Deoxyribonucleic acid (DNA) molecules having a mean length of 5.8 μm were released from purified Bacillus subtilis bacteriophage φ29 with 2 m sodium perchlorate. Small 0.1 to 0.2-μm molecules were also detected in these DNA preparations. Since intact single chains annealed to form linear duplex molecules, phage φ29 DNA was found to be nonpermuted. The molecular weights of single chains of φ29 DNA were approximately half that of native DNA, as determined by analytical band sedimentation in CsCl, indicating that φ29 DNA is composed of two continuous polynucleotide chains. The molecular weight values of native and annealed φ29 DNA from sedimentation agreed with the molecular weight values obtained from electron microscopy. The infectivity of φ29 DNA was reduced to a low level by alkaline denaturation and was partially restored by annealing.

Bacillus subtilis phage 429 has a nonpermuted, duplex deoxyribonucleic acid (DNA) with cohesive ends and a molecular weight of 11 x 106. 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 4)29 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 429 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 CsCI and by electron microscopic examination of selfand mixed-annealed fractions. Pulselabeled ribonucleic acid (RNA) prepared at intervals after infection was hybridized with the self-annealed DNA strands. Preliminary experiments indicated that both strands of 429 DNA are transcribed during the development of the virus. Early transcribed 429-specific RNA hybridizes only with the L strand; at later times, transcription occurs from both the L and H strands. strands were studied by selfand mixed-annealing, banding in CsCl, electron microscopy, and transfection. Analysis of the in vivo transcription of the 4)29 strands demonstrated that both strands are transcribed. MATERIALS AND METHIODS Bacteria and bacteriophage. B. amyloliquefaciens strain H was used for the growth of phage q29 in liquid culture, and B. subtilis strain 12A was the normal plating host. For transfection studies, B.

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Infectious DNA molecules, capable of transforming chicken embryo fibroblasts, can be synthesized by the Rous sarcoma virus-associated reverse transcriptase in vitro. The optimal enzymatic conditions employed for infectious DNA synthesis also facilitate maximum synthesis of genome length DNA. Analysis of the DNA product synthesized by detergent-disrupted Rous sarcoma virus under these conditions indicates that DNA complementary to viral RNA (minus-strand DNA) is genome length in size, whereas DNA complementary to genome length minusstrand DNA (plus-strand DNA) appears as subgenomic-length molecules ranging between 300 and 3,500 nucleotides in length. These features of the DNA product synthesized by the Rous sarcoma virus reverse transcriptase in vitro are similar to those identified in the cytoplasm of cells shortly after infection and lend credence to studies of the mechanism of reverse transcription in vitro and their significance to proviral DNA synthesis in vivo.

Electron microscopic analysis of the DNA product synthesized by detergent-disrupted preparations of Rous sarcoma virus in vitro revealed the presence of several interesting molecular forms including covalently closed circular DNA. The identification of such circular DNA indicates that virions of retroviruses contain all the components necessary to facilitate the complete synthesis of mature forms of viral DNA and therefore provide a useful system to delineate the molecular mechanisms involved in their synthesis.