Size and base composition of RNA segments in complementary strands of supercoiled plasmid ColEl DNA

Abstract

Preparations of ColEl plasmid DNA synthesized in the presence of chloramphenicol were separated into samples having gaps resulting from removal of ribonucleotides in one or the other of the complementary DNA strands. These samples were used as templates for repair resynthesis reactions using DNA polymerase of Rous sarcoma virus and α- 32P-labeled deoxyribonucleoside 5′-triphosphates. Reactions involved the incorporation of each labeled nucleotide in the presence of three unlabeled nucleotides, and also the incorporation of all four labeled nucleotides followed by complete digestion and electrophoretic separation of the products. By these two methods the RNA integrated in the light strand of ColEl DNA was found to comprise an average of 38 ribonucleotides with a base composition of 17G, 5A, 8C, and 8U. The RNA segment in the heavy strand consists of an average of 15 ribonucleotides with a base composition of 5G, 2A, 4C, and 4U.