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Nucleic Acids Research, 1988, Vol. 16, No. 11 4841-4851
© 1988

Articles

Circular DNA of 3T6R50 double minute chromosomes

Alexander M. van der Bliek, Carsten R. Lincke1 and Piet Borst*

Division of Molecular Biology and The Netherlands Cancer Institute Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands 1Department of Biochemistry, University of Amsterdam, Academisch Medisch Centrum Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

*To whom correspondence should be addressed

Received March 16, 1988. Revised May 13, 1988. Accepted May 13, 1988.

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.


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