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.     

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.     

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.     

Reverse differential staining of sister chromatids after substitution with BUdR and incubation in sodium phosphate solution

Chinese hamster strain cells were cultured in the presence of BUdR and air-dried on slides. The chromosome preparations were incubated in 1 M NaH₂PO₄ at 88 °C for 4–6 min and stained with Giemsa. The reverse type of sister chromatid differential staining occurred, in which unifilarly BUdR-substituted chromatids stained faintly and bifilarly substituted chromatids stained darkly. Feulgen reaction performed on the same chromosomes after removing Giemsa stain showed the same type of differential staining.     

On the mechanism of differential giemsa staining of BrdU-substituted chromatids

This paper analyses the effect of acid hydrolysis on the differential Giemsa staining of 5-bromo-2deoxyuridine (BrdU) substituted chromatids in human and plant chromosomes, after treatment with a fluorochrome and light. Human lymphocytes and Allium cepa L. root tips were grown in BrdU for two or three cell cycles. Lymphocyte spreadings and meristem squashes were treated with fluorochrome Hoechst 33258, exposed to sunlight, hydrolysed with 5N HCl and stained with Giemsa. This acid hydrolysis improves the differential staining of BrdU substituted and non-substituted chromatin. It also allows the differentiation of sister chromatids with the DNA specific dye azure-A.     

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.     

An investigation of the mechanism of the reciprocal differential staining of BUdR-substituted and unsubstituted chromosome regions.

After treatment with hot NaH₂PO₄ at pH 9, BUdR-substituted and unsubstituted chromosome regions are palely and intensely stained with Giemsa, respectively; however, after treatment with the same solution at pH 4, the reciprocal staining patterns are produced, i.e. these chromosome regions are intensely and palely stained, respectively. The nature of the mechanisms responsible for this reciprocal differential Giemsa staining of BUdR-substituted and unsubstituted chromosome regions has been investigated by Feulgen staining, electron microscopy, and radioisotope analyses involving scintillation counting and autoradiography. The results indicate that different mechanisms are responsible for the two types of staining effect. The high pH NaH₂PO₄ treatment preferentially extracts BUdR-substituted DNA into the treatment solution, relative to unsubstituted DNA. The collective evidence from this and other work suggests that BUdR-substituted DNA in the chromosomes is partially photolysed by exposure to daylight during the harvesting procedure, and the degraded DNA is subsequently solubilized and extracted during the high pH treatment. This quantitative reduction of DNA in the BUdR-substituted chromosome regions results in pale Giemsa staining of these regions. The low pH NaH₂PO₄ treatment does not produce a significant extraction of either BUdR-substituted or unsubstituted DNA into the treatment solution; rather, there may be a redistribution of the unsubstituted DNA relative to the BUdR-substituted DNA such that the unsubstituted DNA is preferentially dispersed outside the boundaries of the chromosomes onto the surrounding area of the slide. It is suggested that the BUdR-substituted chromosome regions stain relatively intensely with Giemsa after the low pH treatment because the DNA in these regions is less dispersed than that in the unsubstituted regions.     

Differential giemsa staining of sister chromatids and the study of sister chromatid exchanges without autoradiography

Chinese hamster ovary cells grown for two rounds of DNA replication in the presence of BrdUrd contain sister chromatids that fluoresce differentially when stained with Hoechst 33258. If such fluorescent treatments are followed by incubation in 2 X SSC or water at 62° C and staining in 3% Giemsa, the chromosomes now contain one dark (unifilarly substituted) chromatid and one light (bifilarly substituted) chromatid, i.e. are harlequinized. These preparations do not fade and can be studied without resorting to fluorescence microscopy. Sister chromatid exchanges (SCE's) are seen with great clarity and resolution; and all the chromosomes in a cell can be scored, which is contrary to the usual experience with autoradiography. It was found that a) the yield of SCE's is dependent upon the concentration of BrdUrd in which the cells are grown and that the maximum number of SCE's that can occur spontaneously is 0.15 per chromosome per division cycle, b) the yield of SCE's doubles if the cells are exposed to visible light that can cause the photolysis of BrdUrd-containing DNA, and c) chromosomes that appear isolabelled in autoradiographic preparations come from observable multiple exchanges and are not the result of the segregation of DNA from a binemic chromosome. Furthermore, the staining patterns obtained in endoreduplicated cells clearly confirm that the polynucleotide strands of the DNA segregate into sister chromatids as though the newly synthesized strands were laid on the outside of the replicating double helix.     

Differential giemsa staining of sister chromatids after extraction with acids

Chromosomes of Chinese hamster strain cells were air-dried on slides after BrdU substitution for two or three rounds of replication. The preparations were treated with 20% PCA at 55 degrees C for 20-30 min, or 5N HCl at 55 degrees C for 15-20 min. After staining with Giemsa, unifilarly BrdU-substituted chromatids stained faintly and bifilarly substituted chromatids stained darkly. Such a pattern of sister chromatid differential staining was confirmed by the examination of metaphase cells grown with BrdU for three rounds of replication.     

Simple differential Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining

The essential steps of the 33258 Hoechst-Giemsa method for differential chromatid staining consist of (1) 33258 Hoechst treatment, (2) exposure to light, and (3) Giemsa staining. The staining was shown to be a function of the concentration of 33258 Hoechst and the light exposure. The dye was successfully replaced by various metachromatic dyes such as thionine. Two simple methods are proposed. Failure of the pale stained chromatids to restore Giemsa affinity with urea and trypsin and the diminished Feulgen reaction after light exposure suggest that not masking proteins but photolysis of the BrdU-incorporation chromatid components in the present of photosensitive dyes play a role in the differential staining.     

DNA alteration induced by ultraviolet light in human metaphase chromosomes substituted with 5′-bromodeoxy uridine: monitoring by monoclonal antibodies to double-stranded and single stranded DNA

Fixed human metaphase chromosomes, whose DNA had been substituted with 5-bromodeoxyuridine (BrdUrd) for two rounds of replication (TB/BB) or for one round in BrdUrd followed by another round in thymidine (TT/BT), were treated with ultraviolet light (UV), in the presence or in the absence of 33258 Hoechst, to produce sister chromatid differentiation (SCD). Giemsa staining was compared with staining with monoclonal antibodies to double-stranded or single-stranded DNA. We confirmed that UV acts by debrominating BrdUrd-stubstituted DNA but showed that debromination alone cannot explain all our findings. We postulated that UV-induced protein-protein cross-linking, occurring to a different extent in differently BrdUrd-substituted chromatids, may also be invoked in explaining our data. Lastly, the different behaviour of unifilarly substituted TB as opposed to BT chromatids in UV-treated chromosomes, allowed us to hypothesize that such chromatids may differ depending on whether or not newly synthesized DNA is formed on a BrdUrd-containing strand.     

Differential staining of plant chromosomes with Giemsa

Simple Giemsa staining techniques for revealing banding patterns in somatic chromosomes of plants are described. The value of the methods in the recognition of heterochromatin was demonstrated using five monocotyledonous and two dicotyledonous species. In Trillium grandiflorum the stronger Giemsa stained chromosome segments were shown to be identical with the heterochromatic regions (H-segments) revealed by cold treatment. Preferential staining of H-segments was also observed in chromosomes from three species of Fritillaria and in Scilla sibirica. Under suitable conditions the chromosomes of Vicia faba displayed a characteristic banding pattern and the bands were identified as heterochromatin. The Giemsa techniques proved to be more sensitive than Quinacrine fluorescence in revealing a longitudinal differentiation of the chromosomes of Crepis capillaris, where plants with and without B-chromosomes were examined. Again all chromosome types had their characteristic bands but there was no difference in Giemsa staining properties between the B-chromosomes and those of the standard complement.     

Comparison of areas of quinacrine mustard fluorescence and modified Giemsa staining in human metaphase chromosomes

The methods of quinacrine mustard fluorescence and modified Giemsa staining were compared in view of the structural details revealed in human mitotic chromosomes derived from the peripheral blood of normal healthy humans. Over the chromatids both techniques produced a crossbanding pattern where larger segments of heavy staining in the latter technique and the fluorescing bands in the former occurred at similar locations. The centromeric heterochromatin, intensely stained with Giemsa was, however, negative in fluorescence, except for chromosome no. 3 and less often no. 6. The regularly occurring secondary constrictions in chromosomes 1, 9, and 16 behaved generally like areas of centromeric heterochromatin. The area of secondary constriction in the Y chromosome as also that of chromosome 9 in the ASG modification of the Giemsa technique was both non-fluorescent and non-staining.     

Analysis of heterochromatin by combination of C-banding and CMA3 and DAPI staining in two fish species (Pimelodidae,Siluriformes)

The chromosomes of Steindachneridion sp. (2n = 56) and Rhamdia quelen (2n = 58) were analyzed by C-banding (CB) and Chromomycin A₃ (CMA₃) and 4,6-diamidino-2-phenylindole (DAPI) staining, separately and consecutively, in order to understand the role of base-specific fluorochrome treatment after CB. Both species' chromosomes shared common staining profiles as follows. CB with Giemsa (CBG) revealed weak heterochromatic blocks in the telomeric regions of some chromosomes and conspicuous bands on the short arms of one chromosome pair, where nucleolar organizer regions (NORs) were evidenced by silver-staining. Without CB pretreatment, the NORs were stained conspicuously with CMA₃, but not with DAPI. The latter uniformly stained all chromosomes, but leaving the NORs pale. Combination of CMA₃ or DAPI staining with CB showed distinctive fluorescent blocks in the NOR-bearing short arms of the single chromosome pair along with several bright fluorescent signals on other chromosomes, which were not evidenced by single CMA₃ or DAPI staining. These results suggest a modification of chromatin structure by CB treatment, which may increase the stainability of CMA₃ and DAPI.     

Late replicating bands of human chromosomes demonstrated by fluorochrome and Giemsa staining.

The addition of thymidine (TdR) to cells growing in a medium containing 5-bromodeoxyuridine (BUdR) at the end of the first replication cycle results in the incorporation of TdR into the late replicating DNA regions. These sites can be visualized by staining the metaphase chromosomes with the fluorescent dye "33258 Hoechst" or a "33258 Hoechst" Giemsa procedure. A sequence of late replication patterns has been established in metaphase chromosomes of cultured human peripheral lymphocytes. The patterns are in agreement with those obtained by the standard autoradiographic procedures, but are more accurate. As is known from autoradiography, late replicating bands are in the position of G or Q bands. The "33258 Hoechst" Giemsa staining procedure of chromosomes which have replicated in the presence of BUdR first and in TdR for the last 2 hrs of the S phase is preferable to the currently used Giemsa banding techniques: the method yields very well banded metaphases in all preparations examined, as the chromosome structure is not disrupted by the pretreatment. The bands are very distinct, even in the "difficult" chromosomes (e.g. No. 4, 5, 8 and X). In female cells the late replicating X chromosome can be identified by its size and staining pattern. In addition to the replication asynchrony, the sequence of replication within both X chromosomes in female cells is not absolutely identical. The phenomenon of a phase difference in replication between the homologues is not a peculiarity of the X chromosome, but can be found in all autosomes as well as in homologous positions on the chromatids of individual chromosomes.     

Differential Giemsa staining in plants

Interference contrast and scanning electron microscopy studies of BSG treated Tulipa somatic chromosomes revealed transverse ridges coincident with the Giemsa positive areas. No cytological differentiation of the Giemsa banded regions was observed either prior to staining, or following destaining in methanol. Thus, the ridges observed must be interpreted as a result of an accumulation of dye molecules rather than a differential distribution of chromosomal material either before or after staining.     

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.     

Analysis of exchanges in differentially stained meiotic chromosomes of Locusta migratoria after BrdU-substitution and FPG staining

In male mice the X and Y chromosomes are conjoined by a single near-terminal chiasma, but XY bivalents following incorporation of 5-bromodeoxyuridine (BrdU) and fluorescence plus Giemsa (FPG) staining show only one of the two expected configurations, which suggests a preferential involvement of certain non-sister chromatids in crossover formation. To test the possibility that nonrandom chromatid involvement is a general feature of near-terminal crossovers, we reexamined the apparently terminal associations in differentially stained autosomal bivalents of Locusta migratoria. The frequencies of the two configuration types were nearly equal, as would be expected if these terminal associations resulted from conventional near-terminal chiasmata showing the random involvement of non-sister chromatids that characterises interstitial chiasmata.     

The mechanism responsible for reciprocal BrdU-Giemsa staining

Cytological and biochemical experiments were undertaken to elucidate the mechanisms responsible for the reciprocal Giemsa staining of BrdU-substituted and unsubstituted chromosome regions subjected to high or low pH NaH₂PO₄ treatments. These experiments included staining of chromosome preparations with ethidium bromide (EB), acridine orange (AO), or dansyl chloride, digestion of BrdU-substituted and unsubstituted chromatin with pancreatic DNase I, and SDS polyacrylamide gel electrophoresis of the proteins extracted from, and those remaining in isolated, fixed, air-dried nuclei subjected to either NaH₂PO₄ treatment. The collective evidence from this and previous work clearly indicates that, although the staining reactions following the different pH treatments are reciprocal, the mechanisms of induction of the staining effects are not. After the high pH treatment, BrdU-substituted and unsubstituted chromosome regions are palely and intensely stained with Giemsa, respectively. This treatment preferentially solubilizes BrdU-substituted DNA, probably as a result of the photolysis or high temperature hydrolysis of BrdU-DNA. Concomitantly, this treatment selectively denatures the BrdU-DNA. The reduction in the amount of DNA in the BrdU regions leads to a quantitative decrease in Giemsa-dye binding, resulting in pale staining relative to unsubstituted regions. The extraction of BrdU-substituted DNA does not appear to simultaneously extract much chromosomal protein. After the low pH treatment, BrdU-substituted and unsubstituted regions appear intensely and palely stained with Giemsa, respectively. BrdU substitution greatly increases the binding affinity of histone H1 to DNA, and the low pH treatment preferentially extracts the less tightly bound H1 of the unsubstituted chromatin. This extraction of H1 is presumably responsible for the preferential dispersion of unsubstituted DNA outside the boundaries of the chromosome onto the surrounding area of the slide. The unsubstituted chromosome regions subsequently stain relatively palely with Giemsa, because the DNA in these regions is more dispersed than that in the BrdU-substituted regions. The low pH treatment concomitantly denatures the unsubstituted DNA.