and an unpaired metacentric. Comparison of the chromosome sets of all the pectinids studied to date and comparison with a phyletic tree obtained from molecular mitochondrial genes studies yielded good agreement between karyotype morphology and taxonomic classification.     

Cytogenetic and molecular analysis of the pufferfish Tetraodon fluviatilis (Osteichthyes)

In view of their compact genome, pufferfish (Tetraodontiformes) have been proposed as model animal for the study of the vertebrate genome. Despite such interest, cytogenetic information about puffers is still scanty. To fill this gap, a cytogenetic analysis of T. fluviatilis has been performed using both classical and molecular techniques. C-banding, followed by DAPI staining, evidenced that in T. fluviatilis, like all other puffer species so far examined, heterochromatin is essentially AT-rich and it is located at centromeres, whereas staining with CMA₃, silver staining and FISH with a 28S ribosomal RNA gene DNA probe showed 2–4 nucleolar organizing regions (NORs) located in heterochromatic regions in the considered puffer species. FISH with the 5S probe put in evidence both in T. fluviatilis and in T. nigroviridis only a 5S cluster per haploid genome that is physically unlinked with the major ribosomal RNA genes including the 28S rRNA genes. Hybridization with the (TTAGGG)_n probe showed in all the puffers brightly fluorescent signals uniform both in size and intensity at the end of all the chromosomes. Finally, mariner-like elements (MLEs) have been identified in T. fluviatilis and they have located into the NOR-associated heterochromatin.     

Molecular cytogenetic analysis of Haemulidae fish (Perciformes): Evidence of evolutionary conservation

Several fish species belonging to the family Haemulidae present a karyotype consisting of 48 acrocentric chromosomes (FN = 48), and apparently similar chromosomal microstructure, especially in genus Haemulon, representing a striking example of intrafamiliar chromosomal conservation. In this study, a more detailed cytogenetic analysis of the species Conodon nobilis and Pomadasys corvinaeformis was performed using C-banding, Ag-NOR, DAPI/CMA₃ staining, in situ digestion by distinct endonucleases and double-FISH to map the 18S and 5S ribosomal genes. Both species showed a similar karyotypic macrostructure with 2n = 48 acrocentric chromosomes and active ribosomal sites at interstitial position on long arms of chromosomal pair 18 and 24 in P. corvinaeformis and C. nobilis, respectively. These sites were the only CMA₃⁺/DAPI^-regions in the karyotype. Digestion with restriction enzymes revealed a low number of digestion sites in the heterochromatic segments of both species. The data indicate some degree of interspecific evolutionary diversification At the microstructural level, incorporated in a general pattern of extensive karyotypic conservatism. Thus, the interspecific reproductive isolation leading to phyletic diversification apparently occurred without the contribution of conspicuous karyotypic changes.     

Karyotype analysis for diploid and polyploid species of the Solanum L.

Solanum species were cytogenetically analysed to localize species-specific markers. Solanum atropurpureum Schrank, S. dulcamara L., S. gilo Raddi., S. melongena L., S. nitidibaccatum Bitter and S. paniculatum L. showed 2n = 24, whereas S. luteum Mill., S. nigrum L. and S. laciniatum Ait. showed 2n = 48, 2n = 72 and 2n = 92, respectively. All species demonstrated a symmetrical karyotype. The average chromosome size varied between diploid (1.95 μm) and polyploid (1.31 μm) species. Application of the CMA₃/DAPI technique showed two CMA₃ ⁺/DAPI^? terminal blocks in S. dulcamara, S. atropurpureum and S. luteum, indicating one homologous pair. In S. nitidibaccatum two large CMA₃ ⁺/DAPI^? segments were observed apart from several terminal blocks in almost all chromosomes. The same CMA₃ ⁺ telomeric standard was also found in prometaphases of S. luteum. Similar results were obtained with FISH with 45S rDNA probes: fluorochrome CMA₃ ⁺ telomeric blocks associated with satellites, with two blocks in S. atropurpureum, S. dulcamara, S. nitidibaccatum and S. luteum and four blocks in S. nigrum and S. lacinatum. Localization of species-specific markers was successful, allowing recognition of particular cytological features in most of the species analysed.     

Chromosome banding study of European catfish,Silurus glanis (Pisces,Siluridae)

The karyotype of European catfish (Silurus glanis L.) was analyzed sequentially by means of silver staining and the chromomycin A₃ (CMA₃)/distamycin A (DA)/DAPI fluorescence technique and by C-banding, respectively. The nucleolus organizer regions (NORs) were localized on the submetacentric pair No. 14. Brilliant CMA₃ fluorescent heterochromatin blocks corresponded to the NORs visualized by silver staining. No DA/DAPI-bright positive fluorescent patterns were detected while C-banding led to the detection of specific banding patterns on several chromosome pairs.—Using these banding data, the karyotype of S. glanis was redescribed.     

First report on C-banding,fluorochrome staining and NOR location in holocentric chromosomes of Elasmolomus (Aphanus) sordidus Fabricius, 1787 (Heteroptera,Rhyparochromidae)

In spite of various cytogenetic works on suborder Heteroptera, the chromosome organization, function and its evolution in this group is far from being fully understood. Cytologically, the family Rhyparochromidae constitutes a heterogeneous group differing in chromosome numbers. This family possesses XY sex mechanism in the majority of the species with few exceptions. In the present work, multiple banding techniques viz., C-banding, base-specific fluorochromes (DAPI/CMA₃) and silver nitrate staining have been used to cytologically characterize the chromosomes of the seed plant pest Elasmolomus (Aphanus) sordidus Fabricius, 1787 having 2n=12=8A+2m+XY. One pair of the autosomes was large while three others were of almost equal size. At diplotene, C-banding technique revealed, that three autosomal bivalents show terminal constitutive heterochromatic bands while one medium sized bivalent was euchromatic. Microchromosomes (m-chromosomes) were positively heteropycnotic. After DAPI and CMA₃ staining, all the autosomal bivalents showed equal fluorescence, except CMA₃ positive signals, observed at both telomeric heterochromatic regions of one medium sized autosomal bivalent. Silver nitrate staining further revealed that this chromosome pair carries Nucleolar Organizer Regions (NORs) at the location of CMA₃ positive signals. The X chromosome showed a thick C-band, positive to both DAPI /CMA₃ while Y, otherwise C-negative, was weakly positive to DAPI and negative to CMA₃, m-chromosomes were DAPI bright and CMA₃ dull.     

Cytogenetics of the Brazilian Bolitoglossa paraensis (Unterstein, 1930) salamanders (Caudata,Plethodontidae)

Plethodontid salamanders of genus Bolitoglossa constitute the largest and most diverse group of salamanders, including around 20% of living caudate species. Recent studies have indicated the occurrence of five recognized species in the Brazilian Amazon Rainforest. We present here the first cytogenetic data of a Brazilian salamander, which may prove to be a useful by contribution to the cytotaxonomy of the genus. Specimens were collected near the “type” locality (Utinga, Belém, PA, Brazil). Chromosomal preparations from duodenal epithelial cells and testes were subjected to Giemsa staining, C-banding and DAPI/CMA₃ fluorochrome staining. All specimens showed a karyotype with 13 bi-armed chromosome pairs (2n = 26). Nucleolar Organizer Regions, evidenced by CMA₃, were located distally on the long arm of pair 7 (7q). DAPI+ heterochromatin was predominantly centromeric, with some small pericentromeric bands. Although the C-banding patterns of other Bolitoglossa species are so far unknown, cytogenetic studies conducted in other Plethodontid salamanders have demonstrated that pericentromeric heterochromatin is a useful cytological marker for identifying interspecific homeologies. Species diversification is usually accompanied by chromosomal changes. Therefore, the cytogenetic characterization of Bolitoglossa populations from the middle and western Brazilian Amazon Basin could identify differences which may lead to the identification of new species.     

Chromosomal organization of ribosomal genes and NOR-associated heterochromatin, and NOR activity in some populations of Allium commutatum Guss. (Alliaceae)

The position and the number of 18S-5.8S-26S and 5S rDNA loci, characterization of nucleolar organizing region (NOR)-associated heterochromatin and NOR activity assessment are given for six south-eastern Adriatic populations of Allium commutatum Guss. The karyotype characteristics were identical for all the populations studied, even those of distant islands. Diploid karyotypes (2 n = 16) always possessed two NOR-bearing chromosome pairs with pericentric and median secondary constrictions (SCs) on the short arm of the chromosomes VII and VIII. Fluorescent in situ hybridization (FISH) confirmed that these were the only sites of 18S-5.8S-26S rRNA genes. NOR-associated heterochromatin was of the constitutive character as shown after C-banding. Differential fluorochrome banding with Chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) revealed that this heterochromatin comprises both GC- and AT-rich DNA segments. Heteromorphism of C- and CMA-bands was noticed between homologous NOR-bearing chromosomes. The maximum number of four active NORs was correlated with the maximum number of four nucleoli in interphase. Variability of NOR-activity, expressed as number and size of silver stained NORs, existed between cells and between individuals of the same population. The different size of homologous and nonhomologous silver stained NORs was correlated with the extension of SCs. The only 5S rDNA locus was in an intercalary position on short arm of the chromosome VI, at the region of AT-rich constitutive heterochromatin. Dimorphism of C-bands and DAPI/Hoechst(H)-fluorescent bands was noticed between homologous chromosomes VI. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 139 , 99–108.     

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.