Differential Staining Of Sister Chromatids Research Articles

Analysis of SCEs in Vicia faba chromosomes by a simple fluorescent plus Giemsa technique

Cortés, F. and Andersson, H.C. 1987. Analysis of SCEs in Viciafaba chromosomes by a simple fluorescent plus Giemsa technique. —Hereditas 207: 7–13. Lund, Sweden. ISSN 0018–0661. Received October 3, 1986 A modified fluorescent plus Giemsa (FPG) technique for differential staining of sister chromatids and for studying sister chromatid exchanges (SCE) in root tip cells of Viciafaba is described. This new technique is quick and simple and has the advantage of a high reproducibility. The best quality of the sister chromatid differentiation (SCD) is obtained when seedlings are grown for two rounds of chromosome replication in the presence of 5-bromodeoxyuridine (BrdUrd). The technique stains the bifilarly substituted chromatid light and the unifilarly substituted one dark. However. SCD is also obtained after one round of replication in BrdUrd followed by another in the presence of thymidine. There was no difference in the baseline level of SCE in the two types of experimental protocol. The influence of various concentrations of BrdUrd on the yield of SCEs was investigated, as well as the frequency of SCEs in root tip cells grown in the absence and presence of 5-fluorodeoxyuridine. The results of these experiments are discussed.

Sister chromatid exchange assay.

This method results in the differential staining of sister chromatids during replication and permits the direct visualisation of genetic exchanges between sister chromatids. It therefore provides a direct visual readout for crossover recombination. The generation of SCE is dependent on homologous recombination and is elevated in a number of circumstances including following exposure to DNA damaging agents and in some genetic backgrounds that result in increased dependence on recombination based pathways. It is important to remember that SCEs are probably only generated by a fraction of recombination events and their elevation may reflect increased use of recombination-based pathways or an increase in the resolution of recombination intermediates with cross-over.

Atomic force microscopy of human metaphase chromosomes after differential staining of sister chromatids.

Human metaphase chromosomes, in which 5-bromo-deoxyuridine (BrdU) had been incorporated into the DNA, were treated with the fluorescent plus Giemsa (FPG) method. Use of this method distinctly stained one of the paired sister chromatids with the Giemsa solution due to the difference in content of BrdU in the two chromatids. These chromosomes with their differential staining of sister chromatids were observed by atomic force microscopy (AFM). In the air-dried specimens, one of the paired chromatids that was stained strongly with Giemsa solution was about two times higher than the counterpart that was stained faintly with Giemsa solution. In the critical point dried chromosomes, the height of the Giemsa positive chromatid roughly matched that of the Giemsa negative counterpart. These findings imply that the arrangement of the Giemsa negative chromatid after FPG staining is fragile and easily collapses due to the surface tension of water during air-drying. At higher magnifications, the surface structure differed between Giemsa positive and negative chromatids; the Giemsa positive chromatid (i.e., unifilarly BrdU-incorporated chromatid) was composed of fibrous structures while the Giemsa negative chromatid (i.e., bifilarly BrdU-incorporated chromatid) exhibited a fine granular appearance. These structural changes in the sister chromatids are thought to arise from the ultraviolet irradiation and heating of the chromosomes during FPG staining.

Scanning near field optical/atomic force microscopy of bromodeoxyuridine-incorporated human chromosomes.

The present study applied scanning near field optical/atomic force microscopy (SNOM/AFM) to the observation of human chromosomes immunostained with an anti-BrdU antibody after incorporation of BrdU into DNA. Human lymphocytes were cultured in BrdU for 72 h and their chromosomes were prepared with a standard method for light microscopy. After additional fixation with 15% formalin in phosphate buffered saline, the specimens were denatured with 2N HCI with 0.1% Triton-X 100, immunostained with the anti-BrdU antibody, and observed both by fluorescence microscopy and by SNOM/AFM. The preparation technique used in the present study enabled the differential staining of sister chromatids in each chromosome, and sister chromatid exchanges (SCEs) were recognized in some chromosomes of the metaphase spread. Observations of the specimens by SNOM/AFM further provided the simultaneous collection of topographical and fluorescent images of the same portions of BrdU-incorporated chromosomes. The resolution of the fluorescence images by SNOM/AFM was greater than that obtained by fluorescence microscopy. Superimposition of topographical and fluorescent images of the chromosomes is useful for the precise analysis of the fine structure of chromosomes in relation to the SCEs. The application of SNOM/AFM to the BrdU-incorporated chromosomes is thus useful for the analysis of the fine structure of chromosomes in relation to their function.

Cell-cycle kinetics of cell lines from patients with chromosomal mosaicism

Lymphocyte cultures from five patients with chromosomal mosaicism (two 47,XY,+21/46,XY, one 47,XX,+21/46,XX, one 45,X/46,XX, and one 47,XXY/46,XY) were studied using sister chromatid differential staining technique for cell kinetic evaluation. Aneuploid and normal cell lines were compared to identify changes in cellular proliferation in vitro that could be related to cellular selective advantage and cell-line-proportion changes occurring with age. Comparison of the percentage of cells in different cell generations in 48, 72, and 96 h-cultures shows no differences between the aneuploid and normal cell lines indicating that cell-cycle kinetics is similar in these cells in vitro.

Sister chromatid differentiation with biotin-dUTP.

The method of choice to differentiate sister chromatids is to incorporate BrdU in replicating DNA. The disadvantage of BrdU is that its spontaneous or induced radicalization may itself lead to sister chromatid exchanges. Biotin-labelled dUTP is a widely used thymidine analogon for labelling isolated DNA. Its chemical structure suggests that, in contrast to BrdU, it does not give rise to radical formation. We electroporated proliferating Chinese hamster ovary (CHO) cells in the presence of biotin-dUTP which was subsequently detected in metaphase cells with TRITC-conjugated avidin. Microscopic analysis of second mitoses after labelling revealed a clear differential staining of sister chromatids. Thus substitution of thymidine with biotin-dUTP is another method to analyse SCE.

Evaluation of the genotoxicity of municipal sewage effluent using the marine worm Platynereis dumerilii (Polychaeta: Nereidae)

Samples of settled (primary) effluent were collected from a municipal sewage treatment works at Newton Abbot, Devon, UK, a site which discharges primary effluent via long sea pipeline into the English Channel (minimum of 200-fold initial dilution). Sewage samples were collected during the period February–April 1995 and were analysed for standard physico-chemical parameters (ammonia, chemical oxygen demand, conductivity, non-purgeable organic carbon and settled solids). Samples were also tested for cytotoxicity, genotoxicity, and for developmental effects in the embryo-larval stages of the marine worm, Platynereis dumerilii. Exposure to sewage concentrations of ≥10% (v/v) in seawater at 20±1°C led to a marked reduction in normal embryo-larval development (7 h EC 50 values from 10% to 18% v/v, n=5). There was also evidence of a simultaneous delay in the cell cycle progression (as determined by sister chromatid differential staining) following embryo-larval exposures to sewage concentrations of ≥10% (v/v). Following the calculation of the Maximum Tolerated Dose (MTD), based on cytotoxic and developmental effects, cells from the same embryo-larvae were analysed for chromosomal aberrations (CAs). Results were consistent for all samples tested, demonstrating the absence of cytogenetic damage following the in vivo exposure of polychaete embryo-larvae to settled sewage.

Differential involvement of chromosomes 1 and 4 in the formation of chromosomal aberrations in human lymphocytes after X-irradiation

. Whole blood samples from two healthy donors were cultured in the presence of 5-bromo-2-deoxyuridine (BrdU) for a total of 107 h following in vitro X-irradiation with a dose of 2 Gy. Starting from 35 h after culture initiation, every subsequent 12 h a sample was taken from each culture and grown in the presence of demecolcine for another 12 h. At each sampling time, the aberrations involving chromosomes 1 and 4 were analysed using dual-colour fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries. Following differential staining of sister chromatids, the analysed cells were identified to be either in their first, second or third etc. mitosis after irradiation. Cells within the same postirradiation division contained higher frequencies of aberrations when derived from later sampling times, indicating a delay in progression of aberrant cells to mitosis. In contrast, when the aberration frequencies are calculated by sampling time (i.e. independent of the cell cycle) minimal effect of sampling time could be seen. This observation held true for all types of chromosomal aberrations. Analysis of about 2250 first-division cells for each donor (derived from all sampling times) indicates a relative overrepresentation of chromosome 4 in the formation of exchange aberrations/colour junctions. Whereas dicentric frequencies for chromosomes 1 and 4 were close to the expected values based on the DNA content of these chromosomes, frequencies of reciprocal translocations showed a clear overinvolvement of chromosome 4. This resulted in a distinct difference in the reciprocal translocation to dicentric ratio, being 1.12 for chromosome 1 and 2.09 for chromosome 4. These results indicate a non-DNA-proportional distribution of radiation-induced chromosome rearrangements in cultured human lymphocytes.

Detection of chromosomal aberrations by fluorescence in situ hybridization in the first three postirradiation divisions of human lymphocytes

Chromosomal aberrations in human lymphocytes were analyzed by fluorescence in situ hybridization (FISH) in the first 3 postirradiation (0 and 2 Gy) divisions. Cells were grown in the presence of BrdU, collected at different sampling times (47, 70 and 91 h) and analyzed using an alphoid centromeric probe and PCR amplified DNA libraries for chromosomes 2 and 8. Following differential staining of sister chromatids, the analyzed cells were identified to be either in the first, second or third mitosis after irradiation. The frequencies of both dicentrics and fragments showed a reduction of about 50% after each cell generation, whereas translocations were more persistent. Cells within the same postirradiation division showed higher aberration frequencies when derived from later sampling times, indicating a delay in progression of aberrant cells. As a result, the frequencies for dicentrics and fragments remained rather constant at different sampling times if the cell cycle parameter was not taken into account. Thus, the average generation time of the lymphocytes had a clear effect on the obtained aberration frequencies. The described method allows the study of the persistence of chromosome damage using the FISH technique during 3 subsequent cell divisions in vitro.

Do chiasmata disappear? An examination of whether closely spaced chiasmata are liable to reduction or loss

The questions of whether closely spaced crossovers could be misidentified as single chiasmata or could cancel out each other and whether exchange could occur without chiasma formation were examined in Locusta migratoria. Monochiasmate bivalents that showed differential sister chromatid staining following bromodeoxyuridine incorporation were screened for their patterns of label distribution. Half of the chiasmata were associated with an exchange between dark and lightly stained chromatids, as expected if recombination involved any two non-sister chromatids chosen at random. Two variant types of label distribution were also seen: approximately 10% of all monochiasmate bivalents had an anomalous distribution of dark and light chromatids around the chiasma, and in three of the 1365 bivalents screened a second type of anomalous pattern was observed for the first time, in which all four chromatids had a label exchange at the chiasma. The observed incidence of the latter was considerably less than expected if they originated through a four-strand double crossover with closely positioned exchanges. Analysis of label distribution patterns in monochiasmate bivalents did not produce evidence of the other configurations expected if two closely spaced exchanges could be misidentified as single chiasmata or could cancel one another out so as not to form a chiasma. We conclude that analysis of chiasma frequencies and distributions offers an accurate means of assessing recombination in organisms with favourable cytology.

Effect of renutrition on the proliferation kinetics of PHA stimulated lymphocytes from malnourished children

The fraction of lymphocytes that responded to phytohemagglutinin (PHA) stimulation and initiated cellular proliferation (stimulation index or SI) was determined in groups of healthy and severely malnourished children. SI was determined again in the latter group after a period of nutritional recovery. The proportion of interphasic cells showing PHA response was assessed adding bromodeoxyuridine to the culture, so proliferative nuclei appear big and stain light blue, with dispersed granular chromatin and apparent nucleoli, while non-proliferative nuclei look small, stain red, and have compact and homogeneous chromatin. In mitotic nuclei, differential staining of sister chromatids made it possible to distinguish cells that had gone through one, two and three or more proliferation cycles. Based on the data obtained from interphase nuclei and mitosis, the SI was estimated 48 and 72 h of culture. SI were higher in lymphocytes from healthy children than in those from children with severe malnutrition, even after the period of nutritional recovery. However, the SI was significantly higher in lymphocytes from malnourished children after nutritional recovery. Although in these children more cells are stimulated, there seems to be still damage that causes a cycling delay.

Combined FISH painting and harlequin staining for cell cycle-controlled chromosome analysis in human lymphocytes.

We present a simple method that allows scoring of FISH-painted chromosomes exclusively in the first division human lymphocytes. It consists of a combination of FISH with chromosome-specific libraries and a differential sister chromatid staining after BrdU treatment of lymphocyte cultures. The method allows a precise quantification of induced chromosome damage for human biodosimetry.

Chemical induction of quadruple and octuple chromosomes in Chinese hamster CHO-K1 cells and relationship between their three-dimensional structure and spatial distribution of BrdU-labeled chromatids

Double endoreduplication of Chinese hamster CHO-K1 cells that exhibited quadruple chromosomes at metaphase was induced by a combination of rotenone and ammonium vanadate treatments. Analysis of sister chromatid differential staining patterns (using 5-bromo-2'-deoxyuridine) revealed that approximately 50% of the quadruple chromosomes did not keep the scheme of "outside replication" of DNA. Based on the ratio of the staining patterns observed, we suggest that the two diplochromosomes forming a quadruple chromosome are held together by a physical link connecting the two original chromatids. Metaphases with octuple chromosomes were also produced by the same treatment. Each chromosome constituting an octuple chromosome was longer and thinner than ordinary metaphase chromosomes. This suggests incomplete chromosome condensation at metaphase. The majority of octuple chromosomes showed the eight constituent chromosomes to be so enmeshed that a planar alignment could not be observed in air-dried preparations.

Sister-chromatid exchange (SCE) and cell proliferation in lymphocytes from infected and non-infected children with severe protein calorie malnutrition (PCM)

The frequency of sister-chromatid exchanges (SCE) and the rate of cell proliferation were evaluated through differential staining of sister chromatids in mitogen-stimulated cultured lymphocytes sampled from five well-nourished children, from seven severely malnourished children infected with bacterium, and from 10 severely malnourished children following treatment for infection with antimicrobial drugs 2 weeks before blood sampling. The replication indices at 48 h of culture were higher in both groups of malnourished children than in the well-nourished children, indicating either a faster response to PHA and/or a shorter cell cycle in lymphocytes of these patients. The average frequency of SCE per mitosis was also significantly higher than in the control group. The mitotic index was similar in the three groups of children. The lack of significant difference in response between the two groups of malnourished children suggests that the effects observed at the cytogenetic level are caused by severe malnutrition per se, and not by any associated infection.

Methods for the analysis of cellular kinetics in PHA-stimulated blood lymphocytes using BrdU incorporation. A comparative study

The cellular cycle (cc) span was measured by using differential sister-chromatid staining (DSCS) and applying the following methods: cellular cycle time (cct) according to the graphic method of Dutrillaux and Fosse (G-cct); the analytical equation (A-cct) proposed in the present paper, and the average generation time (AGT) suggested by Ivett and Tice. The mean values obtained by the three methods were 12.5, 12.7, and 19.5 h, respectively. A-cct is the more precise method, since the equation of the analytical procedure allows the utilization of numerical data, and when the graphical method is used, the values plotted in a graph may vary according to the employed scale. Cct is the choice over AGT because the first evaluates actively dividing cells and only considers those at M2 or M3. It will be useful to study cell proliferation kinetics in genetic pathological conditions and to investigate with accuracy the effect of cytostatic and cytotoxic drugs.

MAC (Morphology, Antibody, Chromosomes) Method for Study of Cell Proliferation in Unfractionated Human Hematopoietic Cell Cultures

AbstractThe MAC (Morphology, Antibody, Chromosomes) method allows analysis of the karyotype of morphologically and immunologically classified interphase and mitotic cells. Its combination with harlequin staining procedures offers also a direct method for analyzing cellular kinetics. Our studies with mononuclear leukocyte cultures demonstrated that phytohemagglutinin (PHA) is as efficient as pokeweed mitogen (PWM) in inducing B cell proliferation when heterogeneous lymphocyte populations are used and that the combination of human heat-inactivated AB serum and interleukin-2 stimulates CD8 lymphocytes to proliferate almost exclusively. By combining MAC and the differential sister-chromatid staining procedure, we have also shown that human lymphocytes proliferate faster in cultures stimulated by PWM than in those stimulated by PHA. The MAC methodology, thus, appears very useful in studies of the effects of different growth factors and lymphokines on hematopoietic cells and in the recognition of neoplastic and...

Induction of endoreduplication in double minutes of the human neuroblastoma cells and the replication pattern.

Induction of endoreduplication (ERD) using Hoechst 33258 as well as colcemid was carried out in cultured neuroblastoma (NB) line cells. In these endoreduplicated cells, the majority of double minutes (DMs) appeared to take a diplochromosome like configuration to form a cluster consisting of four minute elements, assuming a complex DM. Sister chromatid differential staining (SCD) using 5-bromo-2'-deoxyuridine (BrdUrd) revealed the non-random distribution of the stained chromatids among four chromatids composing each diplochromosome, suggesting the occurrence of so-called "outside replication" of DNA strands during the process of ERD. The same pattern of differential staining was also found in the quadruple minutes of each endoreduplicated DM. Since DMs are acentric, the present results suggest that centromeres do not play any essential role in the formation of diplochromosomes observed in the conventional cytologic preparations and that centromeres are probably not responsible for the phenomenon of the "outside replication" of DNA strands.

Sequential and combined G-banding for identifying breakpoints in sister chromatid exchanges.

A simple new method is described for obtaining sequential and a combination of differential sister chromatid staining and G-banding in the same metaphase. Using this method the sister chromatid exchanges and chromosome lesion breakpoints can be precisely localized in particular bands of individual chromosomes.

The Djungarian hamster,Phodopus sungorus, for the analysis ofin vivosister chromatid exchange

SUMMARYThe Djungarian hamster, Phodopus sungorus, is an organism used extensively in biomedical research. In this investigation, it was examined for its suitability for the in vivo study of sister chromatid exchange (SCE). Bromodeoxyuri- dine (BrdU) incorporation was performed by subcutaneous implantation of a commercially prepared 50 mg slow-release pellet, and sister chromatid differential staining was performed using incandescent light exposure of bone marrow metaphase cells. SCE frequency was markedly increased in animals injected with cyclophosphamide (CP). Cells of P. sungorus treated with this method showed clear and distinct sister chromatid differential staining, indicating that this organism is suitable for the in vivo study of SCE.

Length of cell cycle in vitro and sister-chromatid exchange (SCE) frequency in bone marrow cells from severely malnourished rats

Cell proliferation and SCE frequency were evaluated through differential staining of sister chromatids in cultured bone marrow cells from rats malnourished during the lactation period. Cell proliferation was studied in vitro in sequential analysis every 5 h in cultures from 20 to 40 h of incubation. Results show a longer generation cycle in malnourished rat cells, revealing a delay in cell proliferation. Cells of this group of animals showed a higher percentage of first-cycle metaphases and lacked third-cycle metaphases even after 40 h of culture. This shows that the damage caused to cells of undernourished organisms used in this experiment persists even when they are placed in a nutrient-rich medium. The SCE frequency did not show differences between malnourished rats and their controls.