DNA-SEQUENCES REQUIRED FOR SERUM-RESPONSIVE REGULATION OF EXPRESSION FROM THE MOUSE THYMIDINE KINASE PROMOTER

We have used site-specific mutagenesis and thymidine kinase (TK) promo ter/reporter gene transfection experiments to investigate DNA sequence s required for serum-responsive regulation of expression from the mous e thymidine kinase promoter. Mutations were targeted to each of three previously described protein binding domains (MT1, MT2, and MT3) upstr eam of the TK translation initiation site, as well as to sequences wit hin the TK first exon in order to address each of the following three questions: (a) Do these sequences play any role in regulation? (b) Do all of these sites play the same role? and (c) If any controls are obs erved, do they act positively or negatively on gene expression? The re sults of these experiments indicated that, in the wild-type TK promote r, at least some of these sequences do play a role in regulation, that not all of these sites appear to play the same role, and that some of the targeted elements act positively on gene expression, whereas othe rs appear to act negatively. In particular, mutagenesis of the Spl sit e within MT1 virtually eliminated promoter function, whereas mutations in either the MT2 site or the TK first exon rendered reporter gene ex pression nearly constitutive with respect to serum. Thus, both MT2 and sequences within the TK first exon appear to contain negatively actin g elements. In contrast, mutation or deletion of the MT3 site produced a much less pronounced effect on reporter gene regulation. These resu lts support recent observations from our laboratory (Q-P. Dou et al., manuscript in preparation) indicating that although the protein comple xes that bind to these various sites are similar, they are not identic al. Although the exact roles and interplay of these elements and the f actors that bind them remain unclear, the functional significance of t hese sequences is established.