No abstract

No abstract

The double-stranded form of adeno-associated virus (AAV) DNA has about 20 sites sensitive to endonuclease R *HaeIII from Haemophilus aegyptius; the fragments produced fall into about 13 size classes, 8 of which contain single fragments. The location of the HaeIII-produced AAV fragments relative to the three EcoRi fragments was determined. Using revised figures for the molecular weights of the HaeIII cleavage products of +X174 replicative form DNA, we calculated that AAV DNA contains about 4,000 nucleotides. After HaeIII digestion of duplex DNA terminally labeled with 32P using polynucleotide kinase, the majority of fragments containing a 5' 32P label were about 40 nucleotides in length, and fragments of similar size were generated from each end, suggesting that the Hae site closest to the end is within the terminal repetition. Two moreslowly-migrating cleavage products also bore 5' 32p end label. These three terminally labeled species were also generated from single-stranded AAV DNA by digestion with HaeIII, and evidence that one may have a nonlinear ("rabbitear") structure is presented. The predominant 5' terminal base was identified as thymine for both the plus and minus strands of AAV. Single-stranded AAV molecules could not be efficiently covalently circularized by incubation with polynucleotide ligase or ligase plus T4 DNA polymerase.

No abstract

Linear OX174 single-stranded DNA can be isolated from OX phage particles produced under various conditions. About half of the linear strands have a dGMP residue at the 5' end, the remaining have roughly comparable amounts of dCMP, dTMP, and dAMP. The linear strands can be converted to covalently closed circular molecules by polynucleotide ligase, but only after they have been incubated with T4 DNA polymerase and deoxynucleoside triphosphates. Experiments with endonuclease R, the restriction enzyme from Haemophilus influenzae, indicated that the nucleotides incorporated into the DNA during this reaction were found predominantly in a limited region of the genome. The results suggest that the normal intermediate in single-stranded OX174 DNA synthesis may be a single-stranded linear molecule which is shorter than unit length and is intrinsically capable of circularization.