Induction of Chromosomal Aberrations by the Restriction Endonuclease AluI in Chinese Hamster Ovary (CHO) Cells: Influence of Glycerol on Aberration Frequencies

Abstract

Restriction endonucleases (REs) induce chromosomal aberrations in living cells indicating that the ultimate lesion for the production of chromosomal aberrations is a DNA double-strand break (Obe et al. 1987; Bryant, this Vol.; Morgan et al., this Vol.). It is still not clear how living cells can uptake REs. Different methods have been used to treat cells with REs (for discussion see Bryant 1988; Morgan et al., this Vol.) and all of them are more or less successful when measured as frequencies of chromosomal aberrations induced by REs. From our own results concerning the induction of chromosomal aberrations with AluI following treatment of pellets of 4 × 106 cells (about 10 µl volume) with 8 µl of a suspension of REs and newborn calf serum, we proposed that REs are internalized via an energy-dependent receptor-mediated mechanism (Johannes et al. 1989; Obe et al. 1989; Vasudev and Obe 1987). Analyses of Bryant and Christie (1989) have shown that the uptake of REs using the “pellet technique” is dependent on the shipping buffer in which the REs are suspended. The authors assume that “this may result from a combination of hypertonic shock and the detergent present in the shipping buffer” (Bryant and Christie 1989). In order to avoid a possible influence on the uptake of AluI by the relatively high concentrations of buffer components we analyzed the chromosome breaking effects of AluI by treatment of monolayers in such a way that the concentrations of the buffer components are considerably lower when compared to our earlier method.