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.     

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.     

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.     

Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258

A benzimidazole derivative, Hoechst 33258 can induce decondensation of constitutive heterochromatin in the mouse derived L cell chromosomes when the compound is given in sufficiently high concentration (40 micrograms/ml) to the L cell culture. Hoechst 33258 at low concentration (1 micrograms/ml, 16 h) cannot produce this effect on L cell chromosomes. Bromodeoxyuridine (BUdR) incorporation for one cell cycle simultaneous with the Hoechst 33258 treatment at low concentration could decondense heterochromatin segments in metaphase chromosomes. The heterochromatin decondensation, however, was asymmetric; it was observed only on one chromatid and the other of a chromosome remained in condensed state. The observation of asymmetric decondensation of heterochromatin by Hoechst 33258 after BUdR incorporation for one cell cycle, the association of A-T rich satellite DNA to mouse heterochromatin, and available data on the specific binding of Hoechst 33258 to A-T base pairs of DNA and on the higher affinity of the compound to BUdR substituted DNA than to ordinary DNA implied that the binding of Hoechst 33258 molecules to A-T rich satellite DNA is the cause of heterochromatin decondensation.     

Replacement of serum with a defined medium increases beta-adrenergic receptor number in cultured glioma cells

Lymphocyte chromosomes from a cercopithecoid species, Macaca mulatta, were studied for the occurrence of lateral asymmetry in constitutive heterochromatin. The technique consisted of growing the lymphocytes for one cell cycle in BrdUrd, staining with 33258 Hoechst, exposing them to UV light, treating them with 2 SSC and staining with Giemsa. This procedure revealed asymmetric staining in the region of constitutive heterochromatin of the nucleolar organizer marker chromosome (no. 13 of the complement). In these chromosomes, the darkly staining region was confined at any given point to a single chromatid, while the corresponding region on the sister chromatid was lightly stained. This pattern of asymmetric staining in the constitutive heterochromatic region was not observed in any other chromosome of Macaca mulatta. The lateral asymmetry of constitutive heterochromatin in this species is presumed to reflect the strand bias in the distribution of thymine in the alphoid DNA fractions.     

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.     

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.     

Restrictive distribution of asymmetric C-bands in the chromosome complement of the rhesus (Macaca mulatto)

Lymphocyte chromosomes from a cercopithecoid species, Macaca mulatta, were studied for the occurrence of lateral asymmetry in constitutive heterochromatin. The technique consisted of growing the lymphocytes for one cell cycle in BrdUrd, staining with 33258 Hoechst, exposing them to UV light, treating them with 2 SSC and staining with Giemsa. This procedure revealed asymmetric staining in the region of constitutive heterochromatin of the nucleolar organizer marker chromosome (no. 13 of the complement). In these chromosomes, the darkly staining region was confined at any given point to a single chromatid, while the corresponding region on the sister chromatid was lightly stained. This pattern of asymmetric staining in the constitutive heterochromatic region was not observed in any other chromosome of Macaca mulatta. The lateral asymmetry of constitutive heterochromatin in this species is presumed to reflect the strand bias in the distribution of thymine in the alphoid DNA fractions.     

Fluorometrical detection of thymine base differences in complementary strands of satellite DNA in human metaphase chromosomes.

Using the fluorochrome "Hoechst 33258", intensity of fluorescence was found to differ distinctly between the sister chromatids in the paracentric regions of chromosomes 1, 16, and 19, after one round of replication in medium containing BUdR. Thus the effect of fluorescence asymmetry is not limited to the part of the Y chromosomes that fluoresces intensely with quinacrine; it can also be determined in the weakly Q-fluorescent pericentric regions of chromosomes, which are known to be the sites where highly reiterated sequences of satellite DNA are located. However, an exception is the paracentric region of chromosome 9 which does not show the effect of lateral asymmetry. The difference of fluorescence intensity in the heterochromatic regions of the sister chromatids of human chromosome 1 is measured by densitometric tracement along the long axes of chromosomes; this is obtained from two individuals with an "uncoiler" heterchomatic block (type III) having a relative intensity of 1:1.93 in an average of the total measured blocks. This corresponds to the uneven distribution of thymine base of 22.8 and 43.2 in the two strands of the DNA double hexlix. A chromatid exchange rate of 9 in 100 metaphases per cell cycle was found within the uncoiler region of chromosome 1.     

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.     

In vivo BUdR labeling of mammalian chromosomes

A technique is described for labeling mammalian chromosomes in vivo with BUdR. Rats and mice are given BUdR by tail vein infusion over a 24-h period at a concentration of 25 μg/g wt/h. Metaphase cells that have gone through two or three cycles of DNA synthesis reveal characteristic differential chromatid fluorescence after staining with Hoechst dye. Sister chromatid exchanges can then be easily detected in these cells.     

Differential fluorescence of sister chromatids with 4'-6-diamidino-2-phenylindole.

Differential fluorescence of sister chromatids and sister chromatid exchanges (SCE) in chromosomes from human lymphocytes grown two replication cycles in medium containing 5-bromodeoxyuridine can be detected by fluorescence microscopy after staining with 4'-6-diamidino-2-phenylindole (DAPI). The DAPI fluorescence appears to be more stable than that of the dye 33258 Hoechst and may provide a more sensitive method for the detection of SCE.     

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.     

In vivo BrdU-33258 Hoechst analysis of DNA replication kinetics and sister chromatid exchange formation in mouse somatic and meiotic cells

BrdU (5-bromodeoxyuridine)-33258 Hoechst methods have been adapted for in vivo analyses of replication kinetics, sister chromatid differentiation and sister chromatid exchange (SCE) formation in mice. Sufficient in vivo BrdU substitution for cytological detection was effected with multiple intraperitoneal injections of the analogue. The combination of centromere staining asymmetry and sister chromatid differentiation at metaphase permits unambiguous determination of the number of replications in BrdU and dT (deoxythymidine) undergone by individual cells. Late-replicating regions in marrow and spermatogonial chromosomes are highlighted by bright fluorescence after sequential incorporation of BrdU followed by dT during a single DNA synthesis period. SCEs are analyzed in marrow and spermatogonial metaphases after successive complete cycles of BrdU and dT incorporation. Significant induction of SCE was observed with both mitomycin C and cyclophosphamide; the latter drug requires host-mediated activation to be effective. In meiotic metaphase cells harvested two weeks after BrdU incorporation, satellite DNA asymmetry, sister chromatid differentiation and SCE could be detected in a few chromosomes, most frequently the X and the Y.     

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.     

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.     

Sister chromatid exchange and chromosome organization based on a bromodeoxyuridine Giemsa-C-banding technique (TC-Banding)

Hoechst 33258 fluorescent staining of bromodeoxyuridine substituted chromosomes provided a high resolution technique for following the segregation of replicated chromosomal DNA (Latt, 1973). Modifications have produced the same results after Giemsa staining (Wolff and Perry, 1975). Since this does not necessarily require Hoechst (Korenberg and Freedlander, 1975), we call this bromodeoxyuridine-Giemsa banding (BG-banding). We here describe a further modification which allows one to follow the T-rich strand of the AT-rich satellite DNA of C-band heterochromatin. We call this TC-banding. This technique was used to examine metacentric marker chromosomes found in mouse L-cells that contain many interstitial blocks of centromeric-type heterochromatin in each arm plus the usual two blocks of centromeric heterochromatin. One of the advantages of this technique for such chromosomes is that it is possible to distinguish first from second cell cycle sister chromatid exchange and unambiguously detect centromeric sister chromatid exchange. We found some chromosomes to have high rates of centromeric sister chromatid exchange. After one cycle in bromodeoxyuridine we could examine the satellite polarity of the heterochromatic DNA. Since there was no change in satellite polarity in any of the heterochromatic blocks, marker chromosomes could not have been formed by paracentric inversions, inverted insertions or inverted translocations. These results allow the formulation of several rules of chromosome organization.     

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping

Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-UV irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.     

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.     

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping

Summary Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-U.V irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.In honour of Prof. P. van Duijn