Complementary Strands of Bacteriophage phi29 Deoxyribonucleic Acid: Preparative Separation and Transcription Studies.

Abstract

Bacillus subtilis phage phi29 has a nonpermuted, duplex deoxyribonucleic acid (DNA) with cohesive ends and a molecular weight of 11 x 10(6). Denaturation of this DNA yielded two intact polynucleotide chains. Preferential binding of the polyribonucleotide polyuridylic-guanylic acid (poly UG) to the complementary strands of denatured phi29 DNA permitted separation of the strands in neutral CsCl gradients. In analytical CsCl density gradient centrifugation, the separated strands with poly UG appeared as two symmetrical bands, both heavier than the normal denatured DNA band. The strands differed in density by 11 mg/cc. Preparative separation of the phi29 DNA strands resulted in two fractions, heavy (H) and light (L). The H fraction was essentially free from L contamination, whereas L contained up to 25% of H, as determined both by rebanding the separated fractions in CsCl and by electron microscopic examination of self- and mixed-annealed fractions. Pulse-labeled ribonucleic acid (RNA) prepared at intervals after infection was hybridized with the self-annealed DNA strands. Preliminary experiments indicated that both strands of phi29 DNA are transcribed during the development of the virus. Early transcribed phi29-specific RNA hybridizes only with the L strand; at later times, transcription occurs from both the L and H strands.