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.     

Differential silver carbonate staining of sister chromatids in BrdU-substituted chromosomes

Summary Differential staining of sister chromatids in BrdU-substituted human chromosomes is demonstrated by an ammoniacal silver carbonate procedure. With this method the chromosomes exhibit a subchromatid structure. Because proteolytic treatment indicated that the silver carbonate binds the chromosome proteins, changes of these components may be inferred in the BrdU-substituted chromosomes. Sister chromatid exchanges could be identified.This study was supported in part by research grant 12/119/77 from the Instituto Nacional de Previsión (Seguridad Social).     

Differential gene activity visualized on sister chromatids after replication in the presence of 5-azacytidine

Mitotic chromosomes with sister chromatids bearing differentially active ribosomal gene clusters were recovered from human lymphocytes exposed to 5-azacytidine. The hypothesis was that the differential activity was determined by the hypomethylation of one of the two sister chromatids. The verification was carried out by labeling the 5-azacytidine-substituted chromatid with BUdR, and then checking the location of active clusters by specific staining techniques. Data obtained confirmed that the chromatid bearing the active cluster was indeed the 5-azacytidine-substituted one.     

Differential giemsa staining of sister chromatids after extraction with acids

Chromosomes of Chinese hamster strain cells were air-dried on slides after BrdU substitution for two or three rounds of replication. The preparations were treated with 20% PCA at 55 degrees C for 20-30 min, or 5N HCl at 55 degrees C for 15-20 min. After staining with Giemsa, unifilarly BrdU-substituted chromatids stained faintly and bifilarly substituted chromatids stained darkly. Such a pattern of sister chromatid differential staining was confirmed by the examination of metaphase cells grown with BrdU for three rounds of replication.     

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.     

Factors involved in differential giemsa-staining of sister chromatids

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

Role of 5-bromodeoxyuridine in the formation of sister chromatid exchanges in human lymphocytes exposed to gamma-irradiation at the presynthetic stage of the mitotic cycle

The formation of SCE was studied in human lymphocytes irradiated at the presynthetic stage of the cell cycle in the presence or absence of 5-bromodeoxyuridine. The results obtained showed that 5-bromodeoxyuridine present in the culture medium at the time of exposure or during the postirradiation period increased the yield of SCE. It is suggested that 5-bromodeoxyuridine exerts its effect through interfering with DNA repair.     

Effect of 5-bromodeoxyuridine substitution on sister chromatid exchange induction by chemicals

The fluorescence-plus-Giemsa (FPG) technique for analysis of sister chromatid exchange (SCE) is widely used as an assay for mutagenic carcinogens. There is very little information, however, on whether incorporation of the bromodeoxyuridine (BrdU) necessary for visualization of SCEs affects the sensitivity of the SCE test system to different chemical agents. We have investigated the effect of BrdU incorporation on SCE induction by labeling cells with BrdU for either the first cell cycle or the first and second cell cycles. The cells were then treated with bleomycin, which produces DNA strand breakage; proflavine, which intercalates into DNA; mitomycin C, which produces monoadducts and DNA crosslinks; or aphidicolin, which inhibits DNA polymerase . Chemicals were added before BrdU exposure or during the first, second, or both cell cycles. Only mitomycin C, which induces long-lived lesions, elevated the SCE frequency when cells were treated before BrdU labeling. When bleomycin, proflavine, or mitomycin C was present concurrently with BrdU, the frequency of SCEs was increased independently of the BrdU labeling protocol. Aphidicolin, on the other hand, induced more SCEs when present for the second cell cycle, when DNA replicates on a template DNA strand containing BrdU. We also examined the induction of SCEs in the first cell cycle (twins) and in the second cell cycle (singles) after continuous treatment of cells with BrdU and the test chemicals. Only aphidicolin increased SCE frequency in the second cell cycle. These results indicate that aphidicolin, but not bleomycin, proflavine, or mitomycin C, affects BrdU-substituted DNA and unsubstituted DNA differently. This type of interaction should be taken into consideration when the SCE test is used as an assay system.     

Differentiation of the mechanisms inducing segmentation and asymetry of chromatids after treatment with 5-bromodeoxyuridine]

Rabbit anti-rat mast cell antibody is capable of liberating histamine from rat peritoneal mast cells in the presence of complement. The cytotoxicity of this complement-mediated histamine release mechanism is attested by a substantial reduction of cell ATP, release of ⁵¹Cr and ⁸⁶Rb and lytic ultrastructural changes. Inhibition of complement-dependent cytotoxic histamine release can be achieved by depressing mast cell ATP with 2,4-dinitrophenol (1 mM), antimycin A (0.2 μM) or potassium cyanide (1 mM). Restoration of cell ATP is accompanied by reversal of the inhibition of the cytotoxic histamine release. Ultrastructural analysis and ⁵¹Cr release studies reveal that in mast cells depleted of ATP, cytolysis occurs but perigranule membranes remain intact, thus preventing histamine release.     

Differential regulation of short- and long-tract gene conversion between sister chromatids by Rad51C

The Rad51 paralog Rad51C has been implicated in the control of homologous recombination. To study the role of Rad51C in vivo in mammalian cells, we analyzed short-tract and long-tract gene conversion between sister chromatids in hamster Rad51C(-/-) CL-V4B cells in response to a site-specific chromosomal double-strand break. Gene conversion was inefficient in these cells and was specifically restored by expression of wild-type Rad51C. Surprisingly, gene conversions in CL-V4B cells were biased in favor of long-tract gene conversion, in comparison to controls expressing wild-type Rad51C. These long-tract events were not associated with crossing over between sister chromatids. Analysis of gene conversion tract lengths in CL-V4B cells lacking Rad51C revealed a bimodal frequency distribution, with almost all gene conversions being either less than 1 kb or greater than 3.2 kb in length. These results indicate that Rad51C plays a pivotal role in determining the "choice" between short- and long-tract gene conversion and in suppressing gene amplifications associated with sister chromatid recombination.     

DNA lysis is involved in the simplified fluorescence plus Giemsa method for differential staining of sister chromatids

The DNA labelling of the bifilarly 5-bromodeoxyuridine- to-substituted chromatid decreased relative to that of the unifilarly substituted chromatid with increasing duration of HB pretreatment (Hoechst 33258 plus black light at 55° C). Sister chromatid differential staining was detected by Giemsa as well as a DNA-specific dye, ethidium bromide, after 4 s of HB pretreatment. The contrast of sister chromatid differential staining was improved with increased duration of HB pretreatment or by incubation with exonucleases. Hydrogen donors such as cysteamine, cysteine, and L-ascorbic acid inhibited the HB pretreatment, but this inhibition could be overcome by increasing the duration of HB pretreatment.     

Effect of 5-bromodeoxyuridine concentration on sister chromatid exchange frequency in consecutive replication cycles

A yield of single and twin sister chromatid exchanges (SCE) in Chinese hamster cells incubated at different concentrations of 5'-bromodeoxyuridine (BrdUrd) has been studied. The ratio of SCEs formed in the second and in the first cycle has been discovered to be dependent on the dose of BrdUrd; it is 1.5:1 for the high concentration of BrdUrd and 1:1 for the lowest one. The authors arrived at a conclusion that the observed level of SCEs--0.1 per chromosome per cycle for the lowest concentration is spontaneous.     

Effect of 5-bromodeoxyuridine on incorporation of RNA precursors in sea urchin embryos

Exposure of early sea urchin embryos to 5-bromodeoxyuridine (at concentrations up to 100 μg per ml) severely decreases the uptake of exogenous ³H-uridine into RNA. However, the actual gross rate of DNA or RNA synthesis in these embryos appears not to be affected by the presence of 5-bromodeoxyuridine.