Reverse sister chromatid differential staining by Coomassie Brilliant Blue R-250

A sister chromatid differential staining pattern is observed if chromosomes replicate for two cycles in the presence of 5-bromodeoxyuridine (BUdR) and are subsequently stained in Hoechst 33258, irradiated with black light, and then stained in Coomassie Brilliant Blue R-250. In this pattern the chromatids containing DNA that is bifilarly substituted with BrdUrd are darkly stained and the chromatids with DNA that is unifilarly substituted are lightly stained. This staining pattern is the reverse of that found when slides are stained in Hoechst plus Giemsa. Slides stained with either Giemsa or Coomassie Blue can be destained and restained repeatedly with the other stain to alternate the pattern observed.     

Reverse differential staining of sister chromatids induced by Hoechst plus black light and endonuclease

A differential Giemsa staining between sister chromatids was obtained by treating chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) with Hoechst 33258 plus black light at 55 degrees C (HB pretreatment) and deoxyribonuclease (DNase) I, II, or micrococcal nuclease. In this staining pattern the BrdU bifilarly substituted chromatids were darkly and the unifilarly substituted chromatids lightly stained. This staining pattern was obtained only by staining the HB-DNase I-treated chromosomes with Giemsa and methylene blue, not by several other dyes tested. Relatively more DNA labelling was removed from the non-BrdU-substituted than the BrdU-substituted chromosomes, when the HB-pretreated chromosomes were digested with DNase I. But the protein labelling was not removed appreciably in the same treatment. The differential DNase I sensitivity between the non-BrdU-substituted and BrdU-substituted chromosomes disappeared when the HB-pretreated chromosomes were incubated with proteinase K before The DNase I digestion. Moreover, no differential DNase I sensitivity was found between the HB-pretreated isolated DNA containing and not containing BrdU. We propose that during the HB pretreatment, more DNA-protein cross-linkings are induced in BrdU bifilarly substituted than the unifilarly substituted chromatids. This structure protects the chromosomal DNA against the DNase I digestion. Thus, a reverse differential Giemsa staining between sister chromatids is obtained by the HB-DNase I treatment.     

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.     

A relation between G-, C-, and N-band patterns as revealed by progressive oxidation of chromosomes and a note on the nature of N-bands

Near-ultraviolet irradiation of chromosome preparations mounted in a hydrogen peroxide solution resulted in an oxidative disintegration of the structure of fixed metaphase chromosomes with concomitant production of various band patterns appearing after staining with Giemsa. Neither irradiation nor hydrogen peroxide alone could produce banding. After irradiation in the presence of hydrogen peroxide the gradually increasing effect of oxidation on the chromosomes along the gradient of light intensities from the periphery of the slide towards the radiation focus in the centre of the slide became visible as G-, C-, and N-banding, respectively. Close to the centre only contours of chromosomes were left after this treatment. Although G-banding and differential DNA-extraction often went together, extraction of DNA was not an absolute requirement to obtain a G-band pattern. N-bands appeared to be the chromosomal regions that were most resistant to destruction. Staining methods specific for DNA failed to demonstrate these bands, although with Giemsa an intense staining reaction occurred. On the analogy of the staining behaviour of model protein preparations with Giemsa a phosphoprotein nature is suggested for the N-band material in the chromosomes.     

Involvement of sulfhydryl groups of chromosomal proteins in sister chromatid differentiation

The fluorescence of human lymphocyte chromosomes stained with sulfhydryl group-specific fluorochromes is markedly enhanced by a mild near-ultraviolet irradiation pretreatment, indicating breakage of protein disulfide bonds. When metaphase preparations of cells cultured in the presence of BrdU during two cell cycles are irradiated and subsequently stained with the sulfhydryl group-specific fluorescent reagents used in this study, a differential fluorescence of sister chromatids is observed. After staining with the DNA-specific fluorochrome DAPI an opposite pattern of lateral differentiation appears. It can be concluded that the chromatid containing bifilarly BrdU-substituted DNA has a higher content of sulfhydryl groups than the chromatid containing unifilarly BrdU-substituted DNA. This implies a more pronounced effect of breakage of disulfide bonds in the chromatid with the higher degree of BrdU-substitution. BrdU-containing chromosomes pretreated with the mild near-ultraviolet irradiation procedure used by us, do not show any differentiation of sister chromatids after Feulgen staining. Using sulfhydryl group-specific reagents, differential fluorescence of sister chromatids could still be induced by irradiation with near-ultraviolet light after the complete removal of DNA from the chromosomes by incubation with DNase I. Thus, the protein effect of irradiation of BrdU-containing chromosomes takes place independently of what occurs to DNA.Our results indicate that subsequent to the primary alteration of chromatin structure caused by the incorporation of BrdU into DNA, breakage of disulfide bonds of chromosomal proteins might play an important role in bringing about differential staining of sister chromatids, at least for those procedures that use irradiation as a pretreatment or prolonged illumination during microscopic examination.     

Labelling of DNA and differential sister chromatid staining after BrdU treatment in vivo

A method of labelling DNA in vivo with 5-bromodeoxyuridine (BrdU) is described. After 6 h permanent subcutaneous infusion of BrdU in rodents (adult Microtus agrestis, pregnant NMRI-mice), cell nuclei which have undergone DNA synthesis during the BrdU treatment can be differentiated from the nuclei of other cycle stages by means of their altered staining behaviour after Giemsa. 24 h after the BrdU treatment, mitoses from both bone marrow of the adult animals and tissues from the fetuses showed a differential sister chromatid staining. In male M. agrestis, sister chromatid exchanges were most frequently found in the euchromatic part of the X and in the constitutive heterochromatin of both sex chromosomes.     

Light and scanning electron microscopic observations on the two contrasting types of sister chromatid differential staining after ultraviolet light irradiation

Chinese hamster cells were grown with 50 M 5-bromodeoxyuridine (BrdU) during the penultimate S phase to obtain chromosomes with the TB-TT chromatid constitution. Chromosome preparations made by the air-drying method were used to study the sister chromatid differential staining (SCD) resulting from ultraviolet (UV) irradiation followed by Giemsa staining by light and scanning electron microscopy (SEM). When chromosomes irradiated with UV light (253.7 nm, 5.2 J/m²/s) for more than 5 h were stained with 1% to 4% Giemsa in phosphate buffered saline (PBS) or in distilled water, the resulting SCD invariably belonged to the B-light type in which the TB-chromatid stained lightly. SEM observations of these chromosomes suggested that the B-light SCD was due to the selective photolysis of the TB-chromatid. On the other hand, when chromosomes were irradiated for only 10 min, and stained with 1% Giemsa in PBS, they showed a B-dark type SCD in which the TB-chromatid stained darkly. However, when chromosomes irradiated for 10 min were stained with 4% Giemsa in PBS or 1% Giemsa in distilled water, the resulting SCD again belonged to the B-light type. These findings indicate that when the irradiation dose is small, the resultant SCD is not a simple reflection of selective photolysis in the TB-chromatids and the type of SCD depends not only on the concentration of Giemsa but also on the salinity of the staining solution.     

Different effects of 33258 Hoechst and DAPI in fluorescent staining of sister chromatids differentially substituted with bromodeoxyuridine

Summary After substitution with 5-bromodeoxyuridine (BrdUrd) for two rounds of replication, chromosomes in cytological preparations stained with 33258 Hoechst show upon epiluminescence an immediate differential sister chromatid fluorescence. When stained with DAPI, however, which has a structural resemblance to part of the 33258 Hoechst molecule, such a differential pattern of fluorescence was only induced after some delay. Upon restaining with the same dye the differential fluorescence appeared instantly. In preparations double stained with ethidium bromide and 33258 Hoechst the induction of a differential staining of sister chromatids with 33258 Hoechst was not accompanied by a differential staining with ethidium bromide. Once a differential staining was obtained with DAPI in preparations double stained with ethidium bromide and DAPI, the ethidium bromide pattern also appeared to be differential upon subsequent observation. No differentiation could be obtained with ethidium bromide alone. The observations described in the case of 33258 Hoechst staining are in agreement with a molecular quenching by BrdUrd without gross structural consequences for the DNA. In the case of DAPI staining, however, there occurs a differential photolysis of BrdUrd-substituted DNA. Besides the nature, most likely the size, of the fluorochrome molecules themselves, the state of the fixed chromatin appeared also to play a role in determining the mechanism of the sister chromatid differentiation: after prolonged incubation in buffer, BrdUrd-containing chromosomes stained with 33258 Hoechst showed a differential staining evidently caused by photolysis, indicating that they had become more susceptible to light.     

Differential inhibition of sister chromatid condensation induced by 5-azadeoxycytidine in human chromosomes

The deoxycytidine analogue 5-azadeoxycytidine (5-aza-dC) induces differential inhibition of sister chromatid condensation when cells are treated with this substance for two replication cycles, as the subsequent staining of metaphase chromosomes with Giemsa shows. The bifilarly substituted chromatid is dramatically longer than the unifilar one. A percentage of the metaphases treated with 5-azad-C even show a complete undercondensation of the bifilarly substituted chromatid. The optimum conditions for inducing sister chromatid differentiation were determined. No method has been developed as yet to permit enhancement of the differential staining in 5-aza-dC-treated preparations. The interactions between 5-aza-dC and chromosomal DNA as well as the factors involved in the differential staining of sister chromatids are discussed.     

“Reverse” differential staining of sister chromatids

A technique for the differential staining of sister chromatids with basic fuchsin is described. The resulting staining pattern is the reverse of that obtained with a similar technique using Giemsa dye.     

On the mechanism of differential Giemsa staining of bromodeoxyuridine-substituted chromosomes

Summary Experiments were performed to find out whether different mechanisms are involved in FPG-(fluorescent plus Giemsa) staining for the demonstration of replication patterns and sister chromatid differentiation (SCD) after bromodeoxyuridine (BrdU)-substitution of V79 Chinese hamster chromosomes. The influence of variations of the staining procedure on the quality of both SCD and replication patterns was comparatively investigated and differences in the demonstration of these two phenomena within the same chromosome were studied using various BrdU-labeling protocols. The results show that at least graduated differences exist. For a good differentiation of replication patterns a stronger FPG-treatment is necessary than it is for SCD. Partial BrdU substitution only leads to replication patterns in the next mitosis. A further round of replication either in the presence or absence of BrdU causes a reduced staining of the complete chromatid and three-way differentiation is seen in third generation mitoses. These results support the view that alterations of chromosomal proteins during BrdU-incorporation and replication of BrdU-substituted DNA are decisive for differential staining.     

Topographic examination of sister chromatid differential staining by nomarski interference microscopy and scanning electron microscopy

BrdU-substituted Chinese hamster chromosomes were treated with a hot Na₂HPO₄ solution and stained with Giemsa to produce sister chromatid differential staining (SCD). The process of SCD was examined with the Nomarski differential interference microscope and the scanning electron microscope. After the Na₂HPO₄ treatment alone, unifilarly BrdU-substituted (TB) chromatids appeared somewhat more severely collapsed than the bifilarly substituted (BB) chromatids. Subsequent Giemsa staining, however, brought about pronounced piling up of the Giemsa dye on the TB-chromatids but not on the BB-ones, causing highly distinct differential Giemsa staining as well as a marked differentiation in surface topography between the sister chromatids. Removal of the Giemsa dye from the differentially Giemsa stained chromosomes resulted in a disappearance of such a pronounced topographic differentiation.     

The role of chromosomal proteins in the C-banding of Allium cepa chromosomes.

When chromosomes of Allium cepa are subjected to a C-banding procedure (incubation in saturated barium hydroxide followed by phosphate buffer at 60 degrees C for 1 h) and then treated with Giemsa stain, bands appear at the telomeres of all chromosomes. Microspectrophotometric measurements of Feulgen-DNA content, demonstrated that the C-banding procedure extracted DNA from the nuclei. Staining of banded chromosomes with several DNA-specific stains showed that this loss was differential, with the band DNA exhibiting more resistance to extraction than that of the rest of the chromosome. The C-banding procedure did not extract chromosomal proteins, however, and no difference in mass per unit length could be detected by Nomarski optics between band and interband regions. Several experiments demonstrated that chromosomal proteins play a significant role in C-banding. First, treatment of chromosomes with pronase before C-banding resulted in the elimination of differential staining with Giemsa. Furthermore, in preparations where the DNA was completely hydrolysed with hot TCA, the remaining chromosomal proteins were found to exhibit a differential affinity for Giemsa stain. Amido black staining demonstrated that total chromosomal protein was uniformly distributed after the hot TCA digestion, but the proteins localized in the telomeres had a greater affinity for the Giemsa stain than the bulk of the chromosomal proteins. When the TCA-digested chromosomes were subjected to the C-banding procedure before staining, the differential affinity of the telomeres for the Giemsa stain was lost. Thus, C-banding appears to be the result of a complex interaction between protein and DNA in which the greater resistance to extraction of the band DNA is necessary to stabilize and preserve chromatin protein which exhibits a differential affinity for Giemsa stain.     

Two opposite types of sister chromatid differential staining in BUdR-substituted chromosomes using tetrasodium salt of EDTA

The direct staining of BUdR-substituted Chinese hamster chromosomes in a 4Na-EDTA-Giemsa solution resulted in a B-dark type of sister chromatid differential staining (SCD) in which bifilarly substituted chromatids stained dark. On the other hand, when BUdR-substituted chromosomes were pretreated with a 4Na-EDTA solution and then stained with Giemsa, a B-light type SCD was obtained in which bifilarly substituted chromatids stained light.     

The mechanism of differential sister chromatid fluorescence as studied with the GC-specific DNA-ligand mithramycin

Chromosomes of human blood cells exposed to BUdR for two cell cycles showed an R-band pattern of fluorescence without lateral differentiation after staining with the GC-specific DNA-fluorochrome mithramycin. Differential sister chromatid fluorescence could be induced by a mild near-ultraviolet irradiation pretreatment which was without effect in Feulgen staining. Thus, besides the primary alteration of DNA structure caused by incorporation of BUdR, secondary structural alterations, probably mediated via chromosomal proteins, are required in order to obtain differential mithramycin-fluorescence of sister chromatids. The differential staining pattern was similar to that achieved with the AT-specific DNA-fluorochrome DAPI. Therefore, it may be concluded that the base specificity of fluorochromes does not play any part in the production of differential fluorescence of sister chromatids by this method.     

The differential staining of murine metaphase chromosomes in early embryogenesis after treatment with restrictase AluI in situ]

The picture of differential staining of early mouse embryogenesis metaphasic chromosomes, from the first cleavage up to 10 days of gestation, after digestion by restriction endonuclease AluI was studied. It was shown that depending on the degree of digestion by endonuclease differential bandings of G+C- or C-type were observed. After the least digestion only the first cleavage chromosomes were differently stained. A slight difference in intensity of staining between paternal and maternal chromosomes of the zygote was observed. All the mouse chromosomes were identified after AluI digestion and staining after Giemsa.     

Studies of Chromosome Banding with Giemsa in Vicia faba and Allium cepa

Giemsa dye is a complex mixture containing methylene blue, its oxidation products-azure Ⅰ, Ⅱ, Ⅲ, and their eosinate. The results of our experiments have demonstrated that staining with methylene blue alone can give a faint trace of banding as well as azure Ⅰ, Ⅱ. No bands are obtained with eosin. Nevertheless, good chromosome bandings can be often produced by staining with methylene blue-eosinate or azure Ⅱ-eosinate. These data indicate that eosinate has an important effect for the formation of C-banding on plant chromosomes. In our experiments, the treatments of chromosomes with trypsin or papain have also resulted in good C-banding pattern when slides are stained with Giemsa. We found that the slides untreated with proteinase showed homogeneous intense chromosome staining and, on the contrary, the slides treated with proteinase led to palestaining chromosomes and presenting bandings. It has shown that proteinase, especially trypsin, not only can remove a large amount of chromosomal protein but also can remove DNA and results in C-bandings. Treated properly with trypsin and followed by the Feulgen staining, chromosomes can also produce the C-bandings, but chromosomes treated overtime with trypsin are stained more palely in Feulgen reaction or lead to colourlessness. The above results have further proved that trypsin technique removes large amounts of chromosome DNA and removes less from the C-band regions than from the non-band regions. In this paper we mainly discussed the effects of protein on mechanism of plant chromosome banding. We consider that the production of plant C-banding is probably due to the differential accessibility of nucleoprotein between euehromatin and heteroehromatin regions. It brings about selective removal of nucleoprotein from the chromosome arms. We have compared the effect of trypsin with papain and pepsin on producing bands. Good bands are produced by Giemsa staining chromosomes with trypsin, but no bands are obtained by staining chromosomes treated with pepsin. So the results have expressed that histones are possibly playing more important role in C-bandings.     

Effects on chromosomal proteins in sister chromatid differentiation by incorporation of 5-bromodeoxyuridine into DNA

When fixed metaphase chromosomes of human lymphocytes grown in the presence of BrdUr for two cell cycles were stained with amino group-specific 2-methoxy-2,4-diphenyl-3(2H)furanone (MDPF) after a previous extraction of DNA, sister chromatids showed a light-independent differential staining. Although more faintly differential, a similar staining pattern being just the reverse of the DNA-specific DAPI pattern was obtained without prior removal of DNA. We conclude that the chromatid containing bifilarly BrdUr-substituted DNA has a higher protein content, at least after fixation, than the chromatid containing unifilarly BrdUr-substituted DNA. Possibly, a higher degree of BrdUr substitution leads to a tighter binding of chromosomal proteins. In line with this suggestion we found a markable difference in DNA extractability of BrdUr-containing and normal cytological preparations.     

Recovery of dissected C-band regions in Crepis chromosomes

Summary Chromosome samples were prepared on a plastic coverslip covered with a polyester membrane and were subjected to the C-banding treatment. The C-band pattern was obtained after Giemsa staining. The C-band positive regions of the Crepis chromosomes were identified, dissected out by irradiation with a micro-laser beam and recovered in Eppendorf tubes.     

Giemsa banding,quinacrine fluorescence and DNA-replication in chromosomes of cattle (Bos taurus)

Almost all the 30 chromosome pairs of cattle can be identified by their banding patterns made be visible by a Giemsa staining technique described previously. The banding pattern of the X chromosome shows striking similarities with the banding pattern of the human X chromosome. — The centromeric region of the acrocentric autosomes contains a highly condensed DNA. This DNA is removed by the Giemsa staining procedure as can be shown by interference microscopic studies. If the chromosomes are stained with quinacrine dihydrochloride these centromeric regions are only slightly fluorescent. — Autoradiographic studies with ³H-thymidine show that the DNA at the centromeric regions starts and finishes its replication later than in the other parts of the chromosomes.