Origin of nuclear buds and micronuclei in normal and folate-deprived human lymphocytes

Micronuclei are formed from chromosomes and chromosomal fragments that lag behind in anaphase and are left outside daughter nuclei in telophase. They may also be derived from broken anaphase bridges. Nuclear buds, micronucleus-like bodies attached to the nucleus by a thin nucleoplasmic connection, have been proposed to be generated similarly to micronuclei during nuclear division or in S-phase as a stage in the extrusion of extra DNA, possibly giving rise to micronuclei. To better understand these phenomena, we have characterized the contents of 894 nuclear buds and 1392 micronuclei in normal and folate-deprived 9-day cultures of human lymphocytes using fluorescence in situ hybridization with pancentromeric and pantelomeric DNA probes. Such information has not earlier been available for human primary cells. Surprisingly, there appears to be no previous data on the occurrence of telomeres in micronuclei (or buds) of normal human cells in general. Our results suggest that nuclear buds and micronuclei have partly different mechanistic origin. Interstitial DNA without centromere or telomere label was clearly more prevalent in nuclear buds (43%) than in micronuclei (13%). DNA with only telomere label or with both centromere and telomere label was more frequent in micronuclei (62% and 22%, respectively) than in nuclear buds (44% and 10%, respectively). Folate deprivation especially increased the frequency of nuclear buds and micronuclei harboring telomeric DNA and nuclear buds harboring interstitial DNA but also buds and micronuclei with both centromeric and telomeric DNA. According to the model we propose, that micronuclei in binucleate lymphocytes primarily derive from lagging chromosomes and terminal acentric fragments during mitosis. Most nuclear buds, however, are suggested to originate from interstitial or terminal acentric fragments, possibly representing nuclear membrane entrapment of DNA that has been left in cytoplasm after nuclear division or excess DNA that is being extruded from the nucleus.     

Satellite DNA I in chromatin loops of rat pachytene chromosomes and in spermatids

Biotinylated rat satellite DNA I probe p93-50 was used to visualize the chromatin of surface-spread rat pachytene chromosomes. Fluorescein isothiocyanate (FITC)-conjugated avidin produces a beaded fluorescence pattern along the chromatin loops that insert in the centromeric region of the synaptonemal complex (SC), the paired cores of homologous chromosomes. The number of fluorescent beads ranges from zero for centromeres without satellite DNA I homologous to probe p 93-50, to several hundred for satellite-rich centromeric regions. For the chromosomes that can be identified, the relative amount of satellite DNA is chromosome specific. No satellite DNA I was detected at the non-centromeric ends of the chromosomes or interstitially. DNase-digested nuclei or isolated SCs did not have detectable amounts of satellite DNA in the centromeric regions of the chromosomes or in the residual SCs. The fate of the satellite DNA was followed during spermiogenesis. In the round spermatid the centromeric regions, which appear to be attached to the nuclear envelope, are still distinct and have converging loops of fluorescent chromatin. At later stages there are fewer but still bright fluorescent patches. Satellite DNA I is still detectable in the mature sperm head. These results demonstrate the organization of satellite DNA I in the chromatin loops at the centromeric regions, and they forecast the analysis of chromosome organization in unprecedented detail with a variety of probes in surface spreads of meiotic prophase chromosomes.     

Telomere dysfunction and chromosome structure modulate the contribution of individual chromosomes in abnormal nuclear morphologies

The cytokinesis-block micronucleus assay has emerged as a biomarker of chromosome damage relevant to cancer. Although it was initially developed to measure micronuclei, it is also useful for measuring nucleoplasmic bridges and nuclear buds. Abnormal nuclear morphologies are frequently observed in malignant tissues and short-term tumour cell cultures. Changes in chromosome structure and number resulting from chromosome instability are important factors in oncogenesis. Telomeres have become key players in the initiation of chromosome instability related to carcinogenesis by means of breakage–fusion–bridge cycles. To better understand the connection between telomere dysfunction and the appearance of abnormal nuclear morphologies, we have characterised the presence of micronuclei, nucleoplasmic bridges and nuclear buds in human mammary primary epithelial cells. These cells can proliferate beyond the Hayflick limit by spontaneously losing expression of the p16^INK4a protein. Progressive telomere shortening leads to the loss of the capping function, and the appearance of end-to-end chromosome fusions that can enter into breakage–fusion–bridge cycles generating massive chromosomal instability. In human mammary epithelial cells, different types of abnormal nuclear morphologies were observed, however only nucleoplasmatic bridges and buds increased significantly with population doublings. Fluorescent in situ hybridisation using centromeric and painting specific probes for chromosomes with eroded telomeres has revealed that these chromosomes are preferentially included in the different types of abnormal nuclear morphologies observed, thus reflecting their common origin. Accordingly, real-time imaging of cell divisions enabled us to determine that anaphase bridge resolution was mainly through chromatin breakage and the formation of symmetric buds in daughter nuclei. Few micronuclei emerged in this cell system thus validating the scoring of nucleoplasmic bridges and nuclear buds for measuring chromosome instability in telomere-dysfunction cell environments.     

Laser microirradiation of kinetochores in mitotic PtK2 cells: chromatid separation and micronucleus formation

Irradiation of the kinetochore region of PtK2 chromosomes by laser light of 532 nm was used to study the function of the kinetochore region in chromosome movement and to create an artificial micronuclei in cells. When the sister kinetochores of a chromosome were irradiated at prometaphase, the affected chromosome detached from the spindle and exhibited no further directed movements for the duration of mitosis. The chromatids of the chromosome remained attached to one another until anaphase, at which point they separated. No poleward movement of the chromatids was observed, and at telophase they passively moved to one of the daughter cells and were enclosed in a micronucleus. The daughter cell containing the micronucleus was then isolated by micromanipulation and followed through subsequent mitoses. At the next mitosis, two chromosomes, each with two chromatids, condensed in the micronucleus. These chromosomes did not attach to the spindle and showed chromatid separation, but no poleward movements at anaphase. They were again enclosed in micronuclei at telophase. The third generation mitosis was similar to the second. Occasionally, both the irradiation-produced and naturally occurring micronuclei exhibited no chromosome condensation at mitosis. Feulgen-stained monolayers of PtK2 cells with naturally occurring micronuclei showed that some micronuclei stain positive for DNA and others do not. This finding raises questions about the fate of chromosomes in a micronucleus.     

Laser microirradiation of kinetochores in mitotic PtK2 cells

Irradiation of the kinetochore region of PtK₂ chromosomes by laser light of 532 nm was used to study the function of the kinetochore region in chromosome movement and to create artificial micronuclei in cells. When the sister kinetochores of a chromosome were irradiated at prometaphase, the affected chromosome detached from the spindle and exhibited no further directed movements for the duration of mitosis. The chromatids of the chromosome remained attached to one another until anaphase, at which point they separated. No poleward movement of the chromatids was observed, and at telophase they passively moved to one of the daughter cells and were enclosed in a micronucleus. The daughter cell containing the micronucleus was then isolated by micromanipulation and followed through subsequent mitoses. At the next mitosis, two chromosomes, each with two chromatids, condensed in the micronucleus. These chromosomes did not attach to the spindle and showed chromatid separation, but no poleward movements at anaphase. They were again enclosed in micronuclei at telophase. The third generation mitosis was similar to the second. Occasionally, both the irradiation-produced and naturally occurring micronuclei exhibited no chromosome condensation at mitosis. Feulgenstained monolayers of PtK₂ cells with naturally occurring micronuclei showed that some micronuclei stain positive for DNA and others do not. This finding raises questions about the fate of chromosomes in a micronucleus.     

Chromosome elimination in micronuclei: a common cause of hypoploidy.

An excess of hypoploid cells has repeatedly been reported in studies of aneuploidy and has often been attributed to technical artifact. We have examined at least 200 anaphase or early-telophase cells from each of 28 normal women and found that chromosome or chromatid lagging occurs in an average of 2.43% of cells. In a separate study, we have examined the frequency of micronuclei in cytochalasin B-arrested, binucleate cells and shown that a similar frequency of cells (1.6%) contain one or more micronuclei. Using in situ hybridization of an alpha centromeric probe (alpha R1), which hybridizes to 9 of the 22 human autosomes, we were able to infer that most, if not all, of the micronuclei contain whole chromosomes or chromatids. Since the loss of a chromosome by lagging will induce hypoploid daughter nuclei (two where a chromosome is lost and one where a chromatid is lost), we conclude that lagging is a major mechanism for chromosome loss in human lymphocyte cultures. This loss occurs in the cells of normal individuals under control conditions.     

Labeling of the centromeric region on human chromosome 8 by in situ hybridization

Summary Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.     

Integration of human alpha-satellite DNA into simian chromosomes: centromere protein binding and disruption of normal chromosome segregation.

Centromeres of mammalian and other complex eukaryotic chromosomes are dominated by one or more classes of satellite DNA. To test the hypothesis that alpha-satellite DNA, the major centromeric satellite of primate chromosomes, is involved in centromere structure and/or function, human alpha-satellite DNA was introduced into African green monkey (AGM) cells. Centromere protein binding was apparent at the sites of integrated human alpha-satellite DNA. In the presence of an AGM centromere on the same chromosome, human alpha-satellite was associated with bridges between the separating sets of chromatids at anaphase and an increased number of lagging chromosomes at metaphase, both features consistent with the integrated alpha-satellite disrupting normal chromosome segregation. These experiments suggest that alpha-satellite DNA provides the primary sequence information for centromere protein binding and for at least some functional aspect(s) of a mammalian centromere, playing a role either in kinetochore formation or in sister chromatid apposition.     

Incomplete sister chromatid separation of long chromosome arms

Chromosome segregation ensures the equal partitioning of chromosomes at mitosis. However, long chromosome arms may pose a problem for complete sister chromatid separation. In this paper we report on the analysis of cell division in primary cells from field vole Microtus agrestis, a species with 52 chromosomes including two giant sex chromosomes. Dual chromosome painting with probes specific for the X and the Y chromosomes showed that these long chromosomes are prone to mis-segregate, producing DNA bridges between daughter nuclei and micronuclei. Analysis of mitotic cells with incomplete chromatid separation showed that reassembly of the nuclear membrane, deposition of INner CENtromere Protein (INCENP)/Aurora B to the spindle midzone and furrow formation occur while the two groups of daughter chromosomes are still connected by sex chromosome arms. Late cytokinetic processes are not efficiently inhibited by the incomplete segregation as in a significant number of cell divisions cytoplasmic abscission proceeds while Aurora B is at the midbody. Live-cell imaging during late mitotic stages also revealed abnormal cell division with persistent sister chromatid connections. We conclude that late mitotic regulatory events do not monitor incomplete sister chromatid separation of the large X and Y chromosomes of Microtus agrestis, leading to defective segregation of these chromosomes. These findings suggest a limit in chromosome arm length for efficient chromosome transmission through mitosis.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Structure of DNA near long tandem arrays of alpha satellite DNA at the centromere of human chromosome 7.

The centromeric regions of human chromosomes contain long tracts of tandemly repeated DNA, of which the most extensively characterized is alpha satellite. In a screen for additional centromeric DNA sequences, four phage clones were obtained which contain alpha satellite as well as other sequences not usually found associated with tandemly repeated alpha satellite DNA, including L1 repetitive elements, an Alu element, and a novel AT-rich repeated sequence. The alpha satellite DNA contained within these clones does not demonstrate the higher-order repeat structure typical of tandemly repeated alpha satellite. Two of the clones contain inversions; instead of the usual head-to-tail arrangement of alpha satellite monomers, the direction of the monomers changes partway through each clone. The presence of both inversions was confirmed in human genomic DNA by polymerase chain reaction amplification of the inverted regions. One phage clone contains a junction between alpha satellite DNA and a novel low-copy repeated sequence. The junction between the two types of DNA is abrupt and the junction sequence is characterized by the presence of runs of A's and T's, yielding an overall base composition of 65% AT with local areas > 80% AT. The AT-rich sequence is found in multiple copies on chromosome 7 and homologous sequences are found in (peri)centromeric locations on other human chromosomes, including chromosomes 1, 2, and 16. As such, the AT-rich sequence adjacent to alpha satellite DNA provides a tool for the further study of the DNA from this region of the chromosome. The phage clones examined are located within the same 3.3-Mb SstII restriction fragment on chromosome 7 as the two previously described alpha satellite arrays, D7Z1 and D7Z2. These new clones demonstrate that centromeric repetitive DNA, at least on chromosome 7, may be more heterogeneous in composition and organization than had previously been thought.     

Replication of yeast DNA and novel chromosome formation in mouse cells.

To determine whether yeast DNA can replicate or segregate in mammalian cells, we have transferred genomic DNA from the yeast Saccharomyces cerevisiae into mouse cells. Most of the lines contained stably integrated yeast DNA. However, in two of the lines, the yeast DNA was maintained as numerous small extrachromosomal elements which were still present after 26 cell divisions in selection but which were lost rapidly out of selection. This indicates that, although yeast DNA can replicate in mouse cells, the yeast centromere does not function to give segregation. In one cell line we observed a large novel chromosome consisting almost entirely of yeast DNA. This chromosome segregates well and contains mouse centromeric minor satellite DNA and variable amounts of major satellite DNA which probably comprise the functional centromere. The yeast DNA in the novel chromosome has a compacted chromatin structure which may be responsible for the efficient formation of anaphase bridges. Furthermore, yeast DNA integrated into mouse chromosomes forms constrictions at the point of integration. These features have previously been presumed to be hallmarks of centromeric function in transfection assays aimed at identifying putative centromeric DNA. Hence our results suggest caution be exercised in the interpretation of such assays.     

Interphase cytogenetics reveals somatic pairing of chromosome 17 centromeres in normal human brain tissue, but no trisomy 7 or sex-chromosome loss

Nuclei isolated from normal human brain tissue, collected from six autopsies, were hybridized with a panel of nine satellite DNA probes specific for the centromeric regions of chromosomes 1, 6, 7, 10, 11, 17, 18, and the X and Y chromosomes. The results did not confirm the recently reported trisomy 7 and loss of sex chromosomes observed in metaphases obtained from normal brain tissue after short-term cultures; however, cells of all six brains displayed somatic pairing of the chromosome 17 centromeres in approximately 50% of the nuclei.     

The fate of micronucleated cells post X-irradiation detected by live cell imaging

Micronuclei are closely related to DNA damage. The presence of micronuclei in mammalian cells is a common phenomenon post ionizing radiation. The level of micronucleation in tumor cells has been used to predict prognosis after radiotherapy in many cancers. In order to understand how irradiation-induced micronuclei affect cell fate, we performed extensive long-term live cell imaging on X-irradiated nasopharyngeal carcinoma (NPC) cells. To visualize the dynamics of micronuclei more clearly, chromosomes were stably labeled with red fluorescent protein (RFP) by targeting to human histone H2B. Initially, significantly more micronuclei were observed in radiosensitive cells than in radioresistant cells post irradiation. Additionally, cells with micronuclei were found to be more likely to die or undergo cell cycle arrest when compared with micronucleus-free cells after irradiation, and the more micronuclei the cells contained the more likely they would die or undergo arrest. Moreover, micronucleated cells showed predisposition to produce daughter cells with micronuclei through chromosome lagging. Fluorescence in situ hybridization using human pan-centromeric probes revealed that about 70% of these micronuclei and lagging chromosomes did not contain centromeric signals. Finally, DNA damage was more severe and p38 stress kinase activity was higher in micronucleated cells than in micronucleus-free cells as shown by phospho-H2AX and phospho-p38 immunofluorescence staining. Altogether, our observations indicated that the presence of micronuclei coupled with activated DNA damage response could compromise the proliferation capacity of irradiated cells, providing the evidence and justification for using micronucleus index as a valuable biomarker of radiosensitivity.     

Chromosome nondisjunction and loss induced by protons and X rays in primary human fibroblasts: role of centromeres in aneuploidy

To study the origin of micronuclei induced in human primary fibroblasts by low-energy protons (7.7 and 28.5 keV/microm) and X rays, we have developed a combined antikinetochore-antibody (CREST) and FISH staining with pancentromeric probes. This technique allowed us to analyze the integrity of the kinetochore and centromeric DNA structures and to assess their role in induced aneuploidy. The effect of LET on radiation-induced chromosome nondisjunction was studied in binucleated cells with centromeric-specific DNA probes for chromosomes 7 and 11. Our results indicate that, though more than 90% of radiation-induced micronuclei were CREST(-)/FISH(-), 28.5 keV/microm protons and X rays were also able to induce statistically significant increases in the number of micronuclei that were CREST(-)/FISH(+) and CREST(+)/FISH(+), respectively. One interpretation of these results could be that the protons induced chromosome loss by kinetochore detachment or by breakage in the centromeric DNA region, whereas X rays induced aneuploidy through a non-DNA damage mechanism. Nondisjunction appears to be a far more important mechanism leading to radiation-induced aneuploidy. Irrespective of the higher frequency of micronuclei induced by 28.5 keV/microm protons, the frequency of chromosome loss was markedly higher for X rays than for 28.5 keV/microm protons, strengthening the hypothesis that non-DNA targets, such as components of the mitotic spindle apparatus, may be involved in aberrations in chromosome segregation after X irradiation.     

Age-dependent inclusion of sex chromosomes in lymphocyte micronuclei of man.

Two-color centromeric FISH was used to study the inclusion of the X and Y chromosomes in micronuclei of cultured lymphocytes from 10 men representing two age groups (21-29 years and 51-55 years). In addition, pancentromeric FISH was separately performed to identify any human chromosomes in micronuclei. One hundred micronuclei per probe were examined from each donor. A higher mean frequency of Y-positive micronuclei was observed in the older men than in the younger men. In both age groups, the X chromosome was micronucleated clearly more often than expected by chance, and the Y chromosome was overrepresented in micronuclei among the older men but not among the younger men. In lymphocytes of four women, X-positive micronuclei were more frequent than they were in men, even after the fact that women have two X chromosomes was taken into account. Similar results were obtained in first-division lymphocytes identified by cytochalasin-B-induced cytokinesis block. In comparison with normal cells, these binucleate cells showed a higher frequency (per 1,000 nuclei) of X-positive micronuclei (in the older men) but a lower frequency of micronuclei harboring autosomes or acentric fragments. In conclusion, the results show that both the X chromosome and the Y chromosome are preferentially micronucleated in male lymphocytes, the Y chromosome only in older subjects. Although the X chromosome has a general tendency to be included in micronuclei, it is micronucleated much more often in women than in men, which is probably the main reason for the high micronucleus frequency in women that has been documented in many previous studies.     

PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis.

Alpha satellite DNA is a tandemly repetitive DNA family found at the centromere of every human chromosome. Chromosome-specific subsets have been isolated for over half the chromosomes and have prove useful as markers for both genetic and physical mapping. We have developed specific oligonucleotide primer sets for polymerase chain reaction (PCR) amplification of alpha satellite DNA from chromosomes 3, 7, 13/21, 17, X, and Y. For each set of primers, PCR products amplified from human genomic DNA are specific for the centromere of the target chromosome(s), as shown by somatic cell hybrid mapping and by fluorescence in situ hybridization. These six subsets represent several evolutionarily related alpha satellite subfamilies, suggesting that specific primer pairs can be designed for most or all chromosomal subsets in the genome. The PCR products from chromosome 17 directly reveal the polymorphic nature of this subset, and a new DraI polymorphism is described. The PCR products from chromosome 13 are also polymorphic, allowing in informative cases genetic analysis of this centromeric subset distinguished from the highly homologous chromosome 21 subset. These primer sets should allow placement of individual centromeres on the proposed STS map of the human genome and may be useful for somatic cell hybrid characterization and for making in situ probes. In addition, the ability to amplify chromosome-specific repetitive DNA families directly will contribute to the structural and functional analysis of these abundant classes of DNA.     

Preservation of chromosome integrity during micronucleation induced by colchicine in PtK1 cells

Colchicine induces the formation of small nuclei called micronuclei which contain limited parts of the genome. Some of them exhibit a DNA content equivalent to that of a single chromosome. Our purpose was to study the preservation of chromosome integrity during this micronucleation in PtK₁ cells. Observation of karyotypes obtained after 3 days of cell cycle restoration revealed that micronucleation did not affect chromosome integrity or the presence of each chromosome pair in the surviving cells. In early restoration cells, all the chromosomes included a centromere and were represented in the karyotype, but at variable rates. Furthermore, flow cytometry analysis of micronucleated cells, intermediate in DNA rate between control PtK₁ cells in g₁ and those in G₂/M phases, led us to consider the possibility of selective replication of some chromosomes during micronucleation. Using antibodies against the kinetochore proteins, we derived the presence of one centromeric region (1–2 spots) in the smallest micronuclei. Therefore, these data (karyotypes, number of chromosomes, DNA content and kinetochore proteins) seem to indicate that micronucleation does not induce chromosome damages or translocations. Micronuclei are a convenient tool for investigation of the role of the different chromosomes in the organization of the interphase nuclei.     

A degenerate alpha satellite probe, detecting a centromeric deletion on chromosome 21 in an apparently normal human male, shows limitations of the use of satellite DNA probes for interphase ploidy analysis.

A degenerate alpha satellite DNA probe specific for a repeated sequence on human chromosomes 13 and 21 was synthesized using the polymerase chain reaction (PCR). Fluorescence in situ hybridization (FISH) with this probe to normal metaphase spreads revealed strong probe binding to the centromeric regions of human chromosomes 13 and 21 with negligible cross-hybridization with other chromosomes. FISH to normal interphase cell nuclei showed four distinct domains of probe binding. However, hybridization with probe to interphase and metaphase preparations from one apparently normal human male resulted in only three major binding domains. Metaphase chromosome analysis revealed a centromeric deletion on one chromosome 21 that caused greatly reduced probe binding. The result suggest caution in the interpretation of interphase ploidy studies performed with chromosome-specific alphoid DNA probes.     

Interphase chromosomal abnormalities and mitotic missegregation of hypomethylated sequences in ICF syndrome cells

The immunodeficiency, centromeric region instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disease. Usually, it is caused by mutations in the DNA methyltransferase 3B gene, which result in decreased methylation of satellite DNA in the juxtacentromeric heterochromatin at 1qh, 16qh, and 9qh. Satellite II-rich 1qh and 16qh display high frequencies of abnormalities in mitogen-stimulated ICF lymphocytes without these cells being prone to aneuploidy. Here we show that in lymphoblastoid cell lines from four ICF patients, there was increased colocalization of the hypomethylated 1qh and 16qh sequences in interphase, abnormal looping of pericentromeric DNA sequences at metaphase, formation of bridges at anaphase, chromosome 1 and 16 fragmentation at the telophase–interphase transition, and, in apoptotic cells, micronuclei with overrepresentation of chromosome 1 and 16 material. Another source of anaphase bridging in the ICF cells was random telomeric associations between chromosomes. Our results elucidate the mechanism of formation of ICF chromosome anomalies and suggest that 1qh–16qh associations in interphase can lead to disturbances of mitotic segregation, resulting in micronucleus formation and sometimes apoptosis. This can help explain why specific types of 1qh and 16qh rearrangements are not present at high frequencies in ICF lymphoid cells despite diverse 1qh and 16qh aberrations continuously being generated.     

A human chromosome 9-specific alphoid DNA repeat spatially resolvable from satellite 3 DNA by fluorescent in situ hybridization.

We have isolated a DNA clone (pMR9A) that identifies an alphoid DNA subset specific for chromosome 9. This alphoid subset is characterized by a dimeric organization as revealed by Southern blot analysis after digestion with HaeIII, HinfI, or StuI. Nonradioactive in situ hybridization demonstrated that pMR9A hybridizes only to the centromeric region of chromosome 9 and reveals chromosome 9 aneuploidies in interphase nuclei. In addition, the probe detects quantitative differences in alpha satellite DNA on chromosome 9, but these quantitative differences are not correlated with the size of the heterochromatic region. Double-labeling experiments, using a chromosome 9-specific satellite 3 clone and pMR9A, enabled us spatially to distinguish the alphoid and satellite 3 domains on metaphase chromosomes after treatment of the cultures with 5-azacytidine.