Mechanism of differential staining of BUdR-substituted Vicia faba chromosomes.

Chromosomes of the broad bean Vicia faba were isolated and air-dried on slides after incorporation of BUdR into DNA (BUdR substitution) for two rounds of replication. Then the preparations were embedded in a buffer solution containing trypsin as well as fluorescence dye (acridine orange or Hoechst 33258). We observed chromosomes with a fluorescence microscope at various times after embedding. After about 15 min one sister chromatid of some of the metaphase chromosomes showed enhanced darkening and disintegration within 1–4 min (melting effect) during observation. We suppose that fragmentation of BUdR-substituted DNA by the acridine orange-visible light system in acridine orange staining and by irradiation with wavelengths around the transition from UV to visible light in Hoechst 33258 staining is responsible for this phenomenon. The disintegration of one sister chromatid in BUdR-substituted chromosomes can also be produced by UV irradiation during trypsin treatment when fluorescence dyes are not present.     

Influence of cysteamine on differential staining of BUdR-substituted human chromosomes.

In 5-bromodeoxyuridine (BUdR)-substituted human chromsomes stained with 4'-6-diamidino-2-phenylindole (DAPI) differential staining is suppressed totally by the H+-donor cysteamine (concentration 0.08 M). We propose that differential staining appears because the double BUdR-substituted chromatid will be disintegrated via a photosensitive dye-visible light system. It is suggested that cysteamine prevents the production of strand breaks in DNA and, consequently, differential staining in BUdR-substituted chromosomes. Furthermore it is shown that differential staining with DAPI causes irreversible changes in the double BUdR-substituted chromatid. This finding can be explained with the above mentioned mechanism.     

Further studies on the mechanism involved in differential staining of BUdR-substituted Vicia faba chromosomes

In a preceding publication we reported that photolysis of BUdR-substituted Vicia faba chromatids occurs during observation with a fluorescence microscope when chromosomes were mounted in a solution containing trypsin and a photosensitive dye (Hoechst 33258 or acridine orange). The present investigations support the hypothesis that the rapid dissolving of the double BUdR-substituted (BB) chromatids observed with our method is due to single-strand breaks induced by a photosensitive dye-visible light system. The agents cysteamine and potassium iodide which reduce BUdR radicals and in this way may inhibit single-strand breaks modify the rate of chromosomes showing differential staining. It was totally suppressed by high cysteamine concentrations and markedly reduced by potassium iodide. Several acridine dyes were tested concerning their ability to induce differential staining. Some of them, e.g. aurophosphine and coriphosphine O, yield good results, others, e.g. acriflavine and acridine yellow, give poor differential staining. In an experiment in which the trypsin concentration was varied to induce approximately optimum and non-optimum digestion conditions the necessity of trypsin treatment in our method was confirmed.     

Autoradiographic evidence of differential loss of BUdR-substituted DNA after UV exposure in FPG harlequin staining

Autoradiographic analysis of CHO cells labelled with [³H]TdR or [³H]BUdR shows that only [³H]BUdR label is removed from metaphase chromosomes after FPG staining. [³H]BUdR is differentially removed from the bifilary labelled chromatid (BB) compared with the unifilary labelled chromatid (TB). UV treatment alone removes the label and produces harlequin staining and if the UV step is omitted from the FPG staining technique no loss of label or harlequin staining occurs. The heat treatment step (60 °C in 2 × SSC) removes further label, reducing the ratio of grains BB/TB to 0.8:1.0 and improving the differential staining. Over-treatment with heat produces paler staining chromatids without altering this ratio. The differential loss of BUdR-substituted DNA through UV photolysis and extraction in solution appears to be the cause of the differential harlequin staining of chromatids in this technique.     

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.     

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.     

Specific silver staining of experimentally undercondensed chromosome regions

Treatment of human and mouse cell cultures with DNA binding AT-specific compounds and with some base analogues induced distinct undercondensations in several heterochromatic chromosome regions. All those heterochromatic regions undercondensed by AT-specific DNA ligands (distamycin A, DAPI, Hoechst 33258) could be heavily labeled with the silver(Ag)-staining technique; but the heterochromatic regions undercondensed with the cytidine analogue 5-azacytidine were Ag-negative. In metaphase chromosomes from BrdU-treated human cell cultures, the bifilarly substituted chromatids, which show a slight undercondensation, were also Ag-negative. Cytochemical analyses of the Ag-stained undercondensed heterochromatic regions showed that the Ag-stainable material consisted of nonhistone proteins. The mechanism of Ag staining in the undercondensed heterochromatic regions was compared with Ag staining of the nucleolus organizer regions.     

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.     

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.     

Studies of mammalian chromosome replication. I. BrdU-induced differential staining patterns in interphase and metaphase chromosomes

The patterns of differential staining based on the effects of BrdU-substitution in chromosomal DNA have been examined in both metaphase chromosomes and prematurely condensed chromosomes (PCC) of interphase Chinese hamster cells. Results indicate that differential staining may be obtained in chromosomes from all stages of the cell cycle and correspond to the semi-conservation mode of DNA replication. Such fidelity of differential staining in both interphase and metaphase chromosomes suggests that components essential for induction of differential staining are present throughout the cell cycle and chromosomes may contain similar structures and organization throughout the cycle.     

Considerations on the mechanism of differential Giemsa staining of BrdU-substituted chromosomes

Summary The staining properties of unifilarly bromodeoxyuridine (BrdU)-substituted chromatids were compared using fluorescent-plus-Giemsa (FPG) staining methods. It was found that the staining intensity of chromatids which had incorporated BrdU in the next to last S-phase is less than that of chromatids whose BrdU-containing strand came from the last cell cycle. Thus, FPG-staining is not a function of the number of BrdU-substituted DNA strands alone. These findings lead to the conclusion that the primary point of action of PFG staining leading to sister chromatid differentiation (SCD) are chromosomal proteins which have been altered in the replication of BrdU-substituted DNA and that the demonstration of the SCD and replication patterns with the same staining procedure is based on different mechanisms.     

Sister chromatid differential staining by direct staining in Na2HPO4-Giemsa solution and the mechanism involved

The direct staining of BrdU-substituted Chinese hamster chromosomes in a Na₂HPO₄-Giemsa solution without any pretreatments resulted in a B-dark type SCD in which bifilarly substituted (BB) chromatids stained dark and unifilarly substituted (TB) chromatids stained light. Detailed examinations of the staining process suggested that the Na₂HPO₄ solution acts to collapse chromosomes whereas the Giemsa dye works to reconstruct the collapsed chromosomes, and that during the reconstruction process preferential binding of the Giemsa dye to the BB-chromatids occurs to produce the B-dark SCD. It was revealed that not only the time but the temperature at which chromosome preparations are kept prior to use considerably affect the occurrence of SCD.     

The differential staining pattern of the X chromosome in the embryonic and extraembryonic tissues of postimplantation homozygous tetraploid mouse embryos.

(C57BL x CBA)F1 hybrid female mice were mated with hemizygous Rb(X.2)2Ad males to distinguish the paternal X chromosome. Homozygous tetraploids were produced by blastomere fusion at the 2-cell stage, and 161 of these were transferred to recipients and analysed on the 10th day of gestation. 59 implants contained resorptions and 76 contained either an embryo and/or extraembryonic membranes. 38 (20, XXXX and 18, XXYY) were analysed to investigate their X-inactivation pattern. Embryonic and yolk sac endodermally- and mesodermally-derived samples were analysed by G-banding and by Kanda analysis. In the XX and XY controls, the predicted pattern of X-inactivation was observed, though 12.2% of metaphases in the XX series displayed no X-inactivation. In the XY series the Y chromosome was seen in a high proportion of metaphases. In the XXXX tetraploids, 8 cell lineages were recognized with regard to their X-inactivation pattern, though most belonged to the following 3 categories: (XmXm)XpXp, Xm(XmXp)Xp and XmXm(XpXp). The other categories were only rarely encountered. In the embryonic and mesodermally-derived tissue the ratio of these groups was close to 1:2:1, whereas in the endodermally-derived tissue it was 1:4.11:4.88, due to preferential paternal X-inactivation. A significant but small proportion of all 3 tissues analysed displayed no evidence of X-inactivation. Indirect evidence suggests that this represents a genuine group because of the high efficiency of the Kanda staining. The presence of the Xm(XmXp)Xp category is consistent with the expectation that X-inactivation occurs randomly in 2 of the 4 X chromosomes present. The presence of small numbers of preparations with no evidence of X-inactivation and other unexpected categories suggests that these are probably selected against during development.     

Silver staining the chromosome scaffold

Cytological silver-staining procedures reveal the presence of a core running along the chromatid axes of isolated HeLa mitotic chromosomes. In this communication we examine the relationship between this core and the nonhistone chromosome scaffolding, isolated and characterized in previous publications from this laboratory. When chromosomes on coverslips were subjected to the steps used for scaffold isolation in vitro and subsequently stained with silver, the characteristic core staining was unaffected. Control experiments suggested that the core does not contain large amounts of DNA. When scaffolds were isolated in vitro, centrifuged onto electron microscope grids, and stained with silver, they were found to stain selectively under conditions where specific core staining was observed in intact chromosomes. These results suggest that the nonhistone scaffolding is the principal target of the silver stain in chromosomes.     

Upheaval in the bacterial nucleoid. An active chromosome segregation mechanism

Recent advances have completely overturned the classical view of chromosome segregation in bacteria. Far from being a passive process involving gradual separation of the chromosomes, an active, possibly mitotic-like machinery is now known to exist. Soon after the initiation of DNA replication, the newly replicated copies of the oriC region, behaving rather like eukaryotic centromeres, move rapidly apart towards opposite poles of the cell. They then determine the positions that will be taken up by the newly formed sister nucleoids when DNA replication has been completed. Thus, the gradual expansion of the diffuse nucleoid camouflages an underlying active mechanism. Several genes involved in chromosome segregation in bacteria have now been defined; their possible functions are discussed.     

Selective differential staining of sister chromatids of the facultative heterochromatic X chromosome in the female mouse

Selective differential staining of sister chromatids for the facultative heterochromatic X chromosome in the female mouse has been achieved by the combination of two differential staining techniques; one for the heterochromatic X chromosome and the other for sister chromatids. Thermal hypotonic treatment moderately destroyed the chromosome structure except for the heterochromatic X in BrdU labelled metaphase cells, resulting in the selective sister chromatid differentiation of this X with Giemsa stain. This technique enables us to know the exact frequency of the spontaneous sister chromatid exchanges in the heterochromatic X without using ³H-TdR labelling for detecting the late DNA replication. The results indicate that the sister chromatid exchange frequency of the heterochromatic X chromosome is not affected by its late DNA replication during S phase, or by the genetic inactivation and the resulting heterochromatinization.