Distribution of DNA Cleavages Induced by Bleomycin and Neocarzinostatin in A Defined Sequence of Rat Glioma Cells

Abstract

We have demonstrated the usefulness of a highly reiterated sequence of rat DNA as a probe sequence for evaluating the effect of bleomycin (BLM) and neocarzinostatin (NCS) at the level of individual nucleotides. The 370 base pairs (bp) DNA fragment, purified from rat glioma C6 cells after Hind III digestion, was labeled with ³²P at either the 3′- or the 5′-ends and then divided into 167 bp and 203 bp by Hae III. These end-labeled DNA fragments were reacted in vitro with BLM or NCS, and electrophoresed on the denaturating 8% polyacrylamide gels according to Maxam and Gilbert's sequencing protocol. BLM created DNA strand breaks at the guanine-cytosine and guanine-thymine (5′ → 3′) sequences, and NCS cleaved DNA at the positions of thymines and adenines. The highly reiterated sequence of rat brain tumor DNA therefore provides adequate knowledge of DNA damages induced by BLM and NCS.