Reverse fluorescent chromosome banding with chromomycin and DAPI

Two DNA binding guanine-specific antibiotics, chromomycin A₃ (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI. — In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI. — Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The CMA-banding pattern appears to be similar to the pattern found by R-banding procedures.     

Giemsa banding of a human metacentric chromosome number 9

Summary Giemsa staining procedures have been used on human lymphocyte preparations to locate constitutive heterochromatin (C-bands) and to produce distinct banding patterns for each individual chromosome (G-bands). A metacentric C group chromosome has been shown by both techniques to be chromosome number 9.

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Zusammenfassung An menschlichen Lymphocyten wurde eine Giemsafärbung durchgeführt, mit dem Ziel, konstitutives Heterochromatin (C-Banden) zu lokalisieren und für jedes einzelne Chromosom besondere Bandmuster herauszuarbeiten (G-Banden). Beide Techniken erlaubten, ein metazentrisches C-Chromosom als Nr. 9 zu identifizieren.

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Supported by PHS grants RR-62 and 5-SO1-RRO-5411.     

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.     

Mechanisms of chromosome banding

The interaction of Hoechst 33258 with DNA has been examined to help clarify the mechanisms of banding. 1. In agreement with previous studies Hoechst fluorescence is enhanced to a greater degree in AT-rich compared to GC-rich DNA. 2. Hoechst causes an increase in the DNA Tm which is greater at the higher AT content of the DNA. 3. There is a decrease in extinction coefficient and shift in the adsorption spectra to a higher wavelength when Hoechst binds to DNA. 4. DNA is completely precipitated at a ratio of one dye molecular per base pair, and this precipitation is not affected by salt. 5. There is no increase in viscosity or change in the circular dichroism of DNA when bound to Hoechst. These findings suggest Hoechst does not bind to DNA by intercalation or by ionic interaction with the phosphate groups, but rather binds by an attachment to the outside of the double DNA helix by interacting with the base pairs. This type of binding allows greater sensitivity to the base composition than occurs with intercalating agents. In this respect its binding is similar to that of dibutyl proflavine (Muller et al., 1973).     

Chromosome banding pattern conservatism in birds and nonhomology of chromosome banding patterns between birds,turtles, snakes and amphibians

The G-banded karyotypes of 4 species of birds representing the orders Galliformes, Columbiformes and Musophagiformes were compared. Banding pattern homology between orders was limited t 5o 5 major chromosome arms and the Z chromosome. Even in these major chromosome arms pericentric and paracentric inversions produced alteration of the banding pattern sequences. Addition of constitutive heterochromatin was responsible for changes in banding patterns in the Z chromosome. The chromosome banding patterns of an emydid turtle, Terrepene carolina, 5 species of boid snakes of the genera Liasis, Acrantophis, and Sanzinia and the African clawed-frog. Xenopus muelleri, were also compared to the bird chromosome banding patterns. No homology was observed between any of these major groups: bird, snake, turtle, amphibian. However, intergroup homology was apparent. - The data obtained do not support reports of broad interordinal direct homology of the macrochromosomes of birds and refutes the idea of a primitive bird karyotype with 3 pairs of "Agroup' chromosomes and 3 pairs of "B group' chromosomes. - The major mechanisms responsible for chromosome evolution in birds appear to be centric and tandem fusions, paracentric and pericentric inversions, and addition or deletion of heterochromatin.     

Mechanisms of chromosome banding

Photo-oxidation of mitotic human chromosomes has been used in conjunction with anti-cytosine and anti-adenosine antibodies to produce R-banding. To elucidate the mechanism of this banding procedure we have examined the effect of photo-oxidation alone on chromosomes and nuclei. With short exposures to light in the presence of dilute methylene blue, C-band areas on chromosomes 1, 9, 16 and the terminal segment of the Y stain poorly. We call this phenomena reverse C-banding. After 18 h of exposure to light the chromosomes are swollen and show very little staining with quinacrine or Giemsa. Quantitative autoradiography shows that their DNA is almost completely extracted. Cytophotometric measurements also confirm that nuclear DNA is progressively extracted according to the length of exposure to light. When chromosomes are exposed to dilute methylene blue alone, without light, G-banded chromosomes result. We suggest the following explanation for these observations. In dilute methylene blue, C-band regions take up the greatest amount of dye and after short periods of photo-oxidation the DNA of these regions is preferentially destroyed resulting in reverse C-banding. Autoradiography in photo-oxidized chromosomes suggested that this preferential destruction of C-segments occurred in our experiments. With more prolonged exposure the DNA of the G-bands regions is preferentially destroyed and staining the remaining DNA with sensitive fluorescent labeled anti-C antibodies results in R-banding.     

Mechanisms of chromosome banding

Prior studies on subfractions of mouse and Kangaroo rat DNA have suggested that variations in base concentration within a given genome may not be great enough to account for Q-banding. To examine this with another species, calf DNA was subfractionated by CsCl ultracentrifugation into GC-rich satellites and the main band DNA was further fractionated into AT-rich, intermediate and GC-rich portions. The effect of varying concentrations of these DNAs on quinacrine and Hoechst 33258 fluorescence was examined. Although with both compounds there was less fluorescence in the presence of the GC-rich satellites than main band fractions, these results per se did not answer the question of whether the variation in base composition alone was adequate to account for chromosome banding. To answer this the fluorescence observed in the presence of DNA of a given base composition was related to the fluorescence observed in the presence of DNA of 40% GC content (F/F40). This allowed the derivation of a term B which indicated the relative change in fluorescence per 1% change in base composition of DNA. To determine the percent change in fluorescence observed in Q-banding, the photoelectric recordings of Caspersson et al. (1971) were used. From these data we conclude: 1. Quinacrine is twice as sensitive to changes in base composition as Hoechst 33258. 2. Variation in the base content of DNA along the chromosome is sufficient to account for most Q-banding, except possibly for some of the extremes of quinacrine fluorescence. This was further examined with daunomycin. Even though daunomycin gives good fluorescent banding, DNAs varying in base composition from 100 to 40% GC content all resulted in the same relative fluorescence of 0.03. However, in the presence of poly (dA-dT) the relative fluorescence was 0.85, indicating a great sensitivity to very AT-rich DNA. This suggests that with daunomycin and possibly other fluorochromes, stretches of very AT-rich DNA may be more important in fluorescent banding than simple variation in mean base composition.     

Mechanisms of chromosome banding

A series of biochemical investigations were undertaken to determine the mechanism of Q-banding. The results were as follows: 1. In agreement with previous studies, highly AT-rich DNA, such as poly(dA)-poly(dT), markedly enhanced quinacrine fluorescence while GC containing DNA quenched fluorescence. These effects persisted at DNA concentrations comparable to those in the metaphase chromosome. 2. Studies of quinacrine-DNA complexes in regard to the hypochromism of quinacrine, DNA Tm, DNA viscosity, and equilibrium dialysis, indicated the quinacrine was bound by intercalation with relatively little side binding. 3. Single or double stranded nucleotide polymers, in the form of complete or partial helices, were 1000-fold more effective in quenching than solutions of single nucleotides, suggesting that base stacking is required for quenching. 4. Studies of polymers in the A conformation, such as transfer RNA and DNA-RNA hybrids, indicated that marked base tilting does not affect the ability of nucleic acids to cause quenching or enhancement of quinacrine fluorescence. 5. Salts inhibit the binding of quinacrine to DNA. 6. Spermine, polylysine and polyarginine, which bind in the small groove of DNA, inhibited quinacrine binding and quenching, while histones, which probably bind in the large groove, had little effect. This correlated with the observation that removal of histones with acid has no effect on Q-banding. 7. Mouse liver chromatin was separated into five fractions. At concentrations of quinacrine from 2×10^?6 to 2×10^?5 M all fractions inhibited to varying degrees the ability of the chromatin DNA to bind quinacrine and quench quinacrine fluorescence. At saturating levels of quinacrine two fractions, the 400 g pellet (rich in heterochromatin) and a dispersed euchromatin supernatant fraction, showed a decreased number of binding sites for quinacrine. These two fractions were also the richest in non-histone proteins. 8. DNA isolated from the different fractions all showed identical quenching of quinacrine fluorescence. 9. Mouse GC-rich, mid-band, AT-rich, and satellite DNA, isolated by CsCl and Cs₂SO₄-Ag⁺ centrifugation all showed identical quenching of quinacrine fluorescence, indicating that within a given organism, except for very AT or GC-rich satellites, the variation in base composition is not adequate to explain Q-banding. — We interpret these results to indicate that: (a) quinacrine binds to chromatin by intercalation of the three planar rings with the large group at position 9 lying in the small groove of DNA, (b) most pale staining regions are due to a decrease binding of quinacrine, and (c) this inhibition of binding is predominately due to non-histone proteins.     

Mechanisms of chromosome banding

The binding of methylene blue to DNA and chromatin treated in various ways was examined by equilibrium dialysis. The maximum r value (moles of bound dye/mole of nucleotide) was 1.0 for DNA, 0.6 for unfixed chromatin, and 0.83 for chromatin fixed in methanol-acetic acid. When fixed chromatin was treated with saline-citrate at 60° C for 3 hours, as used for G-banding chromosomes, the r value decreased from 0.83 to 0.55. When unfixed chromatin was treated as for R-banding the r values also dropped. Equilibrium dialysis indicated there was no disproportionate increase of dye binding as the concentration of DNA increased. — These results, and others, suggest that some of the Giemsa negative regions of G- and R-banded chromosomes are due to the denaturation of non-histone proteins so that they more effectively cover the DNA and prevent side binding of the thiazin dyes.     

Mechanisms of chromosome banding

The G-band patterns of mitotic metaphase chromosomes No. 1 and 2 of the Chinese hamster cells correlate closely to the chromomere patterns of the meiotic pachytene bivalents. This is interpreted to indicate that the regions of centromeric and intercalary heterochromatin, which are more tightly condensed or more tightly packaged during interphase, tend to remain so during meiosis and mitosis.     

DNA-protein interactions and chromosome banding

Pancreatic DNase I was used as a probe to study DNA-protein interactions in condensed and extended chromatin fractions isolated from Chinese hamster liver, and in human lymphocyte and mouse L cell metaphase chromosomes in situ. By studying the rate of digestion of chromatin DNA by DNase, we have previously shown that DNA in extended chromatin is more sensitive to DNase digestion than that in condensed chromatin. In the current investigation, we have examined whether this differential sensitivity of the chromatin fractions to DNase is due to differences in protein binding to DNA or differences in the degree of chromatin condensation. By “decondensing” the condensed chromatin and comparing its rate of digestion to that of untreated condensed and extended chromatin, it was found that differences in the degree of binding of proteins to DNA rather than the degree of condensation of the chromatin primarily determines the sensitivity of each fraction to DNase. Extraction of the various classes of chromosomal proteins, followed by DNase digestion of the residual chromatin revealed that both the histone and non-histone proteins protect the DNA in the chromatin fractions from DNase attack; however, the more tightly associated non-histones appear to be specifically responsible for the differential sensitivity of the chromatin fractions to DNase digestion. These non-histones may be more tightly associated with the DNA in condensed than in extended chromatin, thereby protecting the DNA in condensed chromatin against DNase attack to a greater extent than that in extended chromatin. When metaphase chromosomes were briefly digested with DNase in situ and subsequently stained with Feulgen reagent, incontrovertible C-banding and some G-banding was obtained. This DNaseinduced banding demonstrates that the DNA in C-band and possibly G-band regions is less accessible to DNase than that in the interband regions, and our biochemical data suggest that this differential accessibility is caused by differential DNA-protein binding such that the non-histones are more tightly coupled to the DNA in the G- and C-band regions than they are in the interbands. Differences in the binding of non-histones to DNA in different segments of the metaphase chromosome may be involved in the mechanism of G- and C-banding.     

The ultrastructure of the chromosome periphery in human cell lines

We studied the chromosome periphery in human HeLa and TG cells using cryomethods in electron microscopy. A contrasted layer of peripheral chromosomal material (PCM) was visible in cryo-ultrathin sections of mitotic cells. This PCM was composed of closely packed fibrils associated with granules. The PCM did not cover the entire chromosome surface but was found around most of the chromosomes and even between two chromatids. The organization of the PCM was not affected by colchicine treatment of mitotic cells. In cells prepared by quick-freezing, the PCM appeared to be a fibrous material at the chromosome periphery, and was also associated with granules that resembled interchromatin granules in size and shape. At higher magnification, direct contacts between the chromosomes and the fibrils of the PCM were observed. The cryotechniques used are known to preserve the native organization of cells. Therefore, the architecture of the perichromosomal region analysed presumably corresponds to that in vivo during mitosis. These observations show that in HeLa and TG cells, a particular structure present at the chromosome periphery in the form of PCM is persistent and ubiquitous. In addition, we showed by immunolabelling that the PCM is the specific site of accumulation of nucleolar antigens during mitosis. These two results, i.e. the identification of specific morphological structures and the compartmentation of proteins, indicate that this layer is a specific region of mitotic cells.by D. Schweizer     

Karyotype differentiation of four Cestrum species (Solanaceae) revealed by fluorescent chromosome banding and FISH

The karyotypes of four South American species of Cestrum (C. capsulare,C. corymbosum,C. laevigatum and C. megalophylum) were studied using conventional staining, C-CMA/DAPI chromosome banding and FISH with 45S and 5S rDNA probes. The karyotypes showed a chromosome number of 2n = 2x = 16, with metacentric chromosomes, except for the eighth submeta- to acrocentric pair. Several types of heterochromatin were detected, which varied in size, number, distribution and base composition. The C-CMA(+) bands and 45S rDNA were located predominantly in terminal regions. The C-CMA (+) /DAPI (+) bands appeared in interstitial and terminal regions, and the C-DAPI (+) bands were found in all chromosome regions. The 5S rDNA sites were observed on the long arm of pair 8 in all species except C. capsulare, where they were found in the paracentromeric region of the long arm of pair 4. The differences in band patterns among the species studied here, along with data from other nine species reported in the literature, suggest that the bands are dispersed in an equilocal and non-equilocal manner and that structural rearrangements can be responsible for internal karyotype diversification. However, it is important to point out that the structural changes involving repetitive segments did not culminate in substantial changes in the general karyotype structure concerning chromosome size and morphology.     

Molecular analysis of the aberrant replication banding pattern on chromosome 15 in murine T-cell lymphomas

Cytogenetic techniques revealed an altered early replication banding pattern on the distal part of chromosome 15 in some murine T-cell lymphomas. This pattern reverted back to normal replication in somatic cell hybrids that had become non-tumorigenic after fusion of leukemic cells with normal fibroblasts. The altered banding pattern was correlated with malignancy. To investigate the molecular basis of the aberrant pattern in more detail, centrifugal elutriation of cells containing bromodeoxyuridine labeled DNA was used to prepare newly replicated DNA from selected intervals of the S-phase from tumor cells, as well as from hybrid cells with the revertant phenotype. These different DNA fractions were probed for DNA sequences distributed over the distal half of chromosome 15. Only two out of ten chromosome 15 specific genes tested showed a clear change in replication timing between the two different cell lines tested. These two genes were the lymphocyte antigen-6,Ly-6, and the neighboring thyroglobulin gene,Tgn, which replicated at the beginning of S in the tumor cells and later in S in the non-tumorigenic hybrid cells.by J.A. Huberman