The polyomavirus enhancer consists of functionally redundant DNA sub-elements. One such sub-element, element 2, comprises a region with contiguous binding sites, or motifs, for at least three nuclear factors, designated as PEA1, PEA2, and PEA3. Although little is known of PEA2, PEA1 is presumed to be a murine homolog of human transcription activator protein 1 (AP-1), and PEA3 has recently been shown to be encoded by a member of the Ets family of oncogenes. The contributions of each factor to enhancer function are not understood. A cell-free system was devised to assay the individual abilities of the DNA motifs recognized by PEA1, PEA2, and PEA3 to confer transcriptional activation upon a minimal promoter. The motifs were cloned and tested as monomers, as multiple tandem copies, and in paired combinations. The results of these in vitro studies indicate that the PEA1 motif behaves as a low affinity AP-1 binding site; that PEA1 and PEA3, but not PEA2, activate transcription; and that both the PEA1 and PEA3 motifs act synergistically. Band shift titration experiments demonstrated that neither PEA1 nor PEA3 bound to their DNA motifs co-operatively, indicating that synergistic activation of transcription by these factors is not due to cooperative binding. Finally, additional in vitro transcription experiments suggest that PEA1 and PEA3 may co-operate with each other to stimulate transcription. A current model proposes that the minimal sub-units of enhancer structure are small (8-10 base pair) DNA motifs, called enhansons, that act synergistically. I propose that the motifs for PEA1 and PEA3, but not PEA2, are enhansons of the polyomavirus enhancer. / Thesis / Master of Science (MS)

This thesis is missing page 157, the other copies of the thesis did not have the page either. -Digitization Centr / The polyomavirus enhancer is organized into three elements. One of these elements, Element 2, is particularly interesting because the activities of the factors which interact with it are highly regulated. There are at least three cellular proteins, PEA1, PEA2, and PEA3, which bind to adjacent sites in Element 2. These proteins are differentially active in mouse cells at different developmental stages and their activity is modulated by serum, tumor promoting agents and the products of several oncogenes. It is likely, therefore, that these cellular proteins play an important role in interpreting growth stimuli and other physiological cues in the mouse. A plasmid was contructed which can be used to test enhancer elements for their ability to activate both transcription and DNA replication. This plasmid includes the Py origin of replication and a minimal promoter, consisting of a TATA box only, controlling expression of a reporter gene. The activity of the PEA factors was studied by cloning the binding sites for these factors into this reporter plasmid as monomers, multiple tandem copies, and in paired combinations, and testing their ability to activate transcription and DNA replication in vivo. The results of these studies show that PEA1 and PEA3 can function independently and cooperatively to activate both replication and transcription. By contrast, PEA2 is unable to independently activate transcription and represses PEA1-activated transcription when the binding sites for these factors are located adjacent to one another. However, PEA2 functions cooperatively with PEA1 to activate DNA replication, and can weakly activate replication on its own. / Thesis / Master of Science (MS)