Polyadenylation at a cryptic site in the pBR322 portion of pSV2-neo: prevention of its utilization by the SV40 late poly(A) signal

Abstract

Transcripts originating from the SV4O late promoter of pSV2-neo or pSV2-cat contain pBR322 sequences and are polyadenylated at the SV40 late poly(A) site, resulting in an RNA of 3500 nt. If the SV40(L) poly(A) signal is destroyed, late orientation transcripts are polyadenylated at a site within pBR322 sequences, yielding in an RNA of 2500 nt. This cryptic poly(A) site is located 42–46 nucleotides downstream from an AAUAAA. Utilization of the pBR322 poly(A) signal is undetectable in late orientation transcripts from pSV2-neo or pSV2-cat, although it is located 966 nucleotides upstream from the SV40(L) poly(A) signal. The pBR322 site is not utilized when the spacing between the two poly(A) signals is varied from 209 to 1913 nucleotides. The pBR322 poly(A) site was utilized only in constructs in which all or portions of the SV40(L) poly(A) signal were deleted, such as in a construct with a 7 bp deletion into the SV40(L) AATAAA and adjacent sequences.