Genome localization of adeno-associated virus RNA

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Abstract
Previously, linear duplex molecules of adeno-associated virus type 2 (AAV2) DNA were cleaved with the restriction endonucleases R.cntdot.EcoRI, R.cntdot.HindII, and R.cntdot.HindIII. The physical order of the specific fragments obtained was deduced and oriented with respect to the DNA strand polarity and the transcription direction. Stable AAV RNA is transcribed only from 70% of the minus DNA strand. RNA-DNA hybridization experiments using these restriction fragments gave a more accurate map of the AAV genome portion represented in stable RNA. The data obtained with several sets of restriction fragments annealed to whole-cell RNA [human oral carcinoma KB3 cells] or polyA-containing RNA were internally consistent. The AAV RNA annealed with a continuous region of the AAV DNA, beginning at 0.18 map units (18%) from the left end of the molecule and ending at 0.88 map units. The restriction endonuclease BamHI made 1 specific cleavage in AAV2 DNA at 0.22 map units, which is 0.04 map units i.e., 160 nucleotides to the right down stream of the point corresponding to the 5'' end of the viral mRNA.