Circular DNA of 3T6R50 double minute chromosomes

Abstract

In pulsed field gradient gel electrophoresis (PFGE) the intact deproteinized circular DNA of Mycoplasma (800 kb) and Escherichia coli (4700 kb) remains trapped in the slot. We show here that gamma-irradiation of the DNA in agarose plugs is a convenient method to partially convert these circles into full-length linears, migrating with the expected mobility in PFGE. We have used this method to study the structure of Double Minute chromosomes (DMs) from the methotrexate (MTX)-resistant mouse cell line 3T6R50. Intact deproteinized DM DNA is immobile in these gels, but is converted into a single band of about 2500 kb by either gamma-irradiation, DNaseI in the presence of Mn2+, or restriction enzymes. We conclude that the DM DNA in 3T6R50 cells consists of a homogeneous population of 2500-kb circles.