Electron microscopy of differentially BrdU-substituted sister chromatids treated with sodium phosphate

Electron microscopy of unstained BrdU-substituted chromosomes treated with 1.0 M NaH₂PO₄ at high pH and high temperature has demonstrated that there is a structural basis for the light microscopic observation of differentially Giemsa-stained unifilarly and bifilarly BrdU-substituted chromatids and the appearance of chromosome dots. At progressively higher treatment temperatures, sequential structural changes occurred in the chromosomes. After treatment with NaH₂PO₄ at 70–80° C, unifilarly BrdU-substituted chromatids were much more electron opaque than bifilarly substituted chromatids, and the overall data suggest that this difference in electron opacity is a result of the preferential extraction of chromosomal DNA from the bifilarly BrdU-substituted chromatids. NaH₂PO₄ treatment of the BrdU-substituted chromosomes at 80–90° ° C resulted in the formation of highly electron opaque spots (dots) on one or both chromatids. Dots first appeared on the electron lucent bifilarly BrdU-substituted chromatid, indicating that the chromatin with the greatest substitution of BrdU in its DNA is most susceptible to dot formation. At a slightly higher temperature, dots also appeared on the unifilarly BrdU-substituted chromatid concomitant with a disappearance of the electron opacity characterizing this chromatid at the lower treatment temperature. The dots may be formed by an extreme reorganization of residual chromatin or by some kind of interaction or reaction between the chromatin and the salts in the incubation medium. G-band regions may serve as focal points for dot formation.     

Identification of late DNA-replicating regions in Chinese hamster chromosomes using a BrdU-giemsa technique

Asynchronous Chinese hamster cells were labelled with BrdU for 3 h prior to harvesting the metaphase cells. The late DNA replicating sites became unifilarly BrdU-substituted as compared to the earlier replicating sites having a normal DNA constitution. Those late replicating sites were identified by pale coloration or dot formation after treatment with 1.0 M Na-phosphate solution (adjusted to pH 9.0 with supersaturated amount of NaHCO₃ and at a temperature of 69–75° C) and staining with Giemsa dye. Using this technique, nuclei with incorporated BrdU could be distinguished from nuclei that had not incorporated BrdU. — One of the advantages of using this technique for identification of late DNA replicating sites is that cells are treated continuously with BrdU for a short period of time before harvesting and only one sampling, rather than a series of samplings, is required to achieve a clear-cut result.     

DNA extraction during giemsa differentiation of chromatids singly and doubly substituted with BrdU

Human lymphocytes were cultured in ³H-labelled BrdU. Cells were pretreated to induce differentiation, autoradiographed and Giemsastained. DNA extraction was deduced if grain counts were lower in differentiated mitoses compared with untreated controls. — The differentiation method involved sequential pretreatments with short wave UV and 2 × SSC at 60 ° C. This removed 34% of label from first division cells (with TB.TB chromosomes) but relatively more (53%) from second division (TB.BB chromosomes). In second division cells, about two thirds of label was lost from pale (BB) chromatids but only one third from dark (TB) chromatids. The UV and SSC pretreatments acted in collaboration, since neither alone reduced grain counts significantly. — On testing other methods, similar preferential DNA extraction was obtained with Perry and Wolff's FPG method, and with the hot salt pretreatment of Korenberg and Freedlender. However, good Giemsa differentiation could also be obtained using Hoechst 33258 and light pretreatments without any DNA loss. Reverse differentiation patterns (TB pale, BB dark) induced by warm acids resulted in extraction of nearly two thirds of ³H-BrdU label, but relative loss was the same from pale and dark chromatin. Direct reverse staining using alkaline Giemsa did not result in any loss of label. — Thus preferential DNA loss from pale stained chromatin underlies differentiation methods using light plus hot salt pretreatments, but it is not obligatory for good differentiation using other techniques.     

Decrease of DNA synthesis in amniotic fluid cells during the middle part of S-phase revealed by differential chromosome staining after incorporation of BrdU

The time sequence of DNA replication in partially synchronized human amniotic fluid cells has been analysed, employing BrdU incorporation techniques. —Regardless of the interval between removal of the methotrexate/uridine block and addition of BrdU during S-phase, the treatment results in an R-type replication pattern. Conversely, replacement of BrdU containing medium by another one with thymidine yields G-type replication patterns. A thymidine pulse during the first 4 h of S-phase results in R-type replication patterns; from 7–10 h after block removal it produces G-type pattern. In between, only faint red staining dots can be found indicating a marked decrease of replicational activity during the middle part of the S-phase.     

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.     

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.     

Induction of the fragile X on BrdU-substituted chromosomes with direct visualization of sister chromatid exchanges on banded chromosomes

Summary After incorporation of BrdU for one or more replication cycles, the fragile site at Xq27 [fra(X)] was induced by a late pulse with excess thymidine (dT), resulting in the simultaneous visualization of G bands and differentially stained sister chromatids. The degree of BrdU substitution (uni- vs bifilarly substituted DNA) did not affect the expression of the fra(X). Without addition of dT, expression was the same in M1, M2, and M3 cells. With the addition of dT, expression was reduced in M1 cells and increased in M2 and M3 cells. One way to explain this fact would be an increased repair of the fragile site in M1 cells by illegitimate G:BrdU pairing under dCTP-deficient conditions. A preferential depletion of M3 cells, and to a lesser extent also M2 cells, could suggest a synergistic toxic effect of BrdU substitution and dCTP depletion. With this technique, sister chromatid exchanges (SCEs) could be directly localized at band level, facilitating a more detailed study of SCEs at the Xq27 fragile site.     

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.     

G-band-like structures and centromeric asymmetry in the BrdU containing mouse chromosomes

Mouse cells cultured in the presence of BrdU or BrdC for one replication cycle were stained in a 4Na-EDTA Giemsa solution which stains BrdU-containing chromatin preferentially (Takayama and Tachibana, 1980). With this treatment clear bands (B-bands) were revealed along the length of the chromosomes. The B-banding patterns were identical with the G-banding patterns of this species except for the centromeric region in which lateral asymmetry of Giemsa staining was seen. The concomitant occurrence of the lateral asymmetry with the B-banding supports the assumption that the B-bands visualized by the present technique reflect the BrdU-rich chromatin regions differentially localized along the chromosomes. Most of the chromosomes constituting the mouse karyotype showed their own characteristic appearance of the asymmetry, but in some of them the asymmetry was not clear and the Y did not show any specific, centromeric staining. The marked coincidence of the B- and G-banding patterns seems to provide evidence for the involvement of AT-rich chromatin in the induction of positive G-bands. The present technique also seems quite useful to analyze chromosomes of some species in which ordinary G-banding techniques have been known to bring about only unsatisfactory results.     

In vivo sister chromatid differentiation and baseline sister chromatid exchanges in a mouse ascites tumour model.

Induction of differentially stained sister chromatids at G2/M and determination of baseline sister chromatid exchanges (SCEs) in ascites form of mouse sarcoma 180 cell line have been done by in vivo incorporation of 5-bromodeoxyuridine (BrdU) for two consecutive DNA replication cycles. The baseline SCE frequency is 6.24 at log phase of tumour growth.     

Factors involved in differential giemsa-staining of sister chromatids

Microspectrophotometric evaluation of differentially stained sister chromatids made it possible to analyse precisely the factors involved in the Giemsa methods. The concentration of Hoechst 33258, pH of the mounting medium, temperature during UV-exposure and the quality (wavelength) of UV-light influenced the differential staining. Exposure of blacklight of 10^–5 M Hoechst 33528-stained BrdU-labeled chromosome specimens mounted in McIlvaine buffer (pH 8.0) at 50° C reproducibly allowed differential staining of sister chromatids within 15 min. On the other hand, Korenberg-Freedlender's method using no Hoechst 33258 was also UV-light-dependent. Thus, photolysis of BrdU-substituted DNA was considered the basic mechanism of the Giemsa methods where the photosensitive Hoechst 33258 played a role as a sensitizer.     

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.     

Lateral asymmetry in human constitutive heterochromatin

Human lymphocytes were grown for one replication cycle in BrdU, stained with 33258 Hoechst, exposed to UV light and subsequently treated with 2 x SSC and stained with Giemsa. This technique differentially stains the constitutive heterochromatin of chromosomes 1, 9, 15, 16, and the Y. In the heterochromatin of chromosome 9 both sister chromatids stained darkly and symmetrically but in the other four chromosomes the heterochromatin showed lateral asymmetry, one chromatid being darkly stained while its sister chromatid was as pale or paler than the rest of the chromosome. The lateral asymmetry is presumed to reflect an underlying asymmetry in distribution of thymine between the two strands of the DNA duplex in the satellite DNA component of the chromosomes. In some number 1 chromosomes compound lateral asymmetry was seen; darkly staining material was present on both sister chromatids although at any given point lateral asymmetry was maintained so that if one chromatid stained darkly the corresponding point on the sister chromatid was very pale. The pattern of compound lateral asymmetry varied among the number 1 chromosomes studied but was constant for any one homologue from one individual. This technique reveals a previously unsuspected type of polymorphism within the constitutive heterochromatin of man.     

Sister chromatid differentiation and isolabeling of chromosomes

Summary Isolabeling observed during sister chromatid differentiation (SCD) was studied from human skin fibroblasts by the fluorescence-plus-Giemsa (FPG) technique. Bromodeoxyuridine (BrdU) was fed to exponentially dividing cells for 52 h to enable completion of two consecutive cycles of DNA replication. During this period, the late-replicating regions of some chromosomes were able to go through three replication cycles. These chromosome regions had evidently incorporated BrdU bifiliarly in both chromatids and hence, on staining with FPG, appeared isostained (isolabeled). Thus, incubation of exponentially dividing cells with BrdU for a period longer than that required for two cell cycles appears to be a suitable method for revealing the late-replicating regions of the genome, such as the X chromosome in a human female, as isolated.In another experiment with Indian muntjac chromosomes, isolabeled segments were darkly stained, which suggested unifilar incorporation of BrdU. In this case, unequal crossing-over or an unequal distribution of thymine residues probably is responsible for the isolabel.     

Chromosome organisation in the Australian plague locust,Chortoicetes terminifera

In Chortoicetes terminifera, G-banding, produced by the trypsin treatment of air-dried slides followed by Giemsa staining, leads to light staining gaps at the secondary constrictions on autosomal pair 6 and regions proximal to the centromere on the long arms of pair 4. The variable short arms of two of the three smallest pairs were usually flared and lightly stained after treatment. In contrast to the relatively minor response of the normal chromosome set to G-banding, the large supernumerary chromosomes of C. terminifera show a spectacular series of dark bands alternating with lightly stained gaps. Two G-band variants of the B-chromosome were found in a laboratory stock. These patterns of G-banding are discernable both at mitosis in adults and embryos of both sexes and at all stages of male meiosis. Some regions which are gaps after G-banding appear as dark bands after C-banding. Consequently the supernumerary chromosome is mainly darkly stained with C-banding. In addition the centromeres and some telomeres are C-banded along with narrow interstitial bands and polymorphic heterochromatic blocks. — C-banding was not always successful, the technique often yields a mixture of G- and C-banding. The disparity of banding between the normal complement and the B-chromosome implies that whatever the source of origin of the B it has undergone spectacular changes in organisation since its origin.     

Opposite staining effect of two silver-staining techniques on sister chromatids

Opposite differential staining between sister chromatids was obtained by two silver-staining techniques on chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) and pretreated with Hoechst plus black light. Both silver-nitrate and silver-carbonate staining were affected by chemical extraction and enzyme digestion of chromosomal proteins. Prestaining of silver nitrate or silver carbonate also blocked the fluorescences of protein dyes. However, removal of chromosomal DNA affected the silver-carbonate but not the silver-nitrate staining; the fluorescences of DNA dyes were blocked by the prestaining of silver carbonate but not silver nitrate. Chromosomal protein labelling was released only slightly and its relative amount between BrdU bifilarly substituted and unifilarly substituted chromatids was unchanged during pretreatment of Hoechst plus black light. We speculate that chromosomal non-histones are the targets for silver-nitrate stain, and DNA-non-histone complexes for silver-carbonate stain.     

Chromosome banding: specification of structural features of dyes giving rise to G-banding

Summary Metaphase chromosomes were stained in a routine G-banding procedure with 39 basic dyes of varied structures substituted for the Giemsa stain. Staining outcomes were categorized as: iverstained, differentially stained, trivially or unstained. Certain structural features of the dyes were described numerically, namely, largest conjugated fragment (LCF), conjugated bond number (CBN) and cationic weight. The staining, outcomes were compared to these numerical structural parameters, and structure-staining correlations sought. Dyes with large conjugated systems (and high LCF values) were seen to be overstained; dyes with low LCF values were often non-staining. At intermediate LCF values, the more hydrophobic dyes (with high Hansch values) stained differentially; the more hydrophilic dyes failed to stain. Expressed numerically, 89% of the dyes with the following characteristics stained differentially: 30LCF10; Hansch >–5.0. It was concluded that contributions to dye-chromosome affinity included coulombic forces and van der Waals attractions and that the selectivity of G-banding was largely due to hydrophobic bonding. Induction of bands could be due to the loss of hydrophilic, histones, amplifying underlying variations in the hydrophobic-hydrophilic character of the chromosome structure. Relatively hydrophobic sites include AT-rich DNA and disulphide-rich proteins.The effects on Romanowsky G-banding of chemically modifying chromosomes were in keeping with this model. Overstaining resulted from formation of either hydrophobic or conjugated derivatives or both, whereas trivial or non-staining arose from the formation of hydrophilic derivatives. Intriguingly, the efficacy of the dyes used for Q-banding also correlated positively with their hydrophobic character.     

Configurational changes in chromatids from helical to banded structures

Induction of configurational changes in the helical chromatids of air dried chromosomes was used to explore the mechanism of G-banding. From the water-Giemsa stained metaphase spreads of Chinese hamster cells, chromosomes having clearly helical chromatids were selected and photographed. Then the chromosomes were decolorized, treated with trypsin, and restained with saline-Giemsa (1 x SSC). Such procedures were repeatedly carried out upon the same chromosomes. Subsequent examination of the chromosomes showed that configurational changes from a helical structure to a banded structure had occurred. Some chromosomes revealed a variety of transitional changes between these two configurations. During the repeated G-banding treatments, the distances between bands along the same chromatids changed each time. The results obtained seem to indicate that the G-banding results from locally induced compaction of chromosomal materials along the chromatids.     

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.     

Structural organization of the heterochromatic region of human chromosome 9

Giemsa-11, G-banding and Lateral Asymmetry staining techniques were used to define the substructure of the C-band heterochromatin of human chromosome 9, in a sample of 108 different chromosomes 9, from 54 individuals. In this sample, the juxtacentromeric portion of the C-band region stained positive by the G-banding technique while Giemsa-11 delineated a more distally located block. Examination of the pericentric inversions generally revealed that the entire C-band region is changed with the substructural organization left intact; i.e. the G-band is proximal, the G-11 distal to the centromere. The partial pericentric inversions were found to have larger than average amounts of G-band heterochromatin on the short arm. The G-11 staining was in its usual position on the long arm with none on the short arm. Such apparent inversions therefore may not represent true inversions. — Long heterochromatic regions frequently had a segmented appearance when stained with G-11; there was a dark G-band within the pale heterochromatic region when stained with the G-banding technique which corresponded in location to the achromatic gap produced by G-11. This extra G-band may have been derived from the juxtacentromeric G-band by processes analogous to unequal crossing over. — Simple lateral asymmetry was consistently present only in the G-band heterochromatin of those chromosomes 9 containing large blocks of G-band positive material. Examination of the portion of the C-band which would correspond to the G-11 positive material revealed no consistent patterns of asymmetry. Usually both strands were heavily stained and symmetrical but occasionally there were light areas present on one strand suggestive of compound lateral asymmetry.