INDUCTION OF CIRCLES OF HETEROGENEOUS SIZES IN CARCINOGEN-TREATED CELLS - 2-DIMENSIONAL GEL ANALYSIS OF CIRCULAR DNA-MOLECULES

Extrachromosomal circular DNA molecules are associated with genomic in stability, and circles containing inverted repeats were suggested to b e the early amplification products. Here we present for the first time the use of neutral-neutral two-dimensional (2D) gel electrophoresis a s a technique for the identification, isolation, and characterization of heterogeneous populations of circular molecules. Using this techniq ue, we demonstrated that in N-methyl-N'-nitro-N-nitrosoguanidine-treat ed simian virus 40-transformed Chinese hamster cells (CO60 cells), the viral sequences are amplified as circular molecules of various sizes. The supercoiled circular fraction was isolated and was shown to conta in molecules with inverted repeats. 2D gel analysis of extrachromosoma l DNA from CHO cells revealed circular molecules containing highly rep etitive DNA which are similar in size to the simian virus 40-amplified molecules. Moreover, enhancement of the amount of circular DNA was ob served upon N-methyl-N'-nitro-N-nitrosoguanidine treatment of CHO cell s. The implications of these findings regarding the processes of gene amplification and genomic instability and the possible use of the 2D g el technique to study these phenomena are discussed.