Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation

Abstract

The herpes simplex virus (HSV) type 1 thymidine kinase gene (tk) was resected from its 3' end with BAL 31 exonuclease. Two sets of plasmids were isolated that lacked information distal to the two copies of the hexanucleotide 5'-AATAAA-3' located at the 3' end of the HSV tk gene. The presence of a simian virus 40 origin of DNA replication in each plasmid facilitated analysis of patterns of transcription in transfected Cos-1 monkey cells. Transcription analyses were performed with an S1 nuclease protection assay. Efficient processing and polyadenylation at the normal site still occurred when all sequences more than 44 or 46 base pairs (bp) downstream from the first AATAAA were removed (pTK311R/SV010 and pTK209R/SV010). Removal of an additional 7 bp (pTK312R/SV010) decreased the amount of tk mRNA processed at that normal site, and tk mRNA polyadenylated at a cryptic site within pBR322 sequences began to appear. The normal processing and polyadenylation site was not used at all when an additional 12 bp was removed (pTK314R/SV010); the small amount of tk mRNA produced was processed and polyadenylated at the cryptic pBR322 site. The region of the tk gene critical for efficient processing and polyadenylation of tk mRNA is located 20 to 38 bp downstream from the first AATAAA, distal to the polyadenylation site, and as RNA can form a stem-loop structure containing AAUAAA. Similar G + T-rich elements were located in DNA fragments which substitute efficiently for the HSV tk processing and polyadenylation signal and were not found in AATAAA-containing DNA fragments which substitute inefficiently for the HSV tk signal.