We used nuclease digestion to map RNA transcripts encoded in the BamHI M fragment of the Epstein-Barr virus (EBV) genome (strain B95-8). Of the five RNAs, three are rightwardly transcribed, have different cap sites but common 3' termini, and are unspliced. The two remaining RNAs are leftwardly transcribed and are 5' and 3' coterminal. One of these transcripts is spliced, resulting in the removal of a small intron from the 5' region of this RNA. We have previously published data which indicated that the BamHI M region is the first actively transcribed region of the viral genome during the replicative cycle, suggesting that one or more genes in this region is important in the initiation of EBV replication. We have now mapped two large EcoRI restriction fragments which span approximately 75% of the P3HR-1 defective genome and which contain DNA from the BamHI M region of the standard genome. The data indicate that only the coding and 5' flanking sequences for the leftwardly transcribed RNAs are intact within the defective genome. Fewer than 500 bases coding for the 3'-most regions of the rightwardly transcribed RNAs are intact, and it is unlikely that these encode functional native polypeptides. Therefore, it seems that transcriptional activation of the BamHI M-region genes is not mediated directly by the rearrangement of M genes in defective P3HR-1 EBV
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