Thymidylate synthase (TS) is an essential enzyme in proliferating cells and an important target of chemotherapeutic drugs. The goal of this project is to understand the mechanisms for controlling the S phase- specific expression of the mammalian T gene. Earlier studies showed that S phase regulation of the mouse TS genes is controlled primary at the post-transcriptional level. Proper regulation of transfected mouse TS minigenes requires both the TS promoter and a spliceable intron and appears to depend on a novel form of communication between the promoter and splicing machinery. The proposed will examine the mechanism that is responsible for the post-transcriptional regulation of the mammalian TS gene, with particular emphasis on the role of introns and the basis of communication between regulatory elements in the promoter region and the RNA processing machinery. The role of introns in S phase regulation will be further investigated by determining if the efficiency of splicing of TS transcripts increases as cells progress from G1 phase through S phase and if changes in the spacing between the promoter and the intron affect S phase regulation. To determine if the unusual control mechanism has been evolutionary conserved, the human TS promoter regions as well as the requirement for introns for the S phase regulation of the human and rat TS genes. Finally, the mechanism that is responsible for communication between the TS promoter and the splicing machinery will be explored. Two approaches are described in detail. First, the possible role of the RS promoter/regulatory region in controlling the efficiency of capping of TS transcripts in a cell cycle-dependent manner will be examined. Second, a coupled transcription-splicing system will be used to determine if splicing occurs more efficiently with extracts prepared from S phase cells than from quiescent cells and if the changes in splicing efficiency are controlled bu the TS promoter/regulatory region. The results of these studies will provide additional information about the molecular mechanisms by which the TS gene is regulated and how this process may be integrated into the H1 signal transduction cascade.
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