SCHEDULED PERTURBATION IN DNA DURING IN-VITRO DIFFERENTIATION OF MOUSE EMBRYO-DERIVED CELLS

Studies of sister chromatid exchanges (SCE) and recombination rate of certain mini-satellite DNAs have demonstrated that their levels are co nsiderably higher during the preimplantation stage than in latest deve lopmental stages of embryos. It appeared likely that single-strand DNA breaks (SSB) may be relevant to both events during early development. With this in mind, we estimated SSB during in vitro retinoic acid (RA )-induced and spontaneous differentiation of mouse teratocarcinoma (EC ) and embryonic stem (ES) cells. Using the method of nucleoid sediment ation and single-cell DNA electrophoresis, we have observed a dramatic increase in the SSB during the first 2-4 mitoses after beginning of d ifferentiation of EC cells, followed by a gradual return to the basal level characteristic of undifferentiated cells. The increase in the SS B was manifested as the appearance of mass nucleoids with slow sedimen tation rates, as well as the low-weight mass fragments in DNA patterns of most cells. We concluded that not less then half of genomic DNA ha s been nicked at the early steps of differentiation. The decrease in S SB level was observed in spite of continuing differentiation, as judge d by embryonic antigens and morphological criteria. Also, the increase in the SCE level coincided with that of SSB, possibly being its conse quence. The scheduled ''surge'' of SSB may be the earliest event in co mmencing differentiation at steps without a phenotypic manifestation. (C) 1997 Wiley-Liss, Inc.