Cytochemical heterogeneity of the C-band in human chromosome 1

Summary The heterogeneity of the C-band of human chromosome 1 has been evaluated using several selective staining methods: C-banding (CBG), distamycin A plus 4-6-diamidino-2-phenylindole (DA/DAPI) and Giemsa G-11 pattern following the treatment with the restriction endonucleases AluI and HaeIII. The bands produced by each method are characteristic but not identical. The total C-band is resistant to AluI treatment. The bands induced by HaeIII and the one stained by DA/DAPI are markedly similar but smaller than the C-band. The G-11 technique stains yet smaller regions than those of HaeIII and DA/DAPI. Depending on the expression of staining properties, the C-band of chromosome 1 usually consists of three subdivisions: the proximal, intermediate and distal regions, suggesting an extremely heterogeneous nature. The staining variations between different regions are further substantiated by studies of a reciprocal translocation where the proximal region and the remaining C-band of chromosome 1 are separate.     

Heterogeneity of human chromosome 9 constitutive heterochromatin as revealed by sequential distamycin A/DAPI staining and C-banding

Summary Distamycin A/DAPI staining and sequential C-banding of human lymphocyte chromosomes reveals the regular occurrence of differentially staining subfractions of chromosome 9 constitutive heterochromatin. These subfractions are regionally organized as two subsegments: a distal one, which fluoresces brightly with DAPI after preincubation with distamycin A and a proximal one, which stains intensely with Giemsa after sequential C-banding. Observations are presented that indicate an occasionally independent genetic behavior of these heterochromatin subfractions.     

Comparative analysis of micro and macro B chromosomes in the Korean field mouse Apodemus peninsulae (Rodentia, Murinae) performed by chromosome microdissection and FISH

Comparative analysis of micro B and macro B chromosomes of the Korean field mouse Apodemus peninsulae, collected in populations from Siberia and the Russian Far East, was performed with Giemsa, DAPI, Ag-NOR staining and chromosome painting with whole and partial chromosome probes generated by microdissection and DOP-PCR. DNA composition of micro B chromosomes was different from that of macro B chromosomes. All analyzed micro B chromosomes contained clusters of DNA repeats associated with regions characterized by an uncondensed state in mitosis. Giemsa and DAPI staining did not reveal these regions. Their presence in micro B chromosomes led to their special morphology and underestimation in size. DNA repeat clusters homologous to DNA of micro B chromosome arms were also revealed in telomeric regions of some macro B chromosomes of specimens captured in Siberian regions. Neither active NORs nor clusters of ribosomal DNA were found in the uncondensed regions of micro B chromosomes. Possible evolutionary pathways for the origin of macro and micro B chromosomes are discussed.     

Karyotype analysis of Nicotiana kawakamii Y. Ohashi using DAPI banding and rDNA FISH

Prometaphase cells were used to analyze the karyotype of Nicotiana kawakamii Y. Ohashi by means of sequential Giemsa/CMA/DAPI staining and multicolor fluorescence in situ hybridization with 5S and 18S rDNA. Observation of the DAPI-stained prometaphase spreads indicated that N. kawakamii had six pairs of large chromosomes, one pair of medium-sized chromosomes and five pairs of small chromosomes. The six pairs of large chromosomes possessed remarkable DAPI bands, and each could be identified from both the DAPI banding pattern and the length of the short arm. The DAPI banding pattern was approximately identical to the CMA and Giemsa banding patterns. Hybridization signals of the 18S rDNA probe were detected on two pairs of large chromosomes. In addition, two pairs of small chromosomes were identified based on the position of the 5S rDNA signals. An idiogram of N. kawakamii chromosomes was produced based on DAPI bands and rDNA loci. Received: 17 July 2000 / Accepted: 4 September 2000     

Mechanisms of chromosome banding

A thorough understanding of the mechanisms of R-, C-and G-banding will come only from studies of the binding of Giemsa dyes to isolated and characterized preparations of heterochromatin and euchromatin. Since such studies require an exact knowledge of the optical characteristics of Giemsa, the spectral adsorption curves and extinction coefficients of Giemsa and its component dyes at various concentrations in the presence and absence of DNA were determined. — Although Giemsa is a complex mixture of thiazin dyes plus eosin; methylene blue, and azure A, B or C alone gave good banding. Thionin, with no methyl groups, gave poor or no banding. Eosin was not a necessary component for banding. — The most striking characteristic of the thiazin dyes is that they are strongly metachromatic, i.e., their adsorption spectra and extinction coefficients change as the concentration of the dye increases or as they bind to positively charged compounds (chromotropes). These changes, especially for methylene blue, are described in detail and allow a distinction between concentration dependent binding to DNA by intercalation and binding by side stacking.     

The gene for human erythrocyte protein 4.2 maps to chromosome 15q15.

Protein 4.2 (P4.2), one of the major components of the red-blood-cell membrane, is located on the interior surface, where it binds with high affinity to the cytoplasmic domain of band 3. Individuals whose red blood cells are deficient in P4.2 have osmotically fragile, abnormally shaped cells and moderate hemolytic anemia. cDNA clones from both the 5' and the 3' coding regions of the P4.2 gene were used to map its chromosomal location by fluorescence in situ hybridization. The probes, individually or in combination, gave specific hybridization signals on chromosome 15. The hybridization locus was identified by combining fluorescence images of the probe signals with fluorescence banding patterns generated by Alu-PCR (R-like) probe and by DAPI staining (G-like). Our results demonstrate that the locus of the P4.2 gene is located within 15q15.     

Centromeric alpha satellite DNA amplification and translocation in an unusually large chromosome 14p+ variant

Summary We report cytogenetic and molecular studies on a family that carries, in the father, an unusually large chromosome 14p+ variant [WSi-var(14)(p+)] and, in one of his children, a translocation [DSi-der(14)] involving the variant chromosome. Increase in the size of WSi-var(14)(p+) was estimated to be approximately 35% that of a normal chromosome 14. Presence of extra chromosomal material in this variant chromosome was demonstrated by G-banding using trypsin and staining with Leishman, G-banding using bromodeoxyuridine (BrdU) and Giemsa, and R-banding using BrdU and Giemsa. This material was positive using C-banding with BaOH and staining with Giemsa and negative in DAPI/distamycin staining, suggesting that it contained repetitive DNA but probably not of the types found in the heterochromatic regions of chromosomes 1, 9, 15, 16, and Y. Staining of the nucleolus organiser region (NOR) with AgNO₃ indicated the retention of the NOR in WSi-var(14)(p+) but not in DSi-der(14). In situ hybridisation of metaphase cells with an alpha satellite DNA probe specific for human acrocentric chromosomes demonstrated a significantly increased amount of centromeric alpha sequences in WSi-var(14)(p+). Most or all of the extra alpha sequences were retained in DSi-der(14), indicating translocation near the very distal end of the enlarged region. The extra alpha satellite DNA material may have originated through amplification of some centromeric segments. The possible role of the amplified DNA in chromosomal translocations is discussed.     

Interstitial deletion of chromosome 9q with coexistence of the deleted segment as a ring chromosome. A case report.

In a mentally retarded female an interstitial deletion of a chromosome 9 and an additional ring chromosome was shown, which by positive hybridisation with a no 9 library was considered to be the excised segment. The functional centromere and C and DA/DAPI positive material as well on the ring chromosome are explained by a break within the centromere close to the constitutive heterochromatin and supports the hypothesis of "latent" centromere(s).     

An extra idic(15p)(q11) chromosome in Prader-Willi syndrome

Using a nonfluorescent AT-specific oligopeptide antibiotic, Distamycin A, on DAPI fluorescent banding of human chromosome (DA-DAPI) as described by Schweizer et al. (1978), we have detected an additional idic(15p) chromosome in a patient with typical Prader-Willi syndrome. On the basis of the evidence available in previous studies and of our own present results, we suspect that the fundamental genetic error in the syndrome is not caused by a chromosome aberration but by a gene aberration on chromosome 15.     

Tentative assignment of piebald trait gene to chromosome band 4q12

Summary A case of de novo del(4)(q12q21.1) is presented. Three of four patients with comparable deletion show abnormal integumentary pigmentation, which is compatible with the known autosomal dominantly inherited piebald trait. Further analysis of breakpoints of five cases with proximal interstitial 4q deletion suggests the possible localization of the piebald trait gene within the band 4q12.Dedicated to Professor Dr. med. G. G. Wendt, a founding editor of the journal, on the occasion of his 65th birthday     

Rapid irradiation procedure for obtaining permanent differential staining of sister chromatids and aspects of its underlying mechanism

Summary When fixed metaphase preparations of lymphocytes cultured in the presence of BrdU during two cell cycles are subjected to a 1-min simple irradiation treatment with near-ultraviolet light (radiation dose 3×10⁵ J/m²), subsequent Giemsa staining produces differential staining of sister chromatids irrespective of previous exposure to a photosensitizer. The effects of this procedure were analyzed by irradiating single metaphases under the microscope, thus allowing precise dosage of radiation: Metaphase were subsequently stained with Giemsa and then subjected to the Feulgen-Schiff procedure. Whereas in the presence of DAPI as a photosensitizer a differential breakdown of BrdU-containing DNA in the chromatids under the influence of irradiation appeared to be the cause of sister chromatid differentiation, alterations presumably in the higher oeder structure of chromatin, not accompanied by removal of DNA, induced sister chromatid differentiation without DAPI.     

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

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

Banding Human Chromosomes Using a Combined C-Banding-Fluorochrome Staining Technique

Slides pretreated for C-banding and stained with DAPI or CMA3 show different banding patterns in human metaphase chromosomes compared to those obtained with either standard Giemsa C-banding or fluorochrome staining alone. Human chromosomes show C-plus DA-DAPI banding after C-banding plus DAPI and enhanced R-banding after C-banding plus Chromomycin A3 staining. If C-banding preferentially removes certain classes of DNA and proteins from different chromosome domains, C-banding pre-treatment may cause a differential DNA extraction from G- and R-bands in human chromosomes, resulting in a preferential extraction of DNA included in G-bands. This hypothesis is partially supported by the selective cleavage and removal of DNA from R-bands of restriction endonuclease HaeIII with C-banding combined with DAPI or Chromomycin A3 staining. Structural factors relating to regional differences in DNA and/or proteins could also explain these results.     

A modified system for analyzing ionizing radiation-induced chromosome abnormalities

The analysis of dicentric chromosomes in human peripheral blood lymphocytes (PBLs) by Giemsa staining is the most established method for biological dosimetry. However, this method requires a well-trained person because of the difficulty in detecting aberrations rapidly and accurately. Here, we applied a fluorescence in situ hybridization (FISH) technique, using telomere and centromere peptide nucleic acid (PNA) probes, to solve the problem of biological dosimetry in radiation emergency medicine. A comparison by a well-trained observer found that FISH analysis of PBLs for the dose estimation was more accurate than the conventional Giemsa analysis, especially in samples irradiated at high doses. These results show that FISH analysis with centromeric/telomeric PNA probes could become the standard method for biological dosimetry in radiation emergency medicine.     

Chromosome condensation from prophase to late metaphase: relationship to chromosome bands and their replication time

As chromosomes condense during early mitosis, their subbands fuse in a highly coordinated fashion. Subband fusion occurs when two large subbands flanking one minor subband come together to form one band, which takes on the cytological characteristics of the original flanking subbands. Using four different banding techniques--GTG (G-bands obtained with trypsin and Giemsa), GBG (G-bands obtained with BrdU and Giemsa), RHG (R-bands obtained by heating and Giemsa), and RBG (R-bands obtained with BrdU and Giemsa)--we studied subband fusion from prophase (1,250 bands per haploid set) to late metaphase (300 bands). To quantify the condensation process, a fusion index was established. We found that chromosomes contain preferential zones of condensation. From prophase to late metaphase, the early replicating subbands (R-subbands) fuse more readily with each other than do the late-replicating subbands (G-subbands). R-bands usually replicate early and condense late independently of the adjacent G-bands, which replicate late but condense early. Therefore, chromosome bands can undergo DNA replication and chromatin condensation relatively autonomously. Our data suggest that (1) chromosome replication and condensation are closely connected in time, (2) the metaphase bands represent independent units of chromatin condensation, and (3) the condensation process is an important feature of chromosome organization.     

Nuclear Staining of Colletotrichum Gloeosporioides F. SP. Malvae Conidia with Fluorescent and Nonfluorescent Stains

Six different staining techniques were evaluated for their suitability to stain nuclei of Colletotrichum gloeosporioides f. sp. malvae (C.g.m.) spores. Of the three fluorescent stains, DAPI (4',6-diamidino-2-phenylindole) and bisbenzimide (Hoechst 33258) stained spore nuclei well; mithramycin did not. To achieve consistent results with the bisbenzimide staining protocol, the spores had to be fixed prior to staining and the stain had to be supplemented with Triton X-100. Both safranin O and Giemsa were suitable nonfluorescent staining techniques; lomofungin was not. Safranin O staining was simple and rapid. However, reproducibility was better if the spore suspension and KOH droplets were rapidly mixed prior to adding the stain. There was no significant difference in the percentages of uninucleate and binucleate spores observed in spore preparations stained with DAPI, bisbenzimide, safranin O or Giemsa. Bisbenzimide and safranin O were found to be simple, rapid and reliable fluorescent and nonfluorescent techniques, respectively, for staining nuclei of C.g.m. spores.     

Sequential G- to R-banding for high resolution chromosome analysis

Summary A sequential staining protocol for revealing first G-and then R-bands on early metaphase chromosomes is presented. Lymphocyte cultures are synchronized with methotrexate, and released from the S-phase block with bromodeoxyuridine. The resulting early metaphase cells are initially G-banded with Wright's stain and then R-banded by the fluorescence plus Giemsa (FPG) technique.     

Genotoxicity of cadmium chloride in human lymphocytes evaluated by the comet assay and cytogenetic tests

Peripheral blood lymphocytes were tested in vitro for genotoxic effects of cadmium chloride. Whole blood samples of four healthy, non-smoking subjects were preincubated with CdCl2 in concentrations of 10(-4), 10(-3), and 5 . 10(-3) mol/L for three hours before the cells were assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay) or cultivated for chromosomal aberrations (CA), sister chromatid exchanges (SCE), and the micronucleus (MN) test. The comet assay showed notable interindividual differences. The results of the cytogenetic tests showed an increase in the frequency of CA, MN, and SCE with CdCl2 in the treated cultures, yet none was able to show a correlation between concentrations of cadmium chloride and the frequency of damages. The MN slides were stained with Giemsa and with DNA fluorochrome 4', 6'-diamidino-2-phenylindole (DAPI). The frequency of MN in slides stained with DAPI was significantly higher than in those stained with Giemsa, which might be due to an underestimation of small micronuclei in Giemsa-stained slides.