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We have studied RNA transcripts of the defective parvovirus, adeno-associated virus (AAV) present in poly-(A)rich and poly-(A)free fractions of nuclear and cytoplasmic RNA prepared from cells infected together with a helper adenovirus. Cytoplasmic poly-(A)rich RNA contains three overlapping spliced AAV RNAs having sizes of 3.9 x lo3, 3.3 x lo3 and 2.3 x lo3 bases respectively. The nuclear precursors of these RNAs appear to be the coterminal unspliced poly(A)-rich RNAs containing 4.2 x lo3, 3.6 x lo3 and 2.6 x lo3 bases respectively. These unspliced RNAs were also found in the cytoplasm. The nuclear poly(A)-free RNA contained a heterogenous population of AAV RNAs that were generally smaller than 2.3 x lo3 bases. In addition, the Hirt pellet fraction of the nuclear RNA contained two discrete AAV poly(A)-free RNAs having sizes of 2.5 x lo3 and 2.8 x lo3 bases.Thc 4.2 x lo3 and 3.6 x 103-base unspliced RNAs are more abundant than the coterminal 3.9 x lo3 and 3.3 x 103-base spliced RNAs whereas the 2.3 x 103-base spliced RNA is much more abundant than the 2.6 x lo3-base unspliced RNA. Thus, the cytoplasmic abundance of the AAV spliced RNAs appears to be controlled in part by the post-transcriptional events of splicing or message stability.We also analysed the effects of AAV defective-interfering genomes upon AAV transcription. These studies showed that when synthesis of standard AAV genomes was inhibited more than 10-fold by defective-interfering genomes there was no significant effect on the types or amounts of AAV RNA transcripts which accumulated. These observations indicate that interference by defective-interfering genomes occurs mostly at the level of DNA replication rather than transcription.We previously showed that transcription of the defective parvovirus, adeno-associated virus (AAV), genome in human K B cells using a human adenovirus as the helper yielded several cytoplasmic AAV RNA species [l]. All of these RNAs were complementary to overlapping regions of the AAV DNA minus strand. Using DNA probes labeled in vivo we detected three unspliced RNAs (4.2 x lo3, 3.6 x lo3 and 2.6 x lo3 bases) having common 3' termini at map position 96.0 and 5' termini at approximate map positions of 5, 19 and 39 respectively. There were also three co-terminal spliced RNAs (3.9 x lo3, 3.3 x lo3 and 2.3 x lo3 bases respectively) with a single spliced-out region of about 350 nucleotides between map position 40 and 49. Other workers also identified and mapped the unspliced 4.2 x lo3, 3.6 x lo3 and 2.6 x lo3-base RNAs and the spliced 2.3 x 103-base RNA but did not detect the 3.9 x lo3 and 3.3 x 103-base spliced RNAs [2,3]. To resolve these discrepancies we have now performed a more extensive analysis of AAV poly(A)-rich and poly(A)-free RNAs present in both the cytoplasmic and nuclear fractions using both one-dimensional and two-dimensional gel electrophoresis.We show directly the presence of the spliced 3.9 x lo3 and 3.3 x 103-base RNAs in the cytoplasm. In addition, our data shows that splicing of these two RNAs or their accum...

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