Three-way differential staining of sister chromatids in M3 chromosomes: Evidence for spontaneous sister chromatid exchanges in vitro

Abstract

Three types of Giemsa differential staining of sister chromatids were observed in HeLa cells when they were exposed continuously to 5-bromodeoxyuridine (BrdUrd) for three replication cycles. In type-1, about a half set of chromosome complements were composed of pairs of darkly-stained and intermediately-stained chromatids; the other half consisted of pairs of intermediately-stained and lightly-stained chromatids. In type-2, one fourth of chromatids was stained darkly and the remaining ones were stained lightly. In type-3, about a half set of chromosomes consisted of the pairs of darkly-stained and lightly-stained chromatids and the rest of pairs of intermediately-stained and lightly-stained chromatids. Cells showing each differentiation pattern at the third mitotic phase were dependent on the stages of the first DNA synthetic (S) phase at which BrdUrd treatments were initiated. Type-1 cells were observed, when BrdUrd treatment was initiated anywhere from G1 to early S phase, type-2 when treatments were begun in middle S stage, and type-3 when treatments were initiated in the late stages of the first S phase. The appearance of the three types seems to be caused by a different amount of BrdUrd incorporated into DNA between the first (S1) and the second S period (S2). The amount of BrdUrd incorporated is as follows: in type-1 S1>S2, in type-2 S1≒ S2 and in type-3 S2>S1. By analysing type-1 cells, all of the sister chromatid exchanges (SCEs) occurring during each replication cycle can be accurately counted and distinguished from one another. In cells exposed to BrdUrd above 5 μg/ml, the frequencies of SCEs occurring during S1, S2, and S3 are higher than those detected at lower BrdUrd concentrations. On the other hand, at lower concentrations (0.1–1.0 μg/ml) they occurred at the same frequency during S1, S2, and S3. Thus, SCEs detected at low concentrations are free from the incremental effect of BrdUrd incorporated, and enable us to estimate the spontaneous level of SCE frequency.