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    Our research is directed toward understanding the mechanisms that are responsible for the regulation of gene expression in mammalian cells, with particular emphasis on post-transcriptional control mechanisms.  Our current work focuses on the mammalian thymidylate synthase (TS) gene, which codes for an important target enzyme for a variety of cancer chemotherapeutic drugs.  The enzyme is essential for the survival of proliferating cells and is strongly induced when quiescent cells are stimulated to proliferate by growth factors or when they are infected with certain viruses such as CMV.
    We have cloned the cDNA and gene for TS from several mammalian species and have identified a number of unusual structural and regulatory features of the gene, especially its promoter, polyadenylation signal and introns.  We have studied the content, rate of production, and stability of TS mRNA using a variety of biochemical and recombinant DNA techniques and have found that the S phase-specific expression of the TS gene in growth-stimulated cells is controlled primarily at the post-transcriptional level while viral induction of the TS gene is regulated at the transcriptional level.  We have constructed functional TS minigenes and transfected them into cultured cells.  The sequences that are necessary for the production and regulation of TS mRNA have been defined by deletion and site-directed mutagenesis.
    The TS promoter is unusual in that it lacks a TATA box and an initiator element and initiates transcription at many sites over a broad initiation window.  The promoter is also bidirectional.  The proteins that interact with the promoter and that are responsible for its unusual properties are being analyzed.  Introns play an important role in the expression and regulation of the TS gene.  Some (but not all) of the introns of the TS gene stimulate the expression of transfected TS minigenes.  In addition, S phase regulation of transfected TS minigenes requires the presence of an intron in the minigene as well as elements in the promoter region. S phase regulation appears to require some form of communication between the promoter region and the RNA processing machinery, which may occur via the C-terminal domain of RNA polymerase.
    We are also using RNA interference technology to analyze the specific proteins that are important for regulation of TS gene expression.  In addition, we are using this approach to develop alternative strategies for inactivating TS enzyme activity for possible therapeutic applications.