Presence of regulatory sequences within intron 2 of the mouse thymidine kinase gene

The intron 2 of the murine thymidine kinase (TK) gene was observed to contain two DNase hypersensitive site. In vitro footprinting experiments indicated specific binding sites for nuclear proteins which were characterized within the sequence of intron 2. Two GC boxes (binding sites for transcription factor SP1) and two new protein binding regions, one at the promoter proximal end of intron 2, the other one close to the border to exon 3 were found. Oligonucleotides were synthesized comprising the two new binding sites and were shown in gel mobility shift experiments to be capable of forming specific complexes with nuclear proteins. These proteins are present in growing as well as in quiescent cells suggesting that the sites described here do not contribute to growth regulation of TK expression. That they might play a role in upregulation of TK expression is, however, indicated by the results of CAT assays in which inclusion of downstream sequences of the TK gene containing parts or all of intron 2 were found to positively modulate the activity of the TK promoter.