DAPI-banding Patterns Research Articles

Reversible Fluorescent Banding Analysis in Six Wild <i>Corchorus</i> Species from Bangladesh

Fluorescence chromosome bandings with CMA- and DAPI- were analyzed in six wild Corchorus species to find out the diversity in the number and location of AT- and GC-rich sequences. These six wild Corchorus species had variation in somatic chromosome numbers viz. C. brevicornatus (2n=14), C. septentrionalis (2n=14), C. urticifolius (2n=14), C. hirtus (2n=28), C. siliquosus (2n=28) and C. pinnatipartitus (2n=78). A wide range of CMA- and DAPI-positive bands were found in the metaphase chromosomes of six wild Corchorus species. A total number of 35 and 32 terminal CMA- and DAPI-bands were generated which recommended the tendency of accumulation of GC- and AT-rich sequences at the terminal region of chromosomes, respectively. In some species, CMA-banded satellites were extended in prometaphase while contracted at the terminal region of the short arm in the metaphase stage. Reshuffling of chromosomal segments was detected following CMA- and DAPI-banding. After reversible fluorescent banding, C. brevicornatus, C. urticifolius and C. siliquosus had a few CMA-positive and DAPI- negative banded chromosomes which could be used as marker chromosomes. In addition, few chromosomes of C. pinnatipartitus could be used as markers due to their unique DAPI-banding pattern. Each species had its characteristic CMA- and DAPI-banding pattern.

Karyotype characterization of three important aromatic Cinnamomum L. species with special emphasis on reversible chromosome banding

Conventional stain (orcein) and DNA base-specific fluorochromes (CMA and DAPI) were used to characterize three medicinally and economically important aromatic Cinnamomum L. species namely, C. camphora, C. tamala and C. zeylanicum. Despite having identical somatic chromosome numbers 2n = 24, the studied Cinnamomum L. species had distinct centromeric formulae, such as 24m in C. camphora, 18m + 6sm in C. tamala, and 8m + 16sm in C. zeylanicum. When karyotype assessment criteria were taken into account, these three studied species also displayed distinctiveness, and C. zeylanicum represented a more heterogeneous karyotype than the other two species. The karyotypic diversification was reflected in the symmetry classes of Stebbins’ classification, with C. camphora and C. tamala having 1B karyotype while C. zeylanicum was found to have a 2A karyotype. CMA- and DAPI-banding patterns of these species were different and species-specific regarding the number, location and percentages of GC- and AT-rich repetitive sequences in the genome. Reversible fluorescent banding patterns revealed the stain-specific nature of satellite in C. camphora L. By comparing karyotypic traits and reversible fluorescent banding patterns, characterization was thereby encountered for three Cinnamomum L. species from Bangladesh.

DAPI Banding Pattern of Chromosomes in Several Brassicaceae Species

BRASSICACEAE family includes a number of the most important Brassica cultivars such as canola, mustard and cabbage, and Arabidopsis thaliana as plant biology model species. Chromosome banding is the most probable protocol for karyotyping and to distinguish numerical chromosome abnormalities, duplications, inversions or deletions of chromosome parts, or exchanging a part from one to another chromosome as well. The aim of this study is to illustrate the DAPI banding patterns in 16 Brassicaceae species with small chromosome size, and they have different origin, ploidy level (chromosome number varies from 2n= 14 to 2n= 32). These species belong to five tribes (Brassiceae, Sisymbrieae, Lepidieae, Arabideae and Alyssea. The aim of this study is to illustrate the DAPI banding patterns in 16 Brassicaceae species with small chromosome size, and they have different origin and ploidy level (chromosome number varies from 2n= 14 to 2n= 32). In the current study we investigated different genera belong to five tribes of the family in regard to the position of regions as by using DAPI staining. It was excluded that most of plants with small chromosomes have mostly a centromeric bands and the more the chromosome size the more tendency to have interstitial bands.

Agro-Morphological, Microanatomical and Molecular Cytogenetic Characterization of the Medicinal Plant Chelidonium majus L.

Chelidonium majus L. is a medicinal plant well-known as a valuable source of isoquinoline alkaloids, which has a variety of pharmacological properties including anti-viral and anti-bacterial effects. However, considerable intraspecific bio-morphological variability in C. majus complicates raw material identification and verification. For the first time, we have brought into cultivation five populations of C. majus subsp. majus originated from different regions, and performed their agro-morphological, microanatomical and molecular cytogenetic characterization. All examined populations produced high seed (18.6–19.9 kg/ha) and raw material (0.84–1.08 t/ha) yields; total alkaloid contents were within 0.30–0.38%. Nevertheless, significant differences in plant morphology and yield-contributing traits were observed. The performed microanatomical analysis of leaves and flowers in double- and normal-flowered plants revealed micro-diagnostic features (including tissue topography, types of stomata, laticifers, structure of leaf mesophyll, hairs, sepals and petals) important for identification of C. majus raw materials. The analysis of chromosome morphology, DAPI-banding patterns, FISH mapping of 45S and 5S rDNA and also chromosome behavior in meiosis allowed us to identify for the first time all chromosomes in karyotypes and confirm relative genotype stability of the studied plants. Our findings indicate that the examined C. majus populations can be used in further breeding programs.

Molecular cytogenetic characterization, leaf anatomy and ultrastructure of the medicinal plant Potentilla alba L.

Potentilla alba L. is a valuable medicinal plant widely used in folk and traditional medicine and particularly promising in complex treatment of thyroid pathology. Natural resources of this species are insufficient due to ever-growing use in contemporary medicine. Comprehensive investigations of different P. alba populations are essential for the successful extension of P. alba plantings. Aiming for a better understanding of karyotype structure, chromosome behaviour in meiosis and developing new diagnostic characters, we performed molecular cytogenetic characterization and leaf structure and ultrastructure analyses of two introduced P. alba samples originating from different habitats. Based on chromosome morphology, distribution of 45S/5S rDNA and DAPI-banding patterns, all chromosomes in the karyotypes were identified and the P. alba chromosomal idiogram was constructed. Our findings confirmed P. alba karyotype stability and also revealed several diagnostic characters of this species: the features of cells of upper and lower leaf epidermis, the presence of calcium oxalate druses and three types of leaf indumentum, essential for evaluation of genetic diversity in different populations, validation of raw materials and further selection progress. The meiotic abnormalities were detected probably related to low pollen activity and indicated the advantages of vegetative propagation in the development of a P. alba plantation system.

Genetic and ecological niche modeling of Calydorea crocoides (Iridaceae): an endemic species of Subtropical Highland Grasslands.

Evolutionary analyses have been widely used for evaluation of genetic diversity of natural populations and correlate these data to the fitness of the species, especially in the case of threatened species. Calydorea crocoides occurs in a restricted area at altitudes from 800 to 1500 m in southern Brazil and is considered endangered. A study assessing genetic diversity, cytogenetic features and ecological niche was performed aiming to characterize C. crocoides by multidisciplinary approaches. Molecular data highlighted that most of the total variation (76%; p < 0.001) was found within populations and the parameters of genetic diversity were high at the species level (PPB = 98.97%; I = 0.4319; h = 0.2821). Gene flow (Nm) was estimated in 0.97 individuals per generation. Cytogenetically, C. crocoides presents a bimodal karyotype and low asymmetry. DAPI banding pattern was uniform, but the CMA-signal evidenced a pericentric inversion in the population ESC688. The species presents high pollen viability and two different morphologies of pollen grains. Our data showed high levels of polymorphism maintained in this species that could ensure conservationist practices in which the main goal is to preserve the evolutionary potential of the species through the maintenance of genetic diversity.

Karyotype, RAPD and ISSR Analysis of Four Specimens in &lt;i&gt;Gynura nepalensis&lt;/i&gt; DC.

Four specimens of Gynura nepalensis DC. were investigated for authentic characterization by differential cytogenetical and PCR based molecular analysis. Three specimens out of four were collected from different localities of Bangladesh viz. i) from Netrokona district (specimen A), ii) from Dhaka district (specimen B) and iii) from Bogra district (specimen D). On the other hand, specimen C was collected from Canada. The diploid chromosome number of four specimens were 2n=20 with different centromeric formulae such as 14m+6sm in specimens A, B and D while 20m in specimen C. Total length of 2n chromosome complement was 211.61 µm in specimen A, 217.58 µm in specimen B, 168.82 µm in specimen C and 197.80 µm in specimen D. The maximum range of chromosome length was found in specimen B (6.67–14.30 µm) and minimum in specimen C (6.44–10.12 µm). The four specimens have distinct CMA- and DAPI-banding pattern which confined to the terminal regions of respective chromosomes. Karyotype analysis revealed that the number, location and distributions of GC- and AT-rich repeats are different in these four specimens. Heteromorphicity in respect of occurrence of CMA- and DAPI-banding pattern in the homologue members were observed in specimen A, C and D. The heteromorphicity might be a result of deletion of banded region from the respective homologue members. One interstitial CMA band was found on the short arm in a member of pair V in specimen A and in pair IV of specimen C. Two terminal DAPI bands were found in two chromosomes of specimens A and B. These CMA- and DAPI-banded chromosomes were unique and thus could be used as marker chromosomes for the respective specimens. Only specimen C showed RAPD bands with three primers. However, the four specimens had distinguishable ISSR-banding patterns with seven primer combinations. Specimen C showed maximum ISSR bands. The combined RAPD and ISSR analysis placed specimen C in a separate cluster with the highest genetic distance. In spite of very distant location specimens A and D were placed in a sub-cluster with narrow genetic distance.

Characterization of Three Varieties of &lt;i&gt;Lathyrus sativus&lt;/i&gt; L. by Fluorescent Karyotype and RAPD Analysis

Three varieties of Lathyrus sativus L. released from Bangladesh Agricultural Research Institute (BARI), viz., BARI Khesari-1, BARI Khesari-2 and BARI Khesari-3, were studied cytogenetically and at the molecular level by using RAPD for genomic characterization. The three varieties were found to posses 2n=14 chromosomes. The karyotype formulae of BARI Khesari-1 and BARI Khesari-2 are 8m + 6sm, while it is 10 m + 4sm in BARI Khesari-3. After orcein and CMA staining, one to two very small chromosome-like bodies were found in some cells of BARI Khesari-1, whereas no such body was observed after DAPI staining. Due to their unique features these can be considered as GC-rich heterochromatic “B-chromosomes.” In BARI Khesari-2, two pairs of satellites were found, while only one pair was present in BARI Khesari-3. The inverted position of satellited regions indicated the probable occurrence of homozygous inversion. An indication of paracentric inversion regarding DAPI-banding pattern was found in pair V of BARI Khesari-2 and thus could be considered as marker chromosomes for this variety. The three varieties have distinct CMA- and DAPI-banding patterns. In BARI Khesari-2, a total of 12 C-bands were found on 10 chromosomes (out of 14 chromosomes). On the basis of band position and length, a deep correlation between C-banding and DAPI-banding pattern was found suggesting the heterochromatic nature of DAPI bands. Each variety showed different RAPD fingerprinting with 59.09% polymorphisim. In addition, a number of variety-specific unique RAPD bands was observed. Therefore, each variety could be characterized on the basis of karyotype and RAPD analysis.

Intraspecific chromosomal and genetic polymorphism in Brassica napus L. detected by cytogenetic and molecular markers.

The application of DNA intercalator 9-aminoacridine allowed us to increase the resolution of chromosome C-banding and DAPI-banding patterns and to investigate chromosomal polymorphism in karyotypes of seven spring and six winter rape varieties. It was shown that the pericentromeric and intercalary C-bands of most of the chromosomes in spring rape were smaller in size and less polymorphic than those of winter rape. More 26S and 5S rDNA sites were found in the winter rape karyotypes than the spring varieties. Separate or colocalized 26S and 5S rDNA sites were revealed on chromosomes 4, 5, 6, 8, 10, 14, 15, 16 and 18. Intervarietal and intravarietal polymorphism of the number and chromosomal localization of rDNA sites were detected. The generalized idiogram of chromosomes of 13 Brassica napus varieties with account of all possibilities of C-banding patterns as well as localization of 26S and 5S rDNA sites were constructed. Polymorphism of the examined molecular and cytogenetic markers as well as the heterozygosis level of FAE1.1 gene controlling erucic acid synthesis in rapeseed was higher in the winter varieties than in the spring ones. The obtained data were in a atisfactory agreement with increased tolerance to environmental stress conditions of winter rape.

Differential Chromosome Banding and Isozyme Assay of Three Corchorus spp.

Karyotypes of 3 wild Corchorus spp. of Tiliaceae, namely C. tridens, C. fascicularis and C. pseudo-capsularis, were investigated after differential staining with orcein, CMA and DAPI. In addition, the activities of 3 isozymes, esterase, acid phosphatase and peroxidase in these 3 species were compared. These 3 spp. were found to possess 2n=14 metacentric chromosomes. The CMA and DAPI bands were found in either the terminal or interstitial region of the respective chromosomes of 3 species. This indicated a tendency for the accumulation of GC- and AT-rich repeats at terminal or interstitial region of the chromosomes. The number and distribution of CMA- and DAPI-bands were unique for each species. A comparison of CMA- and DAPI-banding pattern indicated the presence of paracentric inversion in the concerned areas of the respective chromosomes. Different types of heteromorphicity regarding CMA- and DAPI-banding revealed the probable occurrence of deletion and tandem duplication. In acid phosphatase, 2 common bands were found in C. fascicularis and C. pseudo-capsularis. A dark, thick peroxidase band was found in C. fascicularis whereas a light, thick band occurred in C. pseudo-capsularis. These light and dark thick bands could be used as markers for these 2 spp. Moreover, a big upper esterase band makes C. tridens unique.

Do polyploids require proportionally less rDNA loci than their corresponding diploids? Examples from Artemisia subgenera Absinthium and Artemisia (Asteraceae, Anthemideae)

Fluorescent in situ hybridisation (FISH) of 5S and 18S-5.8S-26S ribosomal DNA was carried out in two species of the genus Artemisia, belonging to the subgenera Artemisia (A. medioxima) and Absinthium (A. lagocephala), each one showing both low and high ploidy levels (2x, 4x and 16x, and 2x and 6x, respectively). Both species have a base chromosome number of x = 9. Linkage of both rDNA genes has been observed confirming previous results. Diploid A. lagocephala (2n = 18) shows three rDNA loci, and the hexaploid six. Also in A. medioxima, the number of rDNA loci does not increase in the proportion given by the ploidy level, and a relative loss is found. In this species, the diploid population shows two rDNA loci, the tetraploid four, and the hexaidecaploid has around 20. The results evidence a relative loss of rDNA loci and heterochromatin, a phenomenon that is more pronounced at higher ploidy levels. Nevertheless, the DAPI banding pattern of A. lagocephala does not follow this trend, as it shows a spectacular increase of heterochromatic bands at the hexaploid level. These results are discussed in the light of possible chromosome restructuring and gene silencing mechanisms that take place during polyploidy, and more especially allopolyploid formation.

C-Banding/DAPI and in situ Hybridization Reflect Karyotype Structure and Sex Chromosome Differentiation in Humulus japonicus Siebold & Zucc.

Japanese hop (Humulus japonicus Siebold & Zucc.) was karyotyped by chromosome measurements, fluorescence in situ hybridization with rDNA and telomeric probes, and C-banding/DAPI. The karyotype of this species consists of sex chromosomes (XX in female and XY1Y2 in male plants) and 14 autosomes difficult to distinguish by morphology. The chromosome complement also shows a rather monotonous terminal distribution of telomeric repeats, with the exception of a pair of autosomes possessing an additional cluster of telomeric sequences located within the shorter arm. Using C-banding/DAPI staining and 5S and 45S rDNA probes we constructed a fluorescent karyotype that can be used to distinguish all autosome pairs of this species except for the 2 largest autosome pairs, lacking rDNA signals and having similar size and DAPI-banding patterns. Sex chromosomes of H. japonicus display a unique banding pattern and different DAPI fluorescence intensity. The X chromosome possesses only one brightly stained AT-rich terminal segment, the Y1 has 2 such segments, and the Y2 is completely devoid of DAPI signal. After C-banding/DAPI, both Y chromosomes can be easily distinguished from the rest of the chromosome complement by the increased fluorescence of their arms. We discuss the utility of these methods for studying karyotype and sex chromosome evolution in hops.

Different cytotypes in fishes of the genus Hypostomus Lcépède, 1803, (Siluriformes: Loricariidae) from Xingu river (Amazon region, Brazil)

We analyzed the karyotypes of three specimens of fishes of the genus Hypostomus Lacepede, 1803 (Loricaridae) from Xingu River (Amazon region). We used conventional staining techniques, including C-banding, Ag-NOR staining, CMA3- and DAPI-banding. Each specimen had a different cytotype: Hypostomus sp. Xingu-1 had 2n=64 (32M/SM, 32ST/A); Hypostomus sp. Xingu-2 has 2n=66 (32M/SM, 34ST/A), and Hypostomus sp. Xingu-3 had 2n=65 (38M/SM, 26ST/A + 1 B). The three cytotypes showed similar C-, CMA3- and DAPI-banding patterns. The nucleolus organizing regions were located in the short arm of chromosome pair 25 of Hypostomus sp. Xingu-1 and pair 29 of Hypostomus sp. Xingu-2, and in the long arm of pair 30 of Hypostomus sp. Xingu-3, probably because of a pericentric inversion. A fusion/fission rearrangement explains the difference in the diploid number and number of M/SM and ST/A chromosomes between the 2n= 64 and 2n=66 cytotypes. The B chromosome most probably explains the difference between the 2n=64 and 2n=65 cytotypes. The cytotype with 2n=65 had a significantly larger number of M/SM chromosomes, probably because of pericentric inversions. These three cytotypes may represent different species.

NRIP3: a novel translocation partner of MLL detected in a pediatric acute myeloid leukemia with complex chromosome 11 rearrangements

NRIP3: a novel translocation partner of MLL detected in a pediatric acute myeloid leukemia with complex chromosome 11 rearrangements

Physical mapping of rDNA and heterochromatin in chromosomes of 16 Coffea species: A revised view of species differentiation

The chromosome organization among 15 wild diploid Coffea species and cultivated tetraploid C. arabica was determined by fluorochrome banding (CMA, DAPI) and double fluorescence in-situ hybridization (FISH) of 5S and 18S rDNA achieved on the same chromosome plates. Two to five chromosome pairs (plus one putative chromosome B) are marked. Overall, there are two SAT-chromosome pairs for East African species and one for the Malagasy and the West and Central African species. 18S rDNA loci are telomeric and strongly marked the SAT-chromosome pairs. Generally, only one pericentromeric 5S rDNA locus characterized East African species, while an additional minor locus co-localized with the 18S rDNA-SAT locus for the Malagasy species and West and Central African species. A combination of rDNA FISH plus CMA and DAPI banding patterns enables identification of almost all the species, even those for which the genetic or botanical status is still being discussed. C. arabica clearly appears to be an allotetraploid species, including one genome from East Africa and one from West and Central Africa. However, since the minor 5S rDNA-SAT locus present in West/Central African genomes is not detected, two evolutionary hypotheses could be put forward for C. arabica. Considering only the diploid species, global trends are obvious in rDNA signal patterns, genome size variations, and geographic distribution of the species, but there are no clear evolutionary trends. However, complex interactions between these factors and environmental growing conditions exist, which have resulted in loss and gain of rDNA loci and probably also in copy repeat number variations in each rDNA family.

Extensive coloured identification of dog chromosomes to support karyotype studies: the colour code

The identification of individual dog chromosomes is problematic because the 38 pairs of autosomes are small and acrocentric. Here we describe the design and application of a FISH tool that enables definitive identification of each dog autosome in a normal karyotype, without relying on subjective interpretation of DAPI banding patterns. From a high-resolution physical map of the canine genome, we have chosen a panel of 80 canine chromosome-specific BAC clones. DNA from each clone is labeled with one of five different fluorochrome-conjugated nucleotides. By selecting one to three spatially separated BACs per chromosome, and labelling them with a distinctive combination of colours, each autosome can be identified objectively and orientated accurately, irrespective of the quality of DAPI chromosome banding. This tool, or part of it, can be used for any purpose where accurate identification of canine autosomes in a normal karyotype is essential. In this study, we demonstrate use of the ‘colour code’ for chromosome identification following CGH analysis of unbalanced genomic aberrations in a canine brain tumour. Our method is an improvement of an earlier procedure, featuring chromosome-specific BACs and sequential FISH hybridisations, as it enables simultaneous identification of all chromosomes in a single hybridisation.

Fluorescent Banding in Three Varieties of Lens culinaris Medik (Fabaceae)

Differential staining with orcein, CMA and DAPI were used to characterize the karyotypes of three cultivated varieties of Lens culinaris Medik viz. Barimasur-2, Barimasur-3, Barimasur-4. In Barimasur-2 and Barimasur-3, 2n=14 chromosomes were found. Whereas in Barimasur-4, 2n=15 chromosomes were observed indicates the variety as trisomic. Two CMA-positive bands were found at the pericentric region of 2 metacentric chromosomes in Barimasur-2. Barimasur-3 had 2 terminal CMA-positive bands in 2 different sub-metacentric chromosomes. The size of CMA-bands, total CMA-banded area, percentage of CMA-positive banded region of Barimasur-2 and Barimasur-3 were almost similar. The total chromatin length of Barimasur-3 (97.83 μm) was found to be less than that of Barimasur-2 (107.05 μm). These data revealed that deletion near the banded region of metacentric chromosomes of Barimasur-2 might result the formation of terminal CMA-banded sub-metacentric chromosomes in Barimasur-3. No CMA-positive band was found in Barimasur-4. Six DAPI-positive bands were found in Barimasur-2. Barimasur-3 and Barimasur-4 had 11 and 13 DAPI-positive bands, respectively. Heteromorphicity in respect of DAPI bands between the homologue members was found in several chromosome pairs in these three varieties. With the help of DAPI banding it was possible to identify the pair-II of Barimasur-2 and pair-I in Barimasur-4. Therefore, different CMA and DAPI banding patterns were observed in 3 varieties.

Humanized β-Thalassemia Mouse Model Containing the Common IVSI-110 Splicing Mutation

Splicing mutations are common causes of beta-thalassemia. Some splicing mutations permit normal splicing as well as aberrant splicing, which can give a reduced level of normal beta-globin synthesis causing mild disease (thalassemia intermedia). For other mutations, normal splicing is reduced to low levels, and patients are transfusion-dependent when homozygous for the disease. The development of therapies for beta-thalassemia will require suitable mouse models for preclinical studies. In this study, we report the generation of a humanized mouse model carrying the common IVSI-110 splicing mutation on a BAC including the human beta-globin ((hu)beta-globin) locus. We examined heterozygous murine beta-globin knock-out mice ((mu)beta(th-3/+)) carrying either the IVSI-110 or the normal (hu)beta-globin locus. Our results show a 90% decrease in (hu)beta-globin chain synthesis in the IVSI-110 mouse model compared with the mouse model carrying the normal (hu)beta-globin locus. This notable difference is attributed to aberrant splicing. The humanized IVSI-110 mouse model accurately recapitulates the splicing defect found in comparable beta-thalassemia patients. This mouse model is available as a platform for testing strategies for the restoration of normal splicing.

Karyotype analysis ofPsophocarpus tetragonolobus(L.) DC by chromosome banding and fluorescence in situ hybridization

Detailed karyotype of Psophocarpus tetragonolobus (L.) DC was constructed on the basis of chromosome lengths, DAPI and CPD banding, silver staining, together with fluorescence in situ hybridization (FISH) with 45S and 5S rDNA sequences as probes. FISH of the 45S rDNA probe on mitotic chromosomes revealed three pairs of 45S rDNA sites including one major and two minors. The major pair of sites was located in the unique secondary constrictions of the satellite chromosome pair, while the minors were located on two pairs of non-satellite chromo- somes. For 5S rDNA, only one pair of sites was detected using FISH. Distinct DAPI and CPD banding patterns were shown simultaneously after combined PI and DAPI (CPD) staining. CPD bands were shown at all 45S rDNA sites and centromeric regions, suggesting the feature of GC-rich sequences in the centromeres. Each chromosome pair could be identified with DAPI and/or CPD banding patterns in combination with chromosome measurements. Sil- ver staining showed variable number of Ag-NORs on mitotic chromosomes, revealing that the major sites were active in every cell, while the minors were activated only in a proportion of the cells, and the level of expression differed between two pairs of minor NORs.

Fluorescent in situ hybridization with transposable element probes to mitotic chromosomal heterochromatin of Drosophila.

The technique of in situ hybridization of DNA probes to Drosophila chromosomes has been initially applied to the salivary gland polytene chromosomes and is now routinely used for mapping single-copy and repetitive DNA sequences, such as transposable elements, to the euchromatic regions of these chromosomes. However, most of the heterochromatin normally escapes cytogenetic analyses on polytene chromosomes because it is organized in a poorly differentiated cytological structure called the chromocenter. This peculiar organization does not allow a detailed mapping of DNA clones to heterochromatin. Such a limitation can be overcome by the fluorescent in situ hybridization (FISH) technique on mitotic chromosomes of D. melanogaster, where heterochromatin has been extensively characterized by banding techniques and subdivided into several cytologically diverse regions. Digital images of FISH signals and DAPI staining can be separately recorded by CCD camera, pseudocolored, and merged using specific software for image analysis. The visualization of the signals and DAPI banding pattern on a single chromosome enables the mapping of a given sequence to specific cytological regions of mitotic heterochromatin. This method has initially proven successful in the detection and mapping of transposable element clusters in the heterochromatin of D. melanogaster and has been used to study the distribution of repeated and even single-copy sequences.