Fluorescence In Situ Hybridization Signals Research Articles

In Situ Hybridization and Detection Using Nonisotopic Probes

Nonisotopic in situ hybridization can be used to determine the cellular location and relative levels of expression for specific transcripts within cells and tissues. RNA in specimen preparations is hybridized with a biotin- or digoxigenin-labeled probe, which is generally detected by fluorescence or enzymatic methods. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described here, along with amplification of weak FISH signals. Nonisotopic probes can also be detected by enzymatic reactions using horseradish peroxidase or alkaline phosphatase, as described here.

Fully automated FISH examination of amniotic fluid cells

Fluorescence in situ hybridization (FISH) analysis has become a valuable adjunct in cytogenetics, providing a rapid screen for common chromosome abnormalities that is particularly helpful in prenatal diagnosis. FISH analysis using standard microscopy is expensive and labor intensive, requiring both a high skill level and subjective signal interpretation. A reliable fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. The efficacy of an automated system was compared to standard manual FISH analysis. Two sets of slides were generated from each of 152 amniotic fluid samples. Following hybridization with a standard panel of five chromosome FISH probes, one set of slides was evaluated using manual microscopy. The other set was evaluated using an automated microscopy system. A diagnostic outcome was obtained for all 152 samples using manual microscopy and for 146 of 152 (96%) samples using automated microscopy. Three cases of aneuploidy were detected. For those samples for which a diagnostic outcome was determined by both manual and automated microscopy, 100% concordance was observed. All FISH analysis results were confirmed by karyotype. These data suggest that an automated microscopy system is capable of providing accurate and rapid enumeration of FISH signals in amniocytes.

Dissecting mechanisms of nuclear mRNA surveillance in THO/sub2 complex mutants

The nuclear exosome is involved in numerous RNA metabolic processes. Exosome degradation of rRNA, snoRNA, snRNA and tRNA in Saccharomyces cerevisiae is activated by TRAMP complexes, containing either the Trf4p or Trf5p poly(A) polymerase. These enzymes are presumed to facilitate exosome access by appending oligo(A)-tails onto structured substrates. Another role of the nuclear exosome is that of mRNA surveillance. In strains harboring a mutated THO/Sub2p system, involved in messenger ribonucleoprotein particle biogenesis and nuclear export, the exosome-associated 3' --> 5' exonuclease Rrp6p is required for both retention and degradation of nuclear restricted mRNAs. We show here that Trf4p, in the context of TRAMP, is an mRNA surveillance factor. However, unlike Rrp6p, Trf4p only partakes in RNA degradation and not in transcript retention. Surprisingly, a polyadenylation-defective Trf4p protein is fully active, suggesting polyadenylation-independent mRNA degradation. Transcription pulse-chase experiments show that HSP104 molecules undergoing quality control in THO/sub2 mutant strains fall into two distinct populations: One that is quickly degraded after transcription induction and another that escapes rapid decay and accumulates in foci associated with the HSP104 transcription site.

Symmetrical localization of extrachromosomally replicating viral genomes on sister chromatids

In eukaryotes, many latent viruses replicate as extrachromosomal molecules, called episomes, and efficiently segregate to daughter cells by noncovalently attaching to mitotic chromosomes. To understand the mechanism governing the processes, we analyzed the detailed subcellular localization of Epstein-Barr virus (EBV) genomes and a viral protein EBNA1, a bridging molecule between viral genomes and cellular chromatin. In the cells that were infected with a recombinant EBV expressing epitope-tagged EBNA1, EBNA1 localized to intranuclear punctate dots, which coincided with the localization of EBV genomes as revealed by fluorescence in situ hybridization (FISH). A significant number of EBNA1 dots were found to localize symmetrically on sister chromatids of mitotic chromosomes. Such symmetrical localization of EBNA1 dots was observed in prematurely condensed G2 chromosomes as well, correlating with the presence of closely spaced double dots of EBNA1 in G2-phase-enriched cells. The EBNA1 double dots were occasionally interconnected by the FISH signals of EBV episomes, exhibiting a dumbbell-like appearance. Thus, we propose that the partitioning of EBNA1 molecules onto sister chromatids during cellular DNA replication underlies the non-stochastic segregation of extrachromosomally replicating viral genomes.

Sororin Is Required for Stable Binding of Cohesin to Chromatin and for Sister Chromatid Cohesion in Interphase

Sister chromatid cohesion depends on cohesin [1-3]. Cohesin associates with chromatin dynamically throughout interphase [4]. During DNA replication, cohesin establishes cohesion [5], and this process coincides with the generation of a cohesin subpopulation that is more stably bound to chromatin [4]. In mitosis, cohesin is removed from chromosomes, enabling sister chromatid separation [6]. How cohesin associates with chromatin and establishes cohesion is poorly understood. By searching for proteins that are associated with chromatin-bound cohesin, we have identified sororin, a protein that was known to be required for cohesion [7]. To obtain further insight into sororin's function, we have addressed when during the cell cycle sororin is required for cohesion. We show that sororin is dispensable for the association of cohesin with chromatin but that sororin is essential for proper cohesion during G2 phase. Like cohesin, sororin is also needed for efficient repair of DNA double-strand breaks in G2. Finally, sororin is required for the presence of normal amounts of the stably chromatin-bound cohesin population in G2. Our data indicate that sororin interacts with chromatin-bound cohesin and functions during the establishment or maintenance of cohesion in S or G2 phase, respectively.

Spot Counting to Locate Fetal Cells in Maternal Blood and Tissue: A Comparison of Manual and Automated Microscopy

Fetal cell detection in maternal tissue requires an accurate, efficient, and reproducible microscopy method. Our objective was to compare manual scoring to a commercially available automated scanning system for the detection of chromosome signals by fluorescence in situ hybridization (FISH). X and Y chromosome FISH signals were detected on slides of calibrated mixtures of blood, paraffin-embedded liver sections, and post-termination blood. For manual scoring (400x magnification), the number of cells located and duration of scoring were recorded. For automated scanning using the Metasystems Metafer3/Metafer4 Scanning System (200x magnification), duration of scanning, number of gallery images generated, duration of manual review of gallery images, and number of confirmed fetal cells were recorded. From all slides the number of target fetal cells located by manual and automated microscopy was highly correlated (r = 0.90). However, automated scanning required on average 4-fold more time than manual scoring (P < 0.0001), with an average automated scanning time of 9.7 h per slide compared with 2.4 h per slide when scored manually. In general, the accuracy of automated and manual microscopy is comparable, although manual scoring is more efficient because of the level of magnification necessary for automated scanning of cells, and a large number of gallery images generated by automated scanning that must then be reviewed manually. This suggests that when rapid analysis is required (i.e., clinical situations), manual microscopy is preferable. In contrast, automated scanning may have advantages over manual microscopy when time constraints are less imposed (i.e., research situations).

Characterization of Japanese flounder karyotype by chromosome bandings and fluorescence in situ hybridization with DNA markers

The chromosomes of Japanese flounder, Paralichthys olivaceus, were examined by conventional differential staining methods including G-, Q-, C-, silver (Ag)-, fluorochrome, and replication R-bandings and by fluorescence in situ hybridization (FISH) with 5S and 18S rDNAs and telomeric DNA as probes. Replication R-banding substantially made it possible to identify 24 homologous pairs by their RBG-banding pattern and relative length. Both rDNA loci were mapped to chromosome 1, where 5S and 18S rDNA loci were located at the centromeric region and secondary constriction, respectively. C-banding revealed that both rDNA loci were heterochromatic, and 18S rDNA loci were positive for chromomycin A(3) but negative for 4',6-diamidino-2-phenylindole (DAPI) staining. Telomeric FISH signals were observed at all chromosome ends and at the interstitial region of some chromosomes. The observed results were discussed in relation to the karyotype evolution in the order Pleuronectiformes.

Automated detection of residual leukemic cells by consecutive immunolabeling for CD10 and fluorescence in situ hybridization for ETV6/RUNX1 rearrangement in childhood acute lymphoblastic leukemia

Among the various methods available for analyzing minimal residual disease, a new procedure for the cell-based approaches using consecutive phenotypic and genotypic analysis as revealed by immunofluorescent labeling and subsequent fluorescent in situ hybridization (FISH) has been developed. We are introducing a fluorescent microscopy-based technique by which not only cellular targets and immunological marker positivity, but also the FISH pattern was identified by automated scanning. For the latter one translocation-specific FISH pattern recognition was accomplished by using an automated scanning mode for the 3D determination of valid distances between FISH signals, to define the cutoff value for the shortest green–red spot distance differentiating positive cells from negative ones. The procedure was tested with CD10 + acute lymphoblastic leukemia cell line harboring the t(12;21)(p13;q22) resulting in the ETV6/RUNX1 rearrangement (formerly TEL/AML1), as well as peripheral blood lymphocytes of healthy individuals. Using the combined, automated method, a sensitivity of 98.67% and a specificity of 99.97% were obtained. The mean false positivity + 2 standard deviations cutoff level (0.09%) allows detection of leukemic cells with high accuracy, even a bit below the tumor load dilution of 10 −3, a value reported to be critical in clinical decision making.

Wilms' tumor gene (WT1) is predominantly expressed in clonal hematopoietic cells in myelodysplastic syndromes

To determine the expression of Wilms' tumor gene (WT1) in clonal hematopoietic cells in patients with myelodysplastic syndromes (MDS), immunochemistry labelling (alkaline phosphatase anti-alkaline phosphatase) and fluorescence in situ hybridization (FISH) were coperformed on bone marrow cytospins from 18 patients whose abnormal karyotypes had been determined by G-binding analysis. Compared to 12 healthy donors, WT1 positive nucleated cells in MDS marrows were significantly higher (t = 2.30; P = 0.032). Moreover, WT1 was expressed predominantly in MDS clonal cells (with abnormal FISH signals) rather than in non-clonal cells (residual normal cells) (t = 2.19; P = 0.043).

Development of prenatal genetic screening test using fetal cells in maternal blood

Prenatal genetic diagnosis using invasive techniques such as amniocentesis and chorionic villus sampling reveal significant risks to the fetus. The triple marker screening test has low sensitivity and cannot confirm sex chromosomal abnormalities. Prenatal genetic diagnosis using the fetal nucleated RBCs in maternal blood has been studied to overcome this shortage. However, it is difficult to use because of extremely small numbers of fetal RBC in maternal blood. Therefore, we tried to collect nucleated RBCs morphologically by Kleihaur-Betke staining after double gradient centrifugation and magnetic activating cell sorting from maternal blood. Fluorescent in situ hybridization (FISH) analyses with CEP X and CEP Y probes for K-B positive nucleated RBCs were performed to detect whether fetal cells were existed among nucleated RBCs by observation of sex chromosomes. The average number of K-B positive nucleated RBCs separated from 10 ml of maternal blood was 17.3, and the maximum number of nucleated RBCs was 54. We observed FISH signals in nucleated RBCs separated from 18 pregnant women, and Y probe signals were observed in 67.3% of nucleated RBCs separated from 10 pregnant women. We confirmed that separated nucleated fetal RBCs can be used to identify fetal sex and chromosomal aneuploidies by FISH. Since nucleated RBCs form maternal origin were not excluded, further studies are needed to overcome this limitation.

Rapid genome reshuffling induced by allopolyploidization in F1 hybrid in Chrysanthemum remotipinnum (formerly Ajania remotipinna) and Chrysanthemum chanetii (formerly Dendranthema chanetii)

Allopolyploidization was investigated in an artificial F1 hybrid between Chrysanthemum remotipinnum (2n=18) and Chrysanthemum chanetii (2n=36). Homoeologous and non-homologous chromosomes could pair in the PMC of the hybrid that showed complex pairings and configurations at diakinesis. Physical maps of the 5S and 45S rDNA genes were generated by fluorescence in situ hybridization (FISH) for the F1 hybrid that had four signals of the 5S rDNA sites in four median-centromeric chromosomes and were different from that of C. remotipinnum, which had four signals in two median-centromeric chromosomes. The hybrid did not show any of the double signals 5S rDNA like C. remotipinnum did. FISH signals of 45S rDNA showed that the hybrid had eight signals of the 45S rDNA sites and seemed to keep the four signals of C. remotipinnum. Genomic in situ hybridization (GISH) distinguished respective parental chromosomes in the F1 hybrid chromosome complement and showed that approximately 18-yellow-colored chromosomes were hybridized with the probe of C. remotipinnum while the other 18 red-colored chromosomes that were not hybridized could be of C. chanetii. GISH detected translocations at different chromosomal sites and duplication exists in the maternal genome side.

Structure of the Papillomavirus DNA-Tethering Complex E2:Brd4 and a Peptide that Ablates HPV Chromosomal Association

Many DNA viruses that are latent in dividing cells are noncovalent passengers on mitotic chromosomes and require specific viral-encoded and cellular factors for this activity. The chromosomal protein Brd4 is implicated in the hitchhiking of bovine papillomavirus-1 (BPV-1), and the viral protein E2 binds to both plasmids and Brd4. Here, we present the X-ray crystal structure of the carboxy-terminal domain of Brd4 in complex with HPV-16 E2, and with this information have developed a Brd4-Tat fusion protein that is efficiently taken up by different transformed cells harboring HPV plasmids. In cells treated with these fusion proteins for only 2 hr and arrested in metaphase, the HPV DNA, either HPV-16 or -31, is displaced from mitotic chromosomes. Mutant Brd4 peptides are deficient in ablating this association. We suggest that such peptides may lead to the development of inhibitors of latency for many, if not all, papillomaviruses.

Preservation of large-scale chromatin structure in FISH experiments

The nuclear organization of specific endogenous chromatin regions can be investigated only by fluorescence in situ hybridization (FISH). One of the two fixation procedures is typically applied: (1) buffered formaldehyde or (2) hypotonic shock with methanol acetic acid fixation followed by dropping of nuclei on glass slides and air drying. In this study, we compared the effects of these two procedures and some variations on nuclear morphology and on FISH signals. We analyzed mouse erythroleukemia and mouse embryonic stem cells because their clusters of subcentromeric heterochromatin provide an easy means to assess preservation of chromatin. Qualitative and quantitative analyses revealed that formaldehyde fixation provided good preservation of large-scale chromatin structures, while classical methanol acetic acid fixation after hypotonic treatment severely impaired nuclear shape and led to disruption of chromosome territories, heterochromatin structures, and large transgene arrays. Our data show that such preparations do not faithfully reflect in vivo nuclear architecture.Electronic supplementary materialSupplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00412-006-0084-2 and is accessible for authorized users.

Automated Microscopy of Amniotic Fluid Cells: Detection of FISH Signals Using the FastFISH® Imaging System

Objective: FISH (fluorescence in situ hybridization) analysis is a valuable adjunct to cytogenetics that provides a rapid screen for common abnormalities. However, FISH is expensive, labor-intensive, and requires a high skill level and subjective signal interpretation. A fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. Methods: In this study we blindly compared automated FISH signal acquisition and display against standard FISH analysis. A total of 62 amniocentesis samples were prepared using the AneuVysion® multicolor DNA probe kit and probed for chromosomes 13, 18, 21, X, and Y. Two sets of slides were produced from each sample. Fifty cells were scored in each slide. One set was evaluated using standard manual microscopy and the other using the automated image acquisition and display capabilities of the Ikoniscope® fastFISH® amnio Test System. This system uses epifluorescence optics, along with optimized slide management to process slides automatically. Results: A 100% concordance was observed between the results obtained using manual microscopy and the automated system. There was also 100% concordance between the FISH results and those obtained by conventional karyotyping. Conclusion: Our data suggest that the automated system is capable of providing accurate and rapid identification and display of cells and FISH signals.

Tracing B-genome chromatin in Brassica napus × B. juncea interspecific progeny

We used polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques to demonstrate the presence of Brassica B-genome chromosomes and putative B-genome introgressions in B. napus x B. juncea interspecific progeny. The B-genome--specific repeat sequence pBNBH35 was used to generate PCR products and FISH probes. The highest frequencies of viable progeny were obtained when B. napus was the maternal parent of the interspecific hybrid and the first backcross. B-genome--positive PCR assays were found in 34/51 fertile F2 progeny (67%), which was more than double the proportion found in fertile BC(1) progeny. Four B-genome--positive F(2)-derived families and 1 BC(1)-derived family were fixed or segregating for B. juncea morphology in the F(4) and BC(1)S(2), respectively, but in only 2 of these families did B. juncea-type plants exhibit B. juncea chromosome count (2n = 36) and typical B-genome FISH signals on 16 chromosomes. The remaining B. juncea-type plants had B. napus chromosome count (2n = 38) and no B-genome FISH signals, except for 1 exceptional F(4)-derived line that exhibited isolated and weak B-genome FISH signals on 11 chromosomes and typical A-genome FISH signals. B. juncea morphology was associated with B-genome--positive PCR signals but not necessarily with 16 intact B-genome chromosomes as detected by FISH. B-genome chromosomes tend to be eliminated during selfing or backcrossing after crossing B. juncea with B. napus, and selection of lines containing B-genome chromatin during early generations would be promoted by use of this B-genome repetitive marker.

Chromosomal mapping of major ribosomal rRNA genes in the hard clam (Mercenaria mercenaria) using fluorescence in situ hybridization

Karyotype and chromosomal location of the major ribosomal RNA genes were studied in the hard clam (Mercenaria mercenaria Linnaeus) using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos. Internal transcribed spacers (ITS) between major RNA genes were amplified and used as FISH probes. The probes were labeled with digoxigenin-11-dUTP by polymerase chain reaction and detected with fluorescein-labeled anti-digoxigenin antibodies. FISH with the ITS probes produced two to four signals per nucleus or metaphase. M. mercenaria had a haploid number of 19 chromosomes with a karyotype of seven metacentric, four metacentric or submetacentric, seven submetacentric, and one submetacentric or subtelocentric chromosomes (7M + 4M/SM + 7SM + 1SM/ST). Two ITS loci were observed: one located near the centromere on the long arm of Chromosome 10 and the other at the telomere of the short arm of Chromosome 12. FISH signals on Chromosome 10 are strong and consistent, while signals on Chromosome 12 are variable. This study provides the first karyotype and chromosomal assignment of the major RNA genes in M. mercenaria. Similar studies in a wide range of species are needed to understand the role of chromosomal changes in bivalve evolution.

Single-Cell Analysis of Glucocorticoid Receptor Action Reveals that Stochastic Post-Chromatin Association Mechanisms Regulate Ligand-Specific Transcription

The glucocorticoid receptor (GR) dynamically interacts with response elements in the mouse mammary tumor virus (MMTV) promoter to regulate steroid-dependent transcription. In a clonal mammary carcinoma cell line containing a tandem array of MMTV promoter-reporter gene cassettes integrated at a single genomic locus, direct binding of a green fluorescent protein (GFP)-GR fusion protein to the MMTV regulatory elements can be observed in living cells. After ligand treatment, MMTV-dependent transcription in individual cells was detected by RNA fluorescence in situ hybridization (FISH). High-resolution fluorescence images were acquired from large numbers of randomly selected cells. Images were analyzed with a novel automated computer algorithm, measuring the RNA FISH signal and the relative GFP-GR fluorescence intensity at the MMTV array for each cell. Although dexamethasone increased the mean RNA FISH signal approximately 10-fold, RU486 produced only about a 2-fold induction, as expected for this mixed antagonist. For all treatment conditions, the relative GFP-GR fluorescence at the array for the averaged cells paralleled the RNA FISH measurements, suggesting that image analysis accurately detected an increase in steady-state GR association with the MMTV array that was responsible for the increase in transcriptional activity. The antagonist-dependent decreases in GR association with the MMTV promoter were confirmed by chromatin immunoprecipitation experiments, supporting the image analysis results. A pronounced cell-to-cell variability was observed in RNA FISH signal and GR-MMTV association within treatment groups. We observed a nonlinear relationship between GR-MMTV association and RNA FISH in individual cells, indicating that differences in GR-MMTV interaction account for some, but not all, of the transcriptional heterogeneity between individual cells. In selected cell subpopulations with equal levels of GR-MMTV association, there was a decrease in RNA FISH signal with RU486 treatment compared with dexamethasone treatment. These results indicate that stochastic events occurring after GR-promoter association, such as the actions of chromatin remodeling complexes or other cofactors, change in a ligand-dependent manner and regulate heterogeneous transcription in individual cells.

Microbial Diversity in Maras Salterns, a Hypersaline Environment in the Peruvian Andes

Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 x 10(6) to 3 x 10(6) cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4',6'-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon "Haloquadra walsbyi," although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the gamma-proteobacterium "Pseudomonas halophila" DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the "P. halophila" cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.

The Coincidence of Chromosome 15 Aberrations and β2-Microglobulin Gene Mutations Is Causative for the Total Loss of Human Leukocyte Antigen Class I Expression in Melanoma

Total loss of surface presentation of human leukocyte antigen (HLA) class I molecules, protecting tumor cells from the recognition by cytotoxic host CD8+ T cells, is known to be caused by mutations in the beta2-microglobulin (beta2m) gene. We asked whether abnormalities of chromosome 15, harboring the beta2m gene on 15q21, in addition to beta2m gene mutations, are causative for the HLA class I-negative phenotype of melanoma cells. To answer this, we established primary cell lines from the beta2m-negative metastatic melanoma tissues of four different patients and analyzed them for beta2m gene mutations and chromosome 15 aberrations, the latter by loss of heterozygosity analysis, fluorescence in situ hybridization (FISH), and multicolor FISH. Mutations at the beta2m gene level were detected in all cell lines. The loss of heterozygosity analysis of microsatellite markers located on chromosome 15 in three of the four cell lines pointed to an extensive loss of chromosome 15 material. Subsequent molecular cytogenetic analysis revealed the coexistence of apparently normal and rearranged versions of chromosome 15 in three cell lines whereas the fourth cell line solely showed rearranged versions. Two of the four cell lines exhibited a special type of intrachromosomal rearrangement characterized by FISH signals specific for the subtelomeric region of 15q at both ends of the chromosome and one centromeric signal in between. Our data indicate that the complete loss of HLA class I expression in melanoma cells is due to the coincidence of the following mutational events: (a) chromosome 15 instability associated with an extensive loss of genetic material and (b) beta2m gene mutations.

Ribosomal, telomeric and heterochromatin sequences localization in the karyotype of Anemone hortensis

Karyotype of the Mediterranean species Anemone hortensis L. (Ranunculaceae) was characterized with emphasis on heterochromatin distribution and localization of ribosomal (18S-5.8S-26S and 5S rDNA) and telomeric repeats (TTTAGGG). Diploid chromosome complement, 2n = 2x = 16, common to all investigated populations consisted of three acrocentric, one meta-submetacentric and four metacentric chromosomes ranging in size from 6.34 to 10.47 m ; ; m. Fluorescence in situ hybridization (FISH) with 18S and 5S rDNA probes revealed two 18S-5.8S-26S rDNA loci on satellite and secondary constriction (SC) of acrocentric chromosome pair 2 and terminally on accrocentric chromosome pair 3, and two 5S rDNA loci in pericentromeric region of meta-submetacentric chromosome pair 4 and in the proximity to 18S-5.8S-26S rDNA locus on chromosome pair 2. The only GC rich heterochromatin, as revealed by fluorochrome Chromomycin A3 (CMA) staining, was that associated with nucleolar organizer regions (NORs), while AT rich heterochromatin, stained with 4, 6-diamino-2-phenylindole (DAPI), was distributed intercalarly and terminally on the long arm of all three acrocentric chromosomes, and terminally on chromosomes 4 and 5. FISH with Arabidopsis-type of telomeric repeats (TTTAGGG) as a probe revealed two classes of signals, small dot-like and large bands, at chromosome termini exclusively, where they corresponded to terminal DAPI stained heterochromatin. Heteromorphism of chromosome pair 4 which refers to terminal DAPI bands and FISH signals was observed in populations of A. hortensis. Chromosome pairing during meiosis was regular with formation of localized chiasmata proximal to the centromere.