Transcription Vectors That Facilitate the Identification and Mapping of RNA Splice Sites in Genomic DNA

Abstract

Two transcription vectors were constructed that can identify the splice sites at exon–intron boundaries of inserted DNA fragments possessing the complementary splice site. One vector contains the 5′ splice donor site and flanking exon—intron sequences from the 3′ end of the adenovirus first late leader. The other vector contains the 3′ splice acceptor site and the branch acceptor site, plus the flanking exon–intron sequences from the 5′ end of the adenovirus second late leader. Both vectors contain a multiple cloning site for insertion of DNA fragments. DNA fragments supplying the complementary splice site, including the adjacent exon and intron sequences, were inserted into the vectors. The vectors were used as templates for the synthesis of chimeric RNA transcripts that were spliced in in vitro splicing extracts. Chimeric transcripts from the vectors containing complementary splice site boundary regions from the human growth hormone gene were accurately spliced in vitro. A splice site from a human growth hormone intron that is not normally spliced in vitro was spliced when paired with an adenovirus splice site. These vectors can be used to identify splice sites and to determine the lengths of exons and their attached introns within a DNA fragment of unknown coding content.