CONTROL OF MRNA SYNTHESIS IN MAMMALIAN CELLS

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The long term goal of this research effort is to understand the mechanisms by which mRNAs are synthesized in mammalian cells, and to learn how this synthesis is regulated. In particular, we will be concentrating on genes from two cell-studied DNA tumor viruses, SV40 and adenovirus. In this research effort, we will concentrate on several on aspects mRNA synthesis. First, we will exploit our recent observation that exogenously added mRNA precursors can be accurately and efficiently polyadenylated in a HeLa whole-cell lysate. We will study in detail both the genetics and biochemistry of this important reaction. Second, we showed recently that two cloned viral promoters (Ad2 late and SV40 early) have drastically different nucleotide sequence requirements in order to be expressed in two different human cell lines, HeLa and 293. We will determine the genetic basis for these effects, as well as study the biochemical mechanism responsible. Third, we will analyze in depth a series of deletion and point mutations that we constructed in the Ad2 late promoter in order to better understand the molecular basis of transcription initiation and regulation. Fourth, we will study and extend a series of insertion initiation and regulation. Fourth, we will study and extend a series of insertion mutants that we constructed in which the Ad2 late promoter has been inserted at various sites in SV40. These insertions are providing us with important insights into SV40 sites in SV40. These insertions are providing us with important insights into SV40 gene expression and transcriptional control in general. Finally, using antibodies we prepared against HeLa Topoisomerase I, we will study the effects of DNA topology on transcription initiation.