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.     

Incomplete sister chromatid separation of long chromosome arms

Chromosome segregation ensures the equal partitioning of chromosomes at mitosis. However, long chromosome arms may pose a problem for complete sister chromatid separation. In this paper we report on the analysis of cell division in primary cells from field vole Microtus agrestis, a species with 52 chromosomes including two giant sex chromosomes. Dual chromosome painting with probes specific for the X and the Y chromosomes showed that these long chromosomes are prone to mis-segregate, producing DNA bridges between daughter nuclei and micronuclei. Analysis of mitotic cells with incomplete chromatid separation showed that reassembly of the nuclear membrane, deposition of INner CENtromere Protein (INCENP)/Aurora B to the spindle midzone and furrow formation occur while the two groups of daughter chromosomes are still connected by sex chromosome arms. Late cytokinetic processes are not efficiently inhibited by the incomplete segregation as in a significant number of cell divisions cytoplasmic abscission proceeds while Aurora B is at the midbody. Live-cell imaging during late mitotic stages also revealed abnormal cell division with persistent sister chromatid connections. We conclude that late mitotic regulatory events do not monitor incomplete sister chromatid separation of the large X and Y chromosomes of Microtus agrestis, leading to defective segregation of these chromosomes. These findings suggest a limit in chromosome arm length for efficient chromosome transmission through mitosis.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

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.     

A comparative study of DNA synthesis in cotyledon protoplast cultures of Brassica napus,B. campestris and B. oleracea using an immunocytochemical technique

The present study was designed (1) to observe the characterization of 5-bromo-2′-dexoyuridine (BrdU) incorporation into cultured Brassica cotyledon protoplasts and (2) to investigate the genetic differences in the levels of nuclear DNA synthesis (expressed by the percentage of nuclei labelled with BrdU) in cotyledon protoplast cultures from 12 cultivars of three Brassica species (Brassica napus, B. campestris and B. oleracea) at an early stage using immunocytochemistry. Nuclei labelled with BrdU were different from those showing only staining with 4′-6′-diamidino-2-phenylindole (DAPI) under fluorescence and light microscopy. Two to 5% of nuclei were labelled with BrdU after 1 h of culture, indicating that nuclear DNA synthesis occurred at a very early stage of culture. The percentage of nuclei labelled with BrdU increased with time over the length of the culture period. The mean percentage of nuclei labelled with BrdU in the 12 cultivars was about 25% at 24 h after culture initiation. The curve of the increase in percentage of nuclei labelled with BrdU exhibited an S-shape from 1 to 24 h. However, cultivar differences in percentages of nuclei labelled with BrdU were very significant over the time course of 1-24 h from initial culture, with cultivars Eureka (B. napus), Global (B. napus), Narc 82 (B. napus), Bunyip (B. campestris) and Sugar Loaf (B. oleracea) having a consistently higher percentage of nuclei labelled with BrdU than the other cultivars. Species differences were also significant, with cultivars of B. napus showing much higher percentages than the tested cultivars of B. campestris and B. oleracea. The results indicate that the differences in nuclear DNA synthesis in Brassica cotyledon protoplast cultures were most likely at both intra- and interspecies levels.     

Characterisation of a very complex constitutive heterochromatin in two Gerbillus species (Rodentia)

A large amount of heterochromatin is observed in two species of the genus Gerbillus, G. nigeriae and G. hesperinus. The C-band material represents about one-half of the total karyotype length in the former species, and about one-third in the latter. Several banding techniques and various 5-bromodeoxyuridine (BrdU) treatments were used to characterise these heterochromatic segments. After applying the R-banding technique, three different staining responses of the heterochromatin can be distinguished. In G. nigeriae, strongly stained segments (R-band positive) appear in most chromosomes and, in particular, constitute the short arms of all the larger chromosomes. Palely staining heterochromatic segments (R-band negative) are less abundant in G. nigeriae but predominate in G. hesperinus. In addition, in both species an intermediate staining of heterochromatin is observed near the centromere or in the heterochromatic short arms of some acrocentric and small submetacentric chromosomes. Very short BrdU treatment during the end of the last cell cycle results in asymmetrical staining of chromatids in heterochromatic segments after applying the acridine orange or FPG (fluorescence plus Giemsa) technique. The alternating location of strongly staining segments in one or the other chromatid simulates sister chromatid exchanges (pseudo-SCE). This pattern persists after longer BrdU treatment during different stages of the last cell cycle and is independent of the R-staining properties of the heterochromatin. The lateral asymmetric appearance of the large heterochromatic segments in Gerbillus is interpreted as reflecting an uneven distribution of adenine and thymidine between the two strands of DNA.     

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.     

Heterochromatin,repetitive DNS und Nukleolen in Leberzellen von Microtus agrestis

Zusammenfassung Die Zellstruktur von Leberzellen der Erdmaus, Microtus agrestis, wurde nach Giemsafärbung, Feulgenbehandlung, Behandlung mit Ribonuklease und nach Färbung des konstitutiven Heterochromatins untersucht. Das konstitutive Heterochromatin ist in Leberzellen nicht heteropyknotisch, das fakultative Heterochromatin ist im weiblichen Geschlecht als Sexchromatinkörperchen sichtbar. Bestimmungen des relativen DNS-Gehalts ergaben, daß die Zahl der Sexchromatinkörperchen der Ploidie der Zellkerne proportional ist. Die Nukleolen liegen in Hepatozyten oft randständig; in 59% der diploiden Zellkerne sind 2 Nukleolen enthalten. Nach Anfärbung der repetitiven DNS werden oft auch die Nukleolen gefärbt, nach Ribonukleasebehandlung tritt dieser Effekt nicht auf. Das konstitutive Heterochromatin wird in Form von 2 langen fädigen Strukturen sichtbar.

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Heterochromatin, repetitive DNA and nucleoli in liver cells of Microtus agrestis

Summary The nuclear structure of parenchymal liver cells of embryo and adult Microtus agrestis was studied in smear and section preparations after staining with Giemsa solution and treatment according to Feulgen, after treatment with ribonuclease and after specific staining of constitutive heterochromatin. In liver cell nuclei only the facultative heterochromatin is heteropycnotic, a sex chromatin body is observable in female but not in male animals. Constitutive heterochromatin is not heteropycnotic in liver cells. Measurements of the relative DNA content showed that nuclei with one sex chromatin body are diploid; tetraploid nuclei possess two and octoploid nuclei four sex chromatin bodies. Solely in the diploid cell nuclei of the intrahepatic gall ducts two large chromocenters are found. The nucleoli in hepatocytes often lie at the perimeter of the nucleus. 17% of the diploid nuclei contain one nucleolus, 59% two nucleoli, 23% three and 1% four. After staining of repetitive DNA, the nucleoli often become stained as well; after treatment with ribonuclease this effect does not occur. The constitutive heterochromatin becomes visible in form of two long, threadlike structures. After longer periods of dissociation the sex chromatin body ceases to be visible. Sex chromatin and constitutive heterochromatin are contiguous to the nucleoli.

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Mit dankenswerter Unterstützung durch das Bundesministerium für Bildung und Wissenschaft der Bundesrepublik Deutschland.     

Analysis of the Length of S-Phase Required to Show Sister Chromatid Differential Staining

Abstract. In vitro studies of BrdU-dependent sister chromatid differential staining typically employ two cycles of BrdU incorporation. Experiments are described which determined the actual fraction of both S-phases that the rat embryonic fibroblasts (Rat-1) cells had to traverse in order to show distinctive differential staining. Following synchronization of cells by a combination of serum deprivation and hydroxyurea blockage, sister chromatid differential staining, labelling index, mitotic index, and per cent DNA replication are determined. Results indicate that only ≤50% of the first S-phase is necessary in order to show distinctive differential staining. the importance of this finding to studies of cellular proliferation using BrdU incorporation is discussed.     

Sister chromatid exchanges in garlic (Allium sativum L.) callus cells

A technique is described for differential staining of sister chromatids and the study of sister chromatid exchanges (SCEs) in garlic (Allium sativum L.) callus cells. BrdU incorporation into newly synthesized DNA was ensured by culturing calli on medium containing 100 M BrdU+0.01 M FudR+1 M Urd. SCEs were visualized by FPG staining technique and their frequency was analysed. Mean frequency of SCEs in callus cells was higher than that in meristem root-tip cells. Using the same staining method, cell cycle time of callus cells was analysed. It was found that it ranges from 48 to 132 hrs. The method described represents a new approach in the study of genetic instability of plant cells cultured in vitro.Abbreviations BrdU 5-bromo-2-deoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - FPG fluorescent-plus-Giemsa - FudR 5-fluoro-2-deoxyuridine - SCE sister chromatid exchange - SSC 0.15 M NaCl + 0.015 M Na-citrate - T thymidine-containing strand of the DNA duplex - B 5-bromo-2-deoxyuridine-containing strand of the DNA duplex - Urd uridine     

The influence of incorporated bromodeoxyuridine on mutagenicity testing by sister chromatid exchange induction inVicia faba root tip cells

The induction of sister chromatid exchanges (SCEs) inVicia faba root-tip cells after short-term (2 h) and long-term (24 h) treatments with alkylating agents (N-methyl-N-nitrosourea, ethyl methanesulphonate) and maleic hydrazide was studied. The primary roots were treated with mutagens before or after 5-bromodeoxyuridine (BrdU) incorporation into DNA and the influence of mutagen application on SCE induction in the cells with non- and BrdU-substituted chromosomal DNA. On the contrary, application of maleic hydrazide after the incorporation of BrdU into DNA strongly increased the rate of SCEs. The lowest limit concentrations of mutagens capable of significantly increasing SCE frequency in the cells with non-substituted DNA after the long-term treatment were estimated.     

Analysis of the length of S-phase required to show sister chromatid differential staining

In vitro studies of BrdU-dependent sister chromatid differential staining typically employ two cycles of BrdU incorporation. Experiments are described which determined the actual fraction of both S-phases that the rat embryonic fibroblasts (Rat-1) cells had to traverse in order to show distinctive differential staining. Following synchronization of cells by a combination of serum deprivation and hydroxyurea blockage, sister chromatid differential staining, labelling index, mitotic index, and per cent DNA replication are determined. Results indicate that only approximately 50% of the first S-phase is necessary in order to show distinctive differential staining. The importance of this finding to studies of cellular proliferation using BrdU incorporation is discussed.     

The STRUCTURAL MAINTENANCE OF CHROMOSOMES 5/6 Complex Promotes Sister Chromatid Alignment and Homologous Recombination after DNA Damage in Arabidopsis thaliana

Sister chromatids are often arranged as incompletely aligned entities in interphase nuclei of Arabidopsis thaliana. The STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC) 5/6 complex, together with cohesin, is involved in double-strand break (DSB) repair by sister chromatid recombination in yeasts and mammals. Here, we analyzed the function of genes in Arabidopsis. The wild-type allele of SMC5 is essential for seed development. Each of the two SMC6 homologs of Arabidopsis is required for efficient repair of DNA breakage via intermolecular homologous recombination in somatic cells. Alignment of sister chromatids is enhanced transiently after X-irradiation (and mitomycin C treatment) in wild-type nuclei. In the smc5/6 mutants, the x-ray–mediated increase in sister chromatid alignment is much lower and delayed. The reduced S phase–established cohesion caused by a knockout mutation in one of the α-kleisin genes, SYN1, also perturbed enhancement of sister chromatid alignment after irradiation, suggesting that the S phase–established cohesion is a prerequisite for correct DSB-dependent cohesion. The radiation-sensitive51 mutant, deficient in heteroduplex formation during DSB repair, showed wild-type frequencies of sister chromatid alignment after X-irradiation, implying that the irradiation-mediated increase in sister chromatid alignment is a prerequisite for, rather than a consequence of, DNA strand exchange between sister chromatids. Our results suggest that the SMC5/6 complex promotes sister chromatid cohesion after DNA breakage and facilitates homologous recombination between sister chromatids.     

Sister chromatid exchanges in Allium cepa

Differential staining of sister chromatids with Giemsa after BrdU incorporation into DNA was performed in Allium cepa L. chromosomes. A treatment solution containing 10^–7 M FdU, 10^–4 M BrdU and 10^–6 M Urd was found to ensure BrdU incorporation without affecting cell cycle duration. After several procedures before staining the slides with Giemsa had been tested, treatment with the fluorochrome compound 33258 Hoechst, exposure to UV light and heating at 55° C in 0.5×SSC, were found to be essential for good differentiation. The distribution of SCEs per chromosome agrees with the expected Poisson distribution. The mean value of SCEs per chromosome occurring when cells were exposed to the treatment solution for two consecutive rounds of replication (=5.5) was double the mean value observed when cells were exposed to the same treatment for only one round of replication (=2.8). SCEs were found to occur more frequently in those chromosome regions corresponding neither to C-bands nor to late replicating DNA-rich regions. Finally, the occurrence of SCEs involving less than the width of a chromatid is discussed.     

Cell cycle analysis in a multinucleate green alga,Boergesenia forbesii (Siphonocladales,Chlorophyta)*

The cell cycle (nuclear division cycle) of a multinucleate green alga, Boergesenia forbesii (Harvey) Feldmann was studied using microspectrophotometry and BrdU incorporation techniques. Mitosis was observed frequently 1-4 h after the beginning of the light period, on a 16:8 h LD cycle at 25°C. Mitotic nuclei formed discrete patches. Other nuclei remained in the G₁ period. The DNA synthetic phase (S phase) was estimated to last about 12 h from microspectrophotometric study using aphidicolin inhibition just before the S phase and release from it. The G₂ period was estimated to be about 2 h, because a labeled prophase nucleus could be detected when the samples were labeled with BrdU continuously over 3 h. The incorporation pattern of BrdU changed through the S phase nucleus. In early S phase, BrdU staining was detected as many dots in the entire nucleus, while in late S phase, it was detected as several discrete regions along the nuclear membrane. Almost all nuclei in B. forbesii were in the G₁ stage after nuclear division, and the nuclei in several patches of the cell simultaneously initiated DNA synthesis. Once the nuclei entered into S phase, these nuclei continued into G₂ and mitosis. In other words, the cell cycle regulation of entrance into S phase from G₁ is an important factor in the growth and morphogenesis in B. forbesii.     

Studies of mammalian chromosome replication

Sister chromatids of metaphase chromosomes can be differentially stained if the cells have replicated their DNA semiconservatively for two cell cycles in a medium containing 5-bromodeoxyuridine (BrdU). When prematurely condensed chromosomes (PCC) are induced in cells during the second S phase after BrdU is added to the medium, the replicated chromosome segments show sister chromatid differential (SCD) staining. Employing this PCC-SCD system on synchronous and asynchronous Chinese hamster ovary (CHO) cells, we have demonstrated that the replication patterns of the CHO cells can be categorized into G₁/S, early, early-mid, mid-late, and late S phase patterns according to the amount of replicated chromosomes. During the first 4 h of the S phase, the replication patterns show SCD staining in chains of small chromosome segments. The amount of replicated chromosomes increase during the mid-late and late S categories (last 4 h). Significantly, small SCD segments are also present during these late intervals of the S phase. Measurements of these replicated segments indicate the presence of characteristic chromosome fragment sizes between 0.2 to 1.2 m in all S phase cells except those at G₁/S which contain no SCD fragments. These small segments are operationally defined as chromosome replicating units or chromosomal replicons. They are interpreted to be composed of clusters of molecular DNA replicons. The larger SCD segments in the late S cells may arise by the joining of adjacent chromosomal replicons. Further application of this PCC-SCD method to study the chromosome replication process of two other rodents, Peromyscus eremicus and Microtus agrestis, with peculiar chromosomal locations of heterochromatin has demonstrated an ordered sequence of chromosome replication. The euchromatin and heterochromatin of the two species undergo two separate sequences of decondensation, replication, and condensation during the early-mid and mid-late intervals respectively of the S phase. Similar-sized chromosomal replicons are present in both types of chromatin. These data suggest that mammalian chromosomes are replicated in groups of replicating units, or chromosomal replicons, along their lengths. The organization and structure of these chromosomal replicons with respect to those of the interphase nucleus and metaphase chromosomes are discussed.     

Random homologous pairing and incomplete sister chromatid alignment are common in angiosperm interphase nuclei

The chromosome arrangement in interphase nuclei is of growing interest, e.g., the spatial vicinity of homologous sequences is decisive for efficient repair of DNA damage by homologous recombination, and close alignment of sister chromatids is considered as a prerequisite for their bipolar orientation and subsequent segregation during nuclear division. To study the degree of homologous pairing and of sister chromatid alignment in plants, we applied fluorescent in situ hybridisation with specific bacterial artificial chromosome inserts to interphase nuclei. Previously we found in Arabidopsis thaliana and in A. lyrata positional homologous pairing at random, and, except for centromere regions, sister chromatids were frequently not aligned. To test whether these features are typical for higher plants or depend on genome size, chromosome organisation and/or phylogenetic affiliation, we investigated distinct individual loci in other species. The positional pairing of these loci was mainly random. The highest frequency of sister alignment (in >93% of homologues) was found for centromeres, some rDNA and a few other high copy loci. Apparently, somatic homologous pairing is not a typical feature of angiosperms, and sister chromatid aligment is not obligatory along chromosome arms. Thus, the high frequency of chromatid exchanges at homologous positions after mutagen treatment needs another explanation than regular somatic pairing of homologues (possibly an active search of damaged sites for homology). For sister chromatid exchanges a continuous sister chromatid alignment is not required. For correct segregation, permanent alignment of sister centromeres is sufficient.     

The effect of continuous gamma irradiation on formation of sister chromatid exchanges (SCEs) and chromosomal aberrations in meristematic cells ofVicia faba

Germinated seeds ofVicia faba were continuously irradiated at low dose rate of gamma rays (0.05 Gy h-¹) up to a total accumulated dose of 2 Gy. The FPG (fluorescence plus Giemsa) technique of differential chromatid staining was used to monitor the frequency of sister chromatid exchanges (SCEs) in irradiated root tip meristem cells. The results of the experiments have demonstrated that SCE frequency is raised by continuous gamma irradiation only in plant cells containing BrdU in the chromosomal DNA. No effect concerning SCE formation was recorded at continuous irradiation of meristematic cells of Vicia faba with native, i. e. BrdU-nonsubstituted, DNA. In contrast to SCEs, a significant increase was found in the yield of chromosomal aberrations in all variants of irradiation.     

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.     

Sister chromatid exchanges in barley

Summary A mean frequency of 20.6 sister chromatid exchanges (SCEs) per cell has been observed in a reconstructed karyotype of Hordeum vulgare by application of the FPG technique after unifilar incorporation of BrdU into chromosomes. The involvement in SCEs of the 48 segments into which the chromosome set had been subdivided was, with a single deviation, length proportional and independent of the segment's heterochromatin content. Asymmetric bands, indicative of an uneven distribution of adenine and thymidine between the DNA strands in adenine (A)-thymidine (T) rich chromosome regions, could not be detected after incubation of the cells in BrdU for one cycle of DNA replication.     

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.