Differential Staining of Sister Chromatid in BrdU- Substituted Chromosomes after Incubation in Alkalified NaCl Solution

Abstract

(Communicated by Saj iro MAKINO, M. J. A., Dec. 12, 1978) Recently, semiconservative segregation of DNA in eukaryotic chromosomes has elegantly been demonstrated by applying the newly developed technique of sister chromatid differential staining (Latt 1973) . This is substantiated on the fact that when cells are cultured for two successive generations in the presence of 5-bromodeoxyuridine (BrdU) and then stained with Hoechst 33258, the chromatids consisting of unifilarly BrdU-substituted (TB) DNA fluoresce more brightly than the bifilarly substituted (BB) sister chromatids.Sister chromatid differential staining (SCD) of BrdU-substituted chromosomes has also been found to occur after Giemsa staining following a variety of appropriate pretreatments (Korenberg and Freedlender 1974; Perry and Wolf 1974;Goto et al. 1975;Pathak et al. 1975; Sasaki 1976;Scheres et al. 1977 ;Sakanishi and Takayama 1977, ; Sakanishi 1977, 1978;Burkholder 1978). To-date, however, the underlying mechanisms of SCD produced by these various procedures have remained not clear.In the course of investigating the mechanisms, we have found that an incubation of BrdU-substituted chromosomes in the properly alkalified NaCI solution is capable of producing SCD of the type in which TB-chromatids stain darkly while BB-ones lightly. Material and methods.Chinese hamster strain cells of lung origin maintained in Eagle's MEM supplemented with 10% fetal calf serum were grown in the dark with 20 4eM BrdU for 26 hr (two round substitutions) or for 40 hr (three round substitutions). Colchicine (0.5 pg/ml) was added to the cultures 2 hr prior to harvest. Cells were treated with 0.075 M KCI, fixed in ethanol-acetic acid (3: 1) , and airdried. Slides were kept at room temperature for at least 24 hr prior to use, and several-day-old slides were found the best for the present method. Slides aged more than three weeks were not suitable because of the poor contrast in the resulting differential staining.The alkalified NaCI solution was prepared as follows : 1-2 M NaCI was put in a Coplin jar (common soda-lime glass container)