Analysis of Transcripts Derived from Sequences Upstream of the Bidirectional Mouse Thymidylate Synthase Promoter

Abstract

The promoter of the mouse thymidylate synthase (TS) gene lacks a TATAA box and an initiator element and is capable of directing transcriptional initiation with approximately equal strength and over broad initiation windows in both directions. The goal of the present study was to determine if the TS promoter directs the transcription of a second gene that is upstream of the TS gene by characterizing the transcripts that correspond to the upstream sequences. RNA blot analyses revealed the presence of 1.4 and 5 kb cytoplasmic, polyadenylated transcripts that include sequences upstream of the TS promoter. The transcripts were much more abundant in a cell line in which the TS gene is amplified. S1 nuclease protection assays showed that the transcripts have multiple 5′ termini. An exon trap approach identified a potential splice donor site that might correspond to the 3′ end of the first exon of the upstream gene. A cDNA library was probed with a sequence from the putative first exon, and six different cDNA clones were isolated. However, analysis of the sequences of the cDNAs revealed that the upstream transcripts were not spliced at the potential 3′ donor site but instead extended into a repetitive LINE (long interspersed nuclear element) sequence that begins 0.3 kb upstream of the TS promoter. RNase protection assays confirmed that the in vivotranscripts extend into the LINE element. Therefore it appears that the upstream transcripts are unlikely to correspond to a functional mRNA molecule.