Thymidylate synthase (TS) is the target enzyme for the chemotherapeutic drug FdUrd. The overall goal of this project is to understand the biochemical mechanisms for regulating TS gene expression in mammalian cells. Previous work has focused on the regulation of TS mRNA content in growth-stimulated cells and the isolation and sequencing of the cDNA and gene for mouse TS. Several unusal fetaures of the structure and regulation of the gene were noted.
The specific aims for the next grant period are first, to identify the sequences that are responsible for the transcriptional regulation of the TS gene by analyzing the effects of DNA deletions and specific nucleotide changes. The second goal is to study the interactions of proteins with sequences that are important for regulating the transcription of the gene using DNA footprinting assays. If proteins that bind to novel regulatory sequences are identified, they will be purified and characterized. Since the promoter of the TS gene has adjacent binding sites for the cellular transcription factors Sp1 and USF, the possible cooperative or competitive interaction of these factors with their respective binding sites will be examined. Third, the sequences that are responsible for the induction of TS gene transcription by the adenovirus E1A protein will be determined. These may be different from the sequences responsible for growth induction of TS gene transcription. Fourth, an in vitro transcription system for the TS gene will be developed and used to study the regulation of TS gene transcription using purified factors. This will permit a more detailed analysis of the proteins and sequences that control TS gene transcription, and the mechanism by which they function. Finally, the polyadenylation of TS mRNA will be studied in greater detail. The sequences that are responsible for polyadenylation of TS mRNA at the termination codon will be identified, and the efficiency of utilization of the polyadenylation signal in G1 phase versus S phase cells will be determined.
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