Splicing and polyadenylylation at cryptic sites in RNA transcribed from pSV2-neo

Mapping the structures of RNAs transcribed from the chimeric plasmid pSV2-neo in transfected COS cells revealed discontinuities within the neo portion of the transcripts. Two cryptic 5' splice sites and three cryptic 3' splice sites were identified. The cryptic 5' splice sites matched 5 or 7 bases of the 5' consensus sequence. Each cryptic 3' splice site consisted of an AG that was preceded by a 15 nucleotide region which was 73% pyrimidine. They differed from the 3' consensus sequence mainly by the presence of a purine, rather than a pyrimidine, immediately adjacent to the AG at the splice site. Greater than 99% of the transcripts were spliced at the SV40 intron. Approx. 50% of these transcripts were spliced, in a complex pattern, at the additional sites within the neo region. Deletion of the SV40 early polyadenylylation signal from pSV2-neo revealed the presence of a cryptic polyadenylylation signal in a region of pBR322 sequence. It is located in the 5' portion of the beta-lactamase gene and occurs 6 +/- 2 nucleotides downstream from the sequence AATAATAATGAA, which contains three overlapping variant hexanucleotides. The cryptic signal appears to be approx. 10-fold less efficient than the SV40 polyadenylylation signal.