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) promoter/reporter gene transfection experiments to investigate DNA sequences required for serum-responsive regulation of expression from the mouse thymidine kinase promoter. Mutations were targeted to each of three previously described protein binding domains (MT1, MT2, and MT3) upstream of the TK translation initiation site, as well as to sequences within 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 observed, do they act positively or negatively on gene expression? The results of these experiments indicated that, in the wild-type TK promoter, 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 others appear to act negatively. In particular, mutagenesis of the Sp1 site within MT1 virtually eliminated promoter function, whereas mutations in either the MT2 site or the TK first exon rendered reporter gene expression nearly constitutive with respect to serum. Thus, both MT2 and sequences within the TK first exon appear to contain negatively acting elements. In contrast, mutation or deletion of the MT3 site produced a much less pronounced effect on reporter gene regulation. These results support recent observations from our laboratory (Q-P. Dou et al., manuscript in preparation) indicating that although the protein complexes that bind to these various sites are similar, they are not identical.(ABSTRACT TRUNCATED AT 250 WORDS)
Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
J L
JL
Fridovich-Keil
P J
PJ
Markell
J M
JM
Gudas
A B
AB
Pardee
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001790:Blood Physiological Phenomena; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
D013937:Thymidine Kinase